Molecular Systematics of Tribe Cacteae (Cactaceae: Cactoideae): A Phylogeny Based on rpl16 Intron Sequence Variation

Abstract

Parsimony analysis of plastid rpl16 sequences from 62 members of Tribe Cacteae, and four outgroup taxa yielded 1296 equally parsimonious trees of length 666. Strict consensus evaluation of these trees established a highly pectinate topology, which delimited clades within the tribe that correspond to several previously considered generic groups. Aztekium and Geohintonia, which manifest ribs in their stem morphology were shown to represent an early divergence in the tribe, forming a sister group to remaining members of the tribe. Clades containing other genera having ribbed stems also are basal to those that develop tubercles. The most derived clade forms a distinct group of typically small stemmed species with tubercular stem morphology. Within Mammillaria, species formerly placed in the genus Cochemiea and members of the Series Ancistracanthae formed a well-supported, sister clade to the remaining members of Mammillaria. Length variation of the intron in two members of Mammillaria series Stylothelae was also observed. Communicating Editor: Thomas G. Lammers

Full text

257
Systematic Botany (2002), 27(2): pp. 257–270
qCopyright 2002 by the American Society of Plant Taxonomists
Molecular Systematics of Tribe Cacteae (Cactaceae: Cactoideae):
A Phylogeny Based on rpl16 Intron Sequence Variation
C
HARLES
A. B
UTTERWORTH
,
1,3
J. H
UGO
C
OTA
-S
ANCHEZ
,
2
and R
OBERT
S. W
ALLACE
1
1
Department of Botany, Iowa State University, Ames, Iowa 50011-1020;
2
Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK
3
Author for Correspondence (cbutter@iastate.edu)
Communicating Editor: Thomas G. Lammers
A
BSTRACT
.Parsimony analysis of plastid rpl16 sequences from 62 members of Tribe Cacteae, and four outgroup taxa
yielded 1296 equally parsimonious trees of length 666. Strict consensus evaluation of these trees established a highly pectinate
topology, which delimited clades within the tribe that correspond to several previously considered generic groups. Aztekium
and Geohintonia, which manifest ribs in their stem morphology were shown to represent an early divergence in the tribe,
forming a sister group to remaining members of the tribe. Clades containing other genera having ribbed stems also are
basal to those that develop tubercles. The most derived clade forms a distinct group of typically small stemmed species
with tubercular stem morphology. Within Mammillaria, species formerly placed in the genus Cochemiea and members of the
Series Ancistracanthae formed a well-supported, sister clade to the remaining members of Mammillaria. Length variation of
the intron in two members of Mammillaria series Stylothelae was also observed.
Buxbaum (1958b) first described the tribe Cacteae
(as the Echinocacteae) as a ‘clear-cut phylogenetic unit’
in which he included all of the short-columnar or glo-
bose cacti with spineless flowers native to North
America, with the notable exception of the genus As-
trophytum Lemaire, which he considered part of the
Notocacteae. The tribe is in considerable taxonomic
flux, and poor generic delineation means that the exact
number of genera is uncertain. Twenty-three genera
are recognized in the CITES Cactaceae checklist (Hunt
1999), although at least 34 other genera have been de-
scribed. Hunt (1999) accepts 314 species (plus 224 pro-
visionally accepted species) in the tribe, whereas An-
derson (2001) recognizes 26 genera and 384 species.
The geographic range of the Cacteae extends from
western Canada (Escobaria vivipara (Nuttall) Buxbaum)
to Colombia, Venezuela, and the Caribbean (Mammil-
laria colombiana Salm-Dyck and M. mammillaris (L.)
Karsten), with maximal diversity in Mexico.
Characterized as globular or depressed to short co-
lumnar cacti, members of the Cacteae range in size
from dwarf (Turbinicarpus (Backeberg) Backeberg and
Buxbaum and some Mammillaria Haworth) to gigantic
(Ferocactus Britton and Rose and Echinocactus Link and
Otto). Stems may be either ribbed, as in Echinocactus,
or tuberculate as in Coryphantha (Engelmann) Lemaire.
Zimmerman (1985) states that ribs and tubercles are
mutually exclusive terms, although a number of inter-
mediates are found. He recommends the use of the
term podarium, suggesting that in reality ribs are se-
ries of podaria joined end-to-end. Tubercles, however,
represent free or distinct podaria. This terminology al-
lows for intermediacy between ribs and tubercles. The
size and shape of tubercles range from long and leaf-
like (as in Leuchtenbergia Hooker, Obregonia Fric, and
some species of Ariocarpus Scheidweiler) to broad with
shallow axils, as in Turbinicarpus.
Areoles may be borne on the ribs, or in the case of
the tuberculate members, may occur at or near the tu-
bercle apex, or form a groove on the upper surface as
in Coryphantha and some species of Escobaria Britton
and Rose. In a number of genera the tubercles are dif-
ferentiated into spine-bearing areoles at the tubercle
apex and floriferous or vegetative areoles in the axils
of the tubercles. Buxbaum (1958b) suggested that spe-
cies with differentiated tubercles such as Mammillaria
are derived within the tribe. Actinomorphic or, rarely,
zygomorphic (Mammillaria subgenus Cochemiea Bran-
degee) diurnal flowers arise from the areoles. The per-
icarpel (in cacti defined as ovary wall plus stem tissue
external to the ovary wall) ranges from scaly and
woolly to petaloid. Fruits in the Cacteae are fleshy to
juicy berries, and the seeds are borne on short, simple
funiculi. Since the 1920‘s, a number of researchers have
revised the Cacteae, variously interpreting its classifi-
cation based on differing concepts of broadly-defined
or narrowly circumscribed genera. Table 1 lists genera
accepted in a number of key treatments of the Cacteae.
Britton and Rose (1919–1923) did not recognize the
Cacteae as a discrete entity. Within their tribe Cereeae
(equals subfamily Cactoideae), they divided the barrel
cacti into two subtribes, Echinocactinae and Cory-
phanthinae. Their subtribe Echinocactinae included all
ribbed barrel cacti from both North and South Amer-
ica, which manifest a generally low growing, globular
habit. The North American barrel cacti that share the
character of possessing tubercles were placed into the
subtribe Coryphanthinae, although some taxa with
true tubercles or modified tubercles, such as Pediocactus
Britton and Rose, Ariocarpus, and Lophophora Coulter
were placed within the ribbed subtribe Echinocactinae.
It is evident that Britton and Rose (1919–1923) realized
that mutually exclusive suites of morphological char-

258 [Volume 27SYSTEMATIC BOTANY
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1. Comparisons of previous treatments for members of the Cacteae.
This paper Anderson (2001) Hunt (1999) Barthlott & Hunt (1993) Backeberg (1970) Buxbaum (1958b) Britton & Rose (1919–1923)
Acharagma Escobaria Escobaria Escobaria Escobaria, Gymnocactus Escobaria Not Described
Ariocarpus Ariocarpus Ariocarpus Ariocarpus Ariocarpus, Neogomesia, Ro-
seocactus
Ariocarpus Ariocarpus
Astrophytum Astrophytum Astrophytum Astrophytum Astrophytum Astrophytum Astrophytum
Aztekium Aztekium Aztekium Aztekium Aztekium Aztekium Not Described
Coryphantha Coryphantha Coryphantha Coryphantha Coryphantha, Cumarinia,
Lepidocoryphantha
Coryphantha, Cumarinia Coryphantha
Echinocactus Echinocactus Echinocactus Echinocactus Echinocactus Echinocactus Echinocactus
Encephalocarpus Pelecyphora Pelecyphora Pelecyphora Encephalocarpus Encephalocarpus Pelecyphora
Epithelantha Epithelantha Epithelantha Epithelantha Epithelantha Epithelantha Epithelantha
Escobaria Escobaria Escobaria Escobaria Escobaria, Neobesseya Escobaria, Neobesseya Escobaria
Ferocactus
Geohintonia
Ferocactus
Geohintonia
Ferocactus
Geohintonia
Ferocactus
Geohintonia
Ferocactus
Not Described
Ferocactus
Not Described
Ferocactus
Not Described
Glandulicactus Sclerocactus Sclerocactus Sclerocactus Glandulicactus Hamatocactus Hamatocactus
Homalocephala Echinocactus Echinocactus Echinocactus Homalocephala Homalocephala Homalocephala
Leuchtenbergia Leuchtenbergia Leuchtenbergia Leuchtenbergia Leuchtenbergia Leuchtenbergia Leuchtenbergia
Lophophora Lophophora Lophophora Lophophora Lophophora Lophophora Lophophora
Mammillaria Cochemiea, Mammil-
laria, Mammilloydia
Mammillaria, Mam-
milloydia
Mammillaria, Mam-
milloydia
Bartschella, Cochemiea, Dol-
icothele, Krainzia, Mamil-
lopsis, Mammillaria, Phel-
losperma, Solisia
Cochemiea, Dolichothele, Lep-
tocladodia, Mamillopsis,
Mammillaria, Mammilloy-
dia, Oehmea, Pseudomam-
millaria
Bartschella, Cochemiea, Dol-
ichothele, Mamillopsis,
Neomammillaria, Phelos-
perma, Solisia
Neolloydia Neolloydia Neolloydia Neolloydia Neolloydia Neolloydia Neolloydia
Obregonia Obregonia Obregonia Obregonia Obregonia Obregonia Not Described
Ortegocactus Ortegocactus Ortegocactus Ortegocactus Ortegocactus Not Described Not Described
Pediocactus Pediocactus Pediocactus Pediocactus Navajoa, Pediocactus, Pilo-
canthus, Utahia
Pediocactus, Utahia Pediocactus, Utahia
Pelecyphora Pelecyphora Pelecyphora Pelecyphora Pelecyphora Pelecyphora Pelecyphora
Sclerocactus Sclerocactus Sclerocactus Sclerocactus Coloradoa, Echinomastus,
Gymnocactus, Sclerocac-
tus, Toumeya
Ancistrocactus, Coloradoa,
Echinomastus, Sclerocactus
Ancistrocactus, Sclerocactus,
Toumeya
Stenocactus Stenocactus Stenocactus Stenocactus Echinofossulocactus Echinofossulocactus Echinofossulocactus
Strombocactus Strombocactus Strombocactus Strombocactus Strombocactus Strombocactus Strombocactus
Thelocactus Thelocactus Thelocactus Thelocactus Echinomastus, Thelocactus Thelocactus Echinomastus, Thelocactus
Turbinicarpus Turbinicarpus Turbinicarpus Neolloydia Gymnocactus, Turbinicarpus Rapicactus, Toumeya Neolloydia

2002] 259BUTTERWORTH ET AL.: PHYLOGENY OF TRIBE CACTEAE
acters could not be used to delineate subtribes within
their tribe Cereeae, accepting that boundaries between
subtribes Echinocereanae, Echinocactinae and Cory-
phanthinae were not clearly defined.
Using an underlying principle of determining tax-
onomic groups based on inferred phylogenetic relat-
edness, Buxbaum (1958b) described the North Amer-
ican barrel cacti (with minor exceptions) at the rank of
tribe (Cacteae), and defined this group by bringing to-
gether Schumann’s earlier tribe Echinocacteae (Schu-
mann 1899) and Britton and Rose’s subtribes Echino-
cactanae and Coryphanthanae. With the exception of
Astrophytum (which he placed into the tribe Notocac-
teae), Buxbaum (1958b) recognized 36 genera in the
tribe and regarded this group of North American bar-
rel cacti as a distinct phylogenetic unit. Within his tribe
Cacteae, four subtribes were defined based upon seed
morphology: 1) the Echinocactinae, with a smooth,
hard, black testa with conspicuous perisperm; 2) the
Thelocactinae, with a verrucose, mostly black testa be-
coming secondarily smooth or ‘spotted’; 3) the Fero-
cactinae, with a pitted or reticulate testa; and 4) the
Coryphanthinae, with a smooth, brown testa.
In contrast to Buxbaum’s phylogenetically-based
classification, Backeberg’s (1970) classification of the
cacti used a complex system of infrafamilial ranks in-
cluding semitribes, subtribes, groups, and subgroups.
This classification was never intended to be phyloge-
netic. Britton and Rose’s tribe Cereeae was split, large-
ly based on geographic origins of the plants, into the
North and South American semitribes Boreocereeae
and Austrocereeae, respectively. Ignoring Buxbaum’s
(1958b) tribe Cacteae, Backeberg created the subtribe
Boreocactinae to accommodate the North American
barrel cacti, which was further divided into two
groups based on flower position: 1. The Boreoechino-
cacti has flowers borne from undifferentiated (vegeta-
tive vs. flowering) areoles (the Boreoechinocacti were
still further divided into two subgroups, the Eubor-
eoechinocacti and the Mediocoryphanthae) and 2. The
Mammillariae, which has differentiated areoles (e.g.
flowers borne in tubercle axils), with three subgroups,
Coryphanthiae, Mediomammillariae, and Eumammil-
lariae. In total, Backeberg’s subtribe Boreocactinae in-
cluded 48 genera, consistent with his philosophy of
recognizing many genera with few species in each. In
modern taxonomic treatments, many of these ‘‘micro-
genera’’ have been united into more broadly defined
groups; for example Ariocarpus was expanded by An-
derson (1960, 1962) to include Roseocactus Berger and
Neogomesia Castan˜eda, and the genera Porfiria Bo¨ deck-
er, Krainzia Backeberg, Phellosperma Britton and Rose,
Dolichothele (Schumann) Britton and Rose, Bartschella
Britton and Rose, Mamillopsis Morren ex Britton and
Rose, and Cochemiea (Brandegee) Walton were sub-
sumed into the genus Mammillaria by Hunt (1971,
1977a, b; 1981).
Besides the treatment by Buxbaum (1958b), there
has been only one other attempt at a phylogenetic eval-
uation of the tribe Cacteae. In his unpublished Ph.D.
thesis, Zimmerman (1985) presented a cladistic study
of the tribe based on an analysis of morphological
characters, the majority of which are derived from the
study of floral structures. Zimmerman suggested that
the Pachycereeae and the Notocacteae probably rep-
resent the closest outgroups to tribe Cacteae, and that
the tribe likely had its origins in South America, shar-
ing a sister-group relationship with the Notocacteae.
Influenced by Buxbaum’s (1958b) treatment, both
Barthlott (1977) and Zimmerman (1985) questioned the
placement of Astrophytum in the Cacteae, noting sig-
nificant differences in seed morphology. Zimmerman
concluded that, with the possible exception of this ge-
nus, the Cacteae formed a monophyletic unit. Further-
more, Zimmerman (1985) placed Astrophytum within a
clade together with Echinocactus and Homalocephala
Britton and Rose that shows a sister-group relationship
to other members of the tribe. Despite problems as-
sociated with morphological plasticity in the tribe,
Zimmerman made a number of insightful conclusions,
for example that Escobaria,Ortegocactus,Mammillaria,
and Coryphantha sensu stricto are derived from a Mam-
millaria-like rather than a Ferocactus-like ancestor.
In their treatment of the genera of the Cactaceae,
Barthlott and Hunt (1993) united a number of Cacteae
genera, recognizing 22 genera in total. Homalocephala
was included within Echinocactus, and the genera Oeh-
mea Buxbaum, Cochemiea,Dolichothele, and Mamillopsis
were subsumed within Mammillaria. Hunt (pers.
comm.) doubts that the Cacteae are monophyletic, rea-
soning that because the globular growth form has aris-
en independently in several cactus lineages in South
America, it has likely also arisen in North American
lineages independently.
The primary goals of this investigation were to test
monophyly of the tribe, resolve intergeneric relation-
ships in the Cacteae, and to assess monophyly in pre-
viously proposed Cacteae genera using chloroplast
rpl16 intron sequence data. Further, we wished to as-
certain relevant outgroup taxa for an ongoing study of
the genus Mammillaria.
M
ATERIALS AND
M
ETHODS
Taxonomic Sampling. A total of 66 taxa were sampled (Table
2), including 62 representative taxa from the tribe Cacteae. Two
species from tribe Notocacteae and one species from tribe Pachy-
cereeae were also included with members of tribe Cacteae as the
ingroup. Calymmanthium substerile (tribe Browningieae) was used
as the outgroup based upon its basal position within the subfamily
Cactoideae (Wallace 2001). Additional phylogenetic analyses of
chloroplast DNA variation (Butterworth and Wallace, unpub-
lished) were conducted in which representative taxa were exam-
ined from throughout the subfamily, and demonstrated that tribe

260 [Volume 27SYSTEMATIC BOTANY
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2. Species sampled for rpl16 study. CANTE 5CANTE Botanic Garden, Mexico; UCONN 5University of Connecticut; DES
5Desert Botanic Garden, Arizona; ISC 5Ada Hayden Herbarium, Iowa State University; HNT 5Huntington Botanic Garden, Cali-
fornia; HUMO 5Universidad Auto´noma del Estado de Morelos, Mexico; and UNAM 5Universidad Auto´ noma de Mexico, Mexico
City.
Taxon Source/Voucher GenBank No.
Tribe Cacteae
Acharagma aguirreana (Glass & Foster) Glass
Acharagma roseana (Boed.) Glass
Ariocarpus agavoides (Castan˜ eda) Anderson
Ariocarpus retusus Scheidw.
Astrophytum capricorne (Dietrich) Br. & R.
Astrophytum myriostigma Lem.
Aztekium hintoni Glass & Fitz Maurice
Aztekium ritteri (Boed.) Boed.
Coryphantha pallida Br. & R.
Echinocactus grusonii Hildm.
Echinocactus horizonthalonius Lem.
Echinocactus ingens Zucc.
Mesa Garden—ISC
DES 1990-0791-0201—ISC
C. Glass 6889—CANTE
C. Glass 6923—CANTE
HNT 69033—ISC
HNT 69032—ISC
C. Glass 6647—CANTE
C. Staples s.n.—ISC
H. Cota 8050—HUMO
R. Wallace s.n.—UCONN
M. Mendes 186—CANTE
HNT 59498—ISC
AF267915
AF267916
AF267918
AF267919
AF267920
AF267921
AF267922
AF267923
AF267926
AF267927
AF267928
AF267929
Encephalocarpus strobiliformis (Werderm.) Berger
Epithelantha bokei L. D. Benson
Escobaria zilziana (Boed.) Backeb.
Ferocactus cylindraceus (Engelm.) Orcutt
Ferocactus flavovirens (Scheidw.) Br. & R.
Ferocactus glaucescens (DC.) Br. & R.
Ferocactus histrix (DC.) Lindsay
Ferocactus latispinus (Haw.) Br. & R.
Ferocactus robustus (Link & Otto) Br. & R.
HNT 60211—ISC
DES 1993-0717-0101—ISC
DES 1989-0137-0102—DES
Ecker (Slausson) 110—ISC
H. Cota 8051—HUMO
HNT 10339—ISC
H. Cota 8037—CANTE
H. Cota 8039—CANTE
H. Cota 8045—HUMO
AF267930
AF267931
AF267932
AF267933
AF267934
AF267979
AF267935
AF267936
AF267974
Ferocactus wislizenii (Engelm.) Br. & R.
Geohintonia mexicana Glass & Fitz Maurice
Glandulicactus crassihamatus (Weber) Backeb.
Glandulicactus uncinatus (Galeotti) Backeb.
Homalocephala texensis (Hoppfer) Br. & R.
Leuchtenbergia principis Hook.
Lophophora diffusa (Croizat) Bravo
Lophophora williamsii (Lem.) J. M. Coult.
L Slauson 112—ISC
C. Glass 6648—CANTE
C. Glass 5201—CANTE
C. Glass 6846—CANTE
HNT 67080—ISC
HNT s.n.—ISC
Mesa Garden—ISC
D. Martinez s.n.—HUMO
AF267937
AF267938
AF267939
AF267917
AF267940
AF267941
AF267942
AF267943
Mammillaria beneckei Ehrenb.
Mammillaria candida Schweidw.
Mammillaria decipiens Schweidw.
Mammillaria glassii Foster
Mammillaria haageana Pfeiffer
Mammillaria halei Brandegee
Mammillaria jaliscana (Br. & R.) Boed.
Mammillaria karwinskiana Mart.
Mammillaria longimamma DC.
DES 1993-0550-0101—DES
DES 1957-5907-0101—ISC
HNT 68830—ISC
HNT 60162—ISC
H. Cota 8053—HUMO
HNT 72646—ISC
Lau 1050—ISC
H. Cota s.n.—ISC
DES 1992-0049-0203—DES
AF267944
AF267945
AF267946
AF267952
AF267953
AF267947
AF267948
AF267949
AF267950
Mammillaria magnifica Buchenau
Mammillaria plumosa Weber
Mammillaria poselgeri Hildm.
Mammillaria senilis Salm-Dyck
Mammillaria voburnensis Scheer
Mammillaria yaquensis Craig
Neolloydia conoidea (DC.) Br. & R.
Obregonia denegrii Fric
HTN—ISC
HTN 28166—ISC
DES 1983-0746-1018—ISC
Mesa Garden—ISC
Lippold s.n.—UCONN
HNT 7715—ISC
Lippold s.n.—ISC
R. Wallace s.n.—ISC
AF267951
AF267954
AF267955
AF267956
AF267957
AF267958
AF267959
AF267960
Ortegocactus macdougallii Alexander
Pediocactus simpsonii (Engelm.) Br. & R.
Pelecyphora aselliformis Ehrenb.
Sclerocactus brevihamatus (Engelm.) D. R. Hunt
Sclerocactus spinosior (Engelm.) Woodruff & L. Benson
Sclerocactus whipplei (Engelm. & Bigelow) Br. & R.
Stenocactus crispatus Berger
Stenocactus lloydii Berger
Stenocactus vaupelianus (Werdem.) F. M. Knuth
R. Wallace s.n.—ISC
C. Butterworth 60—ISC
DES 1961-6848-0101—DES
DES 1989-0315-0101—DES
Hughes 2—ISC
DES 1993-0925-0103—DES
HNT 46450—HNT
ex Hort. UCONN—UCONN
DES 1948-1289-0101—DES
AF267961
AF267962
AF267963
AF267964
AF267965
AF267966
AF267980
AF267977
AF267978
Strombocactus disciformis (DC.) Br. & R.
Thelocactus conothelos (Reg. & Klein) F. M. Knuth
Thelocactus hastifer (Werderm. & Boed.) F. M. Knuth
Thelocactus macdowellii (Rebut ex Quehl) C. Glass
Turbinicarpus gielsdorfianus (Werdermann) John & Riha
H. Sa´nchez-Mejorada 3603—UNAM
Lippold s.n. (ex Hort)—UCONN
Peter Sharp s.n.
HNT s.n.—ISC
HNT 50008—ISC
AF267967
AF267968
AF267973
AF267969
AF267970

2002] 261BUTTERWORTH ET AL.: PHYLOGENY OF TRIBE CACTEAE
T
ABLE
2. Continued.
Taxon Source/Voucher GenBank No.
Turbinicarpus pseudomacrochele (Backeb.) F. Buxb. & Backeb.
Turbinicarpus schmiedickianus var. schwartzii (Shurly)
Glass & Foster
Brach’s Nursery—ISC
Ex Martiny s.n.—ISC
AF267971
AF267972
Tribe Browningieae
Calymmanthium substerile Ritter HNT 46555—ISC AF267924
Tribe Notocacteae
Corryocactus brachypetalus (Vaupel) Br. & R.
Parodia haselbergii (Haage ex Ru¨ mpler) Brandt
HNT 18015—ISC
ex Hort.—UCONN
AF267925
AF267975
Tribe Pachycereeae
Bergerocactus emoryi (Engelm.) Br. & R. HNT 16514—ISC AF267976
Cacteae was well supported as a monophyletic group. Specimens
were obtained from a number of sources and maintained in the
greenhouse prior to DNA extraction. Institutions in which voucher
specimens are deposited are also listed in Table 2.
DNA Extraction and Purification. Total genomic DNA of rep-
resentative Cacteae samples was isolated using one of two meth-
ods:
1. Modified organelle pellet method suitable for mucilaginous
material. Genomic DNA samples were prepared using previously
published methods (Wallace 1995; Wallace and Cota 1996) for ex-
traction of nucleic acids from highly mucilaginous plants, briefly
summarized as follows: fresh, chlorenchymatous stem tissue was
homogenized in 0.35M sorbitol buffer, filtered through Mira-
clothy(Calbiochem). The organelles were pelleted, supernatant
removed, and pellets were then suspended in 2x CTAB (Doyle and
Doyle 1987) for 1 h at 608C. After partitioning against CHCl
3
:oc-
tanol, 24:1. DNA was isopropanol-precipitated and resuspended
for further purification using isopycnic ultracentrifugation in ce-
sium chloride/ethidium bromide gradients, followed by dialysis
against TE.
2. Nucleon Phytopureyplant and fungal DNA extraction kit for
1g samples (Amersham Life Science). DNA was extracted from
living stem tissue according to the manufacturer’s recommenda-
tions and stored at 2208C in TE buffer.
Amplification and Sequencing. Polymerase chai n reaction
(PCR) amplification of the rpl16 intron was conducted in 100 m1
reactions using GeneAmpyPCR Core Reagents (Perkin Elmer),
and the amplification primers RP71F and RP1661R (Applequist
and Wallace 2000). Each reaction included 20 ng of each primer
and 5 m1 of unquantified DNA template. The PCR reactions were
conducted in a MJ Research PTC-100 thermal cycler using the fol-
lowing temperature cycling parameters: l) initial melting at 958C
for 5 min; 2) 24 cycles of the following protocol: 958C melt for 2
min, 508C annealing for 1 min, ramp temperature increase of 158C
at 0.1258C per sec, 658C extension for 4 min; and a final extension
step at 658C for 10 min.
Agarose electrophoresis in TAE was used to confirm the pres-
ence of 1.1 kb to 1.3 kb PCR amplification products. The amplicons
were cleaned and concentrated in Microcon 100 spin microcon-
centrators (Amicon Inc.) following the manufacturer’s directions.
The products were then quantified in an ultraviolet spectropho-
tometer and diluted to 50mg/ml for use in sequencing reactions.
Sequence data were obtained using the sequencing primers
RP1516R and RP637R (Applequist and Wallace 2000) at concen-
trations of 5 pmol in chain-termination reactions using the ABI
Prism Big DyeyTerminator Cycle Sequencing Ready Reaction Kit
(Perkin Elmer). We found that dilutions of 1:4 of terminator ready
reaction solution gave acceptable reads.
Electrophoresis and automated sequence reading were conduct-
ed using Perkin Elmer/Applied Biosystems automatic sequencing
units (ABI Prism 377) at the Iowa State University Nucleic Acid
DNA Facility. Sequences typically were 650 or more nucleotides in
length. In a small number of taxa, a poly-T region approximately
400bp from the RP1516R priming site caused extremely poor
reads upstream of the RP637R priming site. To overcome this
problem, a new primer, RP543F, (59-TCAAGAAGCGATGGGAAC-
GATGG-39) was designed to run forward from just downstream
of the RP637R priming site, overlapping the unreadable section of
sequence. Due to extensive poly-A and poly-T regions in Domain
I at the 59end, 150–200bp of the intron sequence could not be
obtained using the automated method. Kelchner and Clark (1997)
demonstrated low levels of sequence divergence in this region and
because it is of limited phylogenetic usefulness, further attempts
at obtaining a full length intron sequence were discontinued.
Phylogenetic Analysis. Sequence alignment was carried out
using AutoAssembler (Applied Biosystems 1995) and Se-Al (Ram-
baut 1995). Following an initial Clustal W alignment, sequences
were further aligned manually (e.g., Golenberg et al. 1993). Inser-
tions/deletions considered to be phylogenetically informative
were coded in binary (presence/absence) and added to the end of
the data matrix. There were two regions (totalling 61 nucleotides)
where alignments were of doubtful homology. These regions were
excluded from the analyses. All analyses were carried out using
PAUP* 4.0b2 (Swofford 1999). To test the rpl16 intron dataset for
phylogenetic signal the g-statistic for 10,000 random trees was cal-
culated. According to Hillis and Huelsenbeck (1992) the distribu-
tion of lengths of random trees for all topologies provides a ‘sen-
sitive’ measure of phylogenetic signal within the dataset. Matrices
that contain a strong phylogenetic signal show distributions that
approach a left-skewed gamma distribution as opposed to a more
normal distribution for matrices containing random noise.
Parsimony analyses were done using the heuristic search option.
All substitutions and indels were equally weighted. An initial heu-
ristic search using TBR branch swapping saving multiple parsi-
monious trees (MULTREES ON) was conducted. Random addition
searches of 1,000 replicates, saving 100 most parsimonious trees at
each step, were undertaken to search for islands of shorter trees.
Estimates of decay (Bremer 1988) were obtained using converse
constraint trees as implemented using Autodecay (Eriksson and
Wikstro¨m 1995). Bootstrap values were estimated using the ‘fast
bootstrap’ method for 1,000 replicates. A neighbor-joininganalysis
was also undertaken using the F81 substitution model.
R
ESULTS
Sequence length ranged from 650bp in Mammillaria
glassii and 673bp in M. magnifica and M. haageana to
935bp in Ferocactus glaucescens. Aligned sequence
length for the rpl16 dataset was 1057bp. The full da-
taset (including binary-coded indels) totaled 1069 char-
acters. After exclusion of indels and the two regions of
doubtful homology, the dataset was 953 characters
long, of which 177 were parsimony-informative. The g-

262 [Volume 27SYSTEMATIC BOTANY
statistic for the rpl16 dataset is 0.506. This value falls
well within the 99% confidence interval (C.I.) for da-
tasets of over 25 taxa and 500 characters (Hillis and
Huelsenbeck 1992) and therefore indicates significant
phylogenetic structure within the dataset. The data
matrix of aligned rpl16 sequences is available from the
authors.
A heuristic search using PAUP* found 1296 most
parsimonious trees with length of 666 steps. There ap-
pears to be considerable homoplasy in the rpl16 dataset
with a C.I. of 0.632 (0.494 excluding uninformative
characters). However, a low C.I. may be expected, due
in part to the nature of large datasets, thus the reten-
tion index (R.I.) gives a more suitable indication of
support. In the case of the rpl16 dataset, the R.I. (ex-
cluding uniformative characters) is 0.699. A random
addition search of 100 replicates did not find any is-
lands of shorter trees. The strict consensus tree (Figure
1) supports monophyly of the Cacteae, with a decay
value of 9 and a bootstrap value of 100%.
Within the Cacteae, the general tree topology re-
solves a number of clades nested pectinately within
each other: 1. ‘‘Aztekium Clade’’ consisting of Aztekium
Bo¨ decker and Geohintonia Glass and Fitz Maurice
(bootstrap 100%, decay 7); 2. ‘‘Echinocactus Clade’’—
Astrophytum,Echinocactus, and Homalocephala (boot-
strap 62%, decay 2); 3. Sclerocactus Britton and Rose
(bootstrap 89%, decay 4); 4. ‘‘Lophophora Clade’’—
Acharagma (Taylor) Glass, Lophophora, and Obregonia
(bootstrap 87%, decay 5); 5. Strombocactus disciformis
forms a single lineage; 6. ‘‘ATEP Clade’’—A weakly
supported clade (bootstrap ,50%, decay 1) unites
Ariocarpus,Turbinicarpus,Epithelantha, and Pediocactus;
7. ‘‘Ferocactus Clade’’—consisting of Ferocactus,Ancis-
trocactus Britton and Rose, Leuchtenbergia,Echinocactus
grusonii,Thelocactus (Schumann) Britton and Rose, and
Glandulicactus Backeberg (this clade is poorly resolved
and poorly supported with bootstrap ,50% and decay
1); 8. Stenocactus (Schumann) Hill (bootstrap 100%, de-
cay 4); 9. ‘‘Mammilloid Clade’’ including Pelecyphora
Ehrenberg, Encephalocarpus Berger, Escobaria,Coryphan-
tha,Neolloydia Britton and Rose, Ortegocactus, and
Mammillaria (this terminal clade is well-supported
with bootstrap 60%, and decay 3).
Analysis of the rpl16 data using a neighbor-joining
algorithm with the F81 substitution model resulted in
an initial tree that was topologically quite congruent
with the maximum parsimony tree. There were, how-
ever a number of exceptions. Mammillaria glassii forms
a sister-group to all of the remaining members of the
Cacteae in the neighbor-joining tree. This incongruence
is caused by differences in sequence length of Mam-
millaria glassii of only 650bp due to a large deletion
spanning the region with most informative characters.
Other topological differences between the maximum-
parsimony and neighbor-joining trees were observed
in the placement of members of the Lophophora and
Echinocactus clades of the maximum parsimony tree,
which form a single clade in the neighbor-joining tree.
D
ISCUSSION
Phylogenetic Relationships in the Cacteae. M
ONO
-
PHYLY OF THE
C
ACTEAE
. The phylogeny presented in
this paper supports a monophyletic origin for mem-
bers of the Cacteae as currently circumscribed; no di-
rect relationship was shown with Bergerocactus emoryi
(Pachycereeae), and Parodia haselbergii and Corryocactus
brachypetalus, members of the morphologically similar
South American tribe Notocacteae. A number of syn-
apomorphic substitutions resulted in a decay value of
9 with 100% bootstrap support, providing robust sup-
port for the Cacteae clade. The monophyly of tribe Cac-
teae was further tested using rpl16 intron sequences in
which Austrocylindropuntia Backeberg (subfamily
Opuntioideae) and Maihuenia poepegii (Otto ex Pfeiffer)
Philippi ex Schumann (subfamily Maihuenioideae)
were used as outgroups for comparisons with each ge-
nus of the Cacteae used in this study, together with
representatives of all other tribes in the subfamily Cac-
toideae (Butterworth and Wallace, unpublished data).
Support for monophyly of the tribe Cacteae was also
very strong (98% bootstrap) with this test.
A
ZTEKIUM
C
LADE
. The Aztekium Clade forms the
sister-group to the remaining taxa of the Cacteae.
Plants in this clade typically are globose to subglobose,
rarely short columnar reaching 20 cm by 10 cm in size.
Stem morphology is ribbed, the ribs in Aztekium hav-
ing characteristic transverse wrinkles. Spines are no-
table by their absence from mature areoles; even in
young areoles they are highly reduced and very brittle.
Aztekium and Geohintonia presently are restricted to a
small area of eastern Nuevo Leon in NE Mexico. Hunt
and Taylor (1992) suggested that Geohintonia may rep-
resent an intergeneric hybrid involving Aztekium and
possibly Echinocactus horizonthalonius. Corriveau and
Coleman (1988) demonstrated biparental inheritance of
chloroplast DNA in Rhipsalidopsis Britton and Rose,
and Zygocactus Schumann but maternal inheritance in
Echinocereus Engelmann and Opuntia Miller. If Geohin-
tonia is descended from an ancient intergeneric hybrid,
and its plastid organelles are maternally inherited,
then the maternal parent of the ancient hybrid was
closely related to Aztekium, probably A. hintonii which
is sympatric with Geohintonia. However, if the chloro-
plasts of Geohintonia show biparental inheritance then
Aztekium could represent the descendant of either the
pollen or ovule donor. The relationship between Aztek-
ium and Strombocactus Britton and Rose has been cause
for discussion. In a preliminary list of accepted genera
by the working party of the International Organization
for Succulent Plant Study (IOS) (Hunt and Taylor 1986)
and a follow-up report (Hunt and Taylor 1990), the

2002] 263BUTTERWORTH ET AL.: PHYLOGENY OF TRIBE CACTEAE
F
IG
. 1. Strict consensus of 1296 most parsimonious trees for rpl16 intron sequences. Length 5666 steps, C.I. 50.632, C.I.
(excluding uninformative characters) 50.494, R.I. (excluding uninformative characters) 50.699. Bootstrap values over 50% for
1000 replicates are given above the branches. Decay values are shown below the branches. * 5switch from ribbed to tubercular
stems. l5switch from tubercular to ribbed stems. Boxes indicate clades with dimorphic areoles.

264 [Volume 27SYSTEMATIC BOTANY
F
IG
. 2. Neighbor-joining tree for the tribe Cacteae based on F81 distances.
generic status of Aztekium was accepted, although
among members of the working party, opinion was di-
vided as to whether Aztekium and Strombocactus were
congeneric or convergent. Anderson and Skillman
(1984) concluded that Aztekium and Strombocactus
should each be recognized at the generic level, citing
a number of differences in vegetative, floral, pollen and
seed morphology. The phylogeny presented in this pa-
per strongly supports (bootstrap 100%, decay 7) a
clade containing Aztekium and Geohintonia and does
not support a close relationship between Aztekium and
Strombocactus.

2002] 265BUTTERWORTH ET AL.: PHYLOGENY OF TRIBE CACTEAE
E
CHINOCACTUS
C
LADE
. The clade comprising As-
trophytum,Echinocactus horizonthalonius,E. ingens and
Homalocephala are globose to shortly columnar cacti
with ribbed stems. Areoles are large, and in some spe-
cies of Astrophytum, spines are lacking. Flowers are
shortly funnelform to campanulate, the wooly pericar-
pel having numerous spine-tipped bracts. These cacti
are distributed throughout Mexico and SW United
States. A close relationship between genera of this
clade is also supported by chloroplast restriction-site
data (Wallace 1995). Previous authors (Bravo-Hollis
and Sa´ nchez-Mejorada 1991; Ferguson 1992; Barthlott
and Hunt 1993) have considered Homalocephala as con-
generic with Echinocactus. The rpl16 data (this paper)
does not fully resolve the relationships between Astro-
phytum,Echinocactus, and Homalocephala that were
shown by Wallace (1995), instead displaying a trichot-
omy, such that with the inclusion of Homalocephala, the
genus Echinocactus may be paraphyletic. These data
also corroborate the conclusion of Cota and Wallace
(1997) that Echinocactus grusonii is more closely related
to members of the genus Ferocactus than to other spe-
cies in the Echinocactus clade.
S
CLEROCACTUS
C
LADE
. Porter et al. (2000) attempt-
ed to define generic boundaries for the morphologi-
cally diverse genus Sclerocactus using chloroplast trnL-
trnF sequence data. Although sampling from other
genera of the tribe was not as broad as in the study
presented here, sampling from within the genus Scler-
ocactus clearly contradicted the hypothesized close re-
lationship between Sclerocactus and Pediocactus, sug-
gested by previous authors (Arp 1972 as cited in Porter
et al. 2000; Benson 1982). Our phylogeny places mem-
bers of Sclerocactus in a well-supported clade (boot-
strap 89%, decay 4) and shows no affinity between
Sclerocactus and the genus Pediocactus; thus our results
are consistent with those of Porter et al. (2000). The
working party of the IOS (Hunt and Taylor 1986, 1990)
and Barthlott and Hunt (1993) treat the genus Glan-
dulicactus as a synonym of Sclerocactus. Ferguson (1991
and pers. comm.) disagrees with the placement of
Glandulicactus within Sclerocactus, based on vegetative
and floral morphology. Our data support Ferguson’s
view (see section on Ferocactus Clade).
L
OPHOPHORA
C
LADE
. Although there is strong
support for this clade (87% bootstrap, 5 decay steps),
few morphological features unite this clade. All mem-
bers have napiform or carrotlike tap-root systems, al-
though these features are also found in other members
of the tribe.
The two species of Acharagma have been a source of
taxonomic confusion. Described originally in the genus
Echinocactus,E. roseanus was transferred into the genus
Gymnocactus Backeberg by Glass and Foster (1970),
who later also described G. aguirreanus (Glass and Fos-
ter 1972). However, Anderson and Ralston (1978) felt
that these two species were better placed in the genus
Turbinicarpus, contrary to the views of Glass and Foster
(1977), who felt that despite high degrees of similarity
in distribution, appearance, and flower, fruit and seed
morphology, the larger size and generally heavier spi-
nation of species of Gymnocactus warranted recognition
as a separate genus. In a review of Escobaria, Taylor
(1986) placed G. roseanus and G. aguirreanus as sole
members of the section Acharagma of Escobaria. Unlike
other members of the genus, the axillary areole and
tubercular groove is absent in these two species. Fur-
thermore, the flowers are borne in a zone adjacent to
the spine-bearing areoles, in contrast to the more typ-
ical position in the axils of the tubercles. Glass (1998)
elevated Taylor’s section Acharagma to the rank of ge-
nus following Zimmerman’s provisional generic treat-
ment in which Acharagma was placed in a large clade
containing Ferocactus,Coryphantha,Mammillaria,Orte-
gocactus, and Escobaria, mainly based on foveolate
seeds (Zimmerman 1985). However, Zimmerman
(1985) acknowledged that Acharagma only has weakly
derived character states and so his placement of the
genus was uncertain. The rpl16 intron data suggest the
removal of these two species from Escobaria, placing
them in a well-supported (bootstrap 87%, decay 5)
clade containing Obregonia and Lophophora, the latter
shown to be polyphyletic based on this topology.
S
TROMBOCACTUS
. This monotypic genus from the
states of Queretero and Hidalgo in central Mexico
forms a sister lineage to the ‘‘ATEP’’, Ferocactus,Sten-
ocactus and ‘‘Mammilloid’’ clades according to the
phylogeny presented in this paper. On the basis of
seed morphology, Buxbaum (1958a) suggested that the
genus Strombocactus ought to include the then mono-
typic genus Aztekium. This was in spite of the tuber-
culate stem anatomy of Strombocactus which contrasts
the ribbed anatomy of Aztekium. Buxbaum (1958a) ex-
plained this by suggesting a progression from the
hardened tubercles of Strombocactus to the formation of
ribs in Aztekium. Anderson and Skillman (1984), using
morphological and anatomical data, concluded that
Strombocactus and Aztekium each deserved recognition
at the genus level. No direct relationship between
Strombocactus and Aztekium is demonstrated in our
rpl16 phylogeny.
‘‘A
TEP
’’ C
LADE
. This clade’s acronym-based name
is derived from its included genera—Ariocarpus,Tur-
binicarpus,Epithelantha Weber ex Britton and Rose, and
Pediocactus, and has poor support in our phylogeny
(bootstrap ,50%, decay 1 step). Stem morphologies
are tuberculate, and in Ariocarpus dimorphic areoles
are present, this feature an example of convergence
with members of the ‘‘Mammilloid’’ clade. Turbinicar-
pus is a genus of around sixteen species of small, in-
conspicuous cacti from north-central Mexico. Due to
poor seed dispersal mechanisms, species of Turbinicar-

266 [Volume 27SYSTEMATIC BOTANY
pus are highly localized (Glass and Foster 1977). A
number of species of Turbinicarpus have been allied or
subsumed into other genera such as Gymnocactus
(Backeberg 1970) and Neolloydia (Anderson 1986; Hunt
and Taylor 1990; Barthlott and Hunt 1993). However,
in the CITES Cactaceae checklist, Hunt chose to accept
generic status for species of Turbinicarpus leaving only
two species in Neolloydia (Hunt 1992; 1999). The phy-
logeny presented here supports the exclusion of Tur-
binicarpus from Neolloydia s. str.asN. conoidea (type spe-
cies for the genus) is strongly positioned within the
‘‘Mammilloid’’ clade.
F
EROCACTUS
C
LADE
. This clade contains a number
of seemingly disparate genera with few morphological
affinities (such as conspicuous pericarpel scales) that
unite the entire clade. Members of the genus Ferocactus
possess a number of morphological synapomorphies
including nectar-secreting areolar glands and a ring of
hairs that separate the stamens from the tepals. Al-
though morphologically striking due to elongate, glau-
cous tubercles, the single species of Leuchtenbergia—L.
principis (the ‘‘Agave Cactus’’) is placed in the Ferocac-
tus clade. Barthlott and Hunt (1993) describe the flow-
ers of this species as similar to those of Ferocactus, and
the fruit as being typical of those in subgenus Ferocac-
tus—dry, globose to oblong with thick-walls and de-
hiscing at the base. A close affinity between Ferocactus
and Leuchtenbergia is also demonstrated by the ease
with which these genera hybridize. The phylogenypre-
sented here, as well as chloroplast restriction site data
(Cota 1997; Cota and Wallace 1997), shows that Echin-
ocactus grusonii is more closely related to members of
Ferocactus (particularly F. histrix and F. glaucescens) than
it is to the remaining species of Echinocactus sampled.
These species share a number of distinct character
traits, including straight or slightly curved, terete cen-
tral spines as opposed to hooked spines with flat cross-
sections that are more typical of Ferocactus. In our phy-
logeny, however, F. histrix is positioned outside the Fer-
ocactus clade. If F. histrix is moved and placed sister to
F. glaucescens and E. grusonii, tree-length increases by
only three steps. The slight change in tree-length and
low decay value (decay 51) for the branch separating
F. histrix from the Ferocactus clade implies possible ho-
moplasy in our dataset. Previous molecular studies of
Ferocactus by Cota and Wallace (1997) and Cota (1997)
were only able to partially resolve species relationships
between Ferocactus and its allies, but did recover sim-
ilar clades within the genus Ferocactus.
Glandulicactus uncinatus and G. crassihamatus cur-
rently are recognized as Sclerocactus uncinatus and S.
uncinatus ssp. crassihamatus, respectively, by a number
of authors (Hunt 1992; Barthlott and Hunt 1993; Hunt
1999; Anderson 2001). However, Ferguson (1991) ar-
gued that this genus did not belong in Sclerocactus, cit-
ing a number of morphological differences. Instead, he
allied members of this genus with Ferocactus,Thelocac-
tus, and Leuchtenbergia based on vegetative and floral
morphology. Although the phylogeny presented in this
paper does not necessarily support Ferguson’s view-
point that Glandulicactus should be recognized at genus
level, it does corroborate his conclusions that the mem-
bers of this genus are more closely related to Ferocactus
and Thelocactus than to Sclerocactus.
S
TENOCACTUS
C
LADE
.Comprising about 10 spe-
cies, Stenocactus tends to be separable from the related
Ferocactus clade by two morphological characters: 1)
narrow, fin-like ribs as opposed to wide ribs, and 2)
areoles in which the large spines are subtended by the
smaller spines as opposed to areoles in which the larg-
er spines subtend the smaller spines in Ferocactus.
However, Taylor (1983) argued that despite these mor-
phological differences, flower, fruit and seed morphol-
ogy required a broader generic concept that included
the members of Stenocactus in the genus Ferocactus. Our
rpl16 phylogeny suggests that the Stenocactus clade
(bootstrap 100%, decay 4) is distinct from the Ferocac-
tus clade.
‘‘M
AMMILLOID
’’ C
LADE
. Although support for the
‘‘Mammilloid’’ clade is not particularly strong (boot-
strap 60%, decay 3), members share the morphological
synapomorphies of tuberculate stem anatomy and di-
morphic areoles (the spine-bearing areoles being apical
and the flowering areoles being axillary to the tuber-
cles). Within the clade, generic delimitations have tra-
ditionally been confused. Pelecyphora and Encephalocar-
pus form a well-supported clade (bootstrap 100%, de-
cay 8). These genera have been treated as congeneric
(Pelecyphora) by some previous authors (Anderson and
Boke 1969; Barthlott and Hunt 1993; Anderson 2001),
and are recognized as such in the CITES Cactaceae
Checklist (Hunt 1992, 1999). Relationships between Es-
cobaria and Coryphantha are controversial. Berger (1929
cited in Zimmerman 1985) subsumed Escobaria into
Coryphantha. Taylor (1986) cites a number of character
traits that distinguish the two genera, including pitted
seeds and ciliate outer perianth segments in Escobaria
versus non-pitted seeds and non-ciliate outer perianth
segments in Coryphantha. Taylor (1986) suggests that
Escobaria is more closely related to Mammillaria than it
is to Coryphantha. Indeed, a sister relationship between
Escobaria and Coryphantha is suggested by rpl16 intron
data, although additional data and more intense sam-
pling are required in order to evaluate more robustly
their relationships to Mammillaria.
Based on rpl16 sequences, Mammillaria is not mono-
phyletic as currently circumscribed due to the place-
ment of Neolloydia conoidea and Ortegocactus macdougal-
lii. The working party of the IOS (Hunt and Taylor
1986, 1990) chose to include the genus Turbinicarpus
and Gymnocactus within Neolloydia. Barthlott and Hunt
(1993) followed the same treatment but suggested that

2002] 267BUTTERWORTH ET AL.: PHYLOGENY OF TRIBE CACTEAE
the dimorphic areoles and hence axillary flowers of the
type species of Neolloydia (N. conoidea) were sufficient
to justify recognition of Turbinicarpus at genus level
while continuing to recognize the genus Neolloydia.
Zimmerman (1985) concludes that Neolloydia is distinct
from Coryphantha and its allies (being more closely re-
lated to Ariocarpus,Obregonia,Lophophora,Strombocac-
tus, and Aztekium), and that the tubercular groove, in
this case, is non-homologous with the areolar groove
in Escobaria and Coryphantha.However,Zimmerman
failed to cite which species of Neolloydia were used in
his study, so it is unsure if he used the type—N. con-
oidea or other species referable to Turbinicarpus. Our
data supports the view of Barthlott and Hunt (1993)
that Neolloydia in the strict sense (N. conoidea and N.
matahuelensis Backeberg) are not closely related to ei-
ther Turbinicarpus or Gymnocactus.
Ortegocactus is a monotypic genus, known only from
the state of Oaxaca, Mexico. Although it shares many
morphological features with members of the ‘‘Mam-
milloid’’ clade, taxonomists have had difficulty assess-
ing relationships of this species to other members of
the clade. Bravo-Hollis and Sa´nchez-Mejorada (1991)
placed Ortegocactus in the genus Neobesseya Britton and
Rose. Zimmerman (1985) concluded that Ortegocactus
was a member of a phylogenetically distinct clade con-
taining Coryphantha,Escobaria and Mammillaria. The
rpl16 phylogeny suggests that Ortegocactus is more
closely related to Mammillaria than to other members
of the ‘‘Mammilloid’’ clade, and shows no direct re-
lationship with Coryphantha or Escobaria.
The clade containing M. halei,M. poselgeri, and M.
yaquensis is phylogenetically distinct (bootstrap 83%,
decay 1) from the remaining species sampled in Mam-
millaria sensu stricto. Two of these three species (M.
halei and M. poselgeri) are referable to the genus Coch-
emiea. Mammillaria yaquensis (series Ancistracanthae
Schumann) has been allied to other members of Coch-
emiea by Lu¨ thy (1995). That Cochemiea (represented by
M. halei and M. poselgeri in this study) are found only
in Baja California raises the question of their origin
and dispersal from mainland Mexico. The series An-
cistracanthae are distributed in western Mexico and
Baja California reaching as far north as the southern
USA (California Arizona and New Mexico). A reason-
able hypothesis suggests that ancestral members of the
Ancistracanthae migrated northwards in mainland Mex-
ico, before migrating south through Baja California.
The geologic history of Baja California seems unclear,
but there is evidence that the Gulf of California began
to separate around 4.5 million yr ago (Atwater 1970)
or 5.5 million yr ago according to Riddle et al. (1997).
However, the gulf may have been in existence for the
last 12 million yr (Gastil et al. 1983). Assuming a
north-south migration of ancestral Ancistracanthae, the
phylogeny presented here suggests a more recent ori-
gin for Cochemiea. Hunt (pers. comm.) disputes recog-
nizing Cochemiea as distinct, stating that ornithophilous
flowers (found only in Cochemiea) are derived and con-
tradict a sister-group relationship to other members of
Mammillaria. Our phylogeny does support ornithophi-
ly as being derived and suggests that Cochemiea arose
from an Ancistracanthae-like ancestor.
Within the main clade of Mammillaria, there are a
number of species whose inclusion in the genus has
been disputed by various cactus taxonomists. Mam-
millaria beneckei was considered by Buxbaum (1951, in
Hunt 1977a) as a distinct genus (Oehmea) and argued
that this was an example of morphological conver-
gence with Mammillaria, but that it was actually de-
rived from a Thelocactus-type ancestor. Hunt (1977a)
subsumed the genus Oehmea within Mammillaria, giv-
ing it subgeneric status based upon the rugose/pitted
seed testa, which is also found in other members of
the genus. In their work on the Cactaceae, Britton and
Rose (1919–1923) gave separate generic status to Mam-
millaria senilis by describing it within the genus Mam-
illopsis. Their justifications for this were based on a
number of floral traits that they considered sufficiently
different from Mammillaria to warrant its generic sta-
tus. However, Hunt (1971) concluded that vegetative,
floral, and seed morphology when taken in the sum of
their characters did not support generic status of Mam-
illopsis and that it should only be retained at the sub-
generic level within Mammillaria. Britton and Rose
(1919–1923) also elevated Schumann’s (1899) subgenus
Dolichothele (represented in this study by M. longimam-
ma DC) to genus level, separating it from other species
of Mammillaria due to its very elongate tubercles. As
with Mamillopsis, Hunt (1971) believed that there were
insufficient differences to justify Dolichothele at the rank
of genus, instead accepting it as a subgenus of Mam-
millaria. Our rpl16 phylogeny does not support the
view of Buxbaum (1951, in Hunt 1977a) regarding the
generic status of Oehmea because no direct relationship
between Oehmea and Thelocactus is demonstrated. Al-
though sampling from the genus Mammillaria is lim-
ited, our data also suggest that recognition of Mamil-
lopsis and Dolichothele at the generic rank is unwar-
ranted as they are nested within other Mammillaria
species. Further sampling from Mammillaria is re-
quired and for the present, Oehmea,Mamillopsis, and
Dolichothele should be retained in Mammillaria.
The relationship of Mammillaria candida to other
members of the genus has also been a source of past
debate. Schumann (1899) considered this species to be
within his subgenus Eumamillaria (true mammillarias).
Buxbaum (1951, in Hunt 1977a) elevated this species
to genus status—Mammilloydia candida based soley on
a tuberculate seed testa morphology. Riha and Riha
(1975) disputed Buxbaum’s observations, going so far
as to suggest that Buxbaum had accidentally observed

268 [Volume 27SYSTEMATIC BOTANY
seed material that was not from Mammillaria candida.
Hunt (1977) argued in support of Buxbaum, suggest-
ing that Mammilloydia candida was the product of a sep-
arate evolutionary lineage than that of the remaining
species of Mammillaria. However, he considered the re-
tention of subgenus Mammilloydia a taxonomic com-
promise. The International Cactaceae Systematics
Group recently has accepted that M. candida merits rec-
ognition at the generic rank as Mammilloydia (Hunt
1999) and it is treated as such in Anderson (2001). Se-
quence analysis of the rpl16 intron presented here, and
from a more broad sampling of the ‘‘Mammilloid’’
clade (Butterworth 2000) indicate that recognition of
M. candida at the rank of genus would render Mam-
millaria paraphyletic. Further studies on the ‘‘Mam-
milloid’’ clade are in progress to resolve these issues.
Morphological Evolution. E
VOLUTION OF
T
UBERCLES
IN THE
C
ACTEAE
. Buxbaum (1958a) presented a num-
ber of different scenarios in which tubercular stem
morphologies may have arisen in the Cactaceae. With-
in the tribe Cacteae, he described the convergent evo-
lution (in a number of lineages within the tribe) of
transversely-arranged tubercles formed from ribs in
which the basal portions of the podaria have become
enlarged to form tubercles, implying that a ribbed
stem morphology represents the primitive condition
for the tribe. Gibson and Nobel (1986) also suggest that
the primitive condition for the subfamily Cactoideae is
likely based on ribbed-stem morphology. From our
studies using rpl16 intron sequence data, it appears
that in the Cacteae tubercular stem morphologies rep-
resent a derived condition. The question of multiple
origins of tubercles or reversals to ribbed stems is de-
batable. The most parsimonious explanation based on
the phylogeny presented in this paper is that tuber-
cular stems have arisen independently in a number of
clades, once following the divergence between the
Echinocactus clade and the remaining Cacteae. A rever-
sal to ribbed stems is implicated in Ferocactus histrix,
Stenocactus, and the Ferocactus clade, with secondarily
derived tubercular stem morphologies representing a
zone of transition between ribs and tubercles. The ge-
nus Ferocactus has ribbed stems, while Leuchtenbergia
has very distinct elongate tubercles. Glandulicactus has
deeply notched ribs and may represent the interme-
diate condition, and in Thelocactus, both ribs and tu-
bercles are present—Thelocactus hastifer with distinct,
spiraling ribs divided into tubercles, and the sister
clade of T. conothelos and T. macdowellii having indistinct
ribs and pronounced tubercles (Anderson 1987). There
is also a switch from ribbed to tubercular stems in the
‘‘Mammilloid’’ Clade, whose members also share the
synapomorphy of having dimorphic areoles (see bel-
low).
E
VOLUTION OF
D
IMORPHIC
A
REOLES
. The majority
of genera in the Cacteae produce flowers from the
spine-forming areoles. However, a number of taxa in
the tribe have dimorphic areoles in which spines and
flowers are borne from different regions or even from
separate areoles. To an extent this correlates with tu-
bercular stem morphologies where spines are pro-
duced from apical areoles (the axillary areoles becom-
ing reproductive). Buxbaum (1958a) proposed that the
evolution of dimorphic areoles in the tribe Cacteae oc-
curred along two distinct lines. The first lineage shows
a succession from Leuchtenbergia, which has elongated
tubercles tipped by undifferentiated areoles, to Roseo-
cactus (Ariocarpus fissuratus,A. kotschoubeyanus) with ar-
eoles forming an elongated furrow along the length of
the tubercle with separate floral and spine-bearing re-
gions (Anderson 1960), to Ariocarpus with separate flo-
ral and spine-bearing areoles and some species lacking
spine-bearing areoles altogether (Anderson 1960). Bux-
baum’s second evolutionary lineage occurred from a
non-differentiated ‘‘Thelocactus-type’’ areole in which
growth occurs below the areole causing it to be forced
to the tip of the tubercle. In a number of species,
lengthening growth divides the growing point forcing
the spine producing part towards the tubercle tip, the
flower producing region remains in the tubercle axil.
Species with this form of dimorphic areole may have
a groove running along the adaxial length of the tu-
bercle connecting the vegetative and reproductive ar-
eoles. In Mammillaria, the groove is absent due to di-
vision of the growing point at a very early stage in
development. According to our phylogeny, undiffer-
entiated areoles represent the plesiomorphic condition
for the Cacteae with the evolution of dimorphic areoles
occurring independently in Ariocarpus and the ‘‘Mam-
milloid’’ clade.
In summary, the pattern of evolution that we present
in the tribe Cacteae, as inferred from the rpl16 intron
phylogeny, suggests that the Aztekium-Geohintonia
clade, represents a relictual, yet highly specialized lin-
eage. The remaining members of this North American
barrel cactus tribe have undergone diversification into
several clades, the more derived clades manifesting a
shift from plesiomorphic ribbed stems to those that are
tuberculate, concomitantly undergoing a general re-
duction in plant size. Shifts in floral position and ar-
eole are also inferred from the phylogeny. These
changes occurred in parallel within the Cacteae, fur-
ther adding to the systematic confusion experienced by
many earlier cactologists. Although the inferred evo-
lutionary relationships we present are based on data
from a single molecular marker of the plastid genome,
the resulting tree topology and clades defined are tell-
ing as to the broad-scale, intergeneric relationships
within the tribe that have heretofore only received
‘‘support’’ through speculative conclusions, accompa-
nied by little empirical analyses.
Here we broadly sample representative members of

2002] 269BUTTERWORTH ET AL.: PHYLOGENY OF TRIBE CACTEAE
the tribe Cacteae in a uniformly comparative fashion,
and evaluate the group to determine its primary line-
ages. The intergeneric relationships inferred now lend
themselves to further testing with additional markers
and more intensive sampling for the more species-rich
genera (e.g., Mammillaria,Coryphantha,Escobaria,Fero-
cactus), as well as reexamining those clades that were
not well supported (e.g. the ‘ATEP’ Clade). These in-
vestigations are ongoing at present and will extend the
value of the present study through the use of its con-
clusions in prudent outgroup sampling, identification
of morphological evolutionary trends among the taxa,
and by establishing a baseline phylogeny for integra-
tion with other similar studies being conducted on oth-
er tribes in the Cactaceae.
A
CKNOWLEDGEMENTS
. We would like to thank all the follow-
ing persons and institutions for supplying plant material for this
study. Steven Brack, W.A and Betty Fitz Maurice, Charles Glass,
Lee Hughes, Mario Mendez, Liz Slauson, El Charco Botanic Gar-
den (San Miguel de Allende, Guanajuato, Mexico), The Desert Bo-
tanic Garden, Phoenix, AZ), Mesa Garden (Belen, NM), and The
Huntington Botanic Garden (San Marino, CA). Helpful discussions
regarding intergeneric relationships and morphological variation
within tribe Cacteae were provided by Edward F. Anderson, Wil-
helm Barthlott, Arthur Gibson, Anton Hofer, David Hunt and Ni-
gel Taylor. We would also like to thank Jonathan F. Wendel and
Lynn G. Clark for their comments and suggestions. This study was
supported by grants from the National Science Foundation (NSF
DEB 95–27884) and from the Cactus and Succulent Society of
America Research Fund to R.S.W. We also acknowledge logistic
support from the National Geographic Society (Grant Number
5473–95) and the State University of Morelos, Mexico, which pro-
vided support for field studies by R.S.W. and J.H.C. in Mexico.
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