Blackwell Science, LtdOxford, UKBOJBotanical Journal of the Linnean Society0024-4074The Linnean Society of London, 2003
141
Original Article
Botanical Journal of the Linnean Society, 2003, 141, 349–363. With 5 figures
A. W. MEEROW
ET AL.
MOLECULAR PHYLOGENY OF
CRINUM
Phylogeny and biogeography of Crinum L.
(Amaryllidaceae) inferred from nuclear and limited
plastid non-coding DNA sequences
ALAN W. MEEROW1,2*, DAVID J. LEHMILLER3 and JASON R. CLAYTON1
1
USDA-ARS-SHRS, National Germplasm Repository, 13601 Old Cutler Road, Miami, Florida 33156,
USA
2
Fairchild Tropical Garden, 10901 Old Cutler Road, Miami, Florida 33158 USA
3
550 IH-10 South, Suite 201, Beaumont, Texas 77707 USA
Received June 2002; accepted for publication November 2002
The genus Crinum L. is the only pantropical genus of the Amaryllidaceae. Phylogenetic and biogeographical analyses of nrDNA ITS and plastid trnL-F sequences for all continental groups of the genus Crinum and related African
genera are presented, with the genus Amaryllis used as outgroup. ITS indicates that C. baumii is more closely
related to Ammocharis and Cybistetes than to Crinum sensu stricto. Three clades are resolved in Crinum s.s. One
unites a monophyletic American group with tropical and North African species. The second includes all southern
African species and the Australian endemic C. flaccidum. The third includes monophyletic Madagascar, Australasian
and Sino-Himalayan clades, with southern African species. The trnL-F phylogeny resolves an American and an
Asian/Madagscar clade, and confirms the relationship of C. flaccidum with species endemic to southern Africa. The
salverform, actinomorphic perianths of subg. Crinum appear to have evolved several times in the genus from ancestors with zygomorphic perianths (subg. Codonocrinum), thus neither subgenus is monophyletic. Biogeographical
analyses place the origin of Crinum in southern Africa, as the region is optimized at all ancestral nodes in the tree
topology, and in basal interior nodes of all but one of the major clades. The genus underwent three major waves of
radiation corresponding to the three main clades resolved in our trees. Two entries into Australia for the genus are
indicated, as are separate Sino-Himalayan and Australasian dispersal events. © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 349–363.
ADDITIONAL KEYWORDS: molecular systematics – Africa – geophytes – dispersal – cladistics –
monocotyledons.
INTRODUCTION
The genus Crinum L. is the only pantropical genus of
the Amaryllidaceae, with species occurring in Africa,
America, Asia, and Australia. Crinum has seeds well
adapted for oceanic dispersal (Koshimizu, 1930),
which are considered to have engendered its broad distribution (Arroyo & Cutler, 1984). The genus is most
speciose in Africa, particularly sub-Saharan Africa
(Nordal, 1977), and its systematic affinities are with a
*Corresponding author. E-mail: miaam@ars-grin.gov
group of entirely African endemic genera, constituting
the tribe Amaryllideae (Snijman & Linder, 1996;
Meerow & Snijman, 1998, 2001). This tribe is
extremely well-marked by numerous morphological
synapomorphies, such as extensible fibres in the leaf
tissue, bisulculate pollen with spinulose exines, scapes
with a sclerenchymatous sheath, unitegmic or ategmic
ovules, and nondormant, water-rich, nonphytomelanous seeds with chlorophyllous embryos (Snijman &
Linder, 1996). In plastid DNA sequence analyses of
the Amaryllidaceae (Ito et al., 1999; Meerow et al.,
1999), this tribe is the first branch to resolve in the
family and receives high bootstrap support (>90%).
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 349–363
349
350
A. W. MEEROW ET AL.
Crinum was established by Linneaus (1753).
Herbert (1837) divided the genus into two sections
based on the degree to which the tepals are patent.
Baker (1881) divided the genus into three subgenera:
Stenaster, with salverform, actinomorphic perianths,
straight floral tubes and linear segments; Platyaster,
similar to the former but with lanceolate segments;
and Codonocrinum with funnel-form, zygomorphic
flowers and curved tubes. He later (Baker, 1898) submerged Platyaster into subgenus Stenaster, which
must be called subgenus Crinum since it contains the
type species, C. americanum L.
Crinum was not thoroughly monographed in the
last century, except for a synoptic update of Baker
(1881) by Uphof (1942). It has been the subject of
regional treatments (e.g. Hepper, 1968; Geerinck,
1973; Verdoorn, 1973; Nordal, 1977, 1982, 1986;
Nordal & Wahlstrom, 1980; Lehmiller, 1997a,b) and it
is estimated that the genus contains 60–70 species
(Fangan & Nordal, 1993). Fangan & Nordal (1993)
used DNA RFLPs to investigate the phylogeny of
seven species. Meerow & Snijman (2001) included four
species in their ITS analysis of the tribe Amaryllideae.
In this paper, we investigate the phylogeny of 38 species of Crinum, representing all of the continental
groups within the genus, using nuclear rDNA ITS
sequences (ITS1 spacer, 5.8S intron, ITS2 spacer), and
plastid trnL-F sequences, and discuss the results in a
biogeographical and evolutionary context.
MATERIAL AND METHODS
SAMPLING
Genomic DNA was extracted from silica gel-dried leaf
tissue of the taxa listed in Table 1, as described by
Meerow et al. (2000).
DNA
EXTRACTION, AMPLIFICATION AND SEQUENCING
PROTOCOLS
The trnL-trnF region was amplified and sequenced
using the primers of Taberlet et al. (1991) as described
by Meerow et al. (1999). Amplification of the ribosomal
DNA ITS1/5.8S/ITS2 region was accomplished using
flanking primers (18S, 26S) AB101 and AB102
(Douzery et al., 1999), and the original White et al.
(1990) internal primers ITS2 and 3 to amplify the
spacers along with the intervening 5.8S intron as
described by Meerow et al. (2000). All polymerase
chain reaction (PCR) amplifications were performed
on an ABI 9700 (Perkin-Elmer Applied Biosystems,
Foster City, California, USA).
Amplified products were purified using QIAquick
(Qiagen, Valencia, California, USA) columns, following the manufacturer’s protocols. Cycle sequencing
reactions were performed directly on purified PCR
products on the ABI 9700, using standard dideoxycycle protocols for sequencing with dye terminators on
either an ABI 377 or ABI 310 automated sequencer
(according to the manufacturer’s protocols; Applied
Biosystems, Foster City, California, USA).
SEQUENCE
ALIGNMENT
Both the ITS and trnL-F matrices contained few gaps
and were readily aligned manually using Sequencher
4.1 (Gene Codes, Ann Arbor, Michigan, USA). The
alignment is accessible through GenBank or from the
first author.
PHYLOGENETIC
ANALYSES
The ITS matrix consisted of 43 taxa (38 Crinum species, two species of Amaryllis and Ammocharis, and
Cybistetes longifolia). Amaryllis was designated as
outgroup. Amaryllis is sister to all other members of
the tribe Amaryllideae (Meerow & Snijman, 2001).
Ammocharis and Cybistetes are the only other members of the subtribe Crininae that together form a
sister clade to Crinum (Meerow & Snijman, 2001).
The plastid trnL-F matrix consisted of 19 taxa, 16
species of Crinum and one species each of Amaryllis (A. belladonna), Ammocharis (A. coranica), and
Cybistetes longifolia. We only sampled one to several
taxa from each of the clades resolved by ITS, and
saw little likelihood that additional sampling would
increase information content of the trnL-F data
matrix. In the combined data set, taxa with only one
sequence were coded as missing data for the absent
sequence. Aligned matrices were analysed using the
parsimony algorithm of PAUP* for Macintosh (version
4.0b10; Swofford, 1998), with the MULPARS option
invoked. Tree branches were retained only if unambiguous support was available (i.e. branches were
collapsed if the minimum length = 0). The few gaps
were coded as missing characters, as developing a
binary-coded strict homology gap matrix added no further resolution to the trees in preliminary analyses.
For all matrices, a heuristic search was conducted
under the Fitch (equal) weights (Fitch, 1971) criterion
with 1000 random sequence additions (Maddison,
1991) and tree bisection and reconnection (TRB)
branch swapping. We permitted only 20 trees to be
held at each step to reduce the time spent searching
trees at suboptimal levels. All minimal trees collected
in the 1000 replicates were then swapped to
completion.
We combined the data matrices, opting for the total
evidence approach (Seelanan, Schnabel & Wendel,
1997; Dubuisson, Hebant-Mauri & Galtier, 1998).
Though the trnL-F matrix yielded only a few parsi-
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 349–363
MOLECULAR PHYLOGENY OF CRINUM
mony informative base substitutions, it nonetheless
confirmed some of the relationships well-supported by
the ITS matrix, and inclusion in this study is therefore
warranted. However, before combining the ITS and
trnL-F data sets, we performed a partition homogeneity test on the matrices (Farris et al., 1994, 1995)
to assess the degree of congruence between them. Five
hundred heuristic searches were conducted, each with
ten random addition replications, saving 20 trees from
each for TBR branch-swapping.
Internal support was determined by bootstrapping
(Felsenstein, 1985; 5000 replicates with simple addition) and calculation of Bremer (1988) decay indices
(DI) using the program TreeRot vs. 2.1 (Sorenson,
1996). The cut-off bootstrap percentage is 50. A bootstrap value greater than 75% was considered good
support, 65–75% was designated moderate support,
and less than 65% as weak (Meerow & Snijman, 2001;
Meerow et al., 2002). Five hundred replicate heuristic
searches were implemented for each constraint statement postulated by TreeRot, saving ten trees per replicate. A minimum DI = 2 was considered to represent
good support for a clade.
BIOGEOGRAPHICAL
ANALYSES
The biogeographical patterns inferred from our gene
trees were assessed using both Fitch optimization
(Maddison, Ruvolo & Swofford, 1992) with MacClade
version 4.03 (Maddison & Maddison, 2001) and the
dispersal-variance method of analysis (Ronquist,
1997) using the program DIVA version 1.1 (Ronquist,
1996). The program uses vicariance (i.e. allopatric speciation) in its optimization of ancestral distributions
but takes into consideration dispersal and extinction
events, and indicates their direction (Ronquist, 1996,
1997). The most-parsimonious reconstructions minimize such events. Unlike Fitch optimization, DIVA
does not restrict widespread distributions to terminals
or limit ancestral distributions to single areas
(Ronquist, 1996). DIVA requires a fully bifurcated tree
for analysis, thus one tree (with zero-length branches
not collapsed) from the combined sequence analysis
was used for optimization of 15 coded geographical
areas (Table 1). It must be noted that this tree was one
of 150 equally parsimonious topologies, and contains
zero-length internal branches. Fitch optimization of
area data was performed on the same tree using a single multistate character (Table 1). Two DIVA analyses
were performed, one without limits on the maximum
areas allowed for ancestral nodes (15), and another in
which the limit was set to the minimum (2) to reduce
ambiguities at the more basal nodes of the tree. An
exact optimization (vs. heuristic) was invoked in both
analyses by allowing the maximum number of alternative reconstructions to be held at any node.
351
RESULTS
ITS
Of the 807 characters (ITS1, 5.8S intron, ITS2)
included in the analyses, 122 were parsimony
informative. The search found 119 equally mostparsimonious trees of length = 322, consistency index
(CI) = 0.79 and retention index (RI) = 0.89.
Two very well-supported clades are resolved by ITS
(Fig. 1). The smaller of the two, with a bootstrap of
100% and DI = 11 unites Crinum baumii as sister to
Ammocharis and Cybistetes. The position of Cybistetes
as sister to Ammocharis is only weakly supported
(bootstrap = 58%, DI = 1).
The second, larger clade with strong support (bootstrap = 99, DI = 9) consists of all of the remaining Crinum species. Three subclades of Crinum species form
a trichotomy within this larger group. The first (A),
with a bootstrap = 64 and DI = 2, includes a wellsupported American group (bootstrap = 99%, DI = 7)
embedded among strictly tropical and north African
species. No species endemic to South Africa included
in the analysis resolves within this clade. Crinum distichum, C. humile and C. jagus form a well-supported
group (bootstrap = 100%, DI = 9), as does C. kirkii and
C. politfolium (bootstrap = 90%, DI = 3).
The second subclade (B) is the best supported of
the three (bootstrap = 91%, DI = 4). With the exception of the Australian endemic C. flaccidum, the
clade consists entirely of African species, including
the only South African endemics (e.g. C. acaule,
C. bulbispermum,
C. campanulatum,
C. moorei,
C. variabile) included in the analysis. Outside of
well-supported sister relationships between C. acaule
and C. forbesii, and between C. bulbispermum and
C. variabile, the clade is largely unresolved.
The third subclade (C), with a bootstrap = 63 and
DI = 2, unites the Asiatic species with the Madagascar
endemics. The West African C. fimbriatulum is sister
to all other members of the clade. The Sino-Himalayan
species (C. defixum and C. sp., China) and the Australasian species form separate subclades, both with a
bootstrap of 65% and DI = 1. Three of the four Australasian species form a well-supported monophyletic
group with a bootstrap of 99% and DI = 5. The Madagascar species resolve as monophyletic with moderate
support (bootstrap = 73%, DI = 2). They form a polytomy with the Australasian group, and two broadly
distributed African species, C. subcernuum (Mozambique, Namibia, Tanzania) and C. buphanoides (southwest Africa, Angola, Transvaal region of South Africa).
PLASTID
TRNL-F
Of the 940 characters included in the analysis,
only eight were parsimony informative. Five equally
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 349–363
352
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 349–363
GenBank accession no. or literature
citation
Taxon
Voucher specimen
Provenance
ITS
Amaryllis belladonna L.
M. W. Chase 612 (K)
Western Cape, South Africa
A. paradisicola Snijman
van Jaarsveld 13263
(NBI)
Meerow 2320
Western Cape, South Africa
Eastern Cape, South Africa
Meerow & Snijman
(2001)
Meerow & Snijman
(2001)
Meerow & Snijman
(2001)
AY139116
AY139117
AY139118
AY139119
AY139120
AY139121
AY139122
AY139123
Ammocharis coranica Herb.
A. nerinoides (Baker) Lehmiller
Crinum abyssinicum Hochst. ex A. Rich.
C. acaule Baker
C. americanum L.
C. asiaticum L.
C. baumii Harms.
C. broussonetii (Redouté) Herb.
C. bulbispermum (Burm.) Milne-Redhead
& Schweickerdt
C. buphanoides Welw. ex Baker
C. campanulatum Herb.
Meerow 2321
Meerow 2322
Meerow 2338
Meerow 2323
Meerow 2334
Van Zyl 99.B (PRE)
Meerow 2324
Meerow 2339
Namibia
Ethiopia
Natal, South Africa
Florida, USA
cultivation, Florida, USA
Namibia
Chad
Natal, South Africa
Meerow 2325
Meerow 2337
Transvaal, South Africa
Eastern Cape, South Africa
C. carolo-schmidtii Dinter
C. crassicaule Baker
C. cruentum Ker Gawl.
C. defixum Ker Gawl.
C. distichum Herb.
C. erubescens Sol.
C. fimbriatulum Baker
C. flaccidum Herb.
C. forbesii (Lindl.) Schult. emend. Herb.
Meerow 2340
Meerow 2341
T. M. Howard s. n.
Traub 1235 (MO)
Meerow 2326
T. M. Howard s. n.
Leach 14510 (PRE)
Meerow 2327
Meerow 2328
Namibia
Namibia
Oaxaca, Mexico
Nepal
Chad
Brazil
Angola
Australia
Transvaal, South Africa
AY139124
Meerow & Snijman
(2001)
AY139125
AY139126
AY139127
AY139128
AY139129
AY139130
AY139131
AY139132
AY139133
trnL-F
Area
code1
A
A
AY139152
AY139153
AY139154
AY139155
AY139156
AY139157
AY139158
AY139159
AY139160
AY139161
AY139162
AY139163
ACE
B
D
A
G
M
B
C
A
AB
A
A
A
G
L
C
HI
B
N
A
1
A = South Africa, B = South-west Africa, C = tropical Africa, D = North Africa, E = East Africa, F = Madagascar, G = North America, H = Central America,
I = South America, J = Cuba, K = China, L = Nepal, M = Australasia (south-east Asia and Pacific), N = Australia, O = India.
A. W. MEEROW ET AL.
Table 1. Species, voucher specimens, GenBank sequence accession numbers (or previous citation), and geographical area codes used in the phylogenetic analyses
of Crinum. Vouchers are deposited at FTG unless otherwise stated
Taxon
Voucher specimen
Provenance
ITS
C. humilis A. Chev.
C. jagus Thomps.
C. kirkii Baker
C. latifolium Andr.
C. ligulatum Baker
C. macowanii Baker
Meerow 2329
Meerow 2330
Meerow 2342
Meerow 2343
Hardy 2995 (PRE)
Meerow 2344
Cameroun
cultivation, Florida, USA
Tanzania
India
Madagascar
Kenya
C. mauritianum Lodd.
C. modestum Baker
C. moorei Hook. F.
C. oliganthum Urb.
C. pedunculatum R. Br.
C. politifolium R.Wahlstr.
C. razafindratsiri Lehmiller
Madagascar
Madagascar
Natal, South Africa
Cuba
Australia
Tanzania
Madagascar
C. subcernuum Baker
C. variabile Herb.
Hardy s. n. (PRE)
Meerow 2345
Meerow 2346
Meerow 2336
Meerow 2335
Meerow 2347
Lehmiller 1944
(TAMU)
Meerow 2348
Meerow 2331
AY139134
AY139135
AY139136
AY139137
AY139138
Meerow & Snijman
(2001)
AY139139
AY139140
AY139141
AY139142
AY139143
AY139144
AY139145
C. venosum Baker
C. yemense Deflers
Meerow 2349
M. W. Chase 1595 (K)
Northern Australia
Yemen
C. sp., Borneo
C. sp., SW China
C. sp., Peru
Cybistetes longifolia (L.) Milne-Redh. &
Schweick.
Meerow 2332
Meerow 2333
Schunke 14054
Duncan 304 (NBG)
Borneo
Yunnan, China
Peru
Western Cape, South Africa
Namibia
Western Cape, South Africa
AY139150
Meerow & Snijman
(2001)
AY139146
AY139151
AY139147
AY139148
AY139149
Meerow & Snijman
(2001)
trnL-F
AY139164
AY139165
AY139166
AY139167
Area
code1
C
C
C
O
F
AE
F
F
A
J
N
C
F
BE
A
Meerow et al.
(1999)
AY139168
AY139169
N
D
M
K
I
A
A = South Africa, B = South-west Africa, C = tropical Africa, D = North Africa, E = East Africa, F = Madagascar, G = North America, H = Central America,
I = South America, J = Cuba, K = China, L = Nepal, M = Australasia (south-east Asia and Pacific), N = Australia, O = India.
1
MOLECULAR PHYLOGENY OF CRINUM
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 349–363
GenBank accession no. or literature
citation
353
354
A. W. MEEROW ET AL.
Figure 1. One of 119 most-parsimonious trees found by phylogenetic analysis of nrDNA ITS sequences across 43 species
of Crinum and related genera. Numbers above branches are branch lengths. Numbers below branches are bootstrap
percentages and decay indices (italic), respectively. Dashed lines are zero-length branches. A white bar across a branch
signifies a collapsed node in the strict consensus of all trees.
parsimonious trees were found of length = 42 steps,
CI = 1.00, RI = 1.00 (Fig. 2). The tree is mostly unresolved, but four clades are supported. A tropical Australasian clade (bootstrap = 64%, DI = 1) is resolved
as sister to the single Malagasy species included,
C. ligulatum (bootstrap = 64%, DI = 1). An American
clade is resolved with a bootstrap of 62% and DI = 1.
Finally, South African C. campanulatum and Australian C. flaccidum form a monophyletic group with a
bootstrap of 92% and DI = 2.
COMBINED
total of 150 equally parsimonious trees were found
by the heuristic search of length = 363, CI = 0.81,
RI = 0.89. Not surprisingly, the trees (Fig. 3) are very
similar in topology to those found using ITS alone.
However, bootstrap support for the position of
C. flaccidum as sister to an entirely African clade
rises to 87%, whereas support for the Sino-Himalayan
clade drops to 58%. The position of this clade as sister
to the Australasian–Madagascar–C. buphanoides–
C. subcernuum clade is slightly better supported and
does not collapse in strict consensus.
ANALYSIS
The P-value = 0.995, indicating a high level of congruence between the ITS and trnL-F matrices, though
this value is compromised by the lower sampling level
for trnL-F compared to ITS, and relative paucity of
informative base substitutions in the plastid data. A
BIOGEOGRAPHICAL
ANALYSIS
While allowing the maximum area assignments (15)
to ancestral nodes in DIVA produced an area optimization requiring only 17 dispersal events, it did so by
assigning the more basal nodes every possible area.
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 349–363
MOLECULAR PHYLOGENY OF CRINUM
355
(subg. Codonocrinum, Figs 1,3,4), but at the next
node, a Sino-Himalayan clade with actinomorphic
subg. Crinum flower morphology (Fig. 5) is sister
to an entirely subg. Crinum clade, including two
species from southern Africa (C. buphanoides and
C. subcernuum) whose exact relationships are unresolved. (Figs 1,3).
DISCUSSION
Figure 2. One of five most-parsimonious trees found by
phylogenetic analysis of plastid trnL-F sequences across 19
species of Crinum and related genera. Numbers above
branches are branch lengths. Numbers below branches are
bootstrap percentages and decay indices (italic), respectively. Dashed lines are zero-length branches.
Constraining the program to maxareas = 2 yields a
more realistic scenario, but the cost raises the number
of dispersal events to 23. The latter optimization is
referenced in the discussion (Fig. 4). Even with maxareas set to 2, one node was assigned four equally
parsimonious areas, and one received eight possible
assignments.
Both Fitch optimization and DIVA were largely congruent (Fig. 4). However, Fitch optimization places the
origins of the lineage in South Africa, while DIVA
found an origin in south-western Africa equally parsimonious. The ancestral origin of clade A is equivocal
by Fitch optimization, and DIVA assigns eight possible area assignments, all of which include either South
Africa or south-western Africa and respective dispersal to tropical Africa (C) North Africa (D), North
America (G), and (J) Cuba. However, clade A is the
only one of the three from which South Africa disappears from either the internal nodes or a terminal distribution (the distribution of C. buphanoides in clade
C includes South Africa). In clade B, South Africa persists as an area component through internal nodes as
well (Fig. 4). Clade B contains most of the endemic
South African species included in the analyses. Clade
C is rooted in south-western Africa by C. fimbriatulum
Meerow et al. (1999), using three plastid DNA
sequences, found that the Amaryllideae, the tribe of
Amaryllidaceae to which Crinum belongs, was the
most robustly supported clade in their parsimony
topologies and was sister to the rest of the Amaryllidaceae. Meerow & Snijman (2001), combining morphological characters and ITS sequences, analysed the
Amaryllideae and resolved a well-supported monophyletic subtribe, Crininae, consisting of Crinum,
Ammocharis and Cybistetes. Only four Crinum species
were included in that analysis. Crinum is the largest
genus in the tribe, and the only one to disperse outside
of Africa. The fleshy, floating, and salt-resistant seed of
Crinum has been implicated as the likely agent of its
dispersal success (Koshimizu, 1930; Arroyo & Cutler,
1984).
Fangan & Nordal (1993) performed restriction fragment length polymorphism (RFLP) analysis on seven
species of Crinum, using Pancratium canariensis as
outgroup. Pancratium, as the authors concede, was
probably not the best choice of outgroup for the polarization of character states in Crinum, as it is only distantly related to the ingroup. In fact, it is part of the
monophyletic Eurasian clade of Amaryllidaceae that
branches more terminally than Amaryllideae in plastid DNA based trees of the entire family (Meerow
et al., 1999). Despite the limited sampling, Fangan &
Nordal’s (1993) RFLP topology resolved some geographically congruent clades. A West African species
clade (C. glaucum, C. jagus and C. zeylanicum sensu
Nordal) resolved as a monophyletic sister group to a
clade inclusive of the East African and Indo-Pacific
species that they sampled (2 spp.). Crinum asiaticum
(subg. Crinum) resolved as sister to C. latifolium
(subg. Codonocrinum) within this latter clade. These
results were in contrast to their accompanying cladistic analysis of the same species across 11 morphological characters with the genus Ammocharis used as
outgroup. In these analyses, the species of subgenus
Codonocrinum formed a monophyletic group that was
sister to C. asiaticum, the only representative of subg.
Crinum included. Moreover, morphology placed
C. zeylanicum and C. latifolium in the same trichotomous clade with C. politifolium. The former two species have had a long and confused taxonomic history
(Nordal, 1977; Dassanayake, 1981) in terms of the
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 349–363
356
A. W. MEEROW ET AL.
Figure 3. One of 150 most-parsimonious trees found by phylogenetic analysis of combined nrDNA ITS and plastid
trnL-F sequences across 43 species of Crinum and related genera. Numbers above branches are branch lengths. Numbers
below branches are bootstrap percentages and decay indices (italic), respectively. Dashed lines are zero-length branches.
A white bar across a branch signifies a collapsed node in the strict consensus of all trees.
identity of C. latifolium relative to C. zeylanicum
(Fangan & Nordal, 1993). Crinum latifolium as
treated by Fangan & Nordal (1993) is the only Codonocrinum species found outside Africa other than
C. flaccidum that has not yet been proven to have been
introduced by humans. To confuse matters further, the
Linnean name C. latifolium is sometimes erroneously
applied to a Crinum in southern China with radially
symmetrical, salverform flowers (Zhanhe & Meerow,
2001). In our analyses, C. latifolium, despite its occurrence in India and Sri Lanka, is well nested in the
tropical African/American clade (clade A, Figs 1,3,4).
There are many tropical East African Crinum species
that are similar in morphology to C. latifolium, and a
putative scenario of how this species arrived in India
and Sri Lanka is discussed later in this paper. Finally,
Fangan & Nordal advised that, on the basis of their
RFLP topology, the characteristic floral morphology of
either subgenera Crinum or Codonocrinum may have
evolved more than once or reversals for this character
were possible.
Our ITS (Fig. 1) and combined (Fig. 3) phylogenies
support Fangan & Nordal’s (1993) suggestion based on
RFLPs that neither morphologically based subgenus
Crinum nor Codonocrinum is monophyletic. At issue
is which state is plesiomorphic in the genus. Snijman
& Linder (1996) concluded that zygomorphy is the
ancestral state in tribe Amaryllideae, and Meerow
et al. (1999) hypothesized that perianth symmetry in
Amaryllidaceae is under simple genetic control and
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MOLECULAR PHYLOGENY OF CRINUM
357
Figure 4. One of 150 equally most-parsimonious trees found by phylogenetic analysis of combined nrDNA ITS and plastid
trnL-F sequences across 43 species of Crinum and related genera showing optimization of bigeographical data. Fitch
optimization is indicated by colour or pattern; divergence-vicariance optimization is coded by small letters at ancestral
nodes. Central America and East Africa are not visible in the Fitch optimization because they figure only in ambiguous
area optimizations of terminal taxa.
easily modified. The sister group to Crinum (Ammocharis and Cybistetes; Meerow & Snijman, 2001) has
zygomorphic perianths. However, Crinum baumii,
which resolves strongly as part of the sister group to
Crinum, has floral morphology typical of subg. Crinum. Amaryllis, sister to all remaining genera of tribe
Amaryllideae (Meerow & Snijman, 2001), has zygomorphic perianths. Fitch optimization of this character onto a tree from our combined analysis (Fig. 5)
suggests that actinomorphy is the apomorphic state
and has evolved several times, once within the clade
inclusive of the Asian and Madagascar species, once in
the American clade, and yet again in the sister clade to
Crinum s.s. Both C. buphanoides and C. subcernuum
have subg. Crinum-type flowers, though their exact
relationships to both the Malagasy and Asiatic clades
are not resolved (Figs 1,3). There are only two species
from tropical Africa with subg. Crinum floral morphology, C. natans Baker and C. purpurascens Herb.,
which unfortunately were not available for sequencing. They may well represent the African sister group
to the American clade. Interestingly, both species
are emergent aquatics as are most of the American
species.
Fangan & Nordal (1993) also referred to a plastid
RFLP-supported connection between East African
(C. macowanii and C. politifolium) and ‘Indo-Pacific’
(C. asiaticum) Crinum. As no bootstrap or other confidence estimate was provided, the relative robustness
of this clade could not be determined. However,
our much larger sampling of species resolves
C. macowanii and C. politfolium each in two clades,
respectively (A and B, Figs 1,3,4) other than the one
containing all of the Asian species that we sampled
(clade C, Figs 1,3,4). The African mainland species
within clade C wherein all of the Asian (both SinoHimalayan and Australasian) species resolve are
south-western and southern African in the main
(C. subcernuum, described from Mozambique, has
been collected in Namibia by the second author, and
was collected once in Tanzania according to Nordal,
1977).
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 349–363
358
A. W. MEEROW ET AL.
Figure 5. Fitch optimization of perianth morphology on one of 150 equally most-parsimonious trees found by phylogenetic
analysis of combined nrDNA ITS and plastid trnL-F sequences across 43 species of Crinum and related genera.
Crinum baumii is sister to Ammocharis and
Cybistetes with bootstrap support of 100% and DI = 11
(Figs 1,4). Milne-Redhead & Schweickerdt (1939)
transferred Crinum baumii into Ammocharis without
any detailed justification, and also established the
monotypic genus Cybistetes as a segregate from the
latter, largely on the basis of infructescence structure.
In Cybistetes, the entire infructescence of indehiscent
fruits functions as the dispersal unit (anemogeochory
of Van der Pijl, 1982), whereas in Ammocharis the
fruits are dehiscent and the infrutescence lax
(Snijman & Williamson, 1994). Both Ammocharis and
Cybistetes have biflabellate leaves, which C. baumii
lacks, and zygomorphic perianths. Snijman &
Williamson (1994) went so far as to suggest that
separation of Ammocharis and Cybistetes needed reexamination. To this, we would add that the position of
C. baumii relative to these two genera also requires
re-evaluation, though there is little question that the
species lies outside of Crinum s.s. based on the ITS
phylogeny. This well-supported sister clade to Crinum
is rooted within southern Africa (Fig. 4), but is broadly
dispersed through sub-Saharan Africa via the five species of Ammocharis.
Given the likely role of oceanic dispersal of Crinum
seed in the evolution of the genus, it is not surprising that several geographically cohesive clades are
resolved in the genus by ITS and trnL-F sequences.
The roots of the entire lineage are in southern
Africa, and area analyses places either South Africa
(eastern South Africa in the case of Crinum s.s.) or
south-western Africa within the ancestral node of all
three clades of Crinum (Fig. 4). Each of the clades contains at least one dispersal event out of Africa (albeit
only one in clade B), while clade B also encompasses
continuous and perhaps recent evolution in South
Africa. A monophyletic American clade denotes a single dispersal event into the Western Hemisphere. A
monophyletic Madagascar group indicates a similar
scenario. Two Asiatic clades are resolved as well,
though not as sister groups, suggesting a possible double entry into the region.
A more precise understanding of the exact biogeographical scenario for Clade A is still elusive. The
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 349–363
MOLECULAR PHYLOGENY OF CRINUM
absence of any southern African Crinum species in the
American/North African/tropical African clade, if not
an artifact of our sampling biases, may indicate that
this clade represents the earliest vicariant divergence
from the rest of the genus (Fig. 4). Ostensibly, the decimation of the tropical flora of Africa was a major isolating factor for the genus. Direct connections between
South Africa and tropical Africa were most likely
severely interrupted by the late Oligocene/early
Miocene as uplift and climatic change began to create
the modern landscape of the continent (Axelrod &
Raven, 1978; Goldblatt, 1978; Coetzee, 1993). The
apparent relationships of American species of Crinum
with species from North and tropical Africa, rather
than with the southern African elements of the genus
(Fig. 4), might indicate that the dispersal point of
departure from Africa to America was in the northern
half of the African continent. A land bridge did exist
between the Brazilian Bulge and Nigeria until the end
of the Cretaceous (Rand & Mabesoone, 1982). Again,
sister group relationships are obscured by a basal trichotomy in the American/North African/tropical African clade (Figs 1,3).
Clade C also presents some intriguing biogeographical hypotheses. One subclade represents an Australasian group, i.e. species found in south-east Asia,
the Pacific and Australia (Fig. 4). A well-supported
group within this subclade as presently sampled
(C. asiaticum; C. pedunculatum, and C. sp., Borneo) is
marked morphologically by the obsolescence of the
bulb (the leaves basally form a tight and stout pseudostem which originates directly from a hyper-developed
basal plate). The second, or Sino-Himalayan clade,
represents species from the Himalayan region and
nearby south-west China that form true bulbs, which
may have entered the Indian subcontinent either
directly from Africa or via Madagascar. Both dispersal
pathways have been hypothesized for various taxa of
angiosperms (Raven & Axelrod, 1974). The SinoHimalayan clade barely receives moderate bootstrap
support (65%) and has a DI of only 1. Its ancestral
node has an equivocal Fitch optimization of area, and
two possible area assignments from DIVA. Moreover,
in the strict consensus tree of ITS alone it forms a
polytomy with the Australasian clade, the Madagascar clade, C. buphanoides and C. subcernuum (both
African and with subg. Crinum floral morphology);
thus the sister group relationships of either Asian
clade are not as clear as the single tree used for area
optimization would indicate (Fig. 4). However, in the
bootstrap consensus tree of the ITS matrix and in the
combined analysis, the Sino-Himalayan clade is sister to an Australasian–Madagascar–C. buphanoides–
C. subcernuum polytomy (Fig. 1, bootstrap = 50%;
Fig. 3, bootstrap = 63%), suggesting an earlier dispersal event from Africa, with a likely initial entry
359
into India. Consequently, dispersal to Australasia may
have been an independent and later event.
Van Steenis (1962) hypothesized a land-bridge connection between Madagscar and Sri Lanka incorporating the Seychelles–Comores bank during the mid- to
late Cretaceous, which he named ‘Lemuria.’ He saw no
other way to account for plant distributions that
encompassed the periphery of the Indian Ocean.
Mckenzie & Sclater (1973) refuted the possibility.
However, Haq, Hardenbol & Vail (1988) reported on
the likeliness of increased emergence of the Chagos/
Laccadive Plateau and then contiguous Mascarene
Plateau (including the Seychelles Bank) during the
early Oligocene (c. 30 MYA). Schatz (1996), in his
review of the Indo-Australo-Malesian relationships of
the Malagasy flora, postulates that lower sea levels
allowed these emergences to function as stepping
stones. Schatz’s (1996) ‘Lemurian stepping-stones’
could have engendered migration of Crinum from
Madgascar to western Malaysia. This may have also
been the pathway by which the ancestor of
C. latifolium dispersed from Africa to Sri Lanka and
India. Emergent archipelagos may have existed,
bridging much of the Indian Ocean between India and
Australia as well (McKenzie & Sclater, 1973), which
could have allowed dispersal of Crinum to northern
Australia and from there into south-eastern Asia and
the Pacific. Crinum modestum (Madagascar) and
C. venosum (Australia) have a further morphological
character in common; both have very short stamens
that are atypical for subg. Crinum. Beyond coarse estimates, the relative timing of these events cannot be
inferred, again due to the internal trichotomy formed
by the major subclades of clade C (Figs 1,3).
It is also evident that Crinum entered the
Australian continent at least twice. The other migration (C. flaccidum, ‘Codonocrinum’) was apparently
directly from Africa (in clade B, Fig. 4). The African
relationships of the more southern Australian Crinum
flaccidum are also resolved by both ITS and trnL-F
(Figs 1,2). If homoplasious base substitutions in the
ITS alignment are down-weighted using successive
approximation (Farris, 1969; Wenzel, 1997; Lledó
et al., 1998; Meerow et al., 1999), a sister relationship
between these two species is also resolved (data not
shown). Both species share similar seasonally aquatic
habitats (albeit on different continents) and have terete juvenile leaves. Moreover, like C. flaccidum, the
eastern Cape endemic C. campanulatum, has a campanulate perianth, but not declinate stamens. In the
combined analysis (Fig. 3), the position of C. flaccidum
as sister to an otherwise African clade is better supported than by ITS alone.
How did Crinum get to Australia this first time,
assuming an early introduction for the ancestor(s) of
C. flaccidum, all of whose closest extant relatives are
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 349–363
360
A. W. MEEROW ET AL.
endemic to Africa? Three pathways are possible. Oceanic long distance dispersal directly from Africa is one
scenario. Secondly, Madagascar–India could have provided an intermediate route, followed by oceanic dispersal to Australia, c. 65 MYBP (Raven & Axelrod,
1974). However, the phylogenetic relationships of
C. flaccidum are not with any extant Madagascar or
Indian Crinum species (Fig. 4). It is certainly possible
that the extant Madagascar and Indian species
represent more recent migrations and that the earlier migratory ancestors of C. flaccidum are extinct. A
third possible migration could have been via a more
southerly subtropical route through Antarctica, that
by traditional geophysical hypotheses (Smith &
Hallam, 1970; Smith, Smith & Funnell, 1994) was
available too early in the diversification of the
angiosperms to have figured in the biogeographical
history of a higher asparagoid monocot. However,
more recent hypotheses based on palaeontological evidence (Sampson et al., 1998; Hay et al., 1999; Krause
et al., 1999) suggest that a longer-lived land connection between India and Antarctica–Australia via the
Kerguelen Plateau may have existed as late as
80 MYBP.
Outside of two distinct clades of Crinum in Australia, the continent is home to a distinctive tribal clade
of the family (Meerow & Snijman, 1998; Meerow et al.,
1999), the Calostemmateae (Proiphys Herb. and
Calostemma R. Brown, the latter endemic to Australia, the former also extending into south-east Asia). In
the case of Calostemmateae, one might infer that the
tribe originated in Australia and later migrated to
south-east Asia, since the continent contains both the
generic and species diversity of the lineage. The sister
relationships of this tribe have still not been resolved
(Meerow et al., 1999). In the lower asparagoid family
Iridaceae, all species of the genus Dietes are endemic
to eastern through southern sub-Saharan Africa with
the exception of a single Australian species on Lord
Howe Island (Goldblatt, 1981) that is considered the
least morphologically derived species in the genus
(Goldblatt, 1978). Baum, Small & Wendel (1998) concluded that dispersal between Africa and Australia in
the genus Adansonia (Malvaceae) occurred via ocean
currents considerably after the break-up of Gondwanaland. In the case of Adansonia, the sole Australian species was sister to the rest of the genus
(Madagascar and Africa), thus the direction of the dispersal event was ambiguous. In Crinum, there is no
ambiguity that the genus originated in Africa (Fig. 4).
Southern Africa is also where the sister group to
Crinum originated (Fig. 4), and is the centre of diversity for the genus (Nordal, 1977; Fangan & Nordal,
1993). This is not to suggest that all of this diversity is
necessarily ancient; it may reflect radiation engendered by the more recent palaeoclimatic and geological
history of Africa encompassing Neogene and later
times (Axelrod, 1972; Raven & Axelrod, 1974; Coetzee,
1993). The increased aridity of the African climate and
the uplift of the continental mass that started at the
beginning of the Miocene, further abetted by Quaternary climatic fluctuations (Demenocal, 1995) were catastrophic to many elements of the African flora, but it
may have been a selective pressure for diversity
among groups of geophytes capable of adapting to
increasing drought. The geophyte richness of South
Africa is well documented (Goldblatt, 1978), and the
Cape region has been suggested as a possible refuge
for certain African plant and animal groups as the
tropical flora of the continent was impoverished
(Raven & Axelrod, 1974). C. variabile is the only
Crinum species so far known from the winter-rainfall
region of western South Africa (Verdoorn, 1973). Subtropical forests were still present in the Western Cape
during the Miocene and Pliocene (Scott et al., 1997).
The earliest evidence of modern semiarid, winterrainfall pattern in the Western Cape dates to the Late
Pliocene, but it was not fully established until the
Early Pleistocene (Tankard & Rogers, 1978; Hendey,
1983; Coetzee, 1986). Moreover, the winter-rainfall
region of southern Africa experienced a more recent
pattern of expansion and contraction with concurrent
wetter and drier conditions during glacial and interglacial periods of the Quaternary (Tankard, 1976;
van Zinderen Bakker, 1976; Tyson, 1986; Crockcroft,
Wilkinson & Tyson, 1987). It would appear that a winter rainfall regime is largely inimical to Crinum.
Nonetheless, it is impossible to determine if the genus
ever existed in the Western Cape prior to the establishment of the Mediterranean climate.
In conclusion, nuclear rDNA ITS sequences support
a southern African (eastern South Africa or southwestern Africa) origin for the genus Crinum, and indicate three major waves of radiation corresponding
to the three main clades resolved in our trees
(Figs 1,3,4). Two entries into Australia for the
genus are hypothesized. Asian and Malagasy Crinum
are phylogenetically related, and separate SinoHimalayan and Australasian dispersals are indicated.
The monophyletic American species are allied with
tropical and North African species. Recognition of two
subgenera in Crinum on the basis of floral morphology
is not supported by the molecular phylogeny, as the
apomorphic subg. Crinum floral morphology has
evolved more than once (Fig. 5). Crinum baumii
appears to be more closely related to Ammocharis and
Cybistetes, and the taxonomic standing of this species
and both of these genera needs to be re-evaluated in
light of this relationship.
We sought to augment the incomple resolution of
phylogenetic relationships within Crinum with data
from the plastid atpB-rbcL intergenic spacer (Chiang,
© 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 349–363
MOLECULAR PHYLOGENY OF CRINUM
Schaal & Peng, 1998). The AT-rich spacer proved
extremely hyper-variable, and necessitated the trimming of 360 bp that was impossible to align, even
within clades well-supported by ITS. The resulting
matrix was poorly resolved (data not shown), but after
successive weighting, a few clades in common with the
ITS or trnL-F phylogeny were supported: (1) a tropical
and North African clade (bootstrap = 88%), (2) a Madagascar/Asian clade (71%), albeit with the anomalous
inclusion of C. crassicaule, (3) a C. campanulatum/
C. flaccidum sister relationship (87%), and (4) a
C. kirkii–C. politifolium clade (99%). We are in the
process of obtaining sequences for the plastid ndhF
gene, in the hopes that it will resolve the basal polytomies within the ITS phylogeny and allow stratigraphic estimation of divergence within the genus.
ACKNOWLEDGEMENTS
This work was partially supported by National Science Foundation Grants DEB-968787 and 0129179.
We thank Drs DA Snijman and MW Chase for providing leaf material or DNA of several species, and
an anonymous reviewer whose comments greatly
improved the manuscript.
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