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511
Systematic Botany (2004), 29(3): pp. 511–517
qCopyright 2004 by the American Society of Plant Taxonomists
Pucara (Amaryllidaceae) Reduced to Synonymy with Stenomesson on the
Basis of Nuclear and Plastid DNA Spacer Sequences, and a New Related
Species of Stenomesson
A
LAN
W. M
EEROW
1
and H
ENK VAN DER
W
ERFF
2
1
USDA-ARS-SHRS, 13601 Old Cutler Road, Miami, Florida, 33158 and Fairchild Tropical Garden, 11935 Old
Cutler Road, Miami, Florida 33156;
2
Missouri Botanical Garden, P. O. Box 299, St. Louis, Missouri 63166-0299
Communicating Editor: James F. Smith
A
BSTRACT
.Pucara leucantha is transferred to Stenomesson as Stenomesson leucanthum based on the phylogenetic position
of Pucara resolved by nuclear and plastid DNA sequences. An allied species, Stenomesson chloranthum, is described from
the Departments of Amazonas and Cajamarca in Peru, but at lower elevations. Both of these species release their pollen in
tetrads, unique within Amaryllidaceae, and have tri-lobed stigmas, unique within Stenomesson.
Meerow et al. (1986) described the first known oc-
curence of mature pollen in tetrads within the Amar-
yllidaceae for a species they determined as Stenomesson
elwesii Macbr. from collections by Paul Hutchinson.
Meerow later had the opportunity to examine the type
of S. elwesii [now Clinanthus elwesii (Macbr.) Meerow] at
Kew, and it became obvious that the name was mis-
applied by Meerow et al. (1986). The identity of the
Hutchinson collections remained unknown. While it
clearly bore resemblance to a species of Stenomesson
sensu stricto (Meerow et al. 2000), the palynological
apomorphy and the low elevation of the collection were
unusual.
New collections from the Department of Amazonas
in Peru in 1999 and 2001 were identified as represent-
ing both this unidentified species and the sole species
of Pucara Rav., P. leucantha Rav. (Ravenna 1972), allow-
ing more detailed study. We observed that P. leucantha
has leaf morphology similar to the undescribed spe-
cies, and also releases its pollen in tetrads. In this pa-
per, we show that the phylogenetic position of these
two species based on DNA sequences leaves little
doubt that the genus Pucara should not be recognized
as distinct from Stenomesson (sensu Meerow et al.
2000). We further describe the misidentified taxon as a
new species of Stenomesson, allied to the erstwhile P.
leucantha.
M
ATERIALS AND
M
ETHODS
DNA Sequencing and Alignment. S
AMPLING
. Genomic DNA
was extracted from silica gel dried leaf tissue of the taxa listed in
Appendix 1 as described by Meerow et al. (2000).
DNA E
XTRACTION
,A
MPLIFICATION AND
S
EQUENCING
P
ROTOCOLS
.
The plastid atpß-rbcL spacer was amplified and sequenced using the
primers and polymerase chain reaction (PCR) protocol of Chiang
et al. (1998). 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 gene as described by Meerow et al.
(2000). All PCR amplifications were performed on an ABI 9700
(Perkin-Elmer Applied Biosystems, Foster City, California, USA).
Amplified products were purified using QIAquick (Qiagen, Va-
lencia, California, USA) columns, following manufacturer’s pro-
tocols. Cycle sequencing reactions were performed directly on pu-
rified PCR products on the ABI 9700, using standard dideoxycycle
protocols for sequencing with dye terminators on either an ABI
310 or ABI 3100 automated sequencer (according to the manufac-
turer’s protocols; Applied Biosystems, Foster City, California,
USA).
S
EQUENCE
A
LIGNMENT
. Both the ITS and atp-rbcL spacer ma-
trices were readily aligned manually using Sequencher 4.1 (Gene
Codes, Ann Arbor, Michigan, USA). We also used Clustal X (Hig-
gins and Sharp 1988; Thompson et al. 1997) to align the sequences
as a check against our manual alignments to help highlight any
ambiguous regions. The alignments and the parsimony trees used
in this paper can be accessed at TreeBase (study accession number
S1037, matrix accession numbers M1762–M1765).
Phylogenetic Analyses. Both DNA sequence matrices consisted
of 18 taxa (Appendix 1) representing the Andean tetraploid clade
resolved by Meerow et al. (2000) with ITS sequences. Four tribes
are represented: Clinantheae, Eustephieae, Hymenocallideae, and
Stenomesseae. The Eustephieae (Eustephia darwinii and Chlidanthus
fragrans) were used as outgroup, as this tribe is sister to the rest
of the clade (Meerow et al. 2000).
Aligned matrices were analyzed 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 50). Gaps were coded as missing char-
acters in the initial analyses, but a gap matrix was also constructed
from the indel-rich plastid spacer region using the program PAUP-
GAP (Anthony Cox, formerly RBG Kew), which applies a strict
interpretation of gaps (i.e., only gaps of equal length are consid-
ered homologous among taxa). As Kelchner (2000) has pointed
out, indels within plastid non-coding regions are frequently as, if
not more, informative than the nucleotide sequences themselves.
This binary matrix was added to the sequence alignment and an-
alyzed in combination. For all matrices, a branch and bound (Hen-
dy and Penny 1982) search was conducted under the Fitch (equal)
weights (Fitch 1971) criterion with simple addition sequence.
We also combined the two data matrices, opting for the ‘‘total
evidence’’ approach (Dubuisson et al. 1998; Seelanan et al. 1997).
However, before combining the ITS and atpß-rbcL spacer data sets,
we performed partition homogeneity tests on the matrices (Farris
et al. 1994, 1995) to assess the degree of congruence between them.
One thousand heuristic searches were conducted for each test,
each with 10 random addition replications, saving no more than
20 trees from each for TBR branch swapping.
Internal support was determined by bootstrapping (Felsenstein
1985; 5000 heuristic replicates with simple addition, TRB branch-
swapping, saving 20 trees per replicate) and calculation of Bremer
(1988) decay indices (DI) using the program TreeRot v. 2.1 (Soren-
512 [Volume 29SYSTEMATIC BOTANY
F
IG
. 1. One of four equally most parsimonious trees found
by a cladistic analysis of the plastid atpß-rbcL spacer region
across 18 species of Amaryllidaceae. Numbers above branches
are branch lengths. Numbers below branches are bootstrap
support percentages/decay indices (italic). Large shadowed
numbers adjacent to nodes are ‘‘clade credibility’’ scoresfrom
500,000 generations of Bayesian analysis. A vertical white bar
indicates a branch that collapses in the strict consensus of all
four trees.
F
IG
. 2. One of three equally most parsimonious trees
found by a cladistic analysis of the plastid atpß-rbcL spacer
region plus a binary coded gap matrix across 18 species of
the Amaryllidaceae. Numbers above branches are branch
lengths. Numbers below branches are bootstrap support per-
centages and decay indices (italic).
son 1996). The cut-off bootstrap value is 50%. A bootstrap value
greater than 75% was considered good support, 65–75% was des-
ignated moderate support, and less than 65% as weak (Meerow
and Snijman 2001; Meerow et al. 2002). A branch and bound
search was implemented for each constraint statement postulated
by TreeRot. A minimum DI 52 was considered to represent good
support for a clade.
We also applied Bayesian analysis (Huelsenbeck et al. 2001) to
each sequence matrix, in order to approximate a bootstrap of max-
imum likelihood estimates of the phylogenetic relationships, and
check for congruence with the results of parsimony analysis. The
program MrBayes v. 3.04 (Huelsenbeck and Rohnquist 2001) was
used for this purpose. We first determined which model of nucle-
otide substitution to impose on our data with ModelTest v. 3.06
(Posada and Crandall 1998). We used the Akaike information cri-
terion (Akaike 1974) to choose the model with the best fit. We then
ran 500,000 generations of four simultaneous Markov chains with
Mr. Bayes, retaining the tree from every 100th generation (i.e., a
total of 5000 trees, less the trees produced before log likelihood
scores stabilized) from which a 50% majority rule consensus tree
was constructed. In each case, the log likelihood scores stabilized
after 10,000 generations of Bayesian analysis, but we dropped the
trees from 20,000 generations (200 of 5000) in constructing the 50%
majority consensus trees. The results of the Bayesian analyses are
reported as ‘‘clade credibility’’ (CC) scores, i.e., the percentage of
trees sampled where a given clade is resolved, which is equal to
the posterior probability of the clade existing (Huelsenbeck and
Rohnquist 2001). Only CC scores in excess of 50% are shown in
our trees (Figs. 1–5).
R
ESULTS
Plastid atpß-rbcL Spacer. The atpß-rbcL spacer ma-
trix yielded 37 parsimony informative characters out
of a total of 1276. The percentage of total matrix cells
coded as missing was 22.5%. A branch and bound
search found four equally parsimonious trees of length
5232, consistency index (CI) 50.93 and retention in-
dex (RI) 50.80). The topology (Fig. 1) is congruent
with the major clades of Andean genera resolved in
the large ITS analysis of American Amaryllidaceae by
Meerow et al. (2000), Pucara leucantha and Stenomesson
chloranthum are sister species with a bootstrap of 63%
and decay index (DI) 51. The nucleotide substitution
model that best fit the atpß-rbcL spacer data was the
Kimura 3-parameter model with unequal base fre-
quencies with gamma distribution (K81uf1G; Kimura
1981), which was approximated in Mr. Bayes with the
settings nst 52. The ‘‘Pucara’’ clade within Stenomes-
seae received a clade credibility score of 99% (Fig. 1).
When a binary gap matrix for the spacer region was
added to the sequence alignment, a total of 86 char-
acters were parsimony informative out of 1406 total.
Three equally parsimonious trees were found, of
length 5420, CI 50.82 and RI 50.69 (Fig. 2). Pucara
leucantha still resolves as sister species to Stenomesson
chloranthum but with weak bootstrap support 560
2004] 513MEEROW AND VAN DER WERFF: PUCARA
F
IG
. 3. Single most parsimonious tree found by a cladistic
analysis of the nrDNA ITS region across 18 species of the
Amaryllidaceae. Numbers above branches are branch lengths.
Numbers below branches are bootstrap support percentages/
decay indices (italic). Large shadowed numbers adjacent to
nodes are ‘‘clade credibility’’ scores from 500,000 generations
of Bayesian analysis.
F
IG
. 4. Single most parsimonious tree found by a cladistic
analysis of combined plastid atpß-rbcL spacer and nrDNA ITS
across 18 species of the Amaryllidaceae. Numbers above
branches are branch lengths. Numbers below branches are
bootstrap support percentages/decay indices (italic). Large
shadowed numbers adjacent to nodes are ‘‘clade credibility’’
scores from 500,000 generations of Bayesian analysis.
(though DI 52). Stenomesson flavum resolves as sister
to the Pucara clade with a bootstrap 566 and DI 52.
Pucara and its sister species are embedded within a
monophyletic Stenomesson clade (bootstrap 553, DI 5
1).
ITS. Of 646 total characters, 129 were parsimony
informative. The percentage of total matrix cells coded
as missing was 6.5%. A single most parsimonious tree
was found by the branch and bound search, of length
5401 steps, CI 50.85 and RI 50.85 (Fig. 3). Pucara
leucantha resolves as sister species to Stenomesson chlor-
anthum with bootstrap 571 and DI 51, nested in a
clade with S. miniatum and S. flavum with bootstrap 5
99 and DI 54. Stenomesson is monophyletic with a
bootstrap 571 and DI 52. For the Bayesian analysis,
a general time reversible model with a proportion of
invariant sites (GTR 1I; Lanave et al. 1984) was the
model that best fit our data. Clade credibility for the
sister relationship of P. leucantha and S. chloranthum was
98%, nested within a clade including S. miniatum and
S. flavum (CC 5100%). Stenomesson was monophyletic
with a CC 598%.
atpß-rbcL Spacer and ITS. The partition homoge-
neity test suggested that the two sequence matrices
were largely incongruent (P 50.01). However, if one
compares the trees supported by each of the two gene
regions (Figs. 1, 3), it is clear that the incongruity is
concentrated in the more terminal branches. The same
main clades are resolved by both sets of data. The
combined sequence matrix yielded 166 parsimony in-
formative characters out of 1922 total. Branch and
bound search found a single tree of length 5637 steps,
CI 50.87 and RI 50.83 (Fig. 4). Pucara leucantha and
Stenomesson chloranthum are again sister species, but
with a bootstrap of 86 (DI 52), within the same clade
resolved by ITS alone (bootstrap 581, DI 52). Boot-
strap support for a monophyletic Stenomesson rises to
85 (DI 52). The Bayesian consensus tree of 4800 sam-
pled trees from the analyses is highly congruent with
the parsimony analysis. Pucara leucantha and Stenomes-
son chloranthum are sister species in a clade with S.
flavum and S. miniatum with a CC5100% in both cases.
While the sister relationship of S. flavum to the Pucara
subclade has a CC of only 54%, Stenomesson is mono-
phyletic with a CC of 100%. Adding the atpß-rbcL spac-
er gap matrix to the combined sequence matrix raised
the number of parsimony informative characters to
215, and resulted in two equally parsimonious trees of
828 steps, CI 50.83, RI 50.78 (not shown), differing
only in the resolution of Stenomesson aurantiacum and
S. miniatum (sister species in one tree; forming a grade
514 [Volume 29SYSTEMATIC BOTANY
F
IG
.5. Stenomesson chloranthum and S. leucanthum. A–B. Flowers of S. chloranthum.A.Meerow 2520 (FTG). B. Meerow 1155
(FLAS). C. Leaf of S. leucanthum (Meerow 2522, FTG). D. Leaf detail of S. chloranthum (Meerow 2520, FTG). E. Flowers of S.
leucanthum (Meerow 2522, FTG). F. Pollen tetrad of S. chloranthum (Meerow 1155, FLAS). Scales A–E 51 cm, F 55mm.
with the Pucara subclade in the other). Other than this,
the tree topologies were the same as the one tree found
without the gap matrix added to the sequences.
D
ISCUSSION
The case for treating Pucara as part of the larger ge-
nus Stenomesson is unambiguously supported by both
plastid and nuclear non-coding sequences, whether an-
alyzed by parsimony or likelihood. Both P. leucantha
and the newly described S. chloranthum have shortly
sub-petiolate leaves with a well-developed midrib,
which is characteristic of the genus Stenomesson,the
limits of which were re-assessed by Meerow et al.
(2000) on the basis of nrDNA ITS sequences. The most
significant synapomorphy of P. leucantha and S. chlor-
anthum is the exclusive presence of pollen tetrads at
anthesis (Fig. 5f), a character state that is not known
to occur in any other species of the genus, or within
2004] 515MEEROW AND VAN DER WERFF: PUCARA
F
IG
.6. Stenomesson chloranthum (Meerow 2520, FTG). A. In-
florescence. B. Leaf. C. Longitudinal cross section of flower. D.
Longitudinal view of flower. E. Oblique view of flower. F. Te-
pals, outer (right), inner (left). G. Staminal cup. All scales 5
1 cm.
the entire family. Ravenna (1972) denoted the alter-
nating position of the free staminal filaments in P. leu-
cantha (those opposite the outer tepals are inserted at
the rim of the staminal cup; those opposite the inner
tepals, below the rim) and its tri-lobed stigma as the
main basis for generic recognition. While S. chloran-
thum has a less complex androecium, polymorphism
of staminal cup morphology within the Andean peti-
olate-leafed clade of Amaryllidaceae has been dem-
onstrated within the limits of a single species (Meerow
1989). Transfer of P. leucantha into Stenomesson is thus
warranted. Stenomesson leucanthum is the only white
flowered species of Stenomesson so far known. Nothing
is known about the pollination biology of the genus.
No other Stenomesson species from the interior of
Peru have ever been found below about 2000 m ele-
vation. Low elevation species have only before been
recorded from the coastal lomas of Peru, where max-
imum temperatures approximate those of Andean
high elevations. The same general region in Peru where
S. chloranthum and S. leucanthum are found is also host
to the three species of the bizarre, green-flowered, suc-
culent-leafed endemic genus Rauhia, which resolved in
a different sub-clade of the tribe Stenomesseae in Mee-
row et al.’s (2000) ITS phylogeny. The geographic con-
centration of such novelties suggests that the area bor-
dered by the lower Rı´o Utcubamba and middle Rı´o
Maran˜o´n was a hotspot for diversification in the tribe
Stenomesseae as the Andes rose to their present po-
sition.
T
AXONOMIC
T
REATMENT
Stenomesson leucanthum (Ravenna) Meerow & van
der Werff, comb. nov. (Figs. 5c, e). Pucara leucantha
Ravenna, Ann. Mus. Nat. Valparaiso 5: 85–89
(1972).—Type: Peru, Cajamarca, Jaen, San Anto-
nio (km 81—Pucara), 990 m, 12 Oct 1965, Saga´s-
tegui 5850 (holotype: HUT).
Representative Specimens Examined. PERU. Ama-
zonas: Pedro Ruiz to Chachapoyas, 1500 m, 24 Apr
2002 (pressed from bulbs collected by H. van der Werff
s. n.), Meerow 2523 (FTG); Utcubamba Valley between
Pedro Ruiz and branch to Chachapoyas, 14 Apr 2002
(pressed from bulbs collected by H. van der Werff s.
n.), Meerow 2522 (FTG).
Stenomesson chloranthum Meerow & van der Werff,
sp. nov. (Figs. 5a–b, d, f, 6).—TYPE: PERU. Ama-
zonas: Bagua, near Pongo de Rentema on the Rı´o
Maran˜on, 1 km east of Olmos on the Mesones-
Muro Highway, 370 m, 26 Jan 1964 (pressed from
living material 23 June 1965), Hutchinson and
Wright 3782 (holotype: UC!).
S. leucantho affine sed foliis olivaceis, floribus pallide
viridibus, staminibus insertis pariter margine coronae,
et praesentia altitudine inferiore differt.
Representative Specimens Examined. PERU. Ama-
zonas: Bagua Grande, 500 m, 25 Mar 2002 (pressed
from bulbs collected by H. van der Werff s. n.), Meerow
2520 (FTG). Cajamarca: Jaen, 2 km north of Chamaya
on rd. to Jaen, 450 m, 7 Feb 1964 (pressed 8 Jul 1965
from living material), Hutchinson & Wright 4123 (UC);
Cajamarca: a few km outside Jaen towards San Ignacio,
600 m 8 Apr 2002 (pressed from bulbs collected by H.
van der Werff s. n.), Meerow 2521 (FTG).
Bulbous perennial herb; bulb globose, with a short
(2–3 cm) neck, 2.5–4 cm diam, producing offset bulbils.
Leaves 2–3, hysteranthous or emerging with the flow-
ers, sub-petiolate, lanceolate, obtuse, 25–29 cm long, 2–
2.5 cm wide, olive-green (Royal Horticultural Society
Color Chart Green 137A), somewhat glaucous, with an
obscure midrib on the adaxial surface (pronounced on
the abaxial). Inflorescence scapose; scape terete, solid,
30–40 cm long, 7–8 mm diam proximally, 5 mm diam
distally, terminated by a pseudoumbel of 5–10 flowers
enclosed by two obvolute, greenish-white, marcescent
bracts before anthesis; bracts ovate-lanceolate, 2.5–3 cm
long, 10–11.5 mm wide at the base, acute. Perianth
campanulate-tubular, pale green, 5.8–6.7 cm long
(from base of tube to apex of limb); tube 3.5–4 cm
long, cylindrical and 2.5–3.1 mm wide at base, abrupt-
ly dilating in the distal half to 8.3–9 mm at the throat,
longitudinally striped white, limb spreading 18.5–19
mm wide. Tepals 6, in two series, outer 17.7–18.5 mm
long, 8–8.5 mm wide, elliptic, acute and minutely apic-
516 [Volume 29SYSTEMATIC BOTANY
F
IG
. 7. Map of Peru showing distributions of Stenomesson
chloranthum (open circles) and S. leucanthum (black squares).
ulate; inner 16.8–18.3 mm long, 8.3–8.9 mm wide, el-
liptic, obtuse, margins of both series hyaline. Stamens
6, white to greenish white, fused for the proximal 4–5
mm into a short staminal cup; free filaments broadly
subulate, edentate, 8.3–8.7 mm long, 2.7–3 mm wide,
abruptly tapering to 1 mm in their distal 2 mm, barely
exserted from the limb; anthers ca. 3 mm long, oblong,
dorsifixed, introrse; pollen white, released in tetrads
(Fig. 5f). Style filiform, ca. 6 cm long, exceeding the
stamens by about 5 mm; stigma trilobed. Ovary ellip-
soid, 7.5–8 mm long, 4.3–5 mm wide, locules 3, ovules
axile, numerous per locule, flattened and superposed.
Fruit a turbinate, loculicidal capsule, turning papery at
dehiscence; seeds numerous per locule, black, flat,
obliquely winged. 2n546.
Stenomesson chloranthum is endemic to the lower
slopes of the seasonally dry interandean valleys of the
Maran˜o´n and Utcubamba drainages in northern Ca-
jamarca and west-central Amazonas departments of
Peru (Fig. 7), between 350–600 m elevation, in dry
scrub, often growing with Opuntia and other Cacta-
ceae.
Stenomesson chloranthum can be distinguished from
S. leucanthum by its more glaucous, olive-green leaves
with a more obscure adaxial midrib (Fig. 5d), its fewer
but larger green and white flowers, the more simpli-
fied structure of its androecium, and its lower altitu-
dinal limits. The white-flowered S. leucanthum is not
known from elevations lower than 900 m and occurs
up to 1650 m elevation. The adaxial mibrib is conspic-
uously visible on the less glaucous leaves of this spe-
cies (Fig. 5c).
A
CKNOWLEDGMENTS
. Karen Williams prepared the drawings
of S. chloranthum. This work was partially supported by National
Science Foundation Grants DEB-968787 and 0129179 to AWM.
L
ITERATURE
C
ITED
A
KAIKE
, H. 1974. A new look at the statistical model identification.
IEEE Transactions on Automatic Control 19: 716–723.
B
REMER
, K. 1988. The limits of amino acid sequence data in an-
giosperm phylogenetic reconstruction. Evolution 42: 198–213.
C
HIANG
, T-Y., B. A. S
CHAAL
, and C-I. P
ENG
. 1998. Universal prim-
ers for amplification and sequencing a noncoding spacer be-
tween the atpß and rbcL genes of chloroplast DNA. Botanical
Bulletin of the Academia Sinica 39: 245–250.
D
OUZERY
, J. P., A. M. P
RIDGEON
,P.K
ORES
,H.K
URZWEIL
,P.L
INDER
,
andM.W.C
HASE
. 1999. Molecular phylogenetics of Diseae
(Orchidaceae): a contribution from nuclear ribosomal ITS se-
quences. American Journal of Botany 86: 887–899.
D
UBUISSON
,J.Y.,R.H
EBANT
-M
AURI
,andJ.G
ALTIER
. 1998. Mole-
cules and morphology: conflicts and congruence within the
fern genus Trichomanes (Hymenophyllaceae). Molecular Phy-
logenetics and Evolution 9: 390–397.
F
ARRIS
,J.S,M.K
A
¨LLERSJO
¨
,A.G.K
LUGE
, and C. B
ULT
. 1994. Test-
ing significance of incongruence. Cladistics 10: 315–319.
———, ———, ———, and ———. 1995. Constructing a signifi-
cance test for incongruence. Systematic Biology 44: 570–572.
F
ELSENSTEIN
, J. 1985. Confidence limits on phylogenies: an ap-
proach using the bootstrap. Evolution 39: 783–791.
F
ITCH
, W. M. 1971. Toward defining the course of evolution: min-
imum change for a specific tree topology. Systematic Zoology
20: 406–416.
H
ENDY
,M.D.andD.P
ENNY
. 1982. Branch and bound algorithms
to determine minimal evolutionary trees. Mathematical Biosci-
ence 59: 277–290.
H
IGGINS
,D.G.andP.M.S
HARP
. 1988. CLUSTAL: a package for
performing multiple sequence alignment on a microcomput-
er. Gene 73: 237–244.
H
UELSENBECK
,J.P.,andF.R
ONQUIST
. 2001. MRBAYES: Bayesian
inference of phylogeny. Bioinformatics 17: 754–755.
———, ———, R. N
IELSEN
,andJ.P.B
OLLBACK
. 2001. Bayesian
inference of phylogeny and its impact on evolutionary biol-
ogy. Science 294: 2310–2314.
K
ELCHNER
, S. A. 2000. The evolution of non-coding chloroplast
DNA and its application in plant systematics. Annals of the
Missouri Botanical Garden 87: 482–498.
K
IMURA
, M. 1981. Estimation of evolutionary distances between
homologous nucleotide sequences. Proceedings of the National
Academy of Science, USA 78: 454–458.
L
ANAVE
,C.,G.P
REPARATA
,C.S
ACCONE
,andG.S
ERIO
. 1984. A
new method for calculating evolutionary substitution rates.
Journal of Molecular Evolution 20: 86–93.
M
EEROW
, A. W. 1989. Systematics of the Amazon lilies, Eucharis
and Caliphruria (Amaryllidaceae). Annals of the Missouri Bo-
tanical Garden 76: 136–220.
———, N. B. D
EHGAN
, and B. D
EHGAN
. 1986. Pollen tetrads in
Stenomesson elwesii (Amaryllidaceae). American Journal of Bot-
any 73: 1642–1644.
———, C. L. G
UY
, Q-B. L
I
, and S-L. Y
ANG
. 2000. Phylogeny of the
American Amaryllidaceae based on nrDNA ITS sequences.
Systematic Botany 25: 708–726.
———, ———, ———, and J. R. C
LAYTON
. 2002. Phylogeny of the
tribe Hymenocallideae (Amaryllidaceae) based on morphol-
ogy and molecular characters. Annals of the Missouri Botanical
Garden 89: 400–413.
2004] 517MEEROW AND VAN DER WERFF: PUCARA
——— and D. A. S
NIJMAN
. 2001. Phylogeny of Amaryllidaceae
tribe Amaryllideae based on nrDNA ITS sequences and mor-
phology. American Journal of Botany 88: 2321–2330.
P
OSADA
, D. and K. A. C
RANDALL
. 1998. Modeltest: testing the
model of DNA substitution. Bioinformatics 14: 817–818.
R
AVENNA
, P. 1972. Pucara,ge´nero nuevo de Amaryllidaceae del
norte de Peru. Annales del Museo Nacional de Valparaiso 5: 85–
89.
S
EELANAN
, T., A. S
CHNABEL
,andJ.W
ENDEL
. 1997. Congruence
and consensus in the cotton tribe (Malvaceae). Systematic Bot-
any 22: 259–290.
S
ORENSON
, M. D. 1996. TreeRot. University of Michigan, Ann Ar-
bor.
S
WOFFORD
, D. L. 1998. Phylogenetic analysis using parsimony, v.
4.0 beta. Sutherland: Sinauer Associates,
T
HOMPSON
,J.D.,T.J.G
IBSON
,F.P
LEWNIAK
,F.J
EANMOUGIN
,and
D. G. H
IGGINS
. 1997. The ClustalX windows interface: flexible
strategies for multiple sequence alignment aided by quality
analysis tools. Nucleic Acids Research 24: 4876–4882.
W
HITE
,T.J.,T.B
RUNS
,S.L
EE
,andJ.T
AYLOR
. 1990. Amplification
and direct sequencing of fungal ribosomal RNA genes for
phylogenetics. Pp. 315–322 in PCR protocols: a guide to methods
and applications, eds. M. Innis, D. Gelfand, J. Sninsky, and T.
White. Orlando: Academic Press.
A
PPENDIX
1
Species, vouchers, and Genbank accession numbers (or litera-
ture citations for previously published sequences) of DNA se-
quences used in this paper. All vouchers deposited at FTG unless
otherwise stated.
Clinanthus humilis (Herb.) Meerow—Meerow 2442:atpß-rbcL spac-
er AY460393, ITS Meerow et al. (2000). C. incarnatus (H.B.K.)
Meer ow— Meerow 1120:atpß-rbcL spacer AY460392, ITS Mee-
row et al. (2000). C. mirabilis (Rav.) Meerow—S. Leiva et al.
2000 (HUT): atpß-rbcL spacer AY460391, ITS Meerow et al.
(2000)
Chlidanthus fragrans Herb.—Meerow 2312:atpß-rbcL spacer
AY460390, ITS Meerow et al. (2000)
Eucharis castelnaeana (Baill.) MacBride—Schunke 14156 (FLAS):
atpß-rbcL spacer AY460405, ITS Meerow et al. (2000). E. for-
mosa Meerow—Whitten et al. 95020 (FLAS): atpß-rbcL spacer
AY460406, ITS Meerow et al. (2000)
Eucrosia dodsonii Meerow & Dehgan—Meerow 1115:atpß-rbcL
spacer AY460404, ITS Meerow et al. (2000)
Eustephia darwinii Vargas—Meerow 2436:atpß-rbcL spacer
AY460389, ITS Meerow et al. (2000)
Hymenocallis chiapasiana T. M. Howard—T. M. Howard 1185
(MO): atpß-rbcL spacer AY460396, ITS AY461739. H. glauca
M. Roem.—Meerow 2433:atpß-rbcL spacer AY460395, ITS
Meerow et al. (2000). H. latifolia (Mill.) M. Roem.—Meerow
2438:atpß-rbcL spacer AY460398, ITS Meerow et al. (2000).
H. tubiflora Salisb.—Meerow 2440:atpß-rbcL spacer
AY460399, ITS Meerow et al. (2000)
Ismene vargasii (Velarde) Gereau & Meerow—Meerow 2308:atpß-
rbcL spacer AY460397, ITS Meerow et al. (2000)
Stenomesson aurantiacum Herb.—Meerow 1061 (FLAS): atpß-rbcL
spacer AY460394, ITS Meerow et al. (2000). S. chloranthum
Meerow & van der Werff—Meerow 2520:atpß-rbcL spacer
AY460403, ITS AY461738. S. flavum (R. & P.) Herb.—Meerow
2430:atpß-rbcL spacer AY460402, ITS Meerow et al. (2000). S.
leucanthum (Rav.) Meerow & van der Werff (Pucara leucantha
Rav.)—Meerow 2522:atpß-rbcL spacer AY460401, ITS
AY461737. S. miniatum (Herb.) Ravenna—Meerow 1118:atpß-
rbcL spacer AY460400, ITS AY461736.
