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MOLECULAR PHYLOGENY OF NYCTAGINACEAE:TAXONOMY,
BIOGEOGRAPHY,AND CHARACTERS ASSOCIATED WITH A
RADIATION OF XEROPHYTIC GENERA IN NORTH AMERICA
1
NORMAN A. DOUGLAS
2
AND PAUL S. MANOS
Department of Biology, Duke University, Box 90338, Durham, North Carolina 27708 USA
The four o’clock family (Nyctaginaceae) has a number of genera with unusual morphological and ecological characters, several
of which appear to have a ‘‘tendency’’ to evolve repeatedly in Nyctaginaceae. Despite this, the Nyctaginaceae have attracted little
attention from botanists. To produce a phylogeny for the Nyctaginaceae, we sampled 51 species representing 25 genera (of 28–
31) for three chloroplast loci (ndhF,rps16,rpl16, and nrITS) and included all genera from North America. Parsimony, likelihood,
and Bayesian methods were used to reconstruct the phylogeny for the family. The family is neotropical in origin. A radiation of
woody taxa unites Pisonia and Pisoniella with the difficult tropical genera Neea and Guapira, which also form a clade, though
neither appears to be monophyletic. This group is sister to a clade containing Bougainvillea,Belemia, and Phaeoptilum.A
dramatic radiation of genera occurred in the deserts of North America. The tribe Nyctagineae and its subtribes are paraphyletic,
due to over-reliance on a few homoplasious characters, i.e., pollen morphology and involucre presence. Two notable characters
associated with the desert radiation are cleistogamy and edaphic endemism on gypsum soils. We discuss evolutionary trends in
these traits in light of available data about self-incompatibility and gypsum tolerance in Nyctaginaceae.
Key words: biogeography; cleistogamy; gypsophily; homoplasy; mating system; Nyctaginaceae; phylogeny; pollen
morphology.
Nyctaginaceae Juss. is a family of 28–31 genera and 300–
400 species, that contains the familiar cultivated four o’clocks
(Mirabilis jalapa) and bougainvillea (Bougainvillea spp.).
Nyctaginaceae has long been known to be one of the core
groups of families of Caryophyllales (Centrospermae) on the
basis of the presence of betalain pigments, free-central
placentation, p-type sieve tube elements, and the presence of
perisperm, as well as molecular evidence (Bittrich and Ku¨hn,
1993; Bremer et al., 2003). Within this group, the modern
consensus is that Nyctaginaceae are closely related to certain
monocarpellate members of a paraphyletic Phytolaccaceae,
especially subfam. Rivinoideae (Rodman et al., 1984; Rettig et
al., 1992; Downie and Palmer, 1994; Behnke, 1997; Downie et
al., 1997; Cuenoud et al., 2002), although Sarcobatus
(Sarcobataceae) has also been implicated as a close relative
of this group (Behnke, 1997; Cuenoud et al., 2002).
Nyctaginaceae have a uniseriate petaloid perianth, usually
interpreted as sepalous in origin (Rohweder and Huber, 1974).
In most taxa the lower part of the perianth is fleshy or
coriaceous and encloses the superior ovary, giving it the
appearance of an inferior ovary. This accessory fruit is
persistent and accrescent around the mature achene. While
technically a diclesium (Bogle, 1974; Spellenberg, 2003), it is
typically referred to as an ‘‘ anthocarp.’’
Most genera can be recognized on the basis of fruit structure
alone. In Boldoa,Cryptocarpus,andSalpianthus, the perianth
is persistent but not accrescent, and thus these taxa lack the
anthocarp (Bittrich and Ku¨hn, 1993). In Andradea,Leucaster,
and Reichenbachia, the perianth is variously accrescent but is
not expanded (Bittrich and Ku¨hn, 1993). However, in the
remaining genera the anthocarp completely encloses the fruit
and takes many forms (Willson and Spellenberg, 1977; Bittrich
and Ku¨hn, 1993). In taxa in which anthocarps are ribbed, the
3–10 ribs can be elaborated into wings (Phaeoptilum,
Grajalesia, Tripterocalyx,Abronia, and some Colignonia,
Acleisanthes, and Boerhavia), covered by viscid glandular
hairs or warts (Pisonia,Pisoniella,Cyphomeris,Commicarpus,
and some Boerhavia and Acleisanthes), or unelaborated, to
leave an essentially gravity-dispersed fruit (Mirabilis,Anulo-
caulis,Nyctaginia, and some Colignonia and Boerhavia).
Fleshy anthocarps are probably bird-dispersed in Neea and
Guapira. They are also found in Okenia, though this genus is
geocarpic and the seeds generally germinate at the spot where
they are ‘‘planted’’ by the maternal individual (N. Douglas,
personal observation). The unusual anthocarps of Allionia are
boat-shaped, with two rows of inward-pointing teeth lining the
concave side, suggesting possible exozoochory or wind
dispersal, though no observations on this are available. In
herbaceous taxa, at least, species-level characters are often
found in this structure (Willson and Spellenberg, 1977;
Spellenberg, 2003).
The family was treated by Heimerl in Die Natu¨rlichen
Pflanzenfamilien (Heimerl, 1889, 1934) and by Standley in
several papers (Standley, 1909, 1911, 1918, 1931a, b) by
which time most of the currently recognized genera had been
described. Standley (1931a) formally transferred Oxybaphus
L’He´r. ex Willd., Hesperonia Standl., Quamoclidion Choisy,
and Allionella Rydb. into Mirabilis, though this has been
overlooked in some floras (e.g., Kearney and Peebles, 1960).
1
Manuscript received 22 June 2006; revision accepted 28 February 2007.
The authors thank the members of the Manos Lab at Duke University,
D. Stone, and E. Christine Davis for discussion and helpful comments on
the manuscript; F. Kauff for translations from German; M. Hilda Flores
Olvera and P. Herna´ndez-Ledezma for assistance in the field; the curators
of DUKE, NMC, and NY for permission to sample from their collections;
R. Wilbur, Strybing Arboretum, and the Duke University Botany
Greenhouses for providing plant material used in this study; and Grand
Canyon National Park for permission to collect. The Department of
Biology (Duke), the Consortium in Latin American & Caribbean Studies
(Duke), and the National Science Foundation provided funding for this
project. The authors especially thank R. Spellenberg, who provided
invaluable assistance in many aspects of this project.
2
Author for correspondence (e-mail: nad@duke.edu)
856
American Journal of Botany 94(5): 856–872. 2007.
Heimerl (1934) synthesized the family as it was known,
including in his classification genera that had been recently
described by Standley (i.e., Pisoniella,Cuscatlania). He based
his supergeneric classification on a combination of plant habit,
indumentum, linear vs. capitate stigma, straight vs. curved
embryo, sex distribution, pollen grain morphology, and the
occurrence of bracts or involucre (Bittrich and Ku¨hn, 1993;
Heimerl, 1934). Bittrich and Ku¨hn (1993) provided the most
recent summary of the classification at the tribal and subtribal
level (Table 1). Their treatment broadly followed that of
Heimerl (1934), adjusting ranks and incorporating genera
described after 1934, i.e., Caribea. It recognized six tribes, two
of which, Pisonieae and Nyctagineae, contain the majority of
genera and species (Table 1, Pisonieae: six genera, ca. 200
spp.; Nyctagineae: 14 genera, ca. 100 spp.).
Whereas the bulk of diversity of Pisonieae resides in three
highly similar arborescent genera with poorly differentiated
species, Nyctagineae sensu Bittrich and Ku¨hn (1993) is a
diverse, mainly herbaceous, group recognized largely on the
basis of very large (100–200 lm in diameter), pantoporate
pollen grains, among the largest known in angiosperms
(Stevens, 2001). The original formulation of tribe Mirabileae
subtribe Boerhaaviinae (Heimerl, 1934), the antecedent of tribe
Nyctagineae, was partly diagnosed by the presence of
pantoporate pollen grains. Of four currently recognized
subtribes, the Nyctagininae comprises those taxa with involu-
cres, which may be of connate or distinct bracts. In contrast, the
largest subtribe, Boerhaviinae, is composed of eight genera
united primarily by their lack of involucral bracts. Four of these
(Boerhavia,Anulocaulis,Cyphomeris,Commicarpus) have
occasionally been treated as a single Boerhavia (Fosberg,
1978). This seems merely to reflect a preference for fewer large
genera, because the four segregate genera are as distinct from
each other as any other given pair of genera in the herbaceous
group. The others, Caribea,Okenia,Acleisanthes,and
Selinocarpus (including Ammocodon), were placed in Boerha-
viinae on the basis of pollen morphology and the absence of
involucres subtending flowers or inflorescences (though small
subtending bracts may be present). The remaining two
subtribes, Colignoniiae and the monospecific Phaeoptilinae,
have aberrant morphology compared to Nyctagininae and
Boerhaviinae, for example, pollen grains in Colignonia and
Pisoniella are dramatically smaller, and in Phaeoptilum they
are pantocolpate. In Pisoniella, the embryo is straight, typical
of Pisoneae, instead of a hooked embryo that encircles the
perisperm as found in the remaining Nyctagineae (Bittrich and
Ku¨hn, 1993). Additionally, the shrubby, scandent or lianoid
growth habits of these taxa are rare in the other subtribes,
which are mostly perennial herbs. Though Heimerl placed
Colignonia in a monogeneric tribe Colignonieae, Bittrich and
Ku¨hn (1993) include subtribe Colignoniinae (including
Pisoniella) in tribe Nyctagineae, uniting all taxa with
TABLE 1. Classification of Nyctaginaceae and estimates of species number.
Tribe
Genus
Species
number
Spellenberg (2003),
if different DistributionSubtribe
Leucastereae Leucaster Choisy 1 SA
Reichenbachia Spreng. 2 SA
Andradea Fr. Allema˜o 1 SA
Ramisia Glaz. ex Baillon 1 SA
Boldoeae Boldoa Cav. ex Lagasca 1 SA, CA
Salpianthus Humb. & Bonpl. 1 CA
Cryptocarpus H.B.K. 1 SA
Abronieae Abronia Juss. (incl. Tripterocalyx Hook. ex Standl.) 33 24* NA
Nyctagineae
Colignoniinae Colignonia Endl. 6 SA
Pisoniella (Heimerl) Standl. 1 SA, CA
Boerhaviinae Boerhavia L. 20 ca. 40 Pantropical/subtropical
Anulocaulis Standl. 4–5 5 NA
Cyphomeris Standl. 2 NA
Commicarpus Standl. 25 30–35 Pantropical/subtropical
Caribea Alain 1 Cuba
Acleisanthes A. Gray 7 17** NA
Selinocarpus A. Gray (incl. Ammocodon Standl.) 10 — NA, Africa
Okenia Schldl. & Cham. 1–2 NA, CA
Nyctagininae Mirabilis L. 54 ca. 60 NA, Asia
Cuscatlania Standl. 1 CA
Allionia L. 2 NA, CA, SA
Nyctaginia Choisy 1 NA
Phaeoptilinae Phaeoptilum Radlk. 1 Africa
Bougainvilleeae Bougainvillea Comm. ex Juss. 18 SA
Belemia Pires 1 SA
Pisonieae Pisonia L. 40 10–50 Pantropical/subtropical
Guapira Aubl. 70 10–50 SA, CA
Neea Ruiz & Pavon 83 SA, CA
Neeopsis Lundell 1 CA
Cephalotomandra Karst. & Triana 1–3 SA
Grajalesia Miranda 1 CA
Note: Classification scheme according to Bittrich and Ku¨ hn (1993) and estimates of species number. For those genera treated in Flora of North America
(Spellenberg, 2003), species number reflects newly described species and taxonomic readjustments. SA ¼South America; CA ¼Central America; NA ¼
North America. * As 20 spp. Abronia and four spp. Tripterocalyx. ** Including Selinocarpus &Ammocodon (Levin, 2002).
May 2007] DOUGLAS AND MANOS—PHYLOGENY OF NYCTAGINACEAE 857
pantoporate grains and Phaeoptilum (with its pantocolpate
grains) in one tribe.
Two major centers of distribution have been noted for the
Nyctaginaceae (Standley, 1909). The first is in the neotropics
and Caribbean, characterized by arborescent genera such as
Neea,Guapira,Pisonia, and Bougainvillea, as well as the
herbaceous Colignonia and Salpianthus. The second is in arid
western North America, where several herbaceous or suffru-
tescent genera are native, including Boerhavia,Mirabilis,
Abronia,Acleisanthes sensu Levin (2002), and Commicarpus.
A few genera are widespread in tropical and subtropical
regions of the world (Boerhavia,Commicarpus,Pisonia):
Mirabilis is present in North and South America with one
species in Asia, and Acleisanthes contains the disjunct A.
somalensis from Somalia. Mirabilis (M. jalapa,M. oxy-
baphoides) and Bougainvillea (B. glabra,B. spectabilis,B.
peruviana, and numerous hybrid cultivars) are naturalized in
many parts of the world. Only one genus is restricted to the Old
World, the monospecific Phaeoptilum of southwestern Africa.
The first molecular phylogenetic study of Nyctaginaceae
was presented by Levin (2000). The focus was on species in
certain genera of tribe Nyctagineae sensu Bittrich and Ku¨hn
(1993), including genera in subtribes Nyctagininae (Allionia,
Mirabilis) and Boerhaviinae (Acleisanthes,Selinocarpus,
Boerhavia), as well as Abronia and Pisonia. The study justified
the formal combination of Acleisanthes,Selinocarpus, and
Ammocodon (Levin, 2002; Spellenberg and Poole, 2003), but
due to limited sampling of genera, it was not possible to
evaluate the monophyly of the subtribes of Nyctagineae
(Levin, 2000). The Flora of North America treatment of
Nyctaginaceae (Spellenberg, 2003), while not referring to tribal
classification, reflected these and other taxonomic changes for
the genera and species that occur in North America north of
Mexico (Table 1).
In the herbaceous taxa of Nyctaginaceae found in the deserts
of North America, several unusual characters occur with
notable frequency. As indicated by the common name for the
family, species in several genera (Anulocaulis,Cyphomeris,
Acleisanthes,Mirabilis,Abronia, and Tripterocalyx) flower in
the evening and are adapted to moth pollination (Baker, 1961;
Grant, 1983; Grant and Grant, 1983; Herna´ndez, 1990;
Hodges, 1995; Levin et al., 2001). Internodal bands of viscid
secretions, which may discourage aphid colonization (McClel-
lan and Boecklen, 1993), are present in Anulocaulis,
Cyphomeris, and some species of Boerhavia. As mentioned,
anthocarp morphology is also variable, with wings and viscid
glands being common modifications.
Because these characters are often polymorphic at the
generic level, they would seem to represent evolutionary
‘‘tendencies.’’ Sanderson (1991) discussed evolutionary ten-
dencies in explicit phylogenetic terms: a tendency is a
concentrated distribution of homoplasy within a tree. The
main objection to the study of tendencies is the difficulty in
defining the taxonomic scope at which they operate, in other
words, it is ‘‘. . . biologically inappropriate [when investigating
a hypothesized tendency] to include taxa that cannot under any
circumstances exhibit the states of interest’’ (Sanderson, 1991,
p. 357). Thus, when considering whether a character has a
tendency to evolve, it is first necessary to evaluate the range of
taxa in which it could potentially appear. In some cases, it may
be possible to identify another character upon which the
evolution of the character of interest is dependent. If this other
trait is itself uniquely derived, its occurrence will define the
group in which the tendency may conceivably exhibit itself. If
the independent character is itself derived multiple times, then
the problem is pushed back so that the challenge is first to
explain the tendency for the independent character to evolve in
the group.
In the case of tendencies in Nyctaginaceae, it is not
immediately obvious what sorts of traits may be required to
enable, for instance, a shift to nocturnal pollination or the
development of viscid bands on stem internodes. There are two
traits, however, that seem to have a tendency to evolve in
Nyctaginaceae and that we can reasonably assume are
contingent on other traits: the evolution of cleistogamy is
improbable without prior self-compatibility, and lineages that
specialize on gypsum are unlikely to have arisen from lineages
with no latent or expressed gypsum tolerance.
Cleistogamous (closed, self-fertilizing) flowers are produced
in addition to chasmogamous (open) flowers in four genera of
Nyctaginaceae: Acleisanthes,Cyphomeris,Nyctaginia,and
some Mirabilis (Cruden, 1973; Spellenberg and Delson,
1974; Fowler and Turner, 1977; Levin, 2002). Though species
with cleistogamous flowers have evolved in a number of
angiosperm families, only in much larger families, e.g.,
Poaceae, Fabaceae, and Malpighiaceae, is this trait found in
as many genera (Lord, 1981). Despite a long awareness of this
phenomenon generally (Darwin, 1884), the evolution of this
character has only rarely been investigated with phylogenetic
methods (Desfeux et al., 1996; Bell and Donoghue, 2003).
Second, as in many caryophyllid families, e.g., Amarantha-
ceae and Portulacaeae, there is a propensity in many
Nyctaginaceae to be tolerant of, or specialists of, gypseous
soils. Outcrops of gypsum (hydrous calcium sulfate) are quite
common in arid North America, especially in the Chihuahuan
Desert. These areas have a flora characterized by gypsophiles,
which never occur on other substrates, and gypsum-tolerant
species, which are found on both gypseous and nongypseous
soils (Waterfall, 1946; Parsons, 1976; Meyer, 1986). In the
United States and Mexico, Nyctaginaceae are well represented
in gypsum communities (Parsons, 1976). At least 25 species in
seven genera are known to occur on gypsum. Of these, roughly
half are known gypsophiles, found only on gypsum soils
(Johnston, 1941; Waterfall, 1946; Fowler and Turner, 1977;
Turner, 1991, 1993; Spellenberg, 1993, 2003; Mahrt and
Spellenberg, 1995; Harriman, 1999; Levin, 2002).
Although gypsum soils support a distinct flora, the evolution
of gypsophily is not understood as well as other cases of
edaphic endemism. Gypsum is not an inherently poor substrate
for plants in the same way as soil with, for instance, toxic levels
of heavy metals (Cockerell and Garcia, 1898; Johnston, 1941;
Loomis, 1944; Parsons, 1976; Meyer, 1986; Oyonarte et al.,
2002). Recent experimental work has pointed toward mechan-
ical, rather than chemical, factors to explain the limited flora of
gypsum soils: seedlings of nongypsophiles are unable to
penetrate the hard crust typical of gypseous soils. This indicates
that adaptations of gypsum-tolerant taxa primarily act to
enhance survival in the establishment stage (Meyer, 1986;
Meyer et al., 1992; Escudero et al., 1997, 1999, 2000; Romao
and Escudero, 2005).
Edaphic-endemic species are sometimes found to be related
to species that are merely tolerant: in the case of a serpentine
endemic species of Layia (Asteraceae), certain populations of a
non-endemic progenitor species were found to tolerate
serpentine soils (Baldwin, 2005). Thus, even in the case of
highly toxic soils, saltational speciation (Antonovics, 1971;
858 AMERICAN JOURNAL OF BOTANY [Vol. 94
Kruckeberg, 1986) is not required to explain edaphic
endemism. These lines of evidence, and the fact that roughly
half of the species of Nyctaginaceae found on gypsum are not
restricted to it, make it reasonable to assume that an underlying
ability to survive in gypsum soils is an early stage in the
evolution of this type of edaphic endemism in Nyctaginaceae.
In principle, for both of these examples, the evolution of
both the independent and contingent characters can be
reconstructed on a phylogeny. With an understanding of the
distribution of homoplasy in Nyctaginaceae, we will have a
more robust framework for asking questions about character
evolution and adaptation to xeric environments. In this
phylogenetic study we comprehensively sample the genera of
Nyctaginaceae, with the following goals: (1) to evaluate the
existing classification of Bittrich and Ku¨hn (1993), (2) to
understand the biogeographic history of the family, and (3) to
have a basis for understanding the evolutionary history of
characters of historical taxonomic importance and the potential
adaptive significance as manifested in their ‘‘ tendency’’ to
evolve repeatedly in lineages occurring in the deserts of North
America.
MATERIALS AND METHODS
Sampling—Fifty-one species representing 25 genera of Nyctaginaceae
were sampled. Taxa, voucher information, and GenBank numbers are given in
Appendix 1. Our sampling is nearly comprehensive at the generic level, with
representative species of every genus except Neeopsis,Cephalotomandra,
Grajalesia,Cuscatlania,Boldoa, and Cryptocarpus. The genera omitted are
monotypic, rarely collected, and/or of dubious distinction. For example, Boldoa
purpurascens is often included in Salpianthus (Pool, 2001). All tribes and
subtribes recognized by Bittrich and Ku¨ hn (1993) are included. Because
different taxa have been found to be sister to Nyctaginaceae (Rettig et al., 1992;
Behnke, 1997; Downie et al., 1997; Cuenoud et al., 2002), outgroups were
selected from both Phytolaccaceae and Sarcobataceae. More distantly related
taxa in the ‘‘core Caryophyllales,’’ i.e., Aizoaceae, Molluginaceae, and
Stegnospermataceae (Cuenoud et al., 2002), were also included to enable us
to test the monophyly of Nyctaginaceae and to identify which taxa are sister to
the family. For four species, data were obtained from two different accessions,
and for two, GenBank sequences were used for some loci. ‘‘Phytolacca’’ is a
composite of one GenBank sequence from P. acinosa and three new sequences
from P. americana.
Molecular data—Genomic DNA was extracted from fresh, silica-dried, or
air-dried (herbarium) leaf tissue using either Qiagen DNAeasy Plant Mini Kits
or a modified CTAB method (Doyle and Doyle, 1987). Internal transcribed
spacer (ITS) sequences were obtained using primers ITS4 and ITS5a (White et
al., 1990; Stanford et al., 2000), which amplifies ITS1, 5.8S, and ITS2.
Chloroplast ndhF sequences were obtained as two overlapping fragments using
primers Nyct-ndhF1, ndhF972, Nyct-ndhF13R, and Nyct-ndhF22R. With the
exception of ndhF972 (Olmstead and Sweere, 1994), these were designed based
on GenBank ndhF sequences for Nyctaginaceae and Phytolaccaceae. Many
samples, especially those from herbarium materials, were recalcitrant to PCR of
long (.1 kb) fragments due to DNA degradation; for these, four additional
primers (Nyct-ndhF6F, Nyct-ndhF8R, Nyct-ndhF13F, and Nyct-ndhF16R)
were designed, based on sequences for Nyctaginaceae and Phytolaccaceae, and
used in conjunction with the aforementioned primers, so that the gene was
amplified in four overlapping fragments. The chloroplast intron rps16 was
amplified using primers rpsF and rps2R (Oxelman et al., 1997), and rpl16 was
obtained using primers F71 and R1661 (Jordan et al., 1996). Primer sequences
and references are given in Table 2. PCR products were cleaned with Qiaquick
columns (Qiagen, Valencia, California, USA). Cycle sequencing was
performed using the BigDye Terminator v3.1 Cycle Sequencing Kit, and
sequences were determined with an ABI 3700 DNA Analyzer (Applied
Biosystems, Foster City, California, USA) in the Genetic Analysis facility in
the Department of Biology at Duke University. Raw chromatograms were
edited and assembled in Sequencher 4.1 (Gene Codes Corp., Ann Arbor,
Michigan, USA). Sequence alignment was performed either by eye (ndhF)orin
ClustalX (Thompson et al., 1997) (other regions) followed by manual
adjustment in Se-Al (Rambaut, 1996). Across the entire data set, ITS1 and
ITS2 were too variable to be confidently aligned, although the 5.8S region was
highly conserved. Ambiguously aligned regions were excluded from further
analyses of the entire data set, though they were used in analyses of more
restricted taxon sets (see Restricted analyses).
Caribea littoralis Alain, a Cuban endemic, has been collected only once.
The collection locality is in southeastern Cuba in a dry coastal habitat. The
morphology of the plant is difficult to interpret because it is highly distinct from
any other member of the Nyctaginaceae, and the leaves and flowers are highly
reduced. Few details are clearly visible on the specimen, though the description
appears to have been based on fresh material (Alain, 1960). Due to the age of
the collection, only about 25% of an ndhF sequence was obtainable. This
sequence was unique in our data set, and a BLAST search found that this
sequence fragment was most similar to an existing Bougainvillea ndhF
sequence (GenBank no. AF194825). Preliminary phylogenetic analysis (see
Data analysis) placed this taxon as sister to either Pisoniella or Belemia. These
last two are not closely related to each other, resulting in substantial loss of
resolution in the clade including these taxa. Therefore, Caribea was excluded
from all further analysis, and while this result confirms that this enigmatic taxon
belongs in Nyctaginaceae, further study must await rediscovery of this species.
Unfortunately, repeated attempts to relocate the population at the type locality
TABLE 2. Primer sequences used and original publication.
Region
Sequence ReferencePrimer name
ITS
ITS4 TCCTCCGCTTATTGATATGC White et al., 1990
ITS5a CCTTATCATTTAGAGGAAGGAG Stanford et al., 2000
ndhF
Nyct_ndhF1F TGCCTGGATTATACCCTTCA This study
NdhF972F ATGTCTCAATTGGGTTATATGATG Olmstead and Sweere, 1994
Nyct_ndhF13R CAFCBGGATTACYGCATTT This study
Nyct_ndhF22R CTTGTAACGCCGAAACCATT This study
Nyct_ndhF6F AACGGGBAGTTTYGARTTTG This study
Nyct_ndhF8R AGTAGGCCCCTCCATAGCAT This study
Nyct_ndhF14F TCAATCGTTGCAATCCTTCT This study
Nyct_ndhF16R TTTCCGATTCATGAGGATATGA This study
rps16
rpsF GTGGTAGAAAGCAACGTGCGA Oxelman et al., 1996 (modified)
Rps2R TCGGGATCGAACATCAATTGCAAC Oxelman et al., 1996
rpl16
F71 GCTATGCTTAGTGTGTGACTCGTTG Jordan et al., 1996
R1661 CGTACCCATATTTTTCCACCACGAC Jordan et al., 1996
May 2007] DOUGLAS AND MANOS—PHYLOGENY OF NYCTAGINACEAE 859
in Cuba have proved unsuccessful (D. Stone, Duke University, personal
communication).
Data analysis—Initial maximum parsimony (MP), maximum likelihood
(ML), and Bayesian analyses were performed for each of the four loci. The
5.8S, not surprisingly, had low variation and produced poorly resolved trees;
however, examination of the support values for the topology favored by each
locus revealed no supported nodes in conflict. Therefore, the data sets were
combined for further analyses.
MP analysis was performed using PAUP* version 4.0b10 (Swofford, 2002).
A heuristic search was performed, with 1000 replicates of 10 random-addition
sequences, tree-bisection-reconnection (TBR) branch swapping, MAXTREES
set to autoincrease, MULTREES ¼yes. Support was evaluated using 1000
bootstrap replicates of 10 random addition sequences, TBR branch swapping,
MULTREES ¼YES.
For the ML analysis, the data set was first examined using ModelTest 2.0
(Posada and Crandall, 1998), which selected a complex model of evolution
(GTR þIþC). Ten random-addition replicates (TBR, MAXTREES set to
autoincrease, MULTREES ¼yes) were run in PAUP*. Maximum-likelihood
bootstrap support values were obtained by 100 replicates of single random-
addition sequences, TBR branch swapping, MULTREES ¼yes.
Bayesian analysis was performed using MrBayes 3.1 (Ronquist and
Huelsenbeck, 2003). For exploring the effect of different models for different
partitions of the data, best-fit models for each partition were estimated in
MrModelTest (Nylander, 2004), which selects the best-fit model from those
available in MrBayes. The partitions were as follows: 1, all loci together; 2,
nuclear 5.8S; 3, all chloroplast loci; 4, rpl16;5,rps16;6,ndhF; and 7, 8, 9,
first, second, and third positions of ndhF, respectively. The models selected by
MrModelTest for each partition are given in Table 3. Bayesian searches were
then performed on the entire data set using four partition/model combinations:
‘‘B1,’’ single model for all partitions, (1); ‘‘ B2,’’ nuclear and chloroplast, (2 and
3); ‘‘B4,’’ all loci, (2, 4, 5, and 6); and ‘‘ B6,’’ all loci with separate models for
each codon position of ndhF (2, 4, 5, 7, 8, and 9). For each combination, we
executed four independent runs of 1 310
6
generations each, sampling every
100th tree. After discarding trees from the burn-in (determined by visualizing
the plateau in –lnL scores, approximately after 50 000 generations), we
compared the posterior tree sets from each run by computing a 50% majority
rule tree in PAUP*. No strongly supported topological differences (at posterior
probability 95%) were found between the four runs of each model set.
Therefore, the four posterior tree files for each set of models were combined
into a single posterior tree file for purposes of assessing support values yielded
by each set of models. These preliminary analyses were conducted including
the partial ndhF sequence for Caribea; however, the B6 analysis was repeated
without this sequence.
Sensitivity analyses—Due primarily to the inclusion of GenBank
sequences for outgroup taxa and the failure of certain loci to amplify (mostly
from herbarium material), approximately 17.7% of the data matrix was coded
as ‘‘missing.’’ The potential impact of this was investigated by deleting from the
analysis 18 taxa (Appendix 1) for which one or more sequences were entirely
missing and by combining sequences from Bougainvillea glabra and B. infesta
into a composite operational taxonomic unit ‘‘Bougainvillea.’’ ‘‘Phytolacca’’
and Rivina humilis were the only remaining outgroups in this analysis, which
allowed us to examine the effect of including distant outgroups. The MP, ML,
and corresponding bootstrap searches were performed with the same settings as
in the analysis of the full matrix. The resulting trees were compared to the
topology from the full analysis to see whether the exclusion of missing data led
to a preferred topology that differed substantively from the topology or levels
of support in the analysis of the full matrix.
Restricted analyses—To gain resolution within and between closely related
genera, our selection of loci encompassed a large range of sequence variation.
Because both the ITS1 and ITS2 regions had to be excluded from the analysis
of the complete data set due to questionable alignment (though the highly
conserved 5.8S region was kept in the full matrix), following the analysis of the
full data set, two restricted data sets were constructed to allow us to increase the
number of included characters (Table 3) by reducing the taxon sampling to two
distinct clades found in the full analysis. These restricted data sets comprised all
included nucleotide positions in the full data set, plus sites that were
unalignable across the breadth of taxa included in the full data set, but that were
alignable within each of the restricted sets of taxa. The first restricted analysis
group was comprised of North American herbs representing all taxa in the sister
group to Allionia, whereas the second corresponded to the Pisonieae,
Bougainvillea,Belemia, and Phaeoptilum (the ‘‘B&P’’ clade from the full
analysis). The MP, ML, and corresponding bootstrap analyses were performed
in the same fashion as in the full matrix and sensitivity analyses, with the
exception that the ML models were reestimated in ModelTest.
Character data—The historical taxonomic significance given to pollen
morphology and involucral bracts led us to examine these characters in a
phylogenetic context. Pollen data follows the scheme of Nowicke, who
identified four types in Nyctaginaceae (Nowicke, 1968, 1970, 1975; Nowicke
and Luikart, 1971; Reyes-Salas and Martı´nez-Herna´ndez, 1982; Chavez et al.,
1998). Pollen type was coded as a multistate, unordered character. In many
cases, the exact species included in our study were not examined in the
published studies. If there was no indication of within-genus pollen
polymorphism, that pollen type was assigned to all species in this analysis.
However, multiple pollen types were recorded within Neea and Pisonia. Thus,
only N. psychotrioides, which was examined by Nowicke, was coded
unambiguously; other species of Neea and Pisonia were coded as polymorphic
(states ‘‘1&3’’ and ‘‘1&4,’’ respectively) to reflect this uncertainty in the
assignment of ancestral states. The presence of involucral bracts was scored as
present/absent. If only small subtending bracteoles occur (common in many
taxa), this character was coded as ‘‘absent,’’ mirroring the usage of this
character in defining subtribe Nyctagininae. The occurrence of cleistogamous
flowers was scored based primarily on literature sources (Spellenberg and
Delson, 1974; Bittrich and Ku¨ hn, 1993; Levin, 2002; Spellenberg, 2003).
Gypsophilic taxa were identified in literature sources (Waterfall, 1946; Parsons,
1976; Fowler and Turner, 1977; Turner, 1991; Harriman, 1999; Levin, 2002;
Spellenberg, 2003; N. Douglas, personal observation). Taxa were identified as
full gypsophiles (recorded only from gypseous soils), gypsum tolerant
(recorded from both gypseous and nongypseous soils), or nongypsophilic.
Taxa that do not occur in areas with gypsum outcrops were considered to be
nongypsophilic. It is unlikely that transitions to or from full gypsophily could
evolve with no intermediate gypsum-tolerant step; therefore, this character was
analyzed as both unordered and ordered, with two steps required between
TABLE 3. Summary of sequence statistics by partition for the molecular matrix.
Partition
Full analysis
ndhF
(entire)
ndhF
(1st pos.)
ndhF
(2nd pos.)
ndhF
(3rd pos.) rps16 rpl16 5.8S Entire Chloroplast
No. taxa (full matrix ¼58) 54 54 54 54 51 42 55 55 55
Aligned length 2193 731 731 731 1237 1367 157 5505 4797
Analyzed length 2205 669 668 668 780 792 157 3734 3577
Constant 1348 474 532 342 520 552 136 2556 2420
Uninformative 272 83 69 120 110 139 5 526 521
Parsimony-informative 385 112 67 206 150 101 16 652 636
ML model GTRþIþCGTRþIþCGTRþIþCGTRþCGTRþCGTRþCSYMþIþCGTRþIþCGTRþIþC
Note: Maximum-likelihood (ML) model estimated by ModelTest (Posada and Crandall, 1998): Full, Entire; Sensitivity, Entire; Restricted I & II, Entire.
ML model for remaining partitions (used in Bayesian analyses ‘‘B2,’’ ‘‘B4,’’ and ‘‘ B6,’’ see text) estimated with MrModelTest (Nylander, 2004). Numbers
in parentheses are number of informative characters gained from the inclusion of ITS1 and ITS2 in restricted analyses.
860 AMERICAN JOURNAL OF BOTANY [Vol. 94
nongypsophily and full gypsophily. Parsimony ancestral states of all characters
were reconstructed with the program Mesquite 1.6 (Maddison and Maddison,
2006). Those terminals that were not assigned a single state, and branches that
were not unambiguously resolved, are depicted as ‘‘equivocal.’’
RESULTS
Data matrix—The entire data matrix (Table 3) had a length
of 5505 bp, of which 1771 were excluded due to ambiguous
alignment, mainly due to the presence of length variation in
ITS1 and ITS2 and in the two chloroplast introns, rpl16 and
rps16. Of the remaining 3734 characters, 652 were parsimony
informative.
Phylogenetic analysis of the complete dataset—The MP
analysis resulted in 36 shortest trees (length: 2287, consistency
index [CI]: 0.657, retention index: 0.809, rescaled CI: 0.531);
however, the strict consensus (tree not shown) resolved all but
two ingroup nodes. Thirty-nine nodes were supported with
parsimony bootstrap values (MPBS) 70.
The best-fit model as determined by ModelTest (Table 3)
using both a hiearchical liklihood ratio test (HLRT) and the
Akaike information criterion (AIC) was a general-time-
reversible model with a proportion of invariant sites and a
gamma shape parameter (GTRþIþC). The ML search returned
a single ML tree, which was nearly identical to the MP
topology, except in the placement of the genus Colignonia.
This taxon is placed as sister to the large clade containing
Acleisanthes and Boerhavia in the MP analysis (MPBS ¼80)
and is not resolved with strong support in any ML or Bayesian
analysis. Overall, 38 nodes in the ML analysis were supported
with likelihood bootstrap values (MLBS) 70.
Models determined by MrModelTest for each data partition
in the Bayesian analyses are given in Table 3. On the basis of
our preliminary examination of partitioned models, the signal
in the data set apparently is strong, and the topology is not
contingent on model selection: the tree topologies produced by
the Bayesian B1, B2, B4, and B6 searches were consistent. The
principal difference between them is in the level of support for
the topology, with 37, 39, 40, and 40 nodes, respectively,
supported by posterior probabilities (PP) 95%. Deletion of
Caribea led to the resolution, with support of two additional
nodes in the repeated B6 search, for a total of 42 nodes
supported at greater than 95% PP. The topology of this
Bayesian B6 consensus tree is identical to the ML tree. All
further Bayesian support values refer to the B6 analysis.
The Nyctaginaceae are supported as monophyletic by ML
(MLBS ¼71) and Bayesian (PP ¼100) analyses (Fig. 1).
Interestingly, in the MP bootstrap analysis of this matrix, the
monophyly of the Nyctaginaceae is not supported. Despite the
inclusion of several outgroups, no single sister lineage emerges
with strong support.
Leucastereae, a tribe of four South American genera
(Andradea,Leucaster,Ramisia,andReichenbachia), is
supported as the earliest branching lineage in Nyctaginaceae
(Fig. 1) followed by Boldoeae, represented by Salpianthus.A
clade containing largely neotropical trees and shrubs, and the
African genus Phaeoptilum, receives support from MP and B6
analyses, though not from ML. We will refer to this group as
the Bougainvilleeae and Pisonieae (‘‘B&P’’) clade, (Fig. 2),
recognizing that it also includes Phaeoptilum and Pisoniella,
which are currently classified in Nyctagineae. Bougainvillea,
Belemia, and Phaeoptilum form a clade within this group,
which is sister to a clade containing the Pisonieae and the
genus Pisoniella. Within the Pisonieae, Neea and Guapira
together form a clade but neither genus appears to be
monophyletic.
Strong support is found in all analyses for a clade including
mostly North American xerophytic genera. For the purposes of
this paper, we refer to it as the North American Xerophytic
(‘‘NAX’’ clade) (Fig. 2). The NAX clade is well defined by
geography, habit, and habitat, but it has never been recognized
formally. The earliest branch within this clade leads to
Acleisanthes sensu Levin (2000). It is followed by a clade
representing Abronia and Tripterocalyx (tribe Abronieae).
Phylogenetic relationships of the remaining genera in the
NAX clade are mostly well resolved, with the exception of low
support values for the placement of Commicarpus and Allionia.
Two pairs of genera in this clade are not resolved as
monophyletic. Anulocaulis includes Nyctaginia, and Boerhavia
includes Okenia, though support in both of these cases is weak
or lacking. Examination of the branch lengths (Fig. 2) makes it
clear that Anulocaulis and Nyctaginia are at least very closely
related.
The position of Colignonia is not resolved in the ML and
Bayesian analyses. The ML analysis resolves Colignonia sister
to the B&P and NAX clades but with weak support. A position
sister to only the NAX clade is supported in the MP analysis.
Sensitivity analyses—The deletion of taxa with significant
missing data resulted in a matrix of 39 taxa with only 3.1%
missing data, as compared to 58 taxa with 17.7% missing data
in the full analysis (See Appendix 1). The MP/ML analyses of
this matrix yielded trees (not shown) that had no well-
supported nodes conflicting with the topology of the tree from
the full matrix. The support for the monophyly of the
Nyctaginaceae increased to 94/95 MPBS/MLBS, from /71
in the analysis of the full data set. The high level of support
found in this analysis for the monophyly of Nyctaginaceae
indicates that the inclusion of many outgroups in the full
matrix, including the quite distant Stegnosperma, may have
affected the level of support in the MP analysis. Alternatively,
high levels of missing data in the full data set may be
responsible for low support values at this key node. Support for
the placement of Cyphomeris decreased to 70/66 relative to the
full analysis. Commicarpus and Allionia increased to 73/67 and
87/77, respectively; these nodes had not received strong
support in any analysis of the full data set. The remainder of
the comparable nodes were similarly supported between the
full and sensitivity analyses.
TABLE 3. Extended.
Sensitivity analysis Restricted analysis I Restricted analysis II
Entire Entire Entire
39 19 15
5359 4887 4914
3396 4278 4082
2597 3883 3526
412 160 382
387 235 (122) 174 (76)
GTRþIþCGTRþIþCGTRþIþC
May 2007] DOUGLAS AND MANOS—PHYLOGENY OF NYCTAGINACEAE 861
Fig. 1. Maximum-likelihood (ML) topology from the analysis of the entire data set. Parsimony bootstrap/ML bootstrap support values above branches,
Bayesian posterior probability from the ‘‘B6’’ analysis below branches, ‘‘ -’’ indicates bootstrap support value ,50. tribes of Nyctaginaceae according to
Bittrich and Ku¨hn (1993) are in bold. ‘‘-’’ before unbold name signifies a subtribe of tribe Nyctagineae.
862 AMERICAN JOURNAL OF BOTANY [Vol. 94
Fig. 2. Phylogram of the maximum-likelihood topology from Fig. 1. Major clades referred to in text are highlighted.
May 2007] DOUGLAS AND MANOS—PHYLOGENY OF NYCTAGINACEAE 863
Restricted analyses—For the two restricted analysis groups,
122 and 76 additional informative characters were gained with
the inclusion of ITS1 and ITS2, respectively (Table 3). A small
number of additional sites were gained from the chloroplast
introns rps16 and rpl16 (,5 characters in either data set).
ModelTest 3.7 selected a GTRþIþCmodel for each (Table 3)
data set. For the first group (all taxa in the sister group to
Allionia), MP and ML analyses produced a tree (Fig. 3) with
improved resolution in the Anulocaulis þNyctaginia clade.
Though the placement of A. annulatus differs between the full
matrix topology and the restricted analysis, the monophyly of
Anulocaulis was well supported with bootstrap values of 72/89
MPBS/MLBS. Similarly, the full analysis resolves Okenia
within a paraphyletic Boerhavia with low MP and ML
bootstrap support, but 97% Bayesian posterior probability,
yet the restricted analysis found Boerhavia strongly supported
as monophyletic and sister to Okenia with high support (100/
100). Boerhavia consists of two clades, corresponding to
annual and perennial species, that were also found in the full
analysis.
The restricted analysis of the B&P clade produced a tree (not
shown) that did not conflict with the topology of this clade in
the full matrix analysis. Support values were generally slightly
lower, probably due to the concentration of missing data in this
group and the lower number of additional characters from the
ITS region. Support values remained high for the nodes uniting
Guapira eggersiana and Neea hermaphrodita,forthe
placement of G. discolor in the clade sister to N. psycho-
trioides, and for the monophyly of Neea þGuapira (MPBS/
MLBS bootstrap support of 64/73, 82/94, and 94/92,
respectively).
Character reconstructions—For each character reconstruct-
ed (Fig. 4), multiple state transitions are inferred. Tricolpate-
spinulose pollen (Fig. 4a) appears to be the ancestral condition
in the group, transitioning to a pantoporate-spinulose condition
subsequent to the divergence of Salpianthus from the main
lineage. The latter condition is found in nearly all members of
the NAX clade, yet appears to predate that group. At least eight
transitions among the four pollen types have occurred in the
Nyctaginaceae. Considering the small number of Neea and
Pisonia examined and the polymorphism exhibited by these
genera, the number of transitions could be higher. Reconstruc-
tion of involucral bracts shows five gain/loss steps. This
character is fixed within genera, thus this interpretation is likely
to be affected only by the future inclusion of the remaining
genera in the family. Only the inclusion of Cuscatlania, which
has an involucre, could conceivably change the number of
steps required. Cleistogamous flowers are uniquely derived in
four genera. Gypsophily requires nine or 13 steps to explain,
depending on whether it is considered to be an unordered or an
ordered character. Reconstructions were performed only on the
ML topology from the full analysis. Adjusting the positions of
Okenia and Nyctaginia to reflect the topology from the
restricted analysis (Fig. 3) results in the branches leading to
Nyctaginia þAnulocaulis and Nyctaginia þAnulocaulis þ
Okenia þBoerhavia being resolved as nongypsophilic.
Treating gypsophily as an unordered character has the same
result. Otherwise, the alternative topology has no substantive
effect on the conclusions we make regarding the degree of
homoplasy shown by the remaining three characters shown in
Fig. 4.
DISCUSSION
Phylogeny of Nyctaginaceae—The earliest branching
lineage in Nyctaginaceae, the Leucastereae (Fig. 1), had been
previously recognized as a natural group on the basis of
arborescence, a stellate indumentum, and tricolpate pollen
(Heimerl, 1934; Bittrich and Ku¨hn, 1993). The Boldoeae, an
herbaceous group native from the Galapagos to northwestern
Mexico and the Caribbean, are represented in this study by
Salpianthus. These two lineages had been predicted to be basal
or outside of Nyctaginaceae on the basis of apparent
pleisomorphies such as alternate leaves and bisexual flowers
(Bittrich and Ku¨hn, 1993). The anthocarp structure is absent in
Leucastereae and Boldoeae, although the unexpanded perianth
does persist around the fruit. Persistent tepals are also found in
many Phytolaccaceae. However, the perianth consists of free
tepals in most Phytolaccaceae and all of subfamily Rivinoideae
(except Hilleria, in which three of four tepals are partially
fused, (Rohwer, 1993)). In Nyctaginaceae, including Leucas-
tereae and Boldoeae, tepals are fully connate.
Within the B&P clade (Fig. 2), Phaeoptilum is found to be
sister to Belemia, rendering the Bougainvilleeae paraphyletic.
The Pisonieae are found to be sister to Pisoniella, which had
been included in that tribe by Heimerl (1934) but was removed
Fig. 3. Phylogram of the maximum-likelihood (ML) topology from
the first restricted analysis. MP bootstrap/ML bootstrap support values are
shown. Anulocaulis and Boerhavia are each supported as monophyletic.
864 AMERICAN JOURNAL OF BOTANY [Vol. 94
Fig. 4. Parsimony reconstruction of (A) pollen morphology, (B) involucre presence, (C) cleistogamous flowers, and (D) gypsophilic habit (based on
ordered characters). See text for sources of characters used in reconstructions.
May 2007] DOUGLAS AND MANOS—PHYLOGENY OF NYCTAGINACEAE 865
to subtribe Colignoniinae by Bittrich and Ku¨hn (1993)
following the suggestion of Bohlin (1988). The reasoning
behind this move is mysterious, and in light of our results, it
appears to have been unwarranted. Pisoniella possesses a
straight embryo like other Pisonieae, and the large coriaceous
anthocarps are provided with viscid glands along the ribs,
much like those in Pisonia (Heimerl, 1934).
Within Pisonieae, Neea and Guapira form a clade (Fig. 2).
These genera are distinguished primarily by whether the
stamens are included (Neea) or exserted (Guapira). Our
sampling is extremely limited in these two large genera, with
only five accessions to represent ca. 150 species, though we
were able to include accessions from geographically disparate
locales. Neither genus forms a monophyletic group. This
conclusion has been occasionally anticipated (e.g., Pool, 2001).
It is unclear whether our sampling simply happened to include
misclassified species in otherwise good genera, or whether this
paraphyly is representative of Neea and Guapira generally.
Much more intensive sampling is clearly needed to understand
the relationships of the species in these genera, and it would be
imprudent to attempt to reclassify them until a more detailed
study is made including phylogenetic, morphological, and
distributional data. Unfortunately, collections of these dioe-
cious trees often do not include individuals of both sexes. Also,
the tendency of many Pisonieae to oxidize when dried has left
many descriptions lacking crucial information concerning the
color of fruits. Therefore, the taxonomic literature is quite
confused and species limits are known not much better than
when Standley (1931a, p. 73) wrote that, ‘‘ I know of few
groups of plants in which specific differences are so unstable
and so baffling . . . particularly in Neea,Torrubia [¼Guapira]
and Mirablis, no single character seems to be constant.’’
Finally, in this study we did not attempt to infer the ages of
lineages, yet it appears that the branch lengths in the Neea þ
Guapira clade are comparatively short, especially considering
that this clade can be expected to accommodate as many as 150
species (Fig. 2). A similar pattern has been noted in other
radiations of neotropical trees, e.g., Inga (Fabaceae) (Richard-
son et al., 2001). If the pattern of relatively short branches
inferred between species was upheld with the inclusion of a
larger sample of taxa and more rapidly evolving markers, it
would point to this clade as another example of rapid
diversification in the neotropics.
Tribe Nyctagineae is broadly paraphyletic. As mentioned,
Pisoniella and Phaeoptilum are not found in this study to be
closest relatives of any other Nyctagineae. Based on pollen
morphology, Bohlin (1988) has suggested that Colignonia
(subtribe Colignoniinae) has affinities to the tribe Mirabileae of
Heimerl (1934), which roughly corresponds to the tribe
Nyctagineae and the NAX clade. Colignonia may in fact be
sister to the NAX clade as suggested by the MP analysis or to
the NAX þB&P clade as suggested by the ML analysis (Fig.
1). Tribe Nyctagineae also does not include Abronia or
Tripterocalyx (tribe Abronieae, Fig. 1). There are certain
characters of the Abronieae that are anomalous within the
Nyctagineae (and the NAX clade) and that justified recognition
at a higher taxonomic level, namely, tricolpate pollen and
linear stigmas. The two genera in the tribe have long been
thought to be a natural group and are often synonymized
(Heimerl, 1934; Bittrich and Ku¨hn, 1993), though most authors
have maintained the two genera (Galloway, 1975; Spellenberg,
2003). The Abronia þTripterocalyx clade is characterized by
the combination of an umbellate inflorescence of salverform
flowers with included stamens and style, an involucre,
anthocarps with typically well-developed wings or lobes, and
a mature embryo with a single cotyledon.
Anulocaulis and Nyctaginia are classified in different
subtribes in the classification of Bittrich and Ku¨hn (1993),
presumably based on the presence of an involucre in
Nyctaginia. Both genera are succulent perennial herbs, and
the turbinate fruits with umbonate apices of Nyctaginia
capitata strongly resemble those of Anulocaulis eriosolenus.
They differ in many characters, including flower color (red-
orange in Nyctaginia vs. white to pink in Anulocaulis) and
flowering time (flowers of Nyctaginia are open during the day,
while in Anulocaulis anthesis is at sunset or later and flowers
wilt in the morning). While the full matrix ML tree (Fig. 1)
indicates that Anulocaulis may not be monophyletic, this
relationship is poorly supported (MPBS/MLBS/PP ¼64/55/
63). In the restricted MP and ML analysis (Fig. 3), however, a
monophyletic Anulocaulis is more strongly supported (MPBS/
MLBS ¼72/89). Therefore, we see no compelling reason to
question the taxonomic status of Anulocaulis.
Anulocaulis þNyctaginia are sister to a strongly supported
clade containing Boerhavia and Okenia. Like the previous
instance, Okenia resolves within Boerhavia in the full matrix
ML topology (Fig. 1), but support for this relationship is only
moderately significant in the Bayesian analysis of the full data
set (PP ¼97) and weakly supported by MPBS and MLBS (67/
69). Conversely, Boerhavia is strongly supported as a
monophyletic group in the MP and ML analyses of the
restricted data set (MPBS/MLBS ¼100/100, Fig. 3).
Vegetatively, Okenia strongly resembles most Boerhavia in
its decumbent habit, and subequal opposite leaves with sinuate
or undulate margins. The flowers of Okenia, though larger, are
similar in color to some perennial Boerhavia from the
Chihuahuan Desert. Finally, Okenia is annual, a condition
found in one clade of Boerhavia. However, Okenia is strikingly
different than Boerhavia in its unique reproductive biology: it
produces aerial flowers, but the large, spongy fruits are
geocarpic, with peduncles elongating greatly after fertilization
and the fruits maturing several centimeters belowground. The
relationship between these two genera is deserving of more
study.
Biogeographical patterns—The basal lineages of Nyctagi-
naceae (Boldoeae, Leucastereae, Colignonia, Bougainvilleeae,
and Pisonieae [including Pisoniella]) are fundamentally South
American. Though some taxa have representatives or popula-
tions in (sub)tropical North America, (Salpianthus,Neea,
Guapira,Pisonia,Pisoniella), their distributions all include the
neotropics, and phylogenetically they are interspersed with
neotropical endemics. The widespread tropical genus Pisonia
possesses extremely viscid anthocarps, which aid dispersal,
frequently by seabirds (Burger, 2005). The sole genus not
native to the Americas is Phaeoptilum, endemic to arid
southwestern Africa. This monospecific genus is closely
related to Belemia and Bougainvillea, both from eastern and
southern South America. Phaeoptilum is morphologically quite
distinct from its sister taxon Belemia, though vegetatively it
resembles the xeric-adapted Bougainvillea spinosa. The early
Cretaceous date (130–90 Ma) for the opening of the south
Atlantic (Smith et al., 1994) makes vicariance an unlikely
explanation for this disjunction. Dispersal seems more likely,
and while there is no specialized dispersal structure on the
anthocarp of Belemia, both Bougainvillea and Phaeoptilum
866 AMERICAN JOURNAL OF BOTANY [Vol. 94
have compelling (albeit different) adaptations for wind
dispersal. Phaeoptilum produces winged anthocarps highly
similar to those found in Tripterocalyx and some species of
Acleisanthes.InBougainvillea, most species display three
showy bracts, each fused to a solitary flower. In fruit each
involucral bract remains fused to a fruit and acts as a wing, the
structure functioning as a unit of dispersal (Ridley, 1930).
The North American Xerophytic Clade has diversified in the
deserts of the southwestern United States and northwestern
Mexico. Every genus is confined to or has representatives in
this region. Widespread taxa in this clade, namely Commi-
carpus and Boerhavia, possess glandular fruits, which have
most likely aided bird-dispersal in a manner similar to that of
Pisonia. Two red-flowered Boerhavia,B. coccinea and the
similar B. diffusa are widespread in most tropical and
subtropical areas. Boerhavia diffusa appears to have naturally
dispersed from the Americas, though the confused taxonomy of
this species and B. coccinea in regional floras makes this
difficult to evaluate, and both of these species are frequently
transported by human activity. The ‘‘ repens’’ complex in
Boerhavia (B. repens and related species) is widespread in
coastal habitats throughout the tropical Pacific and Indian
oceans to the Arabian Peninsula, along with B. dominii from
Australia. Like the red-flowered perennial Boerhavia men-
tioned, these species also have viscid glandular anthocarps.
Okenia is found in deep sand dune habitat along the Pacific and
Caribbean coasts of Mexico and Central America, with a
disjunct population in southern Florida. Other authors
(Heimerl, 1934; Fowler and Turner, 1977; Thulin, 1994;
Levin, 2002; Spellenberg and Poole, 2003) have discussed the
remarkable disjunctions of Acleisanthes somaliensis and
Mirabilis himalaicus from east Africa and southern Asia,
respectively. These appear to be attributable to long-distance
dispersal events, due to their derived position within otherwise
exclusively American clades (Levin, 2000; N. Douglas,
unpublished data).
Pollen and involucre evolution—Tribal and subtribal
classifications (Table 1) of the Nyctaginaceae have relied
heavily on a few characters, such as pollen morphology and the
development of an involucre. However, divisions based on
these characters are not supported by our results because these
characters have a high degree of homoplasy among genera.
Parsimony reconstruction of pollen type across Nyctagina-
ceae (Fig. 4a) shows that substantial homoplasy exists (11
changes), involving three of the four types diagnosed by
Nowicke (Nowicke, 1970, 1975; Nowicke and Luikart, 1971).
Pantocolpate grains may constitute a synapomorphy for
Belemia þPhaeoptilum. It has been noted that large,
desiccation-resistant, pantoporate pollen grains, equipped with
pore plates, were found primarily in the herbaceous desert taxa
(Nowicke and Luikart, 1971). Specific correlations between
large and/or polyaperturate grains and habitat in angiosperms
have not been adequately investigated. In a study of ecological
correlates of pollen morphology in a wide selection of
angiosperms (Lee, 1978), there was an extremely weak
correlation of pore number with width and with ‘‘ temperature.’’
According to our reconstructions, the origin of pantoporate-
spinulose pollen predates the major radiation of desert taxa in
the NAX clade. However, Colignonia and Pisoniella have
much smaller grains than do the remaining taxa with
pantoporate-spinulose pollen (Colignonia ¼25–35 lM,
Pisoniella ¼30–37 lM, Nowicke and Luikart, 1971; N.
Douglas, unpublished data). Therefore, it would seem best to
consider grain size as a variable separate from grain shape and
exine structure.
Within Nyctagineae, the subtribes Nyctagininae and Boer-
haviinae were separated by the presence or absence of an
involucre subtending the inflorescence. In subtribe Nyctagini-
nae, the involucre of Mirabilis is comprised of fused bracts; the
remaining genera possess involucres of distinct bracts. The
involucre in Bougainvillea is distinctive; fruits of Bougainvil-
lea retain a large involucral bract as discussed. Involucres have
no known dispersal function in any of the other taxa; they
likely serve merely to protect the flower buds and developing
fruits or discourage nectar-robbing insects (Cruden, 1970).
Parsimony reconstruction of this character on the molecular
topology (Fig. 4b) indicates that, for involucres, there are at
least five gain/loss steps in the family, four in the NAX clade,
which contains the members of the Nyctagineae-Nyctagininae,
Nyctagineae-Boerhaviinae, and Abronieae, reflecting the
artificial nature of this classification. In this analysis, the
character was treated in a very simplistic fashion, reflecting
nothing more than taxonomic convention. Comparative
developmental studies may shed light on deeper homologies
or convergences, especially as they relate to the subtending
bracts found in many genera. The selective benefits involved in
the expression of this structure could be revealed by
appropriate ecological investigations.
Self-compatibility and cleistogamy—The production of
obligately selfing flowers is obviously contingent on the ability
of plants to self-pollinate and produce fertile progeny. Our
incomplete knowledge of reproductive systems in Nyctagina-
ceae means that an unambiguous reconstruction of self-
compatibility is not currently possible. However, several
studies have addressed mating systems in select Nyctagina-
ceae: sporophytic self-incompatibility (SI) is known in
Bougainvillea (Zadoo et al., 1975; Lo´pez and Galetto, 2002).
Some Mirabilis (sect. Quamoclidion) and Abronia macrocarpa
fail to set seed when self-pollinated (Cruden, 1973; Williamson
et al., 1994), but the basis for incompatibility is not known in
these genera. The Pisonieae are usually dioecious and are thus
self-incompatible, although in these genera there are occasional
monoecious or hermaphroditic species (e.g., Pisonia brunoni-
ana) for which the mating system has not been studied (Sykes,
1987). Evidence suggests that many genera in the NAX clade
are self-compatible: in addition to the production of cleistog-
amous flowers in four genera, Boerhavia and some Mirabilis
are known to have a delayed self-pollination mechanism
whereby the style curls and encounters the anthers as the flower
wilts (Chaturvedi, 1989; Herna´ndez, 1990; Spellenberg, 2000).
Finally, flowers protected from pollinators have set viable seed
in Abronia umbellata Lam. (McGlaughlin et al., 2002) and
Colignonia (Bohlin, 1988).
Reasoning from these data, we can make certain inferences
regarding the evolution of mating systems in Nyctaginaceae.
Explanations for current distribution of mating systems family
must incorporate one, or some combination of both, of the
following scenarios. Which one is preferred depends on the
likelihood of self-compatible lineages giving rise to lineages
with an inability to self-fertilize, and the implications of either
scenario are interesting.
One scenario, and the most parsimonious given our current
knowledge, is that there have been at least three independent
derivations of SI from a self-compatible ancestor. A single
May 2007] DOUGLAS AND MANOS—PHYLOGENY OF NYCTAGINACEAE 867
change can account for the Pisonieae and Bougainvillea, one
for the derived Mirabilis sect. Quamoclidion, and one for
Abronia macrocarpa. It is often assumed that outcrossing
species are not derived from selfing ancestors and that selfing
lineages are an evolutionary ‘‘dead end’’ (Fisher, 1941;
Stebbins, 1974; Lande and Schemske, 1985). In the case of
Nyctaginaceae, however, the question is whether it is possible
that self-incompatible species have arisen from self-compatible
ancestors. It would seem that populations making this
transition would be subject to most of the forces that affect
the balance of selfing and outcrossing in self-compatible
populations. A recent study of s-locus polymorphism in
Solanaceae (Igic et al., 2006) has shown that losses of SI are
irreversible in that family. The ‘‘cost’’ of developing the
complex genetic systems necessary for SI would be added to
the transmission advantage of alleles promoting self-fertiliza-
tion (Uyenoyama et al., 1993); these factors must count against
a hypothesis of multiple transitions to SI in one family.
Conversely, if we assume that SI is ancestral and has been
lost repeatedly, transitions from SI to self-compatibility have
occurred a minimum of six times (in Colignonia,Acleisanthes,
some Abronia, two or more times in Mirabilis, and finally in
the clade sister to Mirabilis). This represents a doubling of the
number of evolutionary steps required to explain the
distribution of known Nyctaginaceae mating systems. Other
authors have discussed the merits of parsimony weighting
schemes or maximum-likelihood approaches to testing the
irreversibility of selfing (Barrett et al., 1996; Bena et al., 1998;
Takebayashi and Morrell, 2001). In these cases, it may not be
possible to escape a circular argument employing only
phylogenetic evidence, because a weighting scheme favoring
losses of SI assumes the conclusion. In Solanaceae (Igic et al.,
2006), evidence of ancient polymorphism at the incompatibility
locus itself was required to demonstrate the irreversibility of
the loss of SI. In our case, the most convincing resolution will
come when SI is characterized in Mirabilis sect. Quamoclidion
and SI Abronia. If in these taxa and any others that may yet be
discovered to be self-incompatible the genetic basis for SI can
be identified, homology could be assessed and the ancestral
functionality of the underlying mechanism could be tested.
Assuming the derived state is self-compatibility, of these six
lineages, three have given rise to cleistogamous/chasmoga-
mous lineages, and four gains of cleistogamy are required to
explain the distribution of the character in Nyctaginaceae (Fig.
4c). Interestingly, the cleistogamous genera are all perennial,
which should be less susceptible to selection pressure for
reproductive assurance than annuals (Barrett et al., 1996).
Alternatively, cleistogamous flowers can function to maximize
seed set when resources, rather than pollinators, are limiting
(Schemske, 1978). These hypotheses are both applicable to the
cleistogamous Nyctaginaceae, though distinguishing between
them may be difficult, because pollinators in desert environ-
ments tend to be scarce when water is scarce. Spellenberg and
Delson (1974) found that Acleisanthes (Ammocodon)cheno-
podioides, with a generalized flower morphology and a diurnal
pollinator fauna, produced roughly equal numbers of seeds
from cleistogamous and chasmogamous flowers, and did not
have a strong seasonal pattern in the production of cleistog-
amous flowers. In contrast, Acleisanthes longiflora, a species
with large, specialized hawkmoth-pollinated flowers, produced
the majority of a season’s seeds from cleistogamous flowers
produced preferentially in the dry early summer when sphingid
moths are less active. This may suggest that cleistogamy in this
genus is insurance against reproductive failure due to the
absence of pollinators in some years.
Gypsophily—Parsimony reconstruction of gypsophily in
Nyctaginaceae (Fig. 4d) indicates that gypsophiles and
gypsum-tolerant species are widely dispersed in the NAX
clade. With the current sampling, the ancestor of this clade is
inferred to be nongypsophilic (whether or not the character is
considered ‘‘ordered’’ ), indicating that gypsum tolerance is
derived multiple times. This conclusion is tenuous for two
reasons. First, gypsum outcrops are common in the Chihua-
huan Desert but less so in other parts of the ranges of the NAX
genera. We are unable to rule out the possibility that taxa coded
in this analysis as ‘‘nongypsophilic’’ are actually gypsum-
tolerant, but simply do not occur in areas with gypsum soils.
Second, there are two Mirabilis [M. nesomii Turner and M.
linearis (Pursh) Heimerl] which are gypsophilic (Turner, 1991)
and gypsum-tolerant (R. Spellenberg, New Mexico State
University, personal communication), respectively. These
species, both in section Oxybaphus, are close relatives of the
oxybaphoid M. albida, a nongypsophile included in this study.
It is possible to add gypsophilic taxa as sisters to M. albida on
our topology, so that the resolution of the ancestor of the NAX
clade becomes equivocal, with ACCTRAN reconstruction as
gypsum-tolerant, and DELTRAN as nongypsophilic. The same
reconstruction would be made for the ancestors of Commi-
carpus and Abronia þTripterocalyx. The sensitivity of the
reconstruction at these key nodes to sampling artifacts indicates
that in order to reconstruct the history of gypsophily in this
clade, it will be necessary to undertake more intensive
phylogenetic sampling at the species level, investigating an
appropriate sample of nongypsophilic taxa closely related to
known gypsophiles.
Even if we cannot know the gypsum tolerance of the
ancestor of the NAX clade based on existing data, it is evident
that there are at least four instances of strong gypsophily
evolving in the family. It would be profitable to investigate the
ecology of these gypsophytes and their relatives in the NAX
clade. An experimental approach investigating whether or not
seedlings of nongypsphiles have the latent ability to establish
on gypseous crusts would disentangle the expression of
gypsum tolerance from biogeographic complications, clarify
the phylogenetic distribution of gypsum tolerance and perhaps
reveal the nature of the adaptation(s) involved.
It is possible that establishment on gypsum is facilitated by
some sort of modification to the radicle. Alternatively, because
germination in a desert environment is always risky, adapta-
tions to gypsum soils may differ little from germination
strategies of desert taxa generally. Possible strategies could
serve to optimize the timing of germination, minimize the risk
of all seedlings perishing or increase the length of time a
seedling has to establish itself. These could include high
germination rate at low temperatures and various forms of bet-
hedging, such as seed heteromorphism and variable seed
dormancy (Escudero et al., 1997). The production of mucilage
upon wetting by the seed coat presumably increases the local
availability of water and upon drying, anchors the seed (Romao
and Escudero, 2005). Some of these traits are known in
Nyctaginaceae. For instance, production of mucilage by the
anthocarp is common in both gypsophilic and nongypsophilic
taxa in the NAX clade (Spellenberg, 2003), and fruit/seed
heteromorphism is known in Abronia and Tripterocalyx
(Wilson, 1974).
868 AMERICAN JOURNAL OF BOTANY [Vol. 94
Understanding when in their history Nyctaginaceae became
gypsum-tolerant will clarify whether homoplasy is best
explained by answering the question ‘‘ how do species become
gypsum-tolerant?’’ or ‘‘why are certain species found only on
gypsum?’’ If it turned out that gypsum tolerance was ancestral
in the NAX clade, then experiments may reveal the reasons full
gypsophiles do not occur on more typical soils.
The tendency of Nyctaginaceae to evolve cleistogamy and
gypsophily has been shown to the extent that we have
demonstrated that the high level of homoplasy for these traits
is restricted to the NAX clade. In neither case are we able to
conclusively identify the largest group capable of evolving the
trait. Largely due to the phylogenetic position of Acleisanthes
(with gypsophilic, cleistogamous species), we infer that it is
possible that the ancestor of the entire NAX clade was
predisposed to evolve these traits. In the case of cleistogamy,
the topology indicates either that SI mechanisms develop easily
in Nyctaginaceae, or that once self-compatibility emerges,
there is a high chance of cleistogamy following. If the latter
situation is correct, the explanation for the large number of
cleistogamous species in the NAX clade must ultimately rely
on explaining the frequent loss of SI, though the proximate
cause is more likely related to resource or pollinator limitation
in xeric environments. With gypsophily, it remains to be seen
what trait(s) allow for tolerance of gypsum soils and when they
evolved and what factors act exclude to gypsophiles from
nongypsum soils.
The present study is the first to provide a comprehensive
genus-level examination of the phylogeny of Nyctaginaceae.
Though sampling of Caribea,Cuscatlania,Cephalotomandra,
Grajalesia, and Neeopsis would be desirable, the current level
of sampling is sufficient to draw several useful conclusions
with bearing on future studies of the family. Aside from
providing a framework for future taxonomic revisions, it raises
interesting evolutionary questions regarding biogeography,
reproductive biology, and edaphic endemism. To a degree,
this work may be considered a case study into the practical
issues that may arise in an investigation of tendencies in
character evolution. New insights will be gained with a
combination of phylogenetic work at finer taxonomic scales
and experimental data to better understand the natural history
of individual species, especially those in the xerophytic clade.
LITERATURE CITED
ALAIN, H. 1960. Novedades en la flora cubana, XIII. Candollea 17: 113.
ANTONOVICS, J. 1971. Heavy metal tolerance in plants. Advances in
Ecological Research 7: 1–85.
BAKER, H. G. 1961. The adaptation of flowering plants to nocturnal and
crepuscular pollinators. Quarterly Review of Biology 36: 64–73.
BALDWIN, B. G. 2005. Origin of the serpentine-endemic herb Layia
discoidea from the widespread Layia glandulosa (Compositae).
Evolution 59: 2473–2479.
BARRETT, S. C. H., L. D. HARDER,AND A. C. WORLEY. 1996. The
comparative biology of pollination and mating in flowering plants.
Philosophical Transactions of the Royal Society of London, B,
Biological Sciences 351: 1271–1280.
BEHNKE, H. D. 1997. Sarcobataceae—a new family of Caryophyllales.
Taxon 46: 495–507.
BELL, C. D., AND M. J. DONOGHUE. 2003. Phylogeny and biogeography of
Morinaceae (Dipsacales) based on nuclear and chloroplast DNA
sequences. Organisms Diversity & Evolution 3: 227–237.
BENA, G., B. LEJEUNE, J.-M. PROSPERI,AND I. OLIVIERI. 1998. Molecular
phylogenetic approach for studying life-history evolution: the
ambiguous example of the genus Medicago L. Proceedings of the
Royal Society of London, B, Biological Sciences 265: 1141–1151.
BITTRICH, V., AND U. KU
¨HN. 1993. Nyctaginaceae. In K. Kubitzki, J. G.
Rohwer, and V. Bittrich [eds.], The families and genera of flowering
plants, 473–486. Springer-Verlag, Berlin, Germany.
BOGLE, A. L. 1974. The genera of Nyctaginaceae in the southeastern
United States. Journal of the Arnold Arboretum 55: 1–37.
BOHLIN, J. E. 1988. A monograph of the genus Colignonia (Nyctagina-
ceae). Nordic Journal of Botany 8: 231–252.
BREMER, B., K. BREMER,M.W.CHASE,J.L.REVEAL,D.E.SOLTIS,P.S.
SOLTIS,P.F.STEVENS,A.A.ANDERBERG,M.F.FAY,P.GOLDBLATT,W.
S. JUDD,M.KA
¨LLERSJO
¨,J.KAREHED,K.A.KRON,J.LUNDBERG,D.L.
NICKRENT,R.G.OLMSTEAD,B.OXELMAN,J.C.PIRES,J.E.RODMAN,P.
J. RUDALL,V.SAVOLAINEN,K.J.SYTSMA,M.VAN DER BANK,K.
WURDACK,J.Q.Y.XIANG,AND S. ZMARZTY. 2003. An update of the
Angiosperm Phylogeny Group classification for the orders and
families of flowering plants: APG II. Botanical Journal of the
Linnean Society 141: 399–436.
BURGER, A. E. 2005. Dispersal and germination of seeds of Pisonia
grandis, an Indo-Pacific tropical tree associated with insular seabird
colonies. Journal of Tropical Ecology 21: 263–271.
CHATURVEDI, S. K. 1989. A new device of self pollination in Boerhaavia
diffusa L. Nyctaginaceae. Beitraege zur Biologie der Pflanzen 64: 55–
58.
CHAVEZ, R. P., R. F. NAVA,E.GRAFSTROM,M.VON PFALER,AND S. NILSSON.
1998. On the pollen of Salpianthus (Nyctaginaceae)—a morpholog-
ical and image analysis approach. Grana 37: 352–357.
COCKERELL, T. D., AND F. GARCIA. 1898. Preliminary note on the growth of
plants in gypsum. Science 8: 119–121.
CRUDEN, R. W. 1970. Hawkmoth pollination of Mirabilis multiflora
Nyctaginaceae. Bulletin of the Torrey Botanical Club 97: 89–91.
CRUDEN, R. W. 1973. Reproductive biology of weedy and cultivated
Mirabilis Nyctaginaceae. American Journal of Botany 60: 802–809.
CUENOUD, P., V. SAVOLAINEN,L.W.CHATROU,M.POWELL,R.J.GRAYER,
AND M. W. CHASE. 2002. Molecular phylogenetics of Caryophyllales
based on nuclear 18S rDNA and plastid rbcL,atpB, and matK DNA
sequences. American Journal of Botany 89: 132–144.
DARWIN, C. 1884. The different forms of flowers on plants of the same
species. J. Murray, London, UK.
DESFEUX, C., S. MAURICE,J.P.HENRY,B.LEJEUNE,AND P. H. GOUYON.
1996. Evolution of reproductive systems in the genus Silene.
Proceedings of the Royal Society of London, B, Biological Sciences
263: 409–414.
DOWNIE, S. R., D. S. KATZ-DOWNIE,AND K. J. CHO. 1997. Relationships in
the Caryophyllales as suggested by phylogenetic analyses of partial
chloroplast DNA ORF2280 homolog sequences. American Journal of
Botany 84: 253–273.
DOWNIE, S. R., AND J. D. PALMER. 1994. A chloroplast DNA phylogeny of
the Caryophyllales based on structural and inverted repeat restriction
site variation. Systematic Botany 19: 236–252.
DOYLE, J. J., AND J. L. DOYLE. 1987. A rapid DNA isolation procedure for
small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11–
15.
ESCUDERO, A., L. F. CARNES,AND F. PEREZ-GARCIA. 1997. Seed germination
of gypsophytes and gypsovags in semi-arid central Spain. Journal of
Arid Environments 36: 487–497.
ESCUDERO, A., J. M. IRIONDO,J.M.OLANO,A.RUBIO,AND R. C. SOMOLINOS.
2000. Factors affecting establishment of a gypsophyte: the case of
Lepidium subulatum (Brassicaceae). American Journal of Botany 87:
861–871.
ESCUDERO, A., R. C. SOMOLINOS,J.M.OLANO,AND A. RUBIO. 1999. Factors
controlling the establishment of Helianthemum squamatum,an
endemic gypsophile of semi-arid Spain. Journal of Ecology 87:
290–302.
FISHER, R. A. 1941. Average excess and average effect of a gene
substitution. Annals of Eugenics 11: 53–63.
FOSBERG, F. R. 1978. Studies in the genus Boerhavia Nyctaginaceae, parts
1–5. Smithsonian Contributions to Botany: 1–20.
May 2007] DOUGLAS AND MANOS—PHYLOGENY OF NYCTAGINACEAE 869
FOWLER, B. A., AND B. L. TURNER. 1977. Taxonomy of Selinocarpus and
Ammocodon Nyctaginaceae. Phytologia 37: 177–208.
GALLOWAY, L. A. 1975. Systematics of the North American desert species
of Abronia and Tripterocalyx Nyctaginaceae. Brittonia 27: 328–347.
GRANT, V. 1983. The systematic and geographical distribution of hawk
moth flowers in the temperate North American flora. Botanical
Gazette 144: 439–449.
GRANT, V., AND K. A. GRANT. 1983. Hawkmoth pollination of Mirabilis
longiflora (Nyctaginaceae). Proceedings of the National Academy of
Sciences, USA, Biological Sciences 80: 1298–1299.
HARRIMAN, N. A. 1999. Synopsis of New World Commicarpus
(Nyctaginaceae). SIDA Contributions to Botany 18: 679–684.
HEIMERL, A. 1889. Nyctaginaceae. In A. Engler and K. Prantl [eds.], Die
Natu¨rlichen Pflanzenfamilien, 14–32. Engelmann, Leipzig, Germany.
HEIMERL, A. 1934. Nyctaginaceae. In A. Engler and K. Prantl [eds.], Die
Natu¨rlichen Pflanzenfamilien, 86–134. Engelmann, Leipzig, Ger-
many.
HERNA
´NDEZ, H. 1990. Autopolinizacio´n en Mirabilis longiflora L.
(Nyctaginaceae). Acta Botanica Mexicana 12: 25–30.
HODGES, S. A. 1995. The influence of nectar production on hawkmoth
behavior, self-pollination, and seed production in Mirabilis multiflora
(Nyctaginaceae). American Journal of Botany 82: 197–204.
IGIC, B., L. BOHS,AND J. R. KOHN. 2006. Ancient polymorphism reveals
unidirectional breeding system shifts. Proceedings of the National
Academy of Sciences, USA 103: 1359–1363.
JOHNSTON, I. M. 1941. Gypsophily among Mexican desert plants. Journal
of the Arnold Arboretum 22: 145–170.
JORDAN, W. C., M. W. COURTNEY,AND J. E. NEIGEL. 1996. Low levels of
intraspecific genetic variation at a rapidly evolving chloroplast DNA
locus in North American duckweeds (Lemnaceae). American Journal
of Botany 83: 430–439.
KEARNEY, T. H., AND R. H. PEEBLES. 1960. Nyctaginaceae. In T. H.
Kearney and R. H. Peebles [eds.], Arizona flora, 270–279. University
of California Press, Berkeley, California, USA.
KRUCKEBERG, A. R. 1986. The stimulus of unusual geologies for plant
speciation—an essay. Systematic Botany 11: 455–463.
LANDE, R., AND D. SCHEMSKE. 1985. The evolution of self-fertilization and
inbreeding depression in plants. I. Genetic models. Evolution 39: 24–
40.
LEE, S. 1978. A factor analysis study of the functional significance of
angiosperm pollen. Systematic Botany 3: 1–19.
LEVIN, R. A. 2000. Phylogenetic relationships within Nyctaginaceae tribe
Nyctagineae: evidence from nuclear and chloroplast genomes.
Systematic Botany 25: 738–750.
LEVIN, R. A. 2002. Taxonomic status of Acleisanthes,Selinocarpus, and
Ammocodon (Nyctaginaceae). Novon 12: 58–63.
LEVIN, R. A., R. A. RAGUSO,AND L. A. MCDADE. 2001. Fragrance
chemistry and pollinator affinities in Nyctaginaceae. Phytochemistry
58: 429–440.
LOOMIS, W. E. 1944. Effect of heavy applications of gypsum on plant
growth. Plant Physiology 19: 706–708.
LO
´PEZ, H. A., AND L. GALETTO. 2002. Flower structure and reproductive
biology of Bougainvillea stipitata (Nyctaginaceae). Plant Biology 4:
508–514.
LORD, E. M. 1981. Cleistogamy: a tool for the study of floral mor-
phogenesis, function, and evolution. Botanical Review 47: 421–449.
MADDISON, W. P., AND D. R. MADDISON. 2006. Mesquite: a modular sys-
tem for evolutionary analysis, version 1.1. Website: http://
mesquiteproject.org.
MAHRT, M., AND R. SPELLENBERG. 1995. Taxonomy of Cyphomeris
(Nyctaginaceae) based on multivariate analysis of geographic
variation. SIDA Contributions to Botany 16: 679–697.
MCCLELLAN, Y., AND W.-J. BOECKLEN. 1993. Plant mediation of ant–
herbivore associations: the role of sticky rings formed by Boerhavia
spicata.Coenoses 8: 15–20.
MCGLAUGHLIN, M., K. KAROLY,AND T. KAYE. 2002. Genetic variation and
its relationship to population size in reintroduced populations of pink
sand verbena, Abronia umbellata subsp. breviflora (Nyctaginaceae).
Conservation Genetics 3: 411–420.
MEYER, S. E. 1986. The ecology of gypsophile endemism in the eastern
Mojave Desert. Ecology 67: 1303–1313.
MEYER, S. E., E. GARCI
´A-MOYA,AND L. D. LAGUNES-ESPINOZA. 1992.
Topographic and soil surface effects on gypsophile plant community
patterns in central Mexico. Journal of Vegetation Science 3: 429–438.
NOWICKE, J. 1968. Palynotaxonomic study of the Phytolaccaceae. Annals
of the Missouri Botanical Garden 55: 294–364.
NOWICKE, J. W. 1970. Pollen morphology in the Nyctaginaceae. Part 1:
Nyctagineae, Mirabileae. Grana 10: 79–88.
NOWICKE, J. W. 1975. Pollen morphology in the order Centrospermae.
Grana 15: 51–78.
NOWICKE, J. W., AND T. J. LUIKART. 1971. Pollen morphology of the
Nyctaginaceae. Part 2: Colignonieae, Boldoeae, and Leucastereae.
Grana 11: 145–150.
NYLANDER, J. A. A. 2004. MrModeltest, version 2. Program distributed by
the author, Evolutionary Biology Centre, Uppsala University,
Uppsala, Sweden. Website: http://www.abc.se/;nylander/
mrmodeltest2/mrmodeltest2.html.
OLMSTEAD, R. G., AND J. A. SWEERE. 1994. Combining data in phylogenetic
systematics—an empirical approach using 3 molecular data sets in the
Solanaceae. Systematic Biology 43: 467–481.
OXELMAN, B., M. LIDEN,AND D. BERGLUND. 1997. Chloroplast rps16 intron
phylogeny of the tribe Sileneae (Caryophyllaceae). Plant Systematics
and Evolution 206: 393–410.
OYONARTE, C., G. SANCHEZ,M.URRESTARAZU,AND J. J. ALVARADO. 2002. A
comparison of chemical properties between gypsophile and non-
gypsophile plant rhizospheres. Arid Land Research and Management
16: 47–54.
PARSONS, R. F. 1976. Gypsophily in plants—a review. American Midland
Naturalist 96: 1–20.
POOL, A. 2001. Nyctaginaceae. In W. D. Stevens, C. Ulloa U., A. Pool,
and O. M. Montie [eds.], Flora de Nicaraugua, 1581–1592. Missouri
Botanical Garden Press, St. Louis, Missouri, USA.
POSADA, D., AND K. A. CRANDALL. 1998. Modeltest: testing the model of
DNA substitution. Bioinformatics 14: 817–818.
RAMBAUT, A. 1996. Se-Al: sequence alignment editor. Available at http://
evolve.zoo.ox.ac.uk/.
RETTIG, J. H., H. D. WILSON,AND J. R. MANHART. 1992. Phylogeny of the
Caryophyllales—gene sequence data. Taxon 41: 201–209.
REYES-SALAS, M., AND E. MARTI
´NEZ-HERNA
´NDEZ. 1982. Palynological
catalog for the flora of Veracruz, Mexico. 8. Nyctaginaceae family.
Biotica (Mexico) 7: 423–456.
RICHARDSON, J. E., R. T. PENNINGTON,T.D.PENNINGTON,AND P. M.
HOLLINGSWORTH. 2001. Rapid diversification of a species-rich genus
of neotropical rain forest trees. Science 293: 2242–2245.
RIDLEY, H. N. 1930. The dispersal of plants throughout the world. L.
Reeve & Co., Ashford, UK.
RODMAN, J. E., M. K. OLIVER,R.R.NAKAMURA,J.U.MCCLAMMER JR., AND
A. H. BLEDSOE. 1984. A taxonomic analysis and revised classification
of Centrospermae. Systematic Botany 9: 297–323.
ROHWEDER,O.,AND K. HUBER. 1974. Centrospermen-Studien. 7.
Beobachtungen und Anmerkungen zur Morphologie und Ent-
wicklungsgeschichte einiger Nyctaginaceen. Botanishe Jahrbu¨ cher
94: 327–359.
ROHWER, J. G. 1993. Phytolaccaceae. In K. Kubitski, J. G. Rohwer, and V.
Bittrich [eds.], The families and genera of flowering plants, 506–515.
Springer-Verlag, Berlin, Germany.
ROMAO, R. L., AND A. ESCUDERO. 2005. Gypsum physical soil crusts and
the existence of gypsophytes in semi-arid central Spain. Plant
Ecology 181: 127–137.
RONQUIST, F., AND J. P. HUELSENBECK. 2003. MRBAYES 3: Bayesian
phylogenetic inference under mixed models. Bioinformatics 19:
1572–1574.
SANDERSON, M. J. 1991. In search of homoplastic tendencies—statistical
inference of topological patterns in homoplasy. Evolution 45: 351–
358.
SCHEMSKE, D. 1978. Evolution of reproductive characters in Impatiens
(Balsaminaceae): the significance of cleistogamy and chasmogamy.
Ecology 59: 596–613.
870 AMERICAN JOURNAL OF BOTANY [Vol. 94
SMITH, A. G., D. G. SMITH,AND B. M. FUNNELL. 1994. Atlas of Mesozoic
and Cenozoic coastlines. Cambridge University Press, Cambridge,
UK.
SPELLENBERG, R. 1993. Taxonomy of Anulocaulis (Nyctaginaceae). SIDA
Contributions to Botany 15: 373–389.
SPELLENBERG, R. 2000. Blooming ‘‘behavior’’ in five species of Boerhavia
(Nyctaginaceae). SIDA Contributions to Botany 19: 311–323.
SPELLENBERG, R. 2003. Nyctaginaceae. In F. o. N. A. E. Committee [ed.],
Flora of North America north of Mexico, 14–74. Oxford University
Press, New York, New York, USA.
SPELLENBERG, R., AND R. K. DELSON. 1974. Aspects of reproduction in
Chihuahuan Desert Nyctaginaceae. Transactions of the Symposium
on the Biological Resources of the Chihuahuan Desert Region, United
States and Mexico, 273–287. Sul Ross State University, Alpine,
Texas, USA.
SPELLENBERG, R., AND J. M. POOLE. 2003. Nomenclatural adjustments and
comments in Abronia and Acleisanthes (Nyctaginaceae). SIDA
Contributions to Botany 20: 885–889.
STANDLEY, P. C. 1909. The Allioniaceae of the United States with notes on
Mexican species. Contributions from the United States National
Herbarium 12: 303–389.
STANDLEY, P. C. 1911. The Allioniaceae of Mexico and Central America.
Contributions from the United States National Herbarium 13: 377–
430.
STANDLEY, P. C. 1918. Allioniaceae. In N. L. Britton [ed.], North American
Flora, 171–254. New York Botanical Garden Press, Bronx, New
York, USA.
STANDLEY, P. C. 1931a. Studies of American plants: Nyctaginaceae. Field
Museum Botanical Series 8: 304–311.
STANDLEY, P. C. 1931b. The Nyctaginaceae of northwestern South
America. Field Museum Botanical Series 11: 71–126.
STANFORD, A. M., R. HARDEN,AND C. R. PARKS. 2000. Phylogeny and
biogeography of Juglans (Juglandaceae) based on matK and ITS
sequence data. American Journal of Botany 87: 872–882.
STEBBINS, G. L. 1974. Flowering plants. Evolution above the species level.
Belknap Press of Harvard University Press, Cambridge, Massachu-
setts, USA.
STEVENS, P. F. 2001-onwards. Nyctaginaceae. Angiosperm phylogeny
website http://www.mobot.org/MOBOT/research/APweb/.
SWOFFORD, D. L. 2002. PAUP*: phylogenetic analysis using parsimony
(*and other methods). Sinauer, Sunderland, Massachusetts, USA.
SYKES, W. R. 1987. The parapara, Pisonia brunoniana (Nyctaginaceae).
New Zealand Journal of Botany 25: 459–466.
TAKEBAYASHI, N., AND P. L. MORRELL. 2001. Is self-fertilization an
evolutionary dead end? Revisiting an old hypothesis with genetic
theories and a macroevolutionary approach. American Journal of
Botany 88: 1143–1150.
THOMPSON, J. D., T. J. GIBSON,F.PLEWNIAK,F.JEANMOUGIN,AND D. G.
HIGGINS. 1997. The ClustalX windows interface: flexible strategies for
multiple sequence alignment aided by quality analysis tools. Nucleic
Acids Research 25: 4876–4882.
THULIN, M. 1994. Aspects of disjunct distributions and endemism in the
arid parts of the Horn of Africa, particularly Somalia. Proceedings of
the XIIIth plenary meeting AETFAT, 2: 1105–1119. National
Herbarium and Botanic Gardens of Malawi, Zomba, Malawi.
TURNER, B. L. 1991. A new gypsophilic species of Mirabilis (Nyctagi-
naceae) from Nuevo Leo´ n, Mexico. Phytologia 70: 44–46.
TURNER, B. L. 1993. A new species of Anulocaulis (Nyctaginaceae) from
southern Coahuila, Mexico. SIDA Contributions to Botany 15: 613–
615.
UYENOYAMA, M. K., K. E. HOLSINGER,AND D. M. WALLER. 1993.
Ecological and genetic factors directing the evolution of self-
fertilization. Oxford University Press, Oxford, UK.
WATERFALL, U. T. 1946. Observations on the gypsum flora of southwestern
Texas and adjacent New Mexico. American Midland Naturalist 36:
456–466.
WHITE, T. J., T. BRUNS,S.LEE,AND J. TAYLOR. 1990. Amplifications and
direct sequencing of fungal ribosomal RNA genes for phylogenetics.
In M. Innis, D. Gelfand, J. Sninsky, and T. White [eds.], PCR
protocols: a guide to methods and applications, 315–322. Academic
Press, San Diego, California, USA.
WILLIAMSON, P. S., L. MULIANI,AND G. K. JANSSEN. 1994. Pollination
biology of Abronia macrocarpa (Nyctaginaceae), an endangered
Texas species. Southwestern Naturalist 39: 336–341.
WILLSON, J., AND R. SPELLENBERG. 1977. Observations on anthocarp
anatomy in the subtribe Mirabilinae (Nyctaginaceae). Madrono 24:
104–111.
WILSON, R. C. 1974. Abronia. Part 2. Anthocarp polymorphism and
anatomy for 9 Abronia species found in California. Aliso 8: 113–128.
ZADOO, S. N., R. P. ROY,AND T. N. KHOSHOO. 1975. Cytogenetics of
cultivated bougainvilleas. Part 2. Pollination mechanism and breeding
system. Proceedings of the Indian National Science Academy, Part B,
Biological Sciences 41: 498–502.
APPENDIX 1. Taxa, GenBank accession numbers, and voucher information
used in this study. Regions not sampled are indicated by a dash.
Cultivated plants were obtained from the following sources: DUBG ¼
Duke University Botany Greenhouses, Durham; STRYB ¼Strybing
Arboretum, San Francisco. Vouchers are deposited at the following
herbaria: DUKE ¼Duke University, NMC ¼New Mexico State
University, NY ¼New York Botanical Garden. Accession included
in ‘‘sensitivity’’ analysis. If sequences were downloaded from
GenBank, then no voucher information is given.
Species—GenBank accession numbers: ITS, ndhF,rpl16,rps16,Voucher
specimen, Locality, Year, Herbarium
Abronia bigelovii Heimerl—EF079455, EF079510, EF079564,
EF079606, Douglas 2088, New Mexico, USA, 2001, DUKE;
Abronia carletonii J.M. Coult. & Fisher—EF079456, EF079511,
EF079565, EF079607, Douglas 2091, New Mexico, USA, 2001,
DUKE; Acleisanthes lanceolatus (Wooton) R.A. Levin—EF079454,
EF079509, EF079563, EF079605, Douglas 2072, New Mexico, USA,
2001, DUKE; Acleisanthes longiflora A. Gray—EF079457,
EF079512, —, EF079608, Douglas 2098, New Mexico, USA, 2001,
DUKE; Allionia choisyi Standl.—EF079467, EF079519, EF079574,
EF079618, Douglas 2187, Coahuila, Mexico, 2002, DUKE; Andradea
floribunda Allema˜o—EF079491, EF079545, —, EF079639, Amorim
2294, Brazil, 1998, NY; Anulocaulis annulatus (Coville) Standl.—
EF079503, EF079557, EF079599, EF079650, Spellenberg 3162,
California, USA, 1993, NMC; Anulocaulis leiosolenus (Torr.)
Standley v. leiosolenus Spellenberg—EF079464, EF079517, —,
EF079615, Douglas 2122, Arizona, USA, 2002, DUKE;
Anulocaulis reflexus I.M. Johnst.— EF079468, EF079520, —, —,
Douglas 2192, Chihuahua, Mexico, 2002, DUKE; Anulocaulis
reflexus I.M. Johnst.— —, —, EF079586, EF079629, Spellenberg
10739, Chihuahua, Mexico, 1990, NMC; Belemia fucsioides Pires—
EF079488, EF079542, —, —, Belem 3796, Brazil, 1968, NY;
Boerhavia anisophylla Torr.— EF079469, EF079521, EF079575,
EF079619, Douglas 2194, Durango, Mexico, 2002, DUKE;
Boerhavia ciliata Brandegee— EF079465, —, EF079572,
EF079616, Douglas 2145, Texas, USA, 2002, DUKE; Boerhavia
coccinea Mill.— EF079472, EF079525, EF079579, EF079622,
Spellenberg 13275, Arizona, USA, 2001, DUKE; Boerhavia
coulteri (S. Wats.) v. palmeri Spellenb.— EF079471, EF079524,
EF079578, EF079621, Spellenberg 13273, Arizona, USA, 2001,
DUKE; Boerhavia dominii Meikle & Hewson—EF079487,
EF079540, EF079594, EF079638, Smyth 42, Australia, 1997, MO;
Boerhavia gracillima Heimerl—EF079479, EF079533, EF079587,
EF079630, Spellenberg 12447 , Texas, USA, 1997, NMC; Boerhavia
intermedia M.E. Jones—EF079474, EF079527, EF079581,
EF079624, Spellenberg 13279, Arizona, USA, 2001, DUKE;
Boerhavia lateriflora Standl.—EF079466, EF079518, EF079573,
EF079617, Douglas 2161, Sonora, Mexico, 2002, DUKE; Boerhavia
linearifolia A. Gray—EF079459, EF079514, EF079567, EF079610,
Douglas 2102, New Mexico, USA, 2001, DUKE; Boerhavia
purpurascens A. Gray—EF079470, EF079523, EF079577,
EF079620, Spellenberg 13261, Arizona, USA, 2001, DUKE;
May 2007] DOUGLAS AND MANOS—PHYLOGENY OF NYCTAGINACEAE 871
Boerhavia repens L.—EF079480, EF079534, EF079588, EF079631,
Spellenberg 7183, Sana, Yemen , 1983, NMC; Boerhavia repens L.
—EF079477, EF079531, EF079584, EF079627, Rose 2, Oahu,
Hawaii, USA, 2001, DUKE; Boerhavia spicata Choisy—
EF079473, EF079526, EF079580, EF079623, Spellenberg 13276,
Arizona, USA, 2001, DUKE; Bougainvillea glabra Choisy—
EF079463, —, EF079571, EF079614, Douglas 2121, North
Carolina, USA (DUBG), 2002, DUKE; Bougainvillea infesta
Griseb.—EF079498, EF079551, —, EF079644, Nee 51442, Bolivia,
2000, NY; Caribea litoralis Alain— —, EF079530, —, —, A. H.
Liogier 7013, Cuba, 1959, NY; Colignonia glomerata Griseb.—
EF079495, EF079549, —, EF079642, Nee 52523, Bolivia, 2003, NY;
Colignonia scandens Benth.—EF079502, EF079556, EF079598,
EF079648, Grantham 63, Lojas, Ecuador (STRYB), 2003, DUKE;
Commicarpus coctoris N.A. Harriman—EF079481, EF079535,
EF079589, EF079632, Spellenberg 12883, Oaxaca, Mexico, 1998,
NMC; Commicarpus plumbagineus (Cav.) Standl.—EF079504,
EF079558, EF079600, EF079651, Spellenberg 7374, Ta’izz, Yemen,
1983, NMC; Commicarpus scandens (L.) Standl.—EF079482,
EF079536, EF079590, EF079633, Spellenberg 12887, Puebla,
Mexico, 1998, NMC; Cyphomeris gypsophiloides (M. Martens &
Galeotti) Standl.—EF079458, EF079513, EF079566, EF079609,
Douglas 2100, New Mexico, USA, 2001, DUKE; Guapira discolor
(Spreng.) Little—EF079476, EF079529, EF079583, EF079626,
Spellenberg 13294, Florida, USA, 2001, DUKE; Guapira
eggersiana (Heimerl) Lundell—EF079496, EF079550, —,
EF079643, Mori 25542/40, French Guiana, 2003, NY; Leucaster
caniflorus (Mart.) Choisy— —, EF079541, —, —, Pirani 3602,
Brazil, 1995, NY; Leucaster caniflorus (Mart.) Choisy—EF079497,
—, —, —, Hatschbach 50421, Brazil, 1993, NY; Mirabilis albida
(Walter) Heimerl—EF079451, EF079506, EF079560, EF079602,
Douglas 2035, Arizona, USA, 2001, DUKE; Mirabilis jalapa L.—
EF079461, EF079515, EF079569, EF079612, Douglas 2119, North
Carolina, USA (DUBG), 2002, DUKE; Mirabilis multiflora (Torr.) A.
Gray—EF079452, EF079507, EF079561, EF079603, Douglas 2037,
Arizona, USA, 2001, DUKE; Neea cauliflora Heimerl—EF079493,
EF079547, —, —, Schanke S15106, Peru, 2002, NY; Neea
hermaphrodita S. Moore—EF079489, EF079543, —, —, Nee
51426, Bolivia, 2000, NY; Neea psychotrioides Donn. Sm.—
EF079505, EF079559, EF079601, EF079652, Wilbur 63654,
Heredia, Costa Rica, 1995, DUKE; Nyctaginia capitata Choisy—
EF079478, EF079532, EF079585, EF079628, McIntosh 2049, New
Mexico, USA, 1992, NMC; Okenia hypogaea Schltdl. & Cham.—
EF079483, —, —, EF079634, TR & RK Van Devender 92–1069,
Sonora, Mexico, 1992, NMC; Okenia hypogaea Schltdl. & Cham.—
—, EF079522, EF079576, —, Douglas 2206, Veracruz, Mexico, 2002,
DUKE; Phaeoptilum spinosum Radlk.—EF079490, EF079544, —,
—, Seydel 4077, Namibia, 1964, NY; Pisonia capitata (S. Watson)
Standl.—EF079484, EF079537, EF079591, EF079635, AL Reina G.
(2000—193), Sonora, Mexico, 2000, NMC; Pisonia rotundata Griseb.
—EF079475, EF079528, EF079582, EF079625, Spellenberg 13293,
Florida, USA, 2001, DUKE; Pisoniella arborescens (Lag. & Rodr.)
Standl.—EF079485, —, EF079592, EF079636, LeDuc 231, Oaxaca,
Mexico, 1992, NMC; Pisoniella arborescens (Lag. & Rodr.) Standl.
— —, EF079539, —, —, Anderson 13522, Oaxaca, Mexico, 1988,
NY; Ramisia brasiliensis Oliv.— EF079492, EF079546, —,
EF079640, Jardim 1507, Brazil, 1998, NY; Reichenbachia hirsuta
Spreng.—EF079494, EF079548, EF079595, EF079641, Nee 51972,
Bolivia, 2002, NY; Salpianthus arenarius Humb. & Bonpl.—
EF079486, EF079538, EF079593, EF079637, Spellenberg 12903,
Michoacan, Mexico, 1999, NMC; Tripterocalyx carneus (Greene) L.
A. Galloway—EF079453, EF079508, EF079562, EF079604,
Douglas 2060, New Mexico, USA, 2001, DUKE; Outgroups:
Aptenia cordifolia (L. f.) Schwantes— —, AF194824, —, —, ;
Mollugo verticillata L.— —, AF194827, —, —, ;Mollugo
verticillata L.— —, —, —, EF079649, Wilbur 77788, North
Carolina, USA, 2004, DUKE; Petiveria alliacea L.—EF079499,
EF079552, —, —, AL Reina G. 98–2048, Sonora, Mexico, 1998, NY;
Phytolacca americana Roxb.—EF079460, —, EF079568, EF079611,
Douglas 2118, North Carolina, USA, 2002, DUKE; Phytolacca
acinosa L— —, AF194828, —, —, ;Rivina humilis L.—EF079462,
EF079516, EF079570, EF079613, Douglas 2120, North Carolina,
USA (DUBG), 2002, DUKE; Sarcobatus vermiculatus (Hook.) Torr.
—EF079501, EF079555, EF079597, EF079647, Spellenberg 13312,
Nevada, USA, 2002, DUKE; Stegnosperma cubense A. Rich.—
EF079500, EF079554, EF079596, EF079646, Salas–M. 2649, Oaxaca,
Mexico, 1999, NY; Trichostigma octandrum (L.) H. Walter— —,
EF079553, —, EF079645, Acevedo–Rodriguez 5447 Virgin Islands,
USA 1993, NY.
872 AMERICAN JOURNAL OF BOTANY [Vol. 94