Species Relationships in Krameria (Krameriaceae) Based on ITS Sequences and

Morphology: Implications for Character Utility and Biogeography

Author(s) :Beryl B. Simpson, Andrea Weeks, D. Megan Helfgott, and Leah L. Larkin
Source: Systematic Botany, 29(1):97-108. 2004.
Published By: The American Society of Plant Taxonomists
DOI: http://dx.doi.org/10.1600/036364404772974013
URL: http://www.bioone.org/doi/full/10.1600/036364404772974013

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 Systematic Botany (2004), 29(1): pp. 97–108 q Copyright 2004 by the
American Society of Plant Taxonomists

Species Relationships in Krameria (Krameriaceae) Based on ITS Sequences and

Morphology: Implications for Character Utility and Biogeography

BERYL B. SIMPSON,3 ANDREA WEEKS, D. MEGAN HELFGOTT,1 and LEAH L. LARKIN2

Section of Integrative Biology and Plant Resources Center, The University of Texas, Austin, Texas 78712;
1
Current address: Department of Biological Sciences, University of Illinois, Chicago, Illinois 60607;
2
Current address: Department of Biology, University of New Mexico, Albuquerque, New Mexico 87131
3
Author for Correspondence (beryl@mail.utexas.edu)

Communicating Editor: Paul S. Manos

ABSTRACT. The Krameriaceae is a monotypic family of 18 species distributed in the warm arid and semiarid regions of North
and South America. We have used sequence data from the internal transcribed spacer region of the nuclear ribosomal DNA
repeat (ITS 1, 5.8S, and ITS 2) and morphology to infer relationships within Krameria. Using Kallstroemia parviflora,
Guaiacum angustifolium, and Tribulus terrestris, three North American members of the Zygophyllaceae, as outgroups, our data
provide evidence for two major clades in the genus, each containing a North and a South American subclade. Both the major
clades and subclades are supported by morphological synapomorphies. The phylogeny does not provide an unequivocal
resolution as to the hemisphere in which Krameria arose, but it does indicate an initial split leading to the ancestors of the two
clades, each of which subsequently produced radiations in North and South America. Therefore, there must have been two
independent dispersals to, or two vicariant episodes involving, North and South America.
Krameria Loefling (commonly known as rhatany) has proved to be a taxonomic enigma since its description by Loefling in 1758. Originally
placed in the Polygalaceae by de Jussieu (1789), it was given familial status by Dumortier (1829), and then shifted to the Leguminosae by Taubert
(1892). The current consensus is that the genus is the sole member of the family Krameriaceae (Simpson 1989). It comprises 18 species of perennial
herbs or shrubs that occur in the warm arid and semi-arid regions of the New World. The ten North and Central American species of the genus
range from Kansas across the southwestern USA (with disjunct populations of one species in Florida and adjacent Georgia) to Costa Rica. In South
America, six species range from northern Colombia to east-central Brazil with one species reaching Sinaloa, Mexico, and the Greater and Lesser
Antilles. Two additional species occur in west-central South America in Peru, Bolivia, northern Chile, and Argentina.
The historical ambiguity about the relationships of Krameria to other families is due to its large number of unique floral and fruit characters
(Simpson 1989). All species have zygomorphic flowers superficially reminiscent of Polygalaceae or Caesalpinioideae (Leguminosae). However, the
actual floral structure (Fig. 1A, C) is quite different (Simpson 1982). The showy portion of Krameria flowers consists of five (or four) free purple,
pink, or yellow sepals. The petals are highly reduced with three (or two by suppression) forming a flag over the superior ovary and the remaining
two modified into fleshy secretory structures bilaterally flanking the ovary. These petals secrete fatty oils that are collected by female bees of the
genus Centris Fabricius (Apidae) (Simpson et al. 1977; Simpson et al. 1978). The four anthers dehisce apically. The fruits (Fig. 1B, D) are circular
to heart-shaped in outline and superficially similar in appearance to those of Zuccagnia Cav., an unusual caesalpinioid legume endemic to
Argentina. However, the ovary of Krameria only mimics a legume fruit. It is anatomically bicarpellate with only one carpel developing and
producing ovules. Each fruit has a single exalbuminous seed consisting of an embryo surrounded by a pericarp that splits irregularly rather than
along two sutures. All species that have been studied are hemiparasitic with the ability to use a wide range of host plants. Seedlings lack root hairs
and must attach to a root of a host within the first two months of life or perish (Cannon 1911).
In 1989, Simpson monographed the genus and provided a cladistic treatment based on morphological characters. While the Polygalaceae was
tentatively suggested as the closest relative of the Krameriaceae, members of this family do not have morphological characters homologous to those
of taxonomic utility within Krameria such as glandular petals, flag petals, striate pollen, etc. Consequently, the morphological cladistic treatment
was done without an outgroup. This morphological analysis suggested that the Krameria consists of at least two South American and two North
American clades and that several morphological characters such as sepal position, gland surface sculpturing, and pollen type have states that are
apomorphic for these clades. However, the analysis was done before methods were available for assessing the statistical robustness of individual
clades and rooting was done somewhat arbitrarily (since no outgroups were included) based on pollen aperture type. In addition, Stevens (1991)
criticized some of the continuous characters that were used in this and other papers because there was overlap between character-state boundaries.
97
 98 SYSTEMATIC BOTANY [Volume 29

FIG. 1. Flowers and fruits of Krameria species. A. Flower of Krameria cytisoides showing the free flag petals and the rugose external surface of the glandular petal, 3 3.3. B.
Fruit of K. paucifolia, a relative of K. grayi, showing the large retrorse barbs at the tip of the spines, 3 2.7. C. Flower of K. lanceolata showing the fused flag petals and the
secretory structures concentrated near the distal edge of the gland, 3 2.9. D. Fruit of K. erecta showing the distribution of small barbs along the shafts of the
spines, 3 7.2.

We have subsequently turned to molecular characters as additional evidence for determining the relationships of species within the family and to
see if the pattern of relationships suggested by the previous morphological analysis could be corroborated. In contrast to the situation with
morphological characters, we were able to use three members of the Zygophyllaceae (Guaiacum angustifolium Engelm., Kallstroemia parviflora J.
B. Norton, and Tribulus terrestris L.) as outgroups following the reports of Chase et al. (1993), Gadek et al. (1996), Sheahan and Chase (1996), and
Savolainen et al. (2000) that the Zygophyllaceae sensu stricto and the Krameriaceae are sister families. Here we report the results of this molecular
study using sequences of the nuclear ribosomal DNA internal transcribed spacer regions (ITS 1 and ITS 2) including the intervening 5.8S gene. In
addition we have redone the morphological analysis, omitting ambiguous characters and adding several new informative characters. We then assess
the pattern of evolution of several morphological characters on the phylogeny obtained using the ITS data. Finally, we address the possible events
that could explain the biogeographical patterns we observe among the species of the genus.
 MATERIALS AND METHODS
Molecular Methods. TAXON SAMPLING. We obtained DNA sequence data for nuclear ribosomal ITS regions 1 and 2 and the intervening 5.8S of 17 of the 18 Krameria species,
including the very rare (in collections) K. spartioides (Table 1). We were unable to se-
100 SYSTEMATIC BOTANY [Volume 29

TABLE 1. Species and vouchers of Krameria and outgroups used for molecular analysis (all vouchers housed at TEX). GenBank numbers
for Krameria species include all cloned sequences. Collections marked ** were extracted from fresh material, the remainder from
herbarium material.

K. argentea Spreng.—Brazil: D.F., M. L. Azevedo 646 (AY260975–AY260980). K. cistoidea Hook. & Arn.—Chile: Atacama, C. M.
Taylor et al. 10682 (AY261014–AY261018). K. cytisoides Cav.—Mexico: Nuevo Leo´n, J. Hinton et al. 25159 (AY261023–AY261032).
K. erecta Schultes—Mexico: Sonora, B.
B. Simpson 27-9-95-2** (AY261061–AY261070). K. grandiflora A. St.-Hil—Brazil: D.F., J. A. Lomhardi 2066 (AY260981– AY260990).
K. grayi Rose & Painter—U.S.A.: TX, Brewster Co., M. Butterwick & S. Strong 624 (AY261033–AY261039). K. ixine Loefl.—Mexico:
Oaxaca, M. Mayfield 990 (AY260991– AY261995). K. lanceolata Torr.—USA: TX, Parker Co., E. L. Bridges & K. Kindscher 13622
(AY261071–AY261080). K. lappacea (Dombey) Burdet & B. B. Simpson—Bolivia: Murillo, J. C. Solomon 6703 (AY261019–
AY261022). K. pauciflora DC.— Mexico: Hidalgo, B. B. Simpson 91-VII-18-12 (AY261081– AY261089). K. paucifolia (Rose) Rose—
Mexico: Baja Sur, B. B. Simpson 03-15-80-1 (AY261040–AY261054). K. ramosissima (A. Gray) S. Wats.—U.S.A.: TX, Val Verde Co.,
B. Ertter 5387 (AY261090–AY261095). K. revoluta O. Berg—Mexico: Oaxaca, R. Torres C. 5858 (AY261096–AY261106). K.
secundiflora DC.— Mexico: Coahuila, J. Henrickson 15940 (AY261107–AY261121). K. sonorae Britton—Mexico: Sonora, B. B.
Simpson 27-9-951A** (AY261055–AY261060). K. spartioides O. Berg—Guyana: Rupununi, M. J. Jansen-Jacobs et al. 5041 (AY260996–
AY261006). K. tomentosa A. St.-Hil.—Brazil: Goia´s, M. L. Fonseca et al. 2002 (AY261007–AY261013).
Guaiacum angustifolium Engelm.—USA: TX, Val Verde Co., B. L. Turner 98-192** (AY260974). Kallstroemia parviflora J.B. Norton
—USA: TX, Travis Co., B. B. Simpson 16-VI-00-2** (AY260973). Tribulus terrestris L.—USA: TX, Burnet Co., B. B. Simpson 16-VI-00-
1** (AY260972).

quence the ITS region for K. bahiana B. B. Simpson. We have included in this analysis an additional taxon, K. sonorae, not recognized in
the 1989 monograph (Simpson 1989) because at that time it was considered conspecific with K. grayi. Subsequent extensive fieldwork in
the fall of 1995 by BBS revealed that the two are distinct. In addition we obtained ITS sequences for Guaiacum angustifolium,
Kallstroemia parviflora, and Tribulus terrestris (Zygophyllaceae). The taxa used in the molecular study, the source of the material, and
voucher information are given in Table 1. Sequences were submitted to GenBank under the numbers AY260972–AY261121 and the matrix
of aligned ancestral ITS sequences (see below) and the morphological matrix have been submitted to TreeBase (study accession number
S935, matrix accession numbers M1550 and M1551).
DNA EXTRACTION, AMPLIFICATION, AND SEQUENCING. Total genomic DNA was isolated according to standard protocols without
modification from either fresh field-collected (Doyle and Doyle 1987) or herbarium (Lookerman and Jansen 1996) material of individual
plants (Table 1). Extraction and isolation of Krameria DNA proved to be difficult, perhaps because of the high tannin content known to
occur throughout the plants.
Double-stranded DNA amplification of the ITS regions was carried out by the polymerase chain reaction (PCR). Using the primers listed
in Table 2, the amplification strategy followed the protocol in Kim and Jansen (1994). Each 25 mL PCR mixture contained ca. 15–100 ng
of total genomic DNA, 400 mM each dNTP, 0.1 ml taq polymerase, 1X Triton-X PCR buffer, 2.0 mM MgCl2, and 0.4 mM of each primer.
Initial PCR amplifications of a single fragment comprising all of ITS 1, 5.8S, and ITS 2 were accomplished using primers P1a T ABLE 2.
Primers used for ITS 1 and ITS 2.

ITS 1: Pla (forward) 59GGA AGG AGA AGT CGT AAC

AAG G 39 (Downie & Katz-Downie 1996), P2B (reverse) 59
CTC GAT GGA ACA CGG GAT TCT GC 39 (Helfgott 2000), P2K (reverse) 59 CTC GAT GGT TCA CGG GAT TCT GC 39 (based on
Helfgott 2000).
ITS 2: P3 (forward) 59 GCA TCG ATG AAG AAC GCA GC
39 (White et al. 1990). P3K (forward) 59 GCA TCG ATG AAG AAC GTA GC 39 (Kim and Jansen 1994). P4 (reverse) 59 TCC TCC GCT
CAT TGA TAT GC 39 (based on
White et al. 1990).

and P4 (Table 2). For some taxa, the entire fragment would not amplify and the ITS 1 and ITS 2 regions were amplified separately. Primer pairs
for ITS 1 were either P1a and P2B or P1a and P2K. Primers for ITS 2 were either P3 and P4 or P3K and P4. PCR amplifications in a thermal
cycler followed this protocol: 3 min at 948C; 1 min at 508C; 1 min at 728C; 35 cycles of 1 min at 948C, 1 min at 508C, and 45 sec at 728C with
an additional 3 sec per cycle; 7 min at 728 C and final cooling to 158C. The annealing temperature was adjusted downward for some recalcitrant
taxa.
Due to the presence of multiple copies of ITS and fungal contamination, PCR product for Krameria extracts was immediately cloned into
Escherichia coli (Magula) Castellani & Chalmers bacteria using the TOPO-TA Cloning Kit (Invitrogen Corporation, Carlsbad, CA) following
the manufacturer’s protocols (except that reactions were done at one-third the recommended volumes). White colonies were arbitrarily selected
and used as template DNA in PCR reactions using the protocol outlined above. At least four clones per taxon were sequenced in both directions.
Cloning and sequencing were necessary because in many cases, PCR products of copies of plant ITS and fungal contaminants were
2004] SIMPSON ET AL.: PHYLOGENY OF KRAMERIACEAE 101

indistinguishable using agarose gel electrophoresis. All sequenced products were submitted to BLAST searches that allowed us to recognize
fungal contaminants. Cloning was unnecessary for outgroup taxa.
Final PCR product was purified by filtration through Sephadex G-50 (Sigma Chemical) packed columns. Both complementary strands were
sequenced via dye-terminator cycle sequencing using ABI Prism BigDyey Dye Terminators with 50 ng PCR template, 3.2 pmol primer and
buffer in 20 ml total volume. Terminator reactions were amplified in a thermal cycler equipped with a hotbonnet with an initial denaturing at
968C for 2 min followed by 25 cycles of 10 sec at 968C, 5 sec at 508C and 4 min at 608C and final cooling to 48C. Sequences were visualized
on an MJ BaseStation automated sequencer. Complementary sequences were combined and edited using Sequencher 4.1.2 (Gene Codes
Corporation 2002).
Initial alignments of all cloned Krameria sequences were generated using the default settings of CLUSTAL X (Thompson et al. 1997). The
alignments were then adjusted by eye in SeqApp (Gilbert 1992) and in MacClade 4.0 (Maddison and Maddison 2000). Clones of a single taxon
required between zero and six short indels to align with one exception: a single clone of K. revoluta had a 35 bp deletion. Indels were treated as
missing data in the analysis.
Our initial analysis used all of the cloned sequences and showed that all clones for an individual species of Krameria formed a clade. Mean
pairwise distance for all inter-species comparisons (e.g., omitting comparisons between clones of the same taxon) compared to the mean
pairwise distance between clones of each taxon were: ITS1, 0.11024 between species versus 0.00072–0.06124 within a species and ITS2,
0.08347 between species versus 0.00097– 0.05223 within a species. Consequently, for each taxon and marker, the set of clones was constrained
to monophyly in MrBayes 2.01 (Huelsenbeck and Ronquist 2001) and used to generate a single ancestral sequence. For each ancestral node
designated by a constraint, MrBayes uses a likelihood algorithm to calculate the probabilities of each base in the sequence; it thus generates the
most probable ‘ancestral’ sequence for that node. Ancestral sequences are not consensus sequences and there are no ambiguous sites. We used
the general time-reversible model of evolution (Rodr´ıguez et al. 1990) with rate heterogeneity (G; Yang 1993), GTR1G, which was determined
to fit the clone data best by the likelihood ratio test (LRT; Goldman 1993) as implemented in ModelTest 3.06 (Posada and Crandall 1998). We
explicitly assumed that duplications of the ITS region occurred after speciation for each species and that lineage sorting is not a factor. This
allowed us to combine the ITS 1, ITS 2, and 5.8S sequences for those taxa for which the ITS region could not be sequenced in its entirety. Such
sequences could not otherwise have been combined as we could not know which clone of ITS 1 was contiguous in the actual genome with which
clone of ITS 2. It also drastically reduced the size of the dataset allowing for more computationally intensive analyses. Because MrBayes does
not reconstruct the indels of the ancestral sequences, we artificially inserted them using a simple C 11 program called MissingMasker (written by
Derrick Zwickl, unpublished), which inserts missing nucleotides into simulated data sets to match the pattern of missing data in the real data set.
Where multiple clones of the same taxon had differing gap patterns, the ancestral gap status was resolved using MacClade 4.0 and the 50%
majority rule consensus tree generated by the constrained Bayesian search. Data thus generated were complete for all species and were used for
all subsequent analyses. Outgroups were aligned with these ancestral sequences by eye using SeqApp and MacClade 4.0. This alignment,
available on TreeBase, was 822 bp long, with 5.8S at 358–522 bp. In addition to 5.8S, the alignment was relatively straightforward between bp
1–179, 231–270, 522–541, 577–676, 720–822, although there are gaps in these regions. No regions were excluded from the analyses.
PHYLOGENETIC RECONSTRUCTION. Phylogenetic analyses of DNA data were undertaken using maximum parsimony and maximum likelihood
algorithms using PAUP*4b10 (Swofford 2002) and likelihood-based Bayesian analyses were performed using MrBayes 2.01 (Huelsenbeck and
Ronquist 2001). Base composition TABLE 3. Morphological characters and character states for Krameria species.

1. Petioles (0 5 present; 1 5 absent). Leaf shape (0 5 simple, ovate; 1 5 simple, linear; 2 5 simple, scale-like; 3 5 compound). 3. Flowers (0
5 solitary; 1 5 borne in inflorescences).
4. Peduncles (0 5 long; 1 5 short, almost flush with bracts). 5. Buds (0 5 arcuate in outline; 1 5 ovate in outline). 6. Sepal position (0 5
connivent; 1 5 at a right angle to the pedicel; 2 5 reflexed). 7. Sepal number (0 5 5; 1 5 4). 8. Gland type (0 5 rugose; 1 5 striate over entire
surface; 2 5 striations restricted to petal tip). 9. Petal number (0 5 5; 1 5 4). 10. Petaloid petals (0 5 free, strap-shaped; 1 5 basally fused,
cordate; 2 5 free, spathulate; 3 5 free, broad and oblanceolate). 11. Stamen number (0 5 4; 1 5 3). 12. Stamen lengths (0 5 free from point
of attachment; 2 5 fused beyond point of attachment). 13. Pollen apertures (0 5 tricolporate; 1 5 porate, pores narrow; 2 5 porate, pores
broad). 14. Spine density (0 5 scattered; 1 5 dense with bases touching). 15. Spine type (0 5 thin, black or purple; 1 5 thin, red; 2 5 stout
(conical)). 16. Spine barbs (0 5 with shiny barbs in 2 whorls at the tip; 1 5 with shiny barbs in 1 whorl at the tip; 2 5 with small teeth only
distally; 3 5 irregular barbs along the shaft; 4 5 lacking barbs or teeth). 17. Spine base (0 5 strigose; 1 5 glabrous). 18. Sepal color (0 5 reds
(bright pink to purple); 1 5 rose fading to white; 2 5 yellow with dusky rose). 19. Stamen fusion (0 5 free from point of attachment; 1 5
fused beyond point of attachment). 20. Lateral sepals (0 5 narrower than others; 1 5 broader than others). 21. Shape of fruit (0 5 ovoid; 1 5
heart-shaped). 22. Shoot type (0 5 long only; 1 5 mixed long and short shoots). 23. Pubescence of young leaves (0 5 sericeous; 1 5
canescent; 2 5 strigose; 3 5 tomentose).
bias was calculated in PAUP*4b10.
Phylogenetic information in the data set was assessed using the g 1 statistic based on 100,000 random trees (Hillis 1991; Hillis and Huelsenbeck
1992) and the level of support for clades was gauged using 1000 non-parametric bootstrap pseudoreplicates with a branch and bound search
algorithm (Felsenstein 1985). Pairwise distances between species were calculated using PAUP*. Parsimony searches were done using the
Branch and Bound option of PAUP*4b10.
For maximum likelihood estimations, we used the TrNef1G model (excess transitions, unequal nucleotide frequencies, and rate heterogeneity,
Tamura and Nei 1993) that we found by the likelihood ratio tests (Goldman 1993) to be the best models for the ancestral calculated sequences.
We ran an heuristic search using TBR branch swapping with MULTREES in effect.
We used the program MrBayes to infer a phylogeny in a Bayesian context using the GTR1G model of evolution (MrBayes does not allow
TrNef1 G). Three independent replicates of 2.5 million generations, with trees saved every 100 generations, each converged to the same plateau
by the 25,000th generation. Convergence was confirmed by comparing likelihood scores as well as all estimated parameters from the three
replicate analyses. A majority rule consensus tree was constructed from the trees of the plateau of one of these replicate analyses.
102 SYSTEMATIC BOTANY [Volume 29

We used the SOWH test of Goldman et al. (2000) to test if trees constrained to represent different relationships among the species were
significantly worse (longer) than our most parsimonious estimate. For these tests we constrained to monophyly all of the North American
species, all the South American species, or three of the four major subclades thus testing successively if each of the four major subclades could
be rejected as basal to the remaining members of the genus. Finally we constrained particular pairs of morphologically similar species
(Krameria erecta and K. ramosissima and K. ixine and K. tomentosa) to monophyly. For each of the eight constraints (null models), we used the
most parsimonious resolved constraint tree (or trees) as the model tree (some searches found trees with polytomies, which were not tested) and
simulat-

ed 1000 data sets based on that topology using Seq-Gen v. 1.2.5 (Rambaut and Grassly 1997). Datasets were simulated using the TrNef1G
model. Because Seq-Gen does not recreate the distribution of gaps in the original dataset, gaps were inserted in the simulated datasets using
MissingMasker (Derrick Zwickl, unpublished). This provided us with a data set with gaps and one without. We then analyzed each
simulated data set both with and without the topological constraint to generate a distribution of tree length differences (TLD) between
constrained and unconstrained trees. The TLD for the real data was compared to this distribution to determine if constrained trees were
significantly longer than unconstrained.
Morphological Methods. TAXON SAMPLING. The character states listed in Table 3 were scored for all 18 species of Krameria (including
K. bahiana) and used in a morphological phylogenetic analysis. The morphological character data set (Table 4) was modified from that in
Simpson (1989) by deleting the three continuous characters (i.e., amount of petal fusion, spine base diameter, and leaf length) discussed by
Stevens (1991). In addition four new characters (numbers refer to character numbers in Table 3) were added (spine type [char. 15], vestiture
of the spine base [char. 17], fruit shape [char. 21], and shoot type [char. 22]). More precision was added to the character of the spine tip
morphology [char. 16]. The Zygophyllaceae outgroups were not included in this analysis since they possesses too few homologous
morphological characters states to be scored meaningfully. All characters were scored for every species.
PHYLOGENETIC RECONSTRUCTION. The data set of 18 Krameria species and 23 characters was analyzed using the Branch and Bound option
of PAUP*4.0d64. Strict and majority rule consensus trees were computed from the trees obtained. Bootstrap values were calculated from
1000 pseudoreplicate analyses also using the Branch and Bound option in PAUP*4.0d64.
The morphological data for Krameria species, combined with the molecular data (excluding the Zygophyllaceae outgroups), were also

TABLE 4. Morphological character data matrix for Krameria species.

Character Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Species
K. argentea 0 0 1 1 1 0 1 1 0 2 0 1 2 1 2 4 0/1 0 0 0 0 0 2
K. bahiana 0 0 1 1 1 0 1 1 0 3 0 0 2 0 2 2 0 0 0 0 0 0 3
K. cistoidea 1 0 1 0 1 1 0 0 0 2 0 1 0 0 0 0 0 0 0 0 0 0 0
K. cytisoides 0 3 0 0 1 2 0 0 0 0 0 1 1 0 0 1 0 0 0 0 0 0 2
K. erecta 1 1 0 0 1 0 0 2 0 1 0 1 2 0 2 3 0/1 0 0 0 1 1 2
K. grandiflora 0 0 1 1 1 0 1 1 0 2 0 1 2 0 2 2 0 0 0 1 0 0 2
K. grayi 1 1 0 0 0 2 0 0 0 0 0 1 1 0 0 1 0 0 0 0 0 0 1
K. ixine 0 0 1 1 1 0 1 1 0 2 0 1 2 0 1 3 1 1 0 0 0 0 2/3
K. lanceolata 1 1 1 0 1 1 0 2 0 1 0 0 2 0 2 2 0 0 1 0 0 0 2
K. lappacea 1 0 1 0 1 1 1 0 1 0/2 1 1 0 0 0 0 0 0 0 0 0 0 0
K. pauciflora 1 1 1 0 1 0 0 2 0 1 0 0 2 0 2 3 0 2 0 0 0 0 2
K. paucifolia 1 0 1 0 1 2 0 0 0 0 0 1 1 0 0 1 0 0 0 0 0 0 2
K. ramosissima 1 2 0 0 1 0 0 2 0 1 0 1 2 0 2 4 1 0 0 0 1 1 2
K. revoluta 1 1 1 0 1 0 1 2 0 1 0 1 2 0 1 3 0 2 0 0 0 0 2
K. secundiflora 1 1 1 0 1 0 0 2 0 1 0 0 2 0 2 3 0 0 0 0 0 0 2
K. sonorae 1 1 1 0 0 2 0 0 0 0 0 1 1 0 0 1 0 1 0 0 0 0 2
K. spartioides 0 1 1 1 1 0 1 1 0 2 0 1 2 0 0 2 0 0 0 0 0 0 2
K. tomentosa 0 0 1 1 1 0 1 1 0 2 0 1 2 0 1 3 1 0 0 0 0 0 3
analyzed using the Branch and Bound option of PAUP*
4.0d64.
Morphological characters were optimized on the rooted tree derived from the molecular analysis using MacClade (Maddison and
Maddison 2000) under both DELTRAN and ACCTRAN optimizations.
Biogeographical Methods. Each species was scored for presence or absence in North America and in South America and the resultant
matrix used in a DIVA analysis (Ronquist 1996) to ascertain the possibility of vicariance or dispersal and, if the latter, the minimum
number of events.

RESULTS
The ancestral ITS plus 5.8S sequences were 822 bp long when aligned to the outgroups. The shortest sequence
(without gaps) was Guaiacum (706 bp) and the longest Tribulus (754 bp). The longest Krameria sequences were
from K. revoluta and K. grandiflora (746 bp). Overall, 29.2 percent of the sites (240 of 822) were phylogenetically
2004] SIMPSON ET AL.: PHYLOGENY OF KRAMERIACEAE 103

informative. The overall G 1 C content ranged from 49.3–65.6% with a mean of 54.3%. Within Krameria it ranged
from 49.3–55.2 % with an average of 53.1%. The g 1 value for 100,000 trees of the molecular data with ancestral
sequences was 20.970259 well below the value of 20.15 needed for rejection of random data. Within Krameria
alone, 606 of the 766 characters were constant, 73 were parsimony uninformative, and 143 were parsimony
informative.
Phylogenetic Analysis of ITS Sequences.PARSIMONY ANALYSIS. The parsimony analysis of the combined ITS 1
and ITS 2 plus the 5.8S region yielded two trees of 626 steps (CI50.819, RC50.692, RI50.844, HI50.181). The two
trees differed only in the placement of K. pauciflora and K. revoluta. In one tree the two are sister taxa and in the
other they are in a polytomy with a clade consisting of K. erecta and K. lanceolata (Fig. 2). Both of these trees
consisted of two major clades (1 and 2) each containing two subclades (Figs. 2, 3), labeled for ease of discussion
(Fig. 3) NA-1 (North
America-1), SA-1 (South America-1), NA-2 (North America-2), and SA-2 (South America-2) . Bootstrap values for all
clades were very high (Fig. 2), with the lowest values associated with a clade of Brazilian species (SA-2).
MAXIMUM LIKELIHOOD ANALYSIS. Using the TrNef 1 G model, maximum likelihood analysis yielded a tree identical in
topology (for Krameria) to the parsimony tree shown in Figure 2. The maximum likelihood tree differed from the
parsimony tree in the relationships of the outgroups to one another: Tribulus and Guaiacum were sister to one another
rather than Tribulus and Kallstroemia.
BAYESIAN ANALYSIS. The Bayesian analysis using MrBayes and the GTR1G model again yielded a majority rule
consensus tree with a topology identical to that of maximum likelihood and to one of the parsimony trees in terms of the
relationships of Krameria species to one another. The relationship of the outgroups to one another was the same as in
the maximum likelihood analysis. Posterior probability scores are indicated in parentheses in Fig. 2.
Phylogenetic Analysis of Morphological Data. Eighteen equally parsimonious trees were obtained using an equally
weighted parsimony analysis for the morphological character data set with all species included (tree length 553,
CI50.698, RC50.564, RI50.807, HI50.302). Figure 4 shows one of these trees with branch lengths and bootstrap values
over 50% in parentheses for clades recovered by the bootstrap analysis. All trees showed general relationships
compatible with the parsimony, maximum likelihood, and Bayesian trees derived from molecular data. The morpho-
104 SYSTEMATIC BOTANY [Volume 29

FIG. 2. One of two most parsimonious trees of Krameria species produced from the combined ITS 1 5.8S, and ITS 2 data. The same topology
for Krameria was produced by maximum likelihood under models of GTR1G or TrNef1 G or Bayesian analysis. The maximum likelihood trees
differed from the parsimony trees only in the relationships of the outgroups to one another (Tribulus and Guaiacum were sister to one another
rather than Tribulus and Kallstroemia). The numbers above the branches are the character changes along that branch found in the parsimony
search. The numbers below the branches are the bootstrap value followed by the posterior probabilities (in parentheses) from the Bayesian
analysis. The small insert shows relative branch lengths. Vertical bars on the extreme right indicate the clades discussed in the text.
FIG. 3. Geographical distributions of the four subclades within Krameria (Fig. 2).
FIG. 4. One of the 18 most parsimonious trees using mor-

logical analysis strongly supported a sister relationship phological data from all 18 Krameria species. Numbers indicate
between Krameria erecta and K. ramosissima and branch lengths and numbers in parenthesis are bootstrap values for
clades that appeared in the bootstrap analysis. Dotted lines are
between K. tomentosa and K. ixine. Because these two branches that collapse in the strict consensus tree.
sister relationships were found in the morphological
analysis but not in the molecular analysis, each pair was constrained to a sister relationship in a SOWH test (see below).
In addition, the NA-1 clade formed a polytomy in the strict consensus tree whereas it was resolved in the molecular
trees. The branches on which there were the largest numbers of character state changes correspond to the branches
separating the four major subclades (NA-1, SA-1, NA-2, SA-2) shown in Figs. 2, 3.
Phylogenetic Analysis of Combined Data. Six trees were produced when the molecular and morphological data were
combined. The consensus of these trees (not shown) was congruent with the trees produced by the parsimony, maximum
likelihood, Bayesian, and morphological analyses except that the Brazilian species (clade SA-2, Fig. 2) formed a
polytomy.
Character State Distributions. Optimization of the morphological data on the parsimony tree generated by the
molecular sequence data showed that numerous characters provided good synapomorphic states for the major clades
(Fig. 5), but that some used in Simpson’s 1989 monograph to group one set of North American species are
plesiomorphic in the genus.
Constraint Trees. For each permutation of the SOWH test (presence/absence of gaps) we were able to reject the
monophyly of the North American species and monophyly of the South American
species
(p, 0.
00 1)
.

Likewise we could reject (p,0.05 level) the sister relationships

of Krameria erecta and K. ramosissima, which seemed likely
on the basis of morphological similarities (see below). We
were not able to reject the hypothesis that the North American subclade 2 (NA2) or the South American subclade 2 (SA-
2004] SIMPSON ET AL.: PHYLOGENY OF KRAMERIACEAE 105

2) could be basal in the genus. Likewise, we could not reject a sister relationship between K. ixine and K. tomentosa.
The inclusion or omission of gaps did not affect the results of the constraint analyses (Table 5).
Biogeographical Analysis. The DIVA analysis indicated that there were two dispersals in the history of the genus.

DISCUSSION
Rooting of trees has consistently been a problem with Krameria because of the difficulty in ascertaining a sister taxon
or taxa for the genus. In 1989, Simpson suggested that the Polygalaceae was the closest relative of the Krameriaceae
based on the serological work of Busse-Jung (1979) that had shown cross reactivity between Krameria species and
species of the Polygalaceae. Chase et al. (1993), using rbcL sequences, sug-
106 SYSTEMATIC BOTANY [Volume 29

FIG. 5. Synapomorphic and homoplastic characters mapped onto the tree recovered from the molecular analysis. Morphological character
states are indicated by open boxes with the numbers corresponding to those in Table 3. Characters with ** are homoplastic.
TABLE 5. Results of constraint analyses. Each constraint was done under the TrNef1G model (labeled here simply as TrNef with and
without gaps included (indicated as n.g. 5 no gaps, or g. 5 with gaps). The decision to reject is at the 0.05 level or lower. For constraint
analyses with multiple MP trees, only those that were fully resolved were tested.
Constraint TL # trees Test tree TLD p(TrNefn.g.) p(TrNefg.) Conclusion
2004] SIMPSON ET AL.: PHYLOGENY OF KRAMERIACEAE 107

NA monophyletic 649 4 2, 4 23 p , 0.001 p , p , 0.001 p , reject reject

SA Monophyletic 650 2 2 24 0.001 p 5 0.001 p 5 reject
NA-1 basal 633 2 2 7 0.002 p 5 0.001 p 5 can not reject
NA-2 basal 636 2 2 10 0.231 p 5 0.146 p 5 reject
SA-1 basal SA-2 633 2 2 7 0.033 p 5 0.028 p 5 can not reject can
basal 636 2 2 10 not reject
0.263 p 5 0.157 p 5
tomentosa1ixine 628 2 2 2 reject
0.153 p , 0.120 p ,
erecta1ramosissim 637 1 1 11
0.001 0.001
a
gested a placement near the Zygophyllaceae sensu stricto. However, according to these authors and Chase (pers.
comm.), this relationship is not well supported. More recent work (Savolainen et al. 2000; Sheahan and Chase 1996)
has confirmed this relationship and the three-gene tree of Soltis et al. (2000) had strong support (100% jackknife)
for this sister relationship. However, the two families differ dramatically in morphological characters. In contrast to
the problems encountered using rbcL sequences (Sheahan and Chase 1996), we were able to use the ITS sequences
we obtained from Guaiacum, Tribulus, and Kallstroemia with those of Krameria to produce a strongly supported
phylogenetic hypothesis (Fig. 2).
Species Relationship and Character Evolution. Our data strongly support an hypothesis of two major Krameria
clades (Fig. 2), each containing a subclade of North and South American species (i.e., NA-1, SA-1, NA-2, and SA-2
in Fig. 2). This major pattern is uncovered using parsimony, maximum likelihood, and Bayesian analyses of the
molecular data, and it is consistent with the pattern of species relationships found by analyzing the morphological
data using parsimony (Fig. 4). However, the morphological analysis (Fig. 4) had two strongly supported species pair
relationships not recovered in the molecular analysis. These were between K. erecta and K. ramosissima and
between K. ixine and K. tomentosa. Krameria erecta occurs from southern California to west Texas and K.
ramosissima occurs along the south Texas border. These two share characters of heart-shaped fruit (char. 21–1),
gland morphology, and fusion of non-glandular-petals. Despite these morphological similarities, we were able to
reject that they were sister species. Thus the most striking similarity between the two, heart-shaped fruits, must be
the result of convergence. Krameria ixine occurs from southern Mexico sporadically across Central America and the
West Indies to Venezuela and K. tomentosa from Guyana across Brazil in dry areas. These species do not share any
unique synapomorphies, but for the morphological character states scored, they were identical. We were unable to
reject a sister relationship for these two species so their shared characters could reflect common ancestry or
retention in K. ixine of plesiomorphic character states.
We use the tree in Fig. 5 to discuss the distribution of morphological characters in the genus. The major division
within the Krameriaceae leads to two clades. Clade one (SA-1 1 NA-1) consists of species with rugose dorsal surfaces of
the glandular petals [char. 8– 0], and fruits that have thin purple or black spines [char. 15–0, Fig. 1B]. The second major
clade in the genus, SA-2 1 NA-2, includes species that share characters of connivent sepals [char. 6–0] and broad pollen
apertures [char. 13–2].
Similarly, each of the subclades is well supported by morphological characters. The species in NA-1 have
synapomorphies of reflexed sepals [char. 6–2], free, strap-shaped petaloid petals [char. 10–0], narrow pollen pores [char.
13–1], and fruit spines with barbs in one whorl at the tip [char. 16–1]. The species in SA-1 share sepals borne
perpendicular to the pedicel [char. 6–1], tricolporate pollen [char. 13–0], fruit spines with two terminal whorls of
retrorse hooks [char. 16–0], and sericeous trichomes on the young leaves [char. 23–0].
The morphological synapomorphies that support clade NA-2 are secretory areas of the glandular petals that are
restricted to the distal portion of the gland [char. 8–2, Fig. 1C] and petaloid petals that are basally fused and have
cordate blades [char. 10–1]. The species of SA-2 share only one unique synapomorphy, glandular petals with a striate
dorsal surface [char. 8–1]. All of the species of this clade also have free spathulate petaloid petals [char. 10–2] but this
feature occurs in K. cistoidea (NA-1) as well.
An examination of other morphological characters shown plotted in Fig. 4 indicates that petiolate leaves [char. 1–0],
short peduncles [char. 4–1], reduction of sepal number from five to four [char. 7–1], free spathulate petaloid petals [char.
10–2], thin red spines on the fruit [char. 15–1], glabrous spine bases [char. 17–1], and heart-shaped fruits [char. 21–1]
have all arisen more than once in the genus. The unusual character of yellow sepals with dusky rose overtones [char. 18–
2] constitutes a possible synapomorphy for the sister taxa (in one of the parsimony trees) K. pauciflora and K. revoluta
but a pattern of floral age-related color change from pink to white found only in K. sonorae and K. ixine is clearly
homoplastic [char. 18–1].
Biogeography. Using the phylogenetic reconstruction shown in Fig. 2, one major clade consists of the NA-1 and SA-1
subclades. The two species of the SA1 subclade, K. cistoidea and K. lappacea occur on the coast and in the Andes of
108 SYSTEMATIC BOTANY [Volume 29

western South America. The species of the NA-1 subclade (K. cytisoides, K. grayi, K. paucifolia, and K. sonorae) occur
in North America: three in the southwestern United States and western Mexico (primarily Baja California and Sonora,
Mexico) and the fourth (K. cytisoides) disjunct in the Sierra Madre Oriental (Fig. 3).
The second major clade in the genus also has both a North and a South American subclade. The South American
subclade of this group (SA-2, represented by K. argentea, K. grandiflora, K. ixine, K. spartioides, and K. tomentosa)
contains six species (K. bahiana, not included in the molecular study, is a member of this subclade) that are found
primarily in eastern and east-central Brazil, although one species (K. ixine) has a distribution from Sinaloa, Mexico
across northern Venezuela and into the West Indies. The North American subclade (NA-2) has six North American
members (K. erecta, K. lanceolata, K. pauciflora, K. ramosissima, K. revoluta, and K. secundiflora) that are distributed
across the southern United States and adjacent Mexico as far south as Costa Rica (Fig. 3).
Our SOWH tests allowed us to reject a clade of all of the North American (NA-1 1NA-2) or all of the South American
(SA-11SA-2) species and the possibility that NA-1 is sister to the rest of Krameria. We could not reject the possibility
that either the SA-2 or NA-2 subclade could be basal in the genus (Table 5).
Assuming that the tree shown in Fig. 2 is correct, the presence of a North and a South American subclade in each of
the major clades requires that the initial split in the genus led to the ancestors of each of the two major clades. Each of
these ancestral lineages subsequently gave rise to radiations in both North and South America. Under a vicariance
scenario, the ancestral Krameria would have been present on both North and South America prior to cladogenesis and
the initial split would have been ‘‘longitudinal’’ leading to two species, each of which retained a distribution on both
continents. One or two secondary ‘‘latitudinal’’ vicariant events between North and South American would have
followed leading to the ancestors of the four subclades that were recovered from the data. While such a process is
logically consistent with a vicariant model of speciation, it is unlikely given the distribution of most Krameria species in
deserts or seasonally dry habitats that are not, and have never been, continuous between the continents (Simpson and
Neff 1985). The SA-2 clade does show a patchy distribution pattern with K. ixine occurring in arid pockets across
northern Venezuela and into the West Indies and Central America. However, this species is not sister to the rest of the
NA-21SA-2 clade and probably dispersed into North America in comparatively recent times and so its distribution
probably does not reflect an ancestral distribution of the genus across the tropics.
Looking at the average genetic distance among members within each of the four subclades based on pairwise
sequence comparisons, we find 0.014 for SA1, 0.078 for NA-1, 0.013 for SA-2, and 0.016 for NA-2. These figures
imply a recent divergence of Krameria cistoidea and K. lappacea, consistent with their very similar morphologies
and also between several members of the SA-2 group. The strikingly higher distance among the members of the
NA-1 subgroup is due to the large sequence difference between K. cytisoides and the other members of the NA-1
clade. Krameria cytisoides is the most distinctive species in the genus and was considered the primitive ‘‘link’’ with
the Leguminosae by Taubert (1892). It has trifoliolate leaves in contrast to the simple leaves of the other species of
the genus. In addition it is a large shrub, almost a small tree, whereas the other species are small shrubs or
herbaceous perennials. Finally, it occurs in the semi-arid oak woodlands of the Sierra Madre Oriental of Mexico
rather than in deserts or grasslands. Nevertheless, our analysis indicates that it is not basal in the genus but rather
sister to the other species in the NA-1 clade.
An examination of the branch lengths (Fig. 2) of clade 1 and clade 2 suggests that their sister association could be
the result of long branch attraction (note 70 steps to clade 1 and 98 to clade 2 in Fig. 2). The best way to break up
long-branch attraction is by adding taxa (Felsenstein 1978). However, we have included all of the taxa of
Krameriaceae except Krameria bahiana and morphology leads us to believe that this taxon will appear close to K.
grandiflora in the SA-2 clade and should have little effect on the topology of the tree. The fact that the maximum
likelihood and the Bayesian analyses both generated these same two major clades suggests that the topology
recovered is the true topology since likelihood-based methods are generally less sensitive to long branch attraction
than parsimony.
Our analysis thus does not permit us to say with absolute certainty whether the ancestor of the genus gave rise to
the ancestors of both clade 1 and clade 2 (although all our analyses would suggest this) or whether the genus arose
in one continent and then gave rise to the ancestor of a subclade on another continent plus the remaining two
subclades. The DIVA analysis suggested two dispersal events. It is thus obvious that there were two exchanges
between North and South America but it remains uncertain as to the directions and timings of these exchanges.
ACKNOWLEDGMENTS. We are grateful for the help that Jack Neff provided in collecting material in the field, taking pictures, and reviewing
a draft of the paper. Todd Barkman, Robert K. Jansen, and Jack Neff kindly read the manuscript and made helpful suggestions. We greatly
appreciate the work of M. Grenadier in producing the illustrations. Javier Francisco-Ortega provided guidance and help during the initial
phases of this work. Derrick Zwickl kindly provided us with the programs MissingMasker and LogReader that were used to retrieve tree
2004] SIMPSON ET AL.: PHYLOGENY OF KRAMERIACEAE 109

scores in the SOWH analyses. The manuscript was improved by helpful comments from Paul Manos, Mark Simmons, and an anonymous
reviewer.