See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/262005956

matK DNA Sequences and Phylogenetic

Reconstruction in Saxifragaceae s. str.

Article in Systematic Botany · January 1994

DOI: 10.2307/2419718

CITATIONS READS

469 171

2 authors:

Leigh Johnson Douglas E Soltis

Brigham Young University - Provo Main Campus University of Florida
55 PUBLICATIONS 2,130 CITATIONS 871 PUBLICATIONS 49,383 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Navarretia Systematics View project

Teaching Plant Systematics View project

All content following this page was uploaded by Douglas E Soltis on 29 August 2016.

The user has requested enhancement of the downloaded file.

matK DNA Sequences and Phylogenetic Reconstruction in Saxifragaceae s. str.
Author(s): Leigh A. Johnson and Douglas E. Soltis
Source: Systematic Botany, Vol. 19, No. 1 (Jan. - Mar., 1994), pp. 143-156
Published by: American Society of Plant Taxonomists
Stable URL: http://www.jstor.org/stable/2419718
Accessed: 29-08-2016 17:47 UTC

JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted
digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about
JSTOR, please contact support@jstor.org.

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at
http://about.jstor.org/terms

American Society of Plant Taxonomists is collaborating with JSTOR to digitize, preserve and extend
access to Systematic Botany

This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
All use subject to http://about.jstor.org/terms
Systematic Botany (1994), 19(1): pp. 143-156
(? Copyright 1994 by the American Society of Plant Taxonomists

matK DNA Sequences and Phylogenetic Reconstruction in

Saxifragaceae s. str.

LEIGH A. JOHNSON and DOUGLAS E. SOLTIS

Department of Botany, Washington State University, Pullman, Washington 99164-4238

ABSTRACT. Comparative DNA sequencing of matK, a maturase coding gene located within the
intron of the chloroplast gene trnK, was evaluated for phylogenetic utility using genera of Saxi-
fragaceae s. str. The entire matK gene was sequenced for two members of the family, Sullivantia
sullivantii and Saxifraga integrifolia. Comparison of base substitution rates between these two species
indicated that matK evolves approximately three-fold faster than rbcL. Comparative sequencing of
754 base pairs within matK was subsequently conducted using 25 genera in Saxifragaceae s. str. and
two outgroup taxa. Summed over the 31 taxa sequenced for this 754 base pair region, 40% of the
base positions were variable and 15.6% were potentially informative. Five insertion/deletion events
of three or six base pairs were also detected. Skewness and randomization tests both suggest that
significant non-random structure is present in the matK data set. Parsimony analyses provided 72
most parsimonious trees of 223 steps (excluding autapomorphies) with a consistency index of 0.565.
Several well-supported groups of genera are highly concordant with relationships suggested by
two other chloroplast DNA data sets: chloroplast DNA restriction sites and rbcL sequences.

Because DNA sequencing is relatively fast,
convenient, and offers a large data set of dis-
crete characters, comparative DNA sequencing
has become a widespread tool for phylogenetic
reconstruction. In particular, the chloroplast
gene rbcL has been widely used for inferring
phylogeny at higher taxonomic levels (e.g.,
Chase et al. 1993; Doebley et al. 1990; Donoghue
et al. 1992; Giannasi et al. 1992; Kim et al. 1992;
Olmstead et al. 1992; Qiu et al. 1993; Soltis et
al. 1990). This single copy gene is approximately
1431 base pairs (bp) in length, is free from length
mutations except at the far 3' end, and has a
fairly conservative rate of evolution imposed
by the function of ribulose 1,5 bisphosphate
carboxylase/oxygenase (RUBISCO), the large
subunit of which it encodes. With over 1000
rbcL sequences accumulated to date from di-
verse taxa, it is apparent, however, that the abil-
ity of rbcL to resolve phylogenetic relationships
below the family level is often poor (but see
Conti et al. 1993; Gadek and Quinn 1993; Kron
and Chase 1993; Price and Palmer 1993; Soltis
et al. 1993). Interest thus exists in finding other
useful DNA regions that evolve faster than does
rbcL to facilitate lower-level phylogenetic re-
construction.
One region of the chloroplast genome that
evolves faster than rbcL and appears to have
phylogenetic potential is matK (previously
ORFK; Neuhaus and Link 1987; Wolfe et al.
1992), a maturase encoding gene located in the
intron of the transfer RNA gene for lysine (trnK).
The evolutionary constraints imposed by the
function of the maturase are apparently less
stringent than those on rbcL: Neuhaus and Link
(1987) report a 66% amino acid similarity be-
tween the peptides encoded by matK of Nico-
tiana (Solanaceae) and Sinapis (Brassicaceae),
whereas the large subunits of RUBISCO in these
same two taxa exhibit 93% amino acid similarity.
Comparison of matK DNA sequences for Nico-
tiana and Sinapis (Neuhaus and Link 1987) also
reveals only a few small length mutations, none
of which cause any major problems in align-
ment of the two sequences. The entire trnK in-
tron is approximately 2600 bp in length, of
which approximately 1500 bp constitute matK.
Thus, the length of matK is comparable to that
of rbcL. These factors, coupled with preliminary
data for the Polemoniaceae (Steele 1991), sug-
gest that comparative sequencing of matK may
be appropriate for reconstructing infrafamilial
phylogenies.
To investigate the potential of matK DNA
sequences for retrieving phylogeny in angio-
sperms, we focused on generic relationships in
Saxifragaceae sensu stricto. Phylogenetic trees
from chloroplast DNA (cpDNA) restriction site
data and rbcL sequences already exist for genera
of this family (Soltis et al. 1993). This allowed
us to compare directly the resolution and utility
of matK sequences with the results obtained via
these other two approaches. Herein we report
our successful use of the matK region for ge-
neric-level phylogenetic reconstruction in Sax-

143

This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
All use subject to http://about.jstor.org/terms
 144 SYSTEMATIC BOTANY [Volume 19

TABLE 1. Taxa included in the matK sequencing analyses. Voucher specimens are located at WS unless
otherwise indicated in parentheses following the collection data. * designates DNA's used previously for
rbcL sequencing (see Morgan and Soltis 1993; Soltis et al. 1993). t designates DNA's used previously for
cpDNA restriction site studies (see Soltis et al. 1991a, 1991b, 1993).

Species Collection data

Astilbe japonica x chinesensis original source unknown; cultivar, Johnson s.n.

Astilboides tabularis (Hemsl.) Engl. Univ. of Oslo Bot. Gard., Norway (O)*t
Bensoniella oregona (Abrams & Bacig.) Morton Oregon, Josephine Co., Lang, Soltis & Soltis s.n.t
Bergenia cordifolia (Haw.) A. Br. Komarov Bot. Inst., Leningrad, Russia (LE)*t
Bolandra oregana Wats. Oregon, Multnomah Co., Grable 11587*
Boykinia rotundifolia Parry California, Los Angeles Co., Gornall 0101 (UBC)*t
Chrysosplenium iowense Rydb. Canada, Prince Albert, Wendel s.n. (ISC)*
Darmera peltata (Torr.) Voss Oregon, Gilliam Co., Soltis & Soltis 2083*t
Elmera racemosa (Wats.) Rydb. Washington, Kittitas Co., Soltis & Soltis 2234t
Heuchera micrantha Dougl. California, Humboldt Co., Soltis & Soltis 1949*
Jepsonia parryi (Torr.) Small California, Riverside Co., Rieseberg 1110 (RSA)*t
Leptarrhena pyrolifolia (D. Don) R. Br. Washington, Okanogan Co., Soltis & Soltis 2237*
Lithophragma affine Gray California, Tehama Co., Pellmyr & Thompson s.n.
Mitella diversifolia Greene Oregon, Josephine Co., Soltis & Soltis 1910t
Mitella pentandra Hook. Washington, Chelan Co., Grable 11432
Mukdenia rosii (Oliver) Koidzumi Palmengarten Bot. Gard., Germany, Soltis s.n.*t
Peltoboykinia tellimoides (Maxim.) Hara Nikko Bot. Garden, Japan, Soltis s.n. *t
Ribes aureum Pursh Washington, Whitman Co., Soltis & Soltis 2220*t
Rodgersia pinnata Franch. Palmengarten Bot. Gard., Germany, Soltis s.n. *t
Saxifraga integrifolia Hook. Oregon, Gilliam Co., Soltis & Soltis 2253*
Saxifraga mertensiana Bong. Oregon, Multnomah Co., Grable 11586*t
Suksdorfia ranunculifolia (Hook.) Engl. Washington, Ferry Co., Soltis & Soltis 2308
Suksdorfia violacea A. Gray Washington, Ferry Co., Soltis & Soltis 2309
Sullivantia sullivantii T. & G. Ohio, Hocking Co., Quackenbush s.n.
Tanakaea radicans Franch. Univ. British Columbia Bot. Gard. (UBC)*t
Telesonix heucheriformis Ryd. Wyoming, Teton Co., Wolf 151 *t
Tellima grandiflora (Pursh) Dougl. Alaska, Prince of Wales Island, Soltis & Soltis 2113
[="southern" type]*t
Tellima grandiflora (Pursh) Dougl. British Columbia, Near Port Edward, Soltis & Soltis 2119
[="northern" type]t
Tetracarpaea tasmanica Hook. F. Australia, Jordan s.n. (19 June 1991) (HO)*
Tiarella trifoliata L. California, Mendocino Co., Ness 533
Tolmiea menziesii (Pursh) T. & G. Oregon, Linn Co., Soltis & Soltis 1903*

ifragaceae s. str. We also demonstrate the broad
applicability of the amplification and sequenc-
ing primers developed.
MATERIALS AND METHODS
Plant Samples. Twenty-nine species rep-
resenting virtually all genera of Saxifragaceae
s. str. were included in our analysis (Table 1);
Tetracarpaea and Ribes were chosen as outgroups
based on the results of a recent analysis of rbcL
sequences (Morgan and Soltis 1993) that re-
vealed a close relationship between these gen-
era and Saxifragaceae s. str. Methods of DNA
isolation followed those given in Morgan and
Soltis (1993). In most cases, the same DNA's
used in recent cpDNA restriction site and rbcL
sequencing analyses (Morgan and Soltis 1993;
Soltis et al. 1993) were also used in this inves-
tigation (Table 1).
Amplification and Sequencing Strate-
gies. To obtain sufficient quantities of matK
for sequencing, double-stranded DNA was am-
plified from total DNA via the Polymerase Chain
Reaction (PCR; see below) using Replitherm
DNA polymerase (Epicentre Technologies).
Single-stranded DNA was subsequently syn-
thesized using the double-stranded PCR prod-
uct as template. The single-stranded DNA was
then precipitated with 20% PEG/2.5 M NaCl,

This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
All use subject to http://about.jstor.org/terms
 1994] JOHNSON & SOLTIS: SAXIFRAGACEAE 145

rps16-447F trnK-253F mafK-934F maiK-1506R matK-1848R trnK-2R

trnK-3914F trnK-582F maaK-1470R maK-1412F matK-2200R pabA-R

FIG. 1. Relative position of the PCR amplification and sequencing primers used in this study. Arrows
indicate the direction of strand synthesis. Boxed areas represent coding regions.

washed with 70% and 95% ethanol, and then
resuspended in 1 x TE buffer. Direct sequencing
of these purified, single-stranded PCR products
was accomplished via the dideoxy method us-
ing Sequenase version 2.0 (US Biochemical
Corp.) and 35S dATP. Labeled fragments were
separated in 6% acrylamide gels with 1 x TBE
buffer at 70 watts (constant power).
DNA's of Sullivantia sullivantii and Saxifraga
mately 750 bp were chosen for comparative se-
integrifolia were chosen for initial study to de-
velop a protocol and series of sequencing prim-
ers for use with the remaining taxa. Our strategy
for this first objective involved sequencing the
entire trnK intron. We first amplified the trnK
intron and flanking regions via PCR (Fig. 1).
For the forward strand, primers trnK-3914F and
psbA-R were used for double-stranded ampli-
fication followed by trnK-3914F for single-
stranded amplification. For the reverse strand,
we used primers rpsl6-4547F and trnK-2R for
single-stranded amplification (Fig. 1). The for-
ward strand of DNA (generated with trnK-
3914F) was subsequently sequenced using trnK-
2R as a sequencing primer; for the reverse strand
(generated with trnK-2R), trnK-3914F served as
a sequencing primer. We were able to read be-
tween 300 to 400 bp with each of these two
sequencing primers. These sequences were
aligned with the sequence of tobacco (Sugita et
al. 1985), and new forward and reverse primers
designed in conserved areas near the 3' end of
the sequences. These new primers were then
used to sequence another 300 to 400 bases, with
new primers again designed near the 3' ends
of each of these segments. This stepwise process
was repeated several times until the entire trnK
intron, including matK, was sequenced. For the
remainder of our study, primers trnK-2R and
trnK-3914F were used for double-stranded PCR
amplification and subsequently used individ-
ually to produce single-stranded DNA. The lo-
cation and base composition of each of the prim-
ers used in this study are given in Table 2.
The trnK intron sequences of Saxifraga inte-
grifolia and Sullivantia sullivantii were easily
aligned by hand, and sequence variability was
calculated for each of the regions accessed with
the eight sequencing primers used (Fig. 1; 5'
trnK-3914F, trnK-253F, trnK-582F, matK-934F,
matK-1506R, matK-1848R, matK-2200R, trnK-2R
3'). From this general comparison, two contig-
uous regions comprising a total of approxi-
quencing across virtually all genera of
Saxifragaceae s. str. (Table 1). Relative to the
tobacco trnK map (Sugita et al. 1985), this region
extends from bp 1075 to 1822 and was se-
quenced for all taxa (Table 1) using primers
matK-1506R and matK-1848R, or matK-1470R and
matK-1412F. The latter two primers are located
in fairly conserved regions and were designed
because sequence divergence prevented the use
of matK-1506R or matK-1848R in five taxa in-
cluded in this study.
Data Analysis. With the exception of Saxif-
raga mertensiana, for which 52 bases at the 5' end
were not obtained, sequences varied in length
from 742 to 754 bp due to five separate inser-
tion/deletion events (indels) of three or six bp
occurring in eight taxa. Although all DNA se-
quences were easily aligned by hand, they were
translated into amino acid sequences by the
computer program UWGCG (Devereux et al.
1984) to aid in the positioning of indels and to
assist in the proper determination of the read-
ing frame. Because identical indels can occur
independently in different lineages (e.g., Go-
lenberg et al. 1993), we assessed the phyloge-
netic utility of the indels we encountered by
constructing two different data matrices. In both
matrices missing bases were scored as ambig-
uous ("?") to prevent overweighting. No fur-
ther adjustments were made to the first matrix
(i.e., the indels were ignored). In the second
matrix, taxa were scored for the presence or
absence of the five indels; thus, each indel was
treated conservatively as a single event regard-
less of whether it was three or six bp in length.

This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
All use subject to http://about.jstor.org/terms
 146 SYSTEMATIC BOTANY [Volume 19

TABLE 2. Location and base composition of amplification and sequencing primers used in this study. * We
modified Learn's original primer by removing three nucleotides (ATC) on the 5' end. t Primers designed by
Johnson and Soltis are named based on their approximate position on the trnK map for Sinapis (Neuhaus and
Link 1987). t This primer was synthesized with equal parts "T" or "C" at base position 12.

Primer 5' sequence 3' Designed by

rpsl6-4547F AGG TGC TCA ACC TAC AAG AAC C Jerry Learn
trnK-3914F TGG GTT GCT AAC TCA ATG G * Jerry Learn
trnK-253F TTG GGT CGA GTC AAT AAA T Johnson/Soltist
trnK-582F CTA ACC ATC TTG TTA TCC T Johnson/Soltis
matK-934F ATT TTG GTT ATG ACA ATA A Johnson/Soltis
matK-1412F ATA TAA TTC TTA TGT ATG TG Johnson/Soltis
matK-1470R AAG ATG TTG AT[T/C] GTA AAT GAt Johnson/Soltis
matK-1506R TTC CAT AGA AAT ATA TTC G Johnson/Soltis
matK-1848R TAT CGA ACT TCT TAA TAG C Johnson/Soltis
matK-2200R TCT GTA TAA CCT CCA CAA AG Johnson/Soltis
trnK-2R AAC TAG TCG GAT GGA GTA G Kelly Steele
psbA-R CGC GTC TCT CTA AAA TTG CAG TCA T Kelly Steele

Our data inatrix decreased from 754 to 302
DNA base positions when invariant characters
were-removed. There are 670 character states
distributed among these 302 characters, provid-
ing a minimum-length tree of 365 steps in the
absence of homoplasy. Removing strictly aut-
apomorphic base positions (using the "ignore
uninformative characters" command in PAUP
3.0s; Swofford 1991) reduces these values to 118
potentially informative characters (Appendix
1), 271 character states, and 156 steps in the
absence of homoplasy. However, 30 of these
character states are still autapomorphic, but are
not excluded by PAUP because they occur at
base positions that are otherwise potentially in-
formative. Removing these "hidden" autapo-
morphies provides a data set of 118 characters,
241 character states, and 126 steps in the absence
of homoplasy. This final data set was used to
estimate homoplasy levels because we desired
to compare the consistency index from our data
with that expected using the regression equa-
tion of Sanderson and Donoghue (1989).
Data analyses were performed with PAUP 3.Os
using a Macintosh IIsi computer and the data
set that excluded the binary scoring of indels
(unless otherwise noted).
To evaluate the non-random structure and
robustness of the matK data set, the skewness
test of Hillis (1991; see also Hillis and Huelsen-
beck 1992; Huelsenbeck 1991) and the random-
ization test of Archie (1989a) were performed
(but see also an evaluation of these tests by Kal-
lersjo et al. 1992). For the skewness analyses,
the RANDOM TREES feature of PAUP was used
to generate 10,000 random trees and to calculate
the g, skewness statistic based on the distri-
bution of the lengths of these trees. How and
where non-random structuring of variation is
distributed in the matK data set was determined
by repeating this analysis for all taxa as well as
for subsets of the data matrix derived by re-
moving various clades, once identified. Ob-
served skewness levels were tested for signifi-
cance using null models determined by Hillis
and Huelsenbeck (1992) for the appropriate
number of taxa and characters. For the random-
ization test, the MS-DOS program RANDOM-
IZE (Archie 1989a) was used to construct 50 ran-
dom data sets by permuting character states
within characters among taxa. This program
produces output formatted for direct use by
PAUP 2.4 on MS-DOS computers, so data sets
were reformatted by hand for use by PAUP 3.0s
on Macintosh computer systems. Most parsi-
monious tree lengths were estimated for each
data set by heuristic searches using MULPARS
and TBR branch swapping with CLOSEST and/
or 10 replications of RANDOM addition (time
constraints prevented more replications). The
degree to which the most parsimonious tree
length for the original matK data deviates from
the lengths of the random data sets indicates
the degree of deviation from randomness that
exists in the original data set (Archie 1989a;
Kallersj6 et al. 1992).
To estimate relationships and tree topology,
parsimony analyses were conducted using a

This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
All use subject to http://about.jstor.org/terms
1994] JOHNSON & SOLTIS:
heuristic search with MULPARS, TBR branch
swapping, and CLOSEST addition. To search for
multiple islands of trees (Maddison 1991), 100
replications of RANDOM addition were also
conducted, although no additional trees were
found. Strict and 50% majority rule consensus
trees (with other compatible groups) were con-
structed from all most parsimonious trees. To
estimate homoplasy in the most parsimonious
trees, the consistency index (CI; Kluge and Far-
ris 1969) was calculated by hand to prevent its
inflation by the inclusion of "hidden" autapo-
morphies as described above. The homoplasy
excess ratio maximum (HERM; Archie 1989b,
1990; equivalent to the retention index of Farris
1989a, 1989b), which estimates the homoplasy
excess ratio (HER; Archie 1989b, 1990), was also
calculated, as was HER directly, from parsi-
mony analyses of 50 random permutations of
the matK data set (Archie 1989a; see above).
Phylogenetic analysis using a transversion
(Tv) to transition (Ts) weighting of 1.3:1.0 (based
on the observed Tv: Ts substitution rates with-
in our data set) -gave results identical to the
unweighted analyses and will not be discussed
further.
To determine relative support for various
clades found in the parsimony analysis, both
bootstrap (Felsenstein 1985) and decay analyses
(Bremer 1988; Donoghue et al. 1992) were per-
formed. The bootstrap analysis consisted of 100
replications using CLOSEST addition with no
MAXTREE limit. Due to computer memory lim-
itations and the large number of trees just one
step longer than the most parsimonious tree,
decay analyses were performed using the fol-
lowing approach. The strict consensus of all most
parsimonious trees was used as a constraint tree
for heuristic searches that save only trees of one
additional step that failed to satisfy the con-
straint topology. A strict consensus tree was then
formed from all of the trees found during these
searches as well as the most parsimonious trees
found initially. This strict consensus tree was
then used as a new constraint for saving only
trees of two additional steps that failed to satisfy
the new constraint topology. This method was
repeated for the first three steps of decay, at
which time ingroup monophyly broke down.
Above three steps in our decay analysis, the
number of steps required to decay the few re-
maining branches was estimated by forming a
constraint tree with all branches collapsed other
This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
All use subject to http://about.jstor.org/terms
SAXIFRAGACEAE 147
than the one in question. At least 10 replications
of RANDOM addition were then used to find
trees that failed to satisfy this topology (further
details on this approach to the decay analysis
are available from the authors by request).
Phylogenetic hypotheses resulting from our
analysis of matK sequences were compared with
results from earlier analyses of cpDNA restric-
tion sites and rbcL sequences for Saxifragaceae
s. str. (Soltis et al. 1993). For the rbcL data set
to be more concordant with both cpDNA re-
striction sites and matK sequences, we also ob-
tained an rbcL sequence for Chrysosplenium io-
wense (GenBank accession number L19935). The
rbcL sequences of Soltis et al. (1993) were then
reanalyzed with the Chrysosplenium iowense se-
quence added.
RESULTS
The percentage of unambiguous base differ-
ences within the entire matK region for Sulli-
vantia sullivantii (GenBank accession number
L20130) and Saxifraga integrifolia (GenBank ac-
cession number L20131) is 8.1%, a value three
times greater than the 2.7% sequence diver-
gence for rbcL between these same two species.
Summed over the 31 taxa included in this study
for the 754-bp region of matK sequenced, 40%
of the base positions are variable, and 15.6% are
potentially informative.
Results of the skewness test suggest that con-
siderable non-random structuring of variation
is present and that it is evenly distributed
throughout the matK data set. The g, value for
the entire data set is -0.626, a value well be-
yond the P = 0.01 level of significance (Hillis
and Huelsenbeck 1992). Significant structure as
indicated by the g1 statistic at the P = 0.01 level
was also present in all subsets of taxa examined
until only Saxifraga integrifolia, Darmera, Rodgers-
ia, Bergenia, Mukdenia, and Astilboides remained.
The randomization test of Archie (1989a) also
indicates significant non-random structure in
the matK data set. The most parsimonious tree
lengths of 50 randomized data sets with all aut-
apomorphic character states removed ranged
from 398 to 417 steps, with a mean of 407.40
and standard error of 3.46. The most parsimo-
nious tree length obtrained with actual data (223
steps; see below) is clearly significantly re-
moved from the range and mean of random data
sets with the same character state distributions.
 148 SYSTEMATIC BOTANY [Volume 19

8 Sullivantia sullwantii
3 3 Suksdorfia ranunculyif.
82) 98% Boykinia rotundifol
9 98% 3 8 Bolandra oregana
100% (7) 44% L Suksdorfia violacea :
2 (7) 4 (0) Telesonix heucherif
42% 98% (4) 6 Jepsonia parryi
2 (1) 9 5 Tanakaea radicansl 1
12% 100% (8) Leptarrhena pyrolfi E H
(0) 110 Astilbe jap. X chin.2
2 41 Sfraga integnfolia t
5% 1 2 Darmera peltata
(1) 6%1 2 5 Astilboides tabularis
( 4) 3 (1) Rodgersia pinnata
31 40% (1) 1-1 1-8 Bergenia cordifolia f
( 3%1) ( Mukdenia rosii
4 6 (1) 5 Peltoboykinia tellim.
23% 65% (1) 538 Chrysosplenium iow.
(l) 5 Bensoniella oregona,
3 2 Tolmiea menziesii I
80% (2) 1 5 Lithophragma aff
13 2 Tellima gran. -south-
15 100% (10) 87% I Mitella diversifolia4 1 0
75% (1 Heuchera micrantha4l Q
(3) 4 Mitella pentandra I
9(94) cl2 flarella trifoliata |
Elmera racemosa I
2 Tellima gran. -north J
58 Saxifraga mertens.
36 Ribes aureum
69 Tetracarpaea tasman5

FIG. 2. One of 72 most parsimonious trees derived from matK sequence analysis for Saxifragaceae s. str.
This tree is identical to the 50% majority rule consensus tree (including compatible groups) constructed from
the 72 shortest trees. Numbers above each branch indicate the number of base substitutions. Below branches,
bootstrap support is indicated as percentages based on 100 bootstrap replications; the number of additional
steps required to collapse each branch (decay index) is indicated in parentheses. Well-supported groups of
genera are bracketed with solid lines, while the Heuchera and Tolmiea subgroups are bracketed with broken
lines. Indels are numbered 1 through 5 and their distributions are indicated by these numerals to the right
of the taxa in which they occur.

This suggests significant departure from ran-
dom structuring of the variation in the matK
data set.
Parsimony analyses (excluding all autapo-
morphic character states and indels) provided
72 most parsimonious trees of 223 steps, a value
that increases to 462 steps when autapomor-
phies are included (Fig. 2). The CI of these trees
(excluding autapomorphies) is 0.565, a higher
value than the 0.422 predicted for 31 taxa using
the regression equation of Sanderson and Don-
oghue (1989). Our calculated values for HERM
(=RI) and HER are 0.760 and 0.655, respectively,
indicating that the homoplasy in the matK data
set is approximately 34.5% of that possible in
random data possessing the same character state
distributions, but lacking structure (Archie
1989a).

This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
All use subject to http://about.jstor.org/terms
 1994] JOHNSON & SOLTIS:
Five indels of three or six bp distributed
among eight taxa were found in the 754-bp re-
gion sequenced (Appendix 1). Three of these
indels are autapomorphic, whereas two are po-
tentially phylogenetically informative. When
the latter two are appended as presence/ab-
sence characters to the data matrix, the number
of most parsimonious trees decreased by half to
36. This decrease can be explained by the sta-
bilization of a clade formed by Tellima grandi-
flora (south), Mitella diversifolia, and Heuchera mi-
crantha. These three species share one indel (but
no other synapomorphies) and thus are united
in only 36 of the 72 most parsimonious trees
when indels are not scored; in the other 36 most
parsimonious trees, Tellima grandiflora (south)
and Mitella diversifolia are sister species (sharing
two base substitutions) and part of a large po-
lytomy formed with the other five members of
the Heuchera subgroup included in our study
(Fig. 2). Thus, although identical indels may
occur independently in different lineages (Go-
lenberg et al. 1993), indels detected in matK in
this generic-level inquiry appear to have single
origins and may be useful as informative char-
acters in future analyses.
Support for various clades as determined
through the bootstrap and decay analyses is in-
dicated under each branch in Figure 2. In gen-
eral, branches with high bootstrap percentages
also required a greater number of additional
steps to collapse in the decay analysis than did
branches with lower bootstrap values.
DISCUSSION
Phylogenetic analyses of 754 bp of matK se-
quences representing 31 taxa provide a robust
generic-level phylogeny for Saxifragaceae s. str.
(Fig. 2). Genera of Saxifragaceae s. str. are well
separated from the two outgroup taxa by nine
base substitutions. Our phylogenetic analyses
also reveal the presence of several well-sup-
ported groups of genera: the Boykinia group
(Sullivantia, Suksdorfia, Boykinia, Bolandra, Tele-
sonix, and Jepsonia), the Heuchera group (Benson-
iella, Tolmiea, Lithophragma, Tellima, Mitella, Heu-
chera, Tiarella, and Elmera), TanakaeaILeptarrhena, erally poorly resolved. In all three trees (Figs.
and Peltoboykinia/Chrysosplenium. Less strongly
supported is the Darmera group of genera (Dar-
mera, Astilboides, Rodgersia, Bergenia, and Muk-
denia). Phylogenetic analyses of matK sequences
also suggest that Tanakaea/Leptarrhena are sister
This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
All use subject to http://about.jstor.org/terms
SAXIFRAGACEAE 149
taxa to the Boykinia group. Furthermore, our data
indicate that the two species of Saxifraga ana-
lyzed (S. mertensiana and S. integrifolia) are well
separated phylogenetically; they differ, in fact,
by 95 base substitutions-undoubtedly an un-
derestimate considering 50 bp were not ob-
tained for S. mertensiana. Although phyloge-
netic analyses of matK sequences reveal these
five well-supported groups of genera, relation-
ships among groups, as well as the placement
of some genera, such as Astilbe, are generally
only poorly resolved (Fig. 2).
Results from two other cpDNA data sets, rbcL
sequences and cpDNA restriction sites, were
compared to these results. It is significant that
all three cpDNA-based trees (Figs. 2-4) are
highly concordant in terms of the general pic-
ture of relationships they reveal within Saxi-
fragaceae s. str. For example, all three analyses
(Figs. 2-4) provide strong support for the mono-
phyly of Saxifragaceae s. str., as well as the pres-
ence of the Boykinia, Heuchera, and Leptarrhena /
Tanakaea groups. Furthermore, all three analy-
ses (Figs. 2-4) also reveal that Tanakaea ILeptar-
rhena are the sister to the Boykinia group and
suggest that the Saxifraga species analyzed do
not form a monophyletic group. However, only
the phylogenetic tree based on rbcL sequences
agrees with the matK tree in supporting a close
relationship between Chrysosplenium and Pelto-
boykinia. Conversely, only the cpDNA restric-
tion site and matK trees reveal a strongly sup-
ported Darmera group. The Darmera group was
not revealed by the phylogenetic analysis of
rbcL sequences (Fig. 3), a result that probably
reflects the lower limits of resolution of rbcL
sequence data, rather than a major discrepancy
among the three cpDNA-based phylogenies (see
Soltis et al. 1993). It is readily apparent, for ex-
ample, that very few base differences support
the monophyly of members of the Darmera
group in our matK analysis (Fig. 2).
Not only are the trees derived from matK and
rbcL sequences and cpDNA restriction sites
highly concordant in the generic groups they
reveal, but they are also similar in that rela-
tionships among these generic groups are gen-
2-4), very few mutations support any of the
basal phylogenetic branches. Thus, all three
cpDNA data sets support the findings of Savile
(1975), who, based on a detailed examination of
rust parasites (Puccinia) that use members of Sax-
 150 SYSTEMATIC BOTANY [Volume 19

3 2 Boykinia rot.
2 1 Sullvantia or. *
4 39% 3 53% Bolandra ore.

6 66% 4 ~~~Jepsonia
4Pksonix he] par.
46%1 3 = nakaa rad*
1 78% 2 Uptrrhena
1% I r 15 Bergenaa cor.-
2 14% Saxifraga mert.
1% Ps 7 t Saxifraga opp.
1 100% Saxifraga cem S
IX 3 2 Darmera peL -
3r Mukdenia ro.-<

2X 34 Peltoboykinia] "
3 r 3 Rodgersia pin. -
9b 7e 1 -L Astilboides tab.--

25 99% 7 >3>12% Elmera raCe. 112,Tolmiea mei

4 Tellima
100 Saxfraga integ.
56% 93% 3~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Heuchera mic]
gran a

99% ~~~~~~~~~~~Saxifraga punw.

21 ' _%Ttracaipmae
7 13 23 Myriophyilsn
46_E Penthorum
-~ Kalanchee

FIG. 3. One of six equally most parsimonious trees derived from rb

s. str. Numbers above each branch indicate the number of base substitu
below each branch represent the percentage of occurrence of each mo
bootstrap replicates. Well-supported groups of genera are bracketed, w
which do not form a monophyletic group in this analysis, are indi
Soltis et al. 1993, with Chrysosplenium iowense added).

ifragaceae s. str. as hosts, concluded that the
family radiated rapidly early in its evolutionary
history.
The matK and cpDNA restriction site phy-
logenies are also highly concordant in the re-
lationships they reveal within the well-sup-
ported groups of genera noted above. In contrast,
rbcL sequence data provide little resolution at
that level. Within the Heuchera group of genera,
both matK sequences and cpDNA restriction
sites suggest that presence of two well-sup-
ported lineages: 1) Bensoniella, Tolmiea, and
Lithophragma; and 2) Mitella, Elmera, Heuchera,
Tiarella, Tellima, and Conimitella (although the
latter genus was not included in the matK anal-
ysis; Figs. 2 and 4B). Bootstrap values are high
for each of these lineages in both matK and
restriction site trees.
The two data sets also agree in revealing that
northern and southern populations of the
monotypic Tellima have distinct chloroplast ge-
nomes. Earlier cpDNA restriction site studies
(Soltis et al. 1991a, 1991b) revealed that "north-
ern" populations of Tellima grandiflora have a
distinct chloroplast genome, whereas "south-
ern" populations have a chloroplast genome
identical to that of two species of Mitella (M.
diversifolia and M. trifida Grah.). Nuclear-encod-
ed allozyme data, however, do not distinguish
between "northern" and "southern" popula-
tions of Tellima grandiflora (I = 0.99; Rieseberg
and Soltis 1987), but show that Tellima is well
differentiated from Mitella trifida and M. diver-
sifolia. These data led Soltis et al. (1991b) to sug-
gest that southern populations of Tellima gran-
diflora had captured the chloroplast genome of
a species of Mitella (either M. diversifolia, M.
trifida, or their common ancestor) following an
ancient hybridization event. matK sequence data
corroborate this hypothesis: the matK sequence
of the "southern" population of Tellima gran-
diflora is identical to that of M. diversifolia, and

This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
All use subject to http://about.jstor.org/terms
 1994] JOHNSON & SOLTIS: SAXIFRAGACEAE 151

21 SuUivni oregana
-2 7 20 Suksdorfia ramncuLc
63% 1 8 Boykinia spp. (12)
32 97% 4 9 Bolandra spp. (3)
A 100% Sksdorfia violacea
136 7 | esna parryi
1 36% 93% 18 Telesonix heucher
11% S2 - 7 Saxifraga mertens.
116 , Tanakaea rad4cans_
3 100% 1 Leptarrhena pyrolifill H
24% 1 Heuchera amercana
, 2 MitcUa pentandra
137 89% BensonieUa oregona
100% ~~~~~~~~Mitella ovalis
3 3 Saxfraga punctata
2396 , 6 100% S Saxifraga arguta
3S 5100% 4 Saxifraga LyaUlii
100% 40 Saxifraga ferruginea
2 1 0 Darmera peltata
19% 3 8 Rodgersia spp. (4)
9 79% 4 -Astilboides tabularis '
9 99% 2 23 Bergenia cord&folia
70% 51% 1 9 Mukdenia rosii
43 27
27 Peltoboykinia
ChrysospleniumteUim.
anm
25 Astilbe spp. (2)
100% Outgroup

Conimit
1 ~ MiteUa stauropetala
100% 3 MiteUa diversifolia
B 2 Mitella trifida
2 94%~ = Tellima granx -south
1 9396 Heuchera spp. (25)
1lla1eUa cordifolia ,f
1 93% 4 laeUa trifoliata
Elmera racemosa
3 3 , 1 Mitella pentandra
9 96% L 3 Mitella caulesens
2 100% 9 6 TeUima grani -north
100% 9 6 Heuchera spp. (9)
1 4 Mitela spp. (4) -
1Q096 6 2 Bensoniella oregona
100% Litho~~~~~2~ (phriagma spp. - )~
Tolmiea menziesii (2)

FIG. 4. Phylogenetic trees derived from cpDNA restriction site analysis. Numbers above each branch
indicate the number of restriction site mutations, and numbers below each branch represent the percentage
occurrence of each monophyletic group in the results of 100 bootstrap replicates. Some branches in the
original analyses have been collapsed to emphasize relationships discussed herein; in these cases, the values
in parentheses to the right of the taxon names represent the number of species or populations sampled that
appear along the branch. A. One of 132 equally most parsimonious trees for Saxifragaceae s. str. (Soltis et
al. 1993). Well-supported groups are bracketed. B. Single most parsimonious tree for the Heuchera group
(Soltis et al. 1991a). Brackets indicate the Heuchera and Tolmiea subgroups.

This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
All use subject to http://about.jstor.org/terms
 152 SYSTEMATIC
the sequences of these two collections differ
from that of the "northern" population of T.
grandiflora by a shared deletion and four base
differences.
matK sequences and cpDNA restriction site
data also reveal similar relationships within the
Boykinia group of genera. Phylogenetic analyses
of both data sets strongly support a close rela-
tionship between Telesonix and Jepsonia. This is
a significant finding given that some workers
have considered Telesonix a part of Boykinia
(Gornall and Bohm 1985), a result clearly not
supported by these data sets. Furthermore, be-
cause of its very unusual and distinctive mor-
phology, the affinities of Jepsonia have long been
considered enigmatic (Ornduff 1969). matK se-
quences support (88% bootstrap) the placement
of Sullivantia as the sister to Boykinia, Bolandra,
and Suksdorfia. In contrast, cpDNA restriction
sites place Sullivantia in this position in only
50% of the bootstrap trees; in the other 50% of
the trees, Sullivantia is the sister to Jepsonia and
Telesonix. matK sequences and cpDNA restric-
tion site data both suggest that Suksdorfia is
polyphyletic. In both analyses, Suksdorfia ranun-
culifolia is the sister to Boykina, and S. violacea
is the sister to Bolandra.
Finally, analysis of matK sequences and
cpDNA restriction sites reveals similar patterns
of relationships within the Darmera group of
genera. In both analyses, Mukdenia and Bergenia
are closest allies and appear as the sister group
to a clade comprising Rodgersia, Darmera, and
Astilboides.
In summary, our comparison of phylogenetic
trees based on matK and rbcL sequences and
cpDNA restriction sites has revealed an ex-
tremely high degree of concordance among
these three cpDNA data sets. All three data sets
reveal the same major clades of genera, and matK
sequences and cpDNA restriction site data sup-
port essentially identical relationships within
these clades. This lower taxonomic level is,
however, below the limits of resolution of rbcL
sequence data. This comparison also indicates
the ability of matK sequences to reveal fine-
scale patterns of relationship, such as the pos-
sibility of chloroplast capture in some popula-
tions of Tellima grandiflora and the polyphyly of
some genera (e.g., Suksdorfia). Due to the ease
of comparative sequencing, the matK data set
was compiled in a fraction of the time necessary
to compile the cpDNA restriction site data, even
This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
All use subject to http://about.jstor.org/terms
BOTANY [Volume 19
considering the need to design and synthesize
appropriate sequencing primers for the matK
study. In fairness, however, many more popu-
lations were included in the cpDNA restriction
site analysis (Soltis et al. 1993) than in the matK
study. These comparisons, combined with the
strong and evenly distributed non-random
structure of variation and apparently low levels
of homoplasy, point to the potential utility of
matK sequence data for retrieving phylogeny
below the family level. Futhermore, the results
presented herein were based on only 754 bp of
matK; sequencing the entire reading frame (1500
bp) would undoubtedly provide a better reso-
lution of relationships. Ultimately, we will
combine our cpDNA data and also compare these
with nuclear ITS sequences currently being
generated to provide a robust phylogeny for
Saxifragaceae s. str.
We hope that the results presented for Saxi-
fragaceae s. str. will stimulate the use of matK
sequence data in other groups. To this end, we
have tried to assess the broader applicability of
the amplification and sequencing primers de-
signed for the matK region to families other
than the Saxifragaceae. Because the trnK coding
regions are highly conserved, we found the am-
plification primers trnK-3914F and trnK-2R (Fig.
1; Table 2) to have broad applicability. Among
dicots, we have amplified matK from Apiaceae,
Asteraceae, Brassicaceae, Cornaceae, Grossular-
iaceae, Lauraceae, Magnoliaceae, Polemo-
niaceae, Rosaceae, and Saxifragaceae. Our only
attempt to amplify matK from a monocot family
(Juncaceae) was also successful. Comparison of
the homology of these amplification primers
with the published trnK sequences of Pinus
(Lidholm and Gustafsson 1991) shows good ho-
mology for primer trnK-3914F; however, prim-
er trnK-2R may need slight modification to work
well in conifers. Although we have not widely
tested all of our sequencing primers, we do know
that our most conserved primers, matK-1412F
and matK-1470R, also work in the Apiaceae,
Brassicaceae, Cornaceae, and Polemoniaceae. We
are hopeful that as sequences accumulate for
more diverse taxa, conserved areas throughout
matK will be found that will enable the syn-
thesis of sequencing primers with broad appli-
cability.
ACKNOWLEDGMENTS. We wish to thank Jerry Learn
and Dennis Clark for information regarding the ini-
 1994] JOHNSON & SOLTIS: SAXIFRAGACEAE 153

tial amplification and sequencing primers we used; * 1989b. The retention index and homoplasy
James Archie and David Hillis for helpful informa- excess. Systematic Zoology 38: 406-407.
tion concerning their respective tests for non-random FELSENSTEIN, J. 1985. Confidence limits on phylog-
structure; and Pam Soltis, Brent Mishler, and an anon- enies: An approach using the bootstrap. Evolu-
ymous reviewer for helpful comments on the manu- tion 39: 783-791.
script. This research was supported by NSF Grant GADEK, P. and C. J. QUINN. 1993. An analysis of
9007614. relationships within the Cupressaceae sensu
stricto based on rbcL sequences. Annals of the
Missouri Botanical Garden 80: 581-586.
LITERATURE CITED GIANNASI, D. E., G. ZuRAwsKI, G. LEARN, and M. T.
CLEGG. 1992. Evolutionary relationships of the
ARCHIE, J. W. 1989a. A randomization test for phy-
Caryophyllidae based on comparative rbcL se-
logenetic information in systematic data. System-
quences. Systematic Botany 17: 1-15.
atic Zoology 38: 239-252.
GOLENBERG, E. M., M. T. CLEGG, M. L. DURBIN, J.
. 1989b. Homoplasy excess ratios: New in-
DOEBLEY, and D. P. MA. 1993. Evolution of a
dices for measuring levels of homoplasy in phy-
non-coding region of the chloroplast genome.
logenetic systematics and a critique of the con-
Molecular Phylogenetics and Evolution 2: 52-64.
sistency index. Systematic Zoology 38: 253-269.
GORNALL, R. J. and B. A. BOHM. 1985. A monograph
* 1990. Homoplasy excess statistics and reten-
of Boykinia, Peltoboykinia, Bolandra, and Suksdorfia
tion indices: A reply to Farris. Systematic Zool-
(Saxifragaceae). Botanical Journal of the Linnean
ogy 39: 169-174.
Society 90: 1-71.
BREMER, K. 1988. The limits of amino acid sequence
HILLIS, D. M. 1991. Discriminating between phy-
data in angiosperm phylogenetic reconstruction.
logenetic signal and random noise in DNA se-
Evolution 42: 795-803.
quences. Pp. 278-294 in Phylogenetic analysis of
CHASE, M. W., D. E. SOLTIS, R. G. OLMSTEAD, D. MOR-
DNA sequences, eds. M. M. Miyamoto and J. Cra-
GAN, D. H. LES, B. D. MISHLER, M. R. DUVALL, R.
craft. Oxford: Oxford Univ. Press.
A. PRICE, H. G. HILLS, Y.-L. QIu, K. A. KRON, J.
and J. P. HUELSENBECK. 1992. Signal, noise,
H. RETTIG, E. CONTI, J. D. PALMER, J. R. MANHART,
and reliability in molecular phylogenetic anal-
K. J. SYTSMA, H. J. MICHAELS, W. J. KRESS, K. G.
yses. Journal of Heredity 83: 189-195.
KAROL, W. D. CLARK, M. HEDRtN, B. S. GAUT, R.
HUELSENBECK, J. P. 1991. Tree-length distribution
K. JANSEN, K.-J. KIM, C. F. WIMPEE, J. F. SMITH, G.
skewness: An indicator of phylogenetic infor-
R. FURNIER, S. H. STRAUSS, Q.-Y. XIANG, G. M.
mation. Systematic Zoology 40: 257-270.
PLUNKETT, P. S. SOLTIS, S. M. SWENSEN, S. E. WIL-
KALLERsJo, M., J. S. FARRIS, A. G. KLUGE, and C. BULT.
LIAMS, P. A. GADEK, C. J. QUINN, L. E. EGUIARTE,
1992. Skewness and permutation. Cladistics 8:
E. GOLENBERG, G. H. LEARN, JR., S. W. GRAHAM,
275-287.
S. C. H. BARRETT, S. DAYANANDAN, and V. A. AL-
KIM, K.-J., R. K. JANSEN, R. S. WALLACE, H. J. MICHAELS,
BERT. 1993. Phylogenetics of seed plants: An
and J. D. PALMER. 1992. Phylogenetic implica-
analysis of nucleotide sequences from the plastid
tions of rbcL sequence variation in the Asteraceae.
gene rbcL. Annals of the Missouri Botanical Gar-
Annals of the Missouri Botanical Garden 79: 428-
den 80: 528-580.
445.
CONTI, E. A., A. FISCHBACH, and K. J. SYTSMA. 1993.
KLUGE, A. G. and J. S. FARRIS. 1969. Quantitative
Tribal relationships in Onagraceae: Implications
phyletics and, the evolution of anurans. System-
from rbcL sequence data. Annals of the Missouri
atic Zoology 18: 1-32.
Botanical Garden 80: 672-685.
KRON, K. A. and M. W. CHASE. 1993. Systematics of
DEVEREUX, J., P. HAEBERLI, and 0. SMITHIES. 1984. A
the Ericaceae, Empetraceae, Epacridaceae, and re-
comprehensive set of sequence analysis pro-
lated taxa based upon rbcL sequence data. Annals
grams for the VAX. Nucleic Acids Research 12:
of the Missouri Botanical Garden 80: 735-741.
387-395.
DOEBLEY, J., M. DURBIN, E. M. GOLENBERG, M. T. CLEGG, LIDHOLM, J. and P. GUSTAFSSON. 1991. A three-step
and D. P. MA. 1990. Evolutionary analysis of model for the rearrangement of the chloroplast

the large subunit of carboxylase (rbcL) nucleotide trnK-psbA region of the gymnosperm Pinus con-

sequence among grasses (Gramineae). Evolution torta. Nucleic Acids Research 19: 2881-2887.

44: 1097-1108. MADDISON, D. R. 1991. The discovery and impor-

DONOGHUE, M. J., R. G. OLMSTEAD, J. F. SMITH, and J.
D. PALMER. 1992. Phylogenetic relationships of
Dipsacales based on rbcL sequences. Annals of
the Missouri Botanical Garden 79: 333-345.
FARRIS, J. S. 1989a. The retention index and the re-
scaled consistency index. Cladistics 5: 417-419.
tance of multiple islands of most-parsimonious
trees. Systematic Zoology 40: 315-328.
MORGAN, D. R. and D. E. SOLTIS. 1993. Phylogenetic
relationships among members of Saxifragaceae
s.l. based on rbcL sequence data. Annals of the
Missouri Botanical Garden 80: 631-660.

This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
All use subject to http://about.jstor.org/terms
 154 SYSTEMATIC BOTANY
NEUHAUS, H. and G. LINK. 1987. The chloroplast
tRNALYs (UUU) gene from mustard. Current Ge-
netics 11: 251-257.
OLMSTEAD, R. G., H. J. MICHAELS, K. M. SCOTT, and J.
D. PALMER. 1992. Monophyly of the Asteridae
and identification of their major lineages inferred
from DNA sequences of rbcL. Annals of the Mis-
souri Botanical Garden 79: 333-345.
ORNDUFF, R. 1969. Ecology, morphology, and sys-
tematics of Jepsonia (Saxifragaceae). Brittonia 21:
286-298.
PRICE, R. A. and J. D. PALMER. 1993. Phylogenetic
relationships of the Geraniaceae and Geraniales
from rbcL sequence comparisons. Annals of the
Missouri Botanical Garden 80: 661-671.
Qiu, Y. L., M. W. CHASE, and C. R. PARKS. 1993. Mo-
lecular phylogenetics of the Magnoliidae: Cla-
distic analyses of nucleotide sequences of the
plastid gene rbcL. Annals of the Missouri Botan-
ical Garden 80: 587-606.
RIESEBERG, L. H. and D. E. SOLTIS. 1987. Allozyme
differentiation between Tolmiea menziesii and Tel-
lima grandiflora (Saxifragaceae). Systematic Botany
12: 154-161.
SANDERSON, M. J. and M. J. DONOGHUE. 1989. Pat-
terns of variation in levels of homoplasy. Evo-
lution 43: 1781-1795.
SAVILE, D. B. 0. 1975. Evolution and biogeography
of Saxifragaceae with guidance from their rust
parasites. Annals of the Missouri Botanical Gar-
den 62: 354-361.
SOLTIS, D. E., P. S. SOLTIS, M. T. CLEGG, and M. DURBIN.
1990. rbcL sequence divergence and phyloge-
netic relationships of Saxifragaceae sensu lato.
This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
All use subject to http://about.jstor.org/terms
[Volume 19
Proceedings of the National Academy of Sci-
ences, U.S.A. 87: 4640-4644.
1 , T. G. COLLIER, and M. L. EDGERTON.
1991a. Chloroplast DNA variation within and
among genera of the Heuchera group (Saxifra-
gaceae): Evidence for chloroplast transfer and
paraphyly. American Journal of Botany 78: 1091-
1112.
, M. S. MAYER, P. S. SOLTIS, and M. EDGERTON.
1991b. Chloroplast DNA variation in Tellima
grandiflora (Saxifragaceae). American Journal of
Botany 78: 1379-1390.
, D. R. MORGAN, A. GRABLE, P. S. SOLTIS, and
R. KUZOFF. 1993. Molecular systematics of Sax-
ifragaceae sensu stricto. American Journal of Bot-
any 80: 1056-1081.
STEELE, K. P. 1991. Phylogenetic analyses of angio-
sperms using DNA sequences of the chloroplast
gene ORFK. American Journal of Botany 78: 220
(Abstract).
SUGITA, M., K. SHINOZAKI, and M. SUGIURA. 1985. To-
bacco chloroplast tRNALys (UUU) gene contains
a 2.5-kilobase-pair intron: An open reading frame
and a conserved boundary sequence in the in-
tron. Proceedings of the National Academy of
Sciences, U.S.A. 82: 3557-3561.
SWOFFORD, D. L. 1991. PAUP: Phylogenetic analysis
using parsimony, version 3.0s. Champaign: Illinois
Natural History Survey.
WOLFE, K. H., C. W. MORDEN, and J. D. PALMER. 1992.
Function and evolution of a minimal plastid ge-
nome from a nonphotosynthetic parasitic plant.
Proceedings of the National Academy of Sci-
ences, U.S.A. 89: 10648-10652.
 1994] JOHNSON & SOLTIS: SAXIFRAGACEAE 155

LO \2' C. .*..-.E- EE- E-H H H. H H.

HVC.
C\ E-ECE.-E-E)EC
o E . . . .-.E)
. -E
U
C.) . . 2 H

c\Q a) * H .~ . .... ..
02 00 C.) H- HH.IHHH . . . . . .. . . .1 HHH H

.I.

02t-HOE . .H E.E
> CZ 2 (O O C.*
(A CZ

* C (~O) 02E- . . .. . . . . . H r

02 4)~ C. H.

0c) 1 02 LO to~~~C. .~

41 E- E-~~~1 1 1

a) c to ) E HH. .. .HHH .. HHHH.

CZ C

CO Z 2 o C.OH
H.c.t.

O - H a) . EHH . HH.HHHH-

o 0 ~ ~ ~ ~ ~

C-) ~ ~ C

This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
All use subject to http://about.jstor.org/terms
 156 SYSTEMATIC BOTANY [Volume 19

F t~)C0 C.) H * Cs * Cs Cs * - Cs Cs Cs * Cs O Cs * - - - - O * H O O . . . -
F- Z- HI CC) CC-
(C LO C.* - - - HH

(00) C. ) . .
@ O O CE z z v Co Co * - - - - - ~~~~~~~~~~~~~-9- * -9 - 54 * - - -
(O C), C. .E. H..

0
(0CO
(0 0 V ...-0) , .
CO .......
.....-C) * -1 < E-
C).<
E- El -E-l
* t< .. < <

0) C.) . ... H . H H -
(D (C LO U . ... . .. . . . .C.
()0(011C
. . .. . HEH
E-' - E............ . .................. . .... . .s
(0 00 .C. . EH H -
(0~- H COV.)E ?Ez C.)c

(D? --I LO V *4 : - - -ce - e- -e - - - -4 * e - -e - -E z E - - - - - -: E -

n (O V *E - - E - - - - - - E . C . . . . ): - - H - E- C. - . C ..C'
-1 C. E-' .).HH .. . . . ... - - - -
(C
L O0 a)
LO0)E-
-e C-
-C C-
t *C-)
: -:U C-)
-4 C.) -C
* -C C) --:
-Ct-:C) C)-:C)
--: C)
--: C)--:
--: ... -4
U
L( 0(0 H- C.) C.)C.C.) C.) C.) C.) C.) C.) C.)C ) C) C) *C.) C.)C.)C) C-.)C-) C-) C-)C.)

o 0' - U : : LO(0 H.C)EC- C,: .

(D . E . E- HH

LO n U

>< LO t 0 H . .
LO 00) (- 0 .) . . .HH 4 . . . . . . .
Lo C) C.) . . . . H . . . . . . H. . . . . .

LO
LX ()(C)t
L10 H- U U
OE- - -l C.)
- C.)
- - C.C)C)
E, : : :C)C)U
---*--- 5C. C)

LO L --- -C.) H --: - H . H . E -

1- L O U0 E *- E -- E -E-- E- - E- E-- -V
x ZLO
c0 ? CO
\ . . .E-. Z . . . . . . H *

? ) C C . E. * C -
tV CLO H . . E * - C E E - - - ECC. * C . -)C.)C-
LO CQ C.)

w LO S?)0 C .)-H)H
04 O0) HC.E

~V \00 ) H- C.) (.)(.) C.C))C.C)) .

t CO n 0 H - E- -C - - - -C - - - - - - - -C C -C -. - . - . - . - . - - .

v-00 0CE-'
--- U? * - - --- --
- - -
- - -
- - --- -E
- - -2
- -:E,
- :5
v t nE- - - E5*- E- . . .5

V (1 01) VE -- - : E: * - - ... . . . . . . . . .
v LO C, V- *41 - < 1*- - - - - z *':' ? '
L O E- . . . . . . . .......... C CD

C,, 00 ) 00 1 'S
n 01CO
01 E. -U
25 *Ez
- ~ - ~*- *.
-
- ................
- - E - - -

n LO0 E- C- O U .
) LO (O E- . . . . . . . . * V V c V t - - -

0 =; z E D = E E E E D x~~~~~~~~~~~~~~~~~~~~~~~~~~~~

This content downloaded from 128.227.229.159 on Mon, 29 Aug 2016 17:47:19 UTC
View publication stats
All use subject to http://about.jstor.org/terms