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Molecular Systematics of Saxifragaceae Sensu Stricto

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To circumscribe Saxifragaceae sensu stricto better, as well as to elucidate generic relationships within this group, we sequenced the chloroplast gene rbcL and its 3' flanking region (yielding 1,471 bp) from 19 genera considered to represent core members of Saxifragaceae. In addition, we conducted a restriction site analysis of chloroplast DNA (cpDNA) for 21 core genera using 23 restriction endonucleases. Phylogenetic analyses using both data sets corroborate the results obtained from surveying the distribution of the loss of the intron in the chloroplast gene rp12 in delimiting a well-defined Saxifragaceae sensu stricto. Within the Saxifragaceae s.s. clade, a number of poorly resolved, basal phylogenetic branches supports the hypothesis that Saxifragaceae s.s. radiated rapidly very early in its evolutionary history. Molecular data also indicate the presence of several strongly supported groups of genera, such as the Boykinia group (Boykinia, Suksdorfia, Bolandra, Sullivantia, Jepsonia, and Telesonix), the Heuchera group (Heuchera, Bensoniella, Conimitella, Elmera, Lithophragma, Mitella, Tellima, Tiarella, and Tolmiea) the Leptarrhena/Tanakaea group, and the Darmera group (Darmera, Astilboides, Mukdenia, Bergenia, and Rodgersia). Significantly, molecular data suggest that the very large, taxonomically complex genus Saxifraga may not be monophyletic. DNA data have also helped to resolve the generic relationships of problematic taxa, indicating, for example, that Telesonix and the enigmatic Jepsonia are sister taxa. In addition to its phylogenetic implications, this study provides insight into basic trends in morphological, chemical, and cytological evolution within Saxifragaceae s.s. The molecular-based phylogenies suggest multiple origins and/or losses of several classes of flavonoid compounds, as well as several independent instances of reduction in stamen and petal number, hypanthium-ovary fusion, and aneuploidy. This study also illustrates the ability of rbcL sequence data to resolve generic-level relationships in some taxonomic groups.
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Molecular Systematics of Saxifragaceae Sensu Stricto
Author(s): Douglas E. Soltis, David R. Morgan, Albert Grable, Pamela S. Soltis and Robert
Kuzoff
Source:
American Journal of Botany
, Vol. 80, No. 9 (Sep., 1993), pp. 1056-1081
Published by: Botanical Society of America, Inc.
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American Journal of Botany 80(9): 1056-1081. 1993.
MOLECULAR SYSTEMATICS OF SAXIFRAGACEAE SENSU STRICTO
DOUGLAS E. SOLTIS,2 DAVID R. MORGAN, ALBERT GRABLE,
PAMELA S. SOLTIS, AND ROBERT KuzoFF
Department of Botany, Washington State University, Pullman, Washington 99164-4238
To circumscribe Saxifragaceae sensu stricto better, as well as to elucidate generic relationships within this group, we
sequenced the chloroplast gene rbcL and its 3' flanking region (yielding 1,471 bp) from 19 genera considered to represent
core members of Saxifragaceae. In addition, we conducted a restriction site analysis of chloroplast DNA (cpDNA) for 21
core genera using 23 restriction endonucleases. Phylogenetic analyses using both data sets corroborate the results obtained
from surveying the distribution of the loss of the intron in the chloroplast gene rpl2 in delimiting a well-defined Saxifragaceae
sensu stricto. Within the Saxifragaceae s.s. clade, a number of poorly resolved, basal phylogenetic branches supports the
hypothesis that Saxifragaceae s.s. radiated rapidly very early in its evolutionary history. Molecular data also indicate the
presence of several strongly supported groups of genera, such as the Boykinia group (Boykinia, Suksdorfia, Bolandra,
Sullivantia, Jepsonia, and Telesonix), the Heuchera group (Heuchera, Bensoniella, Conimitella, Elmera, Lithophragma,
Mitella, Tellima, Tiarella, and Tolmiea) the Leptarrhena/Tanakaea group, and the Darmera group (Darmera, Astilboides,
Mukdenia, Bergenia, and Rodgersia). Significantly, molecular data suggest that the very large, taxonomically complex genus
Saxifraga may not be monophyletic. DNA data have also helped to resolve the generic relationships of problematic taxa,
indicating, for example, that Telesonix and the enigmatic Jepsonia are sister taxa. In addition to its phylogenetic implications,
this study provides insight into basic trends in morphological, chemical, and cytological evolution within Saxifragaceae s.s.
The molecular-based phylogenies suggest multiple origins and/or losses of several classes of flavonoid compounds, as well
as several independent instances of reduction in stamen and petal number, hypanthium-ovary fusion, and aneuploidy. This
study also illustrates the ability of rbcL sequence data to resolve generic-level relationships in some taxonomic groups.
Few families of angiosperms present the magnitude of
systematic problems posed by Engler's (1930; see also
Schulze-Menz, 1964) broadly defined Saxifragaceae (=
Saxifragaceae sensu lato). From a morphological stand-
point the family has been almost impossible to charac-
terize clearly; hence, it represents one of the major phy-
logenetic problems at higher taxonomic levels in the
angiosperms (Spongberg, 1972; Dahlgren, 1980; Takh-
tajan, 1 987). Following Engler's (1930) interpretation, the
family is a large, morphologically diverse assemblage of
annual, biennial, and perennial herbs, shrubs, trees, and
vines comprising 15 subfamilies. The number of subfam-
ilies was later increased to 17 in Engler's Syllabus (Schulze-
Menz, 1964).
The morphological diversity encompassed by Saxifra-
gaceae sensu lato is so great that subsequent investigators
(e.g., Cronquist, 1968, 1981; Thome, 1968, 1976, 1983,
1992; Takhtajan, 1969, 1980, 1987; Dahlgren, 1975, 1980,
1983; Dahlgren, Jensen, and Nielsen, 1981) provided sub-
stantially modified concepts of relationships. These more
recent treatments differ dramatically, and little agreement
exists regarding the circumscription, taxonomic rank, or
relationships of Engler's subfamilies (reviewed in Morgan
I Received for publication 10 December 1992; revision accepted 5
April 1993.
The authors thank Jeff and Jane Doyle, Christian Brochmann, Loren
Rieseberg, Richard Gornall, Paul Wolf, Charles Werth, Tetsukazu Ya-
hara, Terry McIntosh, and the following Botanical Gardens for supplying
plant material: University of British Columbia, Komarov, Botanical
Institute, Nikko Botanical Garden, Palmengarten Botanical Garden,
University of Oslo Botanical Garden, and University of Tokyo Botanical
Gardens; Mike Edgerton for technical assistance; Ken Sytsma and Bruce
Bohm for helpful comments on the manuscript; and Yin-Long Qiu and
Mark Chase for the use of unpublished rbcL sequences. This work was
supported by NSF grants BSR 8717471, and BSR 9007614.
2 Author for correspondence (FAX: 509-335-3517).
and Soltis, 1993). As a result, definitions of Saxifra-
gaceae have varied greatly from author to author. Most
authors agree, however, that Engler's treatment is too
broad to be useful and have provided more restricted
views of Saxifragaceae (e.g., Dahlgren, 1975, 1980, 1983;
Cronquist, 1981; Takhtajan, 1987; Thome, 1992).
Not only has it been difficult to define Saxifragaceae,
but generic relationships among core members of the fam-
ily (i.e., Saxifragaceae sensu stricto) are also poorly un-
derstood. In part, this confusion may be the result of a
high degree of morphological similarity, particularly in
vegetative features. Genera are often distinguished by one
or a few pronounced differences in floral and/or fruit mor-
phology. As a result, monotypic genera abound in this
group; over one-third of the genera of Saxifragoideae
(Schulze-Menz, 1964), which approximates Takhtajan's
(1987) narrowly defined Saxifragaceae, each comprise a
single species (e.g., Bensoniella, Conimitella, Elmera, Tel-
lima, Tolmiea, Darmera, Tanakaea, Mukdenia, Astil-
boides, Oresitrophe, Peltoboykinia, Saxifragella, Zahl-
brucknera, and Leptarrhena). In striking contrast to the
numerous monotypic genera, however, is the very large
(approximately 300 species) genus Saxifraga, which is
ecologically, cytologically, morphologically, and taxo-
nomically complex.
In one of the first attempts to delimit generic groups
within Saxifragaceae s.s., Engler (1930) considered the
group (his tribe Saxifragaceae) to consist of four subtribes:
Astilbinae, Leptarrheninae, Saxifraginae, and Vahliinae.
Later, Schulze-Menz (1964) recognized Vahlia as a dis-
tinct subfamily (Vahlioideae), and the remaining taxa as
three tribes (Astilbeae, Leptarrheneae, and Saxifrageae)
of subfamily Saxifragoideae (Table 1). Klopfer (1973), in
contrast, recognized two large groups within Saxifragoi-
deae: those genera centered around Heuchera, having pa-
rietal placentation, and those centered around Saxifraga,
1056
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September 1993] SOLTIS ET AL.-SAXIFRAGACEAE SENSU STRICTO 1057
TABLE 1. Tribes of Saxifragoideae following Schulze-Menz (1964), which
are essentially equivalent to Engler's (1930) subtribes of Saxifrageae.
Generic names and boundaries in Saxifrageae have been updated
to show the complete cast of characters.
Astilbeae Leptarrheneae Saxifrageae
Astilbe Leptarrhenaa Bensoniellaa Mukdeniaa
(Aceriphyllum)
Rodgersia Tanakaeaa Conimitellaa Telesonixb
Astilboidesa Elmeraa Hieronymusiaax
Heuchera Bolandra
Lithophragma Boykinia
Mitella Suksdorfia
Tolmieaa Peltoboykiniaa
Tellimaa Saxifragellaa
Jepsonia Zahlbruckneraa
Oresitrophea Chrysosplenium
Bergenia Saxifraga
Darmeraa
(Peltiphyllum)
a Designates a monotypic genus.
bSubmerged into Boykinia by Gornall and Bohm (1985).
c Submerged into Suksdorfia by Gornall and Bohm (1985).
having axile placentation (Table 2). However, data from
diverse sources, including gross morphology, cytology,
karyology, flavonoid chemistry, and chloroplast DNA re-
striction site analysis have called into question most of
these generic groupings (e.g., Bohm, Dovevan, and Bhat,
1986; Soltis, 1986). Although these same data have sug-
gested several natural groups of genera (e.g., Gomall and
Bohm, 1980; Soltis, 1987; Soltisetal., 1991), phylogenetic
relationships at the generic level in Saxifragaceae s.s. still
remain largely uncertain.
A recent phylogenetic analysis of sequence data from
the chloroplast gene rbcL (which encodes the large subunit
of ribulose- 1 ,5-bisphosphate carboxylase-oxygenase) in-
dicates clearly that Saxifragaceae sensu lato are a poly-
phyletic assemblage of distantly related taxa representing
perhaps as many as ten different evolutionary lineages
(Morgan and Soltis, 1993). These data further suggest
that a more restricted definition of Saxifragaceae (= Sax-
ifragaceae sensu stricto) is clearly more appropriate. In
that large analysis, a clade of six genera was identified
that likely represents, in part, Saxifragaceae s.s. For the
present analysis, we obtained rbcL sequence data for ad-
ditional genera typically considered to represent the core
of Saxifragaceae in both broad and narrow definitions of
the family. In addition, representatives of 21 genera tra-
ditionally ascribed to Saxifragaceae s.s. were subjected to
a phylogenetic analysis of chloroplast DNA restriction
site data. We used these two molecular data sets to: 1)
ascertain the proper circumscription of a narrowly defined
Saxifragaceae (i.e., Saxifragaceae s.s.); 2) provide hypoth-
eses of phylogenetic relationships at the generic and, in
some instances, the specific level within Saxifragaceae s.s.;
and 3) provide an initial test of the monophyly of the
large, taxonomically complex genus Saxifraga. The phy-
logenetic hypotheses generated afforded the opportunity
to address basic questions of morphological, cytological,
and chemical evolution in this well-studied family (e.g.,
Jay, 1970; Bensel and Palser, 1975; Hideux and Ferguson,
1976; Soltis, 1986, and cytological references therein;
Bohm, Chalmers, and Bhat, 1988, and numerous flavo-
noid references therein).
MATERIALS AND METHODS
rbcL sequence analysis -In an earlier sequence analysis
of Saxifragaceae s.l., we obtained 1,407 bp of rbcL from
genera representing 16 of 17 subfamilies of Saxifragaceae
s.l. and compared these to a broad array of non-Magnoliid
dicot rbcL sequences (Morgan and Soltis, 1993). This
earlier analysis demonstrated that Saxifragaceae s.l. com-
prised at least ten distinct lineages and also identified a
clade of six taxa (Heuchera, Astilbe, Boykinia, Darmera,
Leptarrhena, and Saxifraga) that we considered to rep-
resent, in part, core genera of a narrowly defined family
Saxifragaceae. In the current study, we sequenced the
remainder of the rbcL gene, as well as the region between
the 3' end of rbcL and the reverse amplification primer,
for these same six taxa (providing a total of 1,471 bp) and
also obtained sequence data (1,471 bp) for 17 additional
taxa, thus providing rbcL sequences for a total of 19 genera
and 23 species (Table 3). All 23 species included herein
are traditionally considered core members of Saxifraga-
ceae in all taxonomic treatments of the family. Our se-
quencing of 1,471 bp, instead of the 1,407-1,431 bp stan-
dardly employed in rbcL sequence analyses, was done
with the hope of increasing the resolution of generic-level
relationships by including the more rapidly evolving re-
gion flanking the 3' end of the gene.
For all of the taxa for which rbcL sequences were ob-
tained, DNAs were isolated via the miniprep procedure
of Saghai-Maroofet al. (1984) and Doyle and Doyle (1987)
as modified by Soltis et al. (1991). Many of the DNAs
used for comparative sequencing were also used in the
cpDNA restriction site study (compare Tables 3, 4). Five
species of Saxifraga were included in the rbcL sequence
TABLE 2. Groups of genera within Saxifragoideae following Klopfer (1973). According to Klopfer, the affinities of Chrysosplenium are uncertain
(see text).
Centered on Heuchera Centered on Saxifraga
Bensoniella Astilbe Telesonix
Conimitella Rodgersia Hieronymusia
Elmera Astilboides Bolandra
Heuchera Chrysosplenium Leptarrhena Boykinia
Lithophragma Tanakaea Suksdorfia
Mitellc Jepsonia Peltoboykinia
Tolmiea Oresitrophe Saxifragella
Tellima Bergenia Zahlbrucknera
Tiarella Darmera (Peltiphyllum) Chrysosplenium
Mukdenia (Aceriphyllum) Saxifraga
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1058 AMERICAN JOURNAL OF BOTANY [Vol. 80
TABLE 3. Taxa for which rbcL sequence data were obtained in this study. Vouchers at WS.
Species Collection data, location of voucher specimen
aSaxifraga integrifolia Hook. OR, Gilliam Co., Soltis & Soltis 2253
Saxifraga punctata L. WA, Okanogan Co., Soltis & Soltis 2234
Saxifraga mertensiana Bong. OR, Multnomah Co., Grable 11586
Saxifraga oppositifolia L. AK, near Valdez, Soltis & Soltis 2160
Saxifraga cernua L. AK, near Valdez, Soltis & Soltis 2161
Sullivantia oregana Wats. OR, Hood River Co., Grable 11214
aBoykinia rotundifolia Parry CA, Los Angeles Co., Gornall 0101
Tellima grandiflora (Pursh) Dougl. AK, Craig, Prince of Wales Island, Soltis & Soltis 2113
Telesonix heucheriformis Rydb. WY, Teton Co., Wolf 151
Tanakaea radicans Franch. University of British Columbia Botanical Garden
Jepsonia parryi (Torr.) Small CA, Riverside Co., Rieseberg 1110
aLeptarrhena pyrolifolia (D. Don) R. Br. WA, Okanogan Co., Soltis & Soltis 2237
aDarmera peltata (Torr.) Voss OR, Gilliam Co., Soltis & Soltis 2083
Bergenia cordifolia (Haw.) A. Br. Komarov Botanical Institute Leningrad U.S.S.R.
Rodgersia pinnata Franch. Palmengarten Botanical Garden GERMANY
Peltoboykinia tellimoides (Maxim.) Hara Nikko Botanical Garden JAPAN
Mukdenia rosii (Oliver) Koidzumi Palmengarten Botanical Garden GERMANY
Astilboides tabularis (Hemsl.) Engl. University of Oslo Botanical Garden NORWAY
aHeuchera micrantha Dougl. See Soltis et al. (1990)
aAstilbe taquetii (Leveille) Koidzumi See Soltis et al. (1990)
Tolmiea menziesii T. & G. OR, Linn Co., Soltis & Soltis 1903
Elmera racemosa (Wats.) Rydb. WA, Kittitas Co., Soltis & Soltis 2179
Bolandra oregana Wats. OR, Multnomah Co., Grable 11587
a Taxon sequenced earlier (Soltis et al., 1990; Morgan and Soltis, 1993), but for which additional sequencing was conducted to obtain 1,471 bp
of rbcL sequence data.
analysis: S. integrifolia, S. punctata, and S. mertensiana
(sect. Micranthes); S. oppositifolia (sect. Porphyrion); S.
cernua (sect. Saxifraga) [sections follow those of Engler
and Irmscher (1916-1919)]. Of these five species, only S.
mertensiana and S. punctata were also used in the re-
striction site analysis portion of this study because we
were unable to obtain sufficient quantities of DNA for
restriction site analysis using the small amounts of leaf
material available to us of the remaining four taxa. The
difficulties encountered in applying miniprep procedures
to species of Saxifraga are discussed below in the section
on restriction site analysis. Amplification of rbcL, prep-
aration of single-stranded DNAs, and dideoxy sequencing
all follow methods described earlier (e.g., Morgan and
Soltis, 1993; Xiang et al., 1993).
Because one goal of this study was to ascertain whether
rbcL sequence data circumscribe a natural group of core
Saxifragaceae (i.e., Saxifragaceae s.s.), we included ad-
ditional rbcL sequences in our phylogenetic analysis. These
sequences were selected based on an earlier analysis of
Saxifragaceae s.l. (Morgan and Soltis, 1993) and in-
cluded many taxa identified as possible close relatives of
Saxifragaceae s.s. The total number of rbcL sequences
included in this analysis was 42. For comparison to the
1,471 bp obtained for all members of Saxifragaceae s.s.
included (see above), we also obtained 1,471 bp of se-
quence data for several additional taxa: Lambertia, Ptero-
stemon, Tetracarpaea, Myriophyllum, Rosa, Crossosoma,
Cephalotus, and Lepuropetalon. Thus, for 31 of the 42
species involved in this analysis, 1,471 bp were compared.
For the remaining taxa (sequenced by other investigators),
1,428 base pairs were available. The sources of these
sequences are as follows: Trochodendron, Platanus,
Daphniphyllum, and Hamamelis (Albert, Williams, and
Chase, 1992); Gossypium, Sabia, and Tetracentron (Qiu,
Chase, and Parks, 1993); Cercidiphyllum (Olmstead et
al., 1992).
Sequence data were analyzed with PAUP version 3.0s
(Swofford, 199 1) with MULPARS and TBR branch-swap-
ping. Albert, Chase, and Mishler (1993) have proposed
a method to weight differentially codon positions as well
as transitions and transversions. They concluded that their
weighting model ". . . actually supports the use of the
computationally simpler Fitch criterion for DNA se-
quence data." In all analyses of rbcL sequence data, there-
fore, all characters were specified as unweighted. Multiple
"islands" or separate clusters of most parsimonious trees
are likely to be present for a given data set, especially if
it includes a large number of taxa (Maddison, 1991). To
ensure that all groups of shortest trees were found, 100
replicate tree searches with random taxon addition were
therefore conducted, each analysis beginning with a dif-
ferent taxon addition order. To obtain estimates of reli-
ability for monophyletic groups, bootstrap (Felsenstein,
1985) and decay analyses were conducted. For the boot-
strap analysis (100 replicates) PAUP was instructed to
ignore all invariant nucleotide positions; the analysis was
performed using simple taxon addition, TBR branch-
swapping, and unweighted characters. To conduct the
decay analysis, TBR branch-swapping was initiated on
the set of shortest trees, and as many trees as possible up
to five steps longer were saved. The largest number of
trees that can be saved by PAUP (32,767) was reached
in this analysis. By examining in succession all trees up
to one, two, three, four, and five steps longer than the
shortest, the number of extra steps required for mono-
phyletic groups to decay (cease to be monophyletic) was
determined.
cpDNA restriction site analysis -Chloroplast DNA re-
striction site data for 41 species representing the nine
genera of Saxifragaceae known as the "Heuchera group"
(Heuchera, Tiarella, Mitella, Elmera, Conimitella, Tel-
lima, Lithophragma, Bensoniella, and Tolmiea) were pre-
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September 1993] SOLTIS ET AL. -SAXIFRAGACEAE SENSU STRICTO 1059
TABLE 4. Taxa analyzed for cpDNA restriction site data. Vouchers at WS.
Species Collection data
Astilbe taquetii (Leveille) Koidzumi See Soltis et al. (1990)
Astilbe microphylla Knoll. Botanical Gardens, University of Tokyo
Bergenia cordifolia (Haw.) A. Br. Komarov Botanical Institute, Leningrad
Bolandra californica A. Gray CA, Yosemite Co., Soltis & Soltis 2076
Bolandra oregana Wats. OR, Multnomah Co., Grable 11668
OR, Multnomah Co., Grable 11671
Boykinia aconitifolia Nuttall VA, Giles Co., Werth s.n.
Boykinia occidentalis Torrey & Gray WA, Mason Co., Grable 11636
CA, Placer Co., Grable 11386
Boykinia intermedia (Piper) G. N. Jones WA, Grays Harbor Co., Grable 11638
Boykinia lycoctonifolia (Maxim.) Engl. JAPAN, Tateyama, Soltis & Soltis 2225
Boykinia major A. Gray MT, Ravallii Co., Grable 11620
CA, Plumas Co., Grable 11398
ID, Idaho Co., Grable 11452
CA, Madera Co., Soltis & Soltis 2084
ID, Idaho Co., Grable 11450
CA, Yosemite Co., Soltis & Soltis 2077
Boykinia rotundifolia Parry CA, Los Angeles Co., Gornall 0101
Chrysosplenium americanum Schw. NY, Tompkins Co., Doyle & Doyle 1286
Darmera peltata (Torr.) Voss CA, Madera Co., Soltis & Soltis 2083
CA, Trinity Co., Soltis & Soltis 2104
CA, Mariposa Co., Soltis & Soltis 2081
Francoa sonchifolia Cav. UC Berkeley Botanical Garden
Jepsonia parryi (Torr.) Small CA, Riverside Co., Rieseberg 1110
Leptarrhena pyrolifolia (D. Don) R. Br. BC, CANADA, Vancouver Island, McIntosh s.n.
Peltoboykinia tellimoides (Maxim.) Hara Nikko Botanical Garden, JAPAN
Rodgersia aesculifolia Batalin Nikko Botanical Garden, JAPAN
Palmengarten Botanical Garden, GERMANY
Rodgersia pinnata Franch. Palmengarten Botanical Garden, GERMANY
Rodgersia podophylla A. Gray Palmengarten Botanical Garden, GERMANY
University of British Columbia Bot. Garden
Rodgersia sambucifolia Hemsl. Palmengarten Botanical Garden, GERMANY
Ribes aureum Pursh WA, Whitman Co., Soltis & Soltis 2220
Ribes sanguineum Pursh UC, Riverside Botanical Garden
Philadelphus lewisii Pursh WA, Whitman Co., Soltis & Soltis 2218
Saxifraga arguta D. Don WA, Yakima Co., Grable 11639
Saxifragaferruginea Grah. var. mancounii, Engl. & Irmsch. WA, Okanogan Co., Soltis & Soltis 2233
Saxifraga lyallii Engl. WA, Okanogan Co., Soltis & Soltis 2233
Saxifraga mertensiana Bong. OR, Multnomah Co., Grable 11586
Saxifraga punctata L. WA, Okanogan Co., Soltis & Soltis 2217
Suksdorfia ranunculifolia (Hook.) Engl. WA, Ferry Co., Soltis & Soltis 2169
WA, Chelan Co., Grable 11574
Suksdorfia violacea A. Gray WA, Ferry Co., Soltis & Soltis 2169
WA, Ferry Co., Soltis & Soltis 2216
MT, Ravalli Co., Grable 11627
Sullivantia oregana Wats. OR, Hood River Co., Grable 11214
Tanakaea radicans Franch. University of British Columbia Botanical Garden
Telesonix heucheriformis Rydb. WY, Teton Co., Wolf 151
Mukdenia rosii (Oliver) Koidzumi University of British Columbia Botanical Garden
Astilboides tabularis (Hemsl.) Engl. Palmengarten Botanical Garden, GERMANY
University of British Columbia Botanical Garden
Heuchera micrantha Dougl. From Soltis et al. (1991)
Tellima grandiflora (Pursh) Dougl. From Soltis et al. (1991)
Tolmiea menziesii Torrey & Gray From Soltis et al. (1991)
Elmera racemosa (Wats.) Rydb. From Soltis et al. (1991)
sented in an earlier investigation (Soltis et al., 1991). Re-
striction site data for four representatives of the Heuchera
group were combined with the restriction site data pre-
sented here (Table 4). Mutations reported herein as char-
acteristic of the Heuchera group typify all nine genera of
that alliance. The present analysis included 36 species in
21 genera. These taxa, together with those analyzed in an
earlier investigation of the Heuchera group (Soltis et al.,
1991), represent virtually all genera considered core mem-
bers of Saxifragaceae in all systematic treatments; they
also represent nearly all members of Saxifragaceae sensu
Takhtajan (1987), which parallels Engler's (1930) Saxi-
fragaceae and the Saxifragoideae of Schulze-Menz (1964).
The only possible core genera of Saxifragaceae not in-
cluded are the monotypic Oresitrophe, Saxifragella, Hi-
eronymusia (which is sometimes included in Suksdorfia;
Gornall and Bohm, 1985), and Zahlbrucknera. We have
thus far been unable to obtain living material of these
genera due to their localized and geographically remote
distributions.
Most genera were thoroughly sampled in this portion
of the study. Many of the genera examined are monotypic
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1060 AMERICAN JOURNAL OF BOTANY [Vol. 80
(Table 1). Four of six species of Rodgersia and six of seven
species of Boykinia were examined, as were two of the
three species of Suksdorfia (Gomall and Bohm, 1985).
Telesonix, Sullivantia, and Jepsonia were also well rep-
resented given that these genera each comprise only two
or three species (see Omduff, 1969; Gomall and Bohm,
1985; Soltis, 1991). The most poorly represented genera
are Astilbe (two of approximately 25 species were includ-
ed), Chrysosplenium (one of perhaps 55 species), and Sax-
ifraga (five of approximately 300 species). However, this
broad, generic-level phylogenetic analysis was not meant
to be a comprehensive study of the latter three genera,
particularly the large and taxonomically complex Saxi-
fraga.
Mature plants typically were collected in the field or
obtained from botanical gardens (Table 1) and subse-
quently cultured in the greenhouse. For most of the taxa
analyzed, total DNAs were isolated using the modifica-
tions of the protocols of Doyle and Doyle (1987) and
Saghai-Maroofet al. (1984) provided in Soltis et al. (1991).
However, these miniprep methods for DNA isolation did
not provide suitable quantities of digestible DNAs for
some of the taxa investigated (e.g., members of the Boy-
kinia group and species of Saxifraga). For these taxa a
large-scale DNA isolation procedure (Rieseberg, Soltis,
and Palmer, 1988) requiring at least 20 g (fresh weight)
of leaf material was used. Because such large amounts of
leaf material were required for these taxa, we were only
able to include a few species of Saxifraga that occur locally
in the Pacific Northwest: S. punctata, S. arguta, S. lyallii,
S. mertensiana, and S. ferruginea (Section Micranthes).
The broader sample of Saxifraga species included in the
rbcL sequence analysis could not be examined for cpDNA
restriction site variation because sufficient quantities of
high-quality DNA could not be isolated.
DNAs were digested with 23 endonucleases following
the specifications of the suppliers: ApaI, BamHI, BanI,
BanlI, BglI, BglII, BstEII, BstNI, CfoI, EcoRI, EcoRV,
HaeII, HindIII, HpaII, NciI, PstI, PvuII, Sacl, SaclI,
Sall, SmaI, XbaI, and XhoI. DNA fragments were sep-
arated in 1.0% agarose gels, denatured, and transferred
to a nylon membrane (ZETABIND, Cuno Laboratory
Products, Meriden, CT) following Palmer (1986). Het-
erologous cpDNA probes from lettuce (Jansen and Palm-
er, 1987) and petunia (used in place of the 22-kb inversion
in the chloroplast genome of lettuce) were labeled using
the Random Prime Labelling Kit (U.S. Biochemical Corp.,
Cleveland, OH) and hybridized to the membrane-bound
DNA fragments (Palmer, 1986). Chloroplast DNA probes
were kindly provided by J. D. Palmer and R. K. Jansen.
Ribes aureum, R. sanguineum, Francoa sonchifolia, and
Philadelphus lewisii were used as outgroups in the restric-
tion site analysis. Ribes has traditionally been considered
a close relative of Saxifragaceae s.s. (e.g., Thorne, 1983;
Dahlgren, 1983; Takhtajan, 1987). Francoa and Phila-
delphus were chosen as additional outgroups because of
their traditional placement in Saxifragaceae s.l. Francoa,
in fact, is often included in narrow circumscriptions of
the family (e.g., Cronquist, 1981). However, rbcL se-
quence data suggest that Francoa is not as closely related
to Saxifragaceae s.s. as is Ribes (Chase et al., 1993;
Morgan and Soltis, 1993). Philadelphus is even more
distantly related to Saxifragaceae s.s., having as its closest
allies members of Comaceae (Chase et al., 1993; Mor-
gan and Soltis, 1993). Not surprisingly, therefore, Phila-
delphus proved to be too distantly related to serve as a
suitable outgroup in our restriction site analysis. The po-
larity of each restriction site mutation was therefore hy-
pothesized by comparison to a consensus outgroup con-
dition determined from restriction site maps of Ribes
aureum, R. sanguineum, and Francoa sonchifolia.
Methods of phylogenetic analysis for the cpDNA re-
striction site data (using PAUP 3.0s) were the same as
those used to analyze the rbcL sequence data: 100 random-
taxon-addition tree searches were performed with MUL-
PARS, TBR branch-swapping, and unweighted characters
(in which gains and losses are weighted equally). Bootstrap
and decay analyses of the restriction site data were con-
ducted as described above for rbcL sequences (the limit
of 32,767 trees was again reached in the decay analysis).
Additional analyses were performed with character-state
weighing (gains weighted 1.5:1 and 2.0:1 over losses) (Al-
bert, Mishler, and Chase, 1992), using MULPARS and
TBR branch-swapping.
Combined restriction site and rbcL sequence analysis -
The restriction site and rbcL sequence data were also
combined for a joint analysis of 21 taxa for which both
types of data were available. This analysis was conducted
using unordered and unweighted characters for both re-
striction site and sequence data. Tree searches, as well as
bootstrap and decay analyses for the combined data ma-
trix, were performed in the same way as were the separate
analyses (see above).
Assessment of secondary chemical evolution -To deter-
mine patterns of evolution of secondary chemical char-
acteristics, the distributions of these chemical features
were mapped onto the majority-rule consensus tree that
resulted from analysis of restriction site data (see Fig. 2).
The Heuchera group was expanded to include the more
complete results of Soltis et al. (199 1) (see Figs. 5, 6). This
tree was then used as a constraint tree in PAUP for sep-
arate analyses of each chemical feature. With the con-
straint tree specified, each chemical characteristic was
analyzed separately as a two-state (present or absent) char-
acter. In this way the most parsimonious explanation for
the distribution of each chemical feature (given the con-
straint tree topology) was determined.
RESULTS
rbcL sequence data-All taxa for which 1,471 bp of
sequence data were obtained were found to have colinear
sequences from base positions 1,428-1,471 except Astilbe,
which has a seven-nucleotide insertion (AATATTG) be-
ginning at position 1459. A total of 418 variable nucle-
otide positions was present in this analysis. Ofthis number
397 are within the rbcL gene, with an average variation
of 0.28 variable positions per base pair. Twenty-one of
the variable positions occur between the terminus of rbcL
and the reverse amplification primer. The average se-
quence variability in this region is 0.49 variable positions
per base pair, which is almost twice as high as that for
the rbcL gene.
Phylogenetic analysis of the rbcL sequence data resulted
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19 1 Trochodendron
100, >5 Tetracentron
11 Platanus 1 J&
18 62, Lambertia ]
85, 3 1 Sabia
12 Daphniphyllum
13 Hamamelis
918 1 Cercidiphyllum
93, >5 21 Ribes
11 ~~~27
5 17 Tetracarpaea
42, 1 18, 2 1 70, 3 37 Myriophyllum
71,3 Kalanchoe
6 8 52 Rs
171 18 Ros
5 ~ ~~~~~~~~~ ~~~5 43, 2 Gossypium
45'1 22, 2 28 Crossosoma
37
45 4 20 Cephalotus
64, 5 70 Lepuropetalon
10
12 I Pterostemon
98, >5 2 Itea
4 |16 Saxifraga integrifolia
39,1 0 1 |1 Saxifraga punctata j
6, 0~1 Astilbe taquetEii.
3 1 2 Tolmiea menziesii 1
7 Elmera racemosa
1 I >51 99 ' 25 Heuchera micrantha I
3 Tellima grandiflora j
2 Li I Rodgersia pinnata
6 4,1 4811 Astilboides tabularis
2 ~~~~~Mukdenia rosii
,<3, 1 l 61, 1 3 Peltoboykinia tellimoides k
3, 1 2 Darmera peltata
Bergenia cordifolia
6 Boykinia rotundifolia
2 2 Sullivantia oregana
3I3 04 Bolandra oregana 4
83,4 512 3 Jepsonia parryi
6 4 7 Telesonix heucheriformis j
89, 5 62, 3 1 3 7 - Leptarrhena pyrolifolia
73, 3 Tanakaea radicansain
27 Saxifraga oppositifolia
1 12 100, >5 Saxifraga cernua
87,1>2 15
Saxifraga mertensiana
Fig. 1. One of four most-parsimonious trees resulting from phylogenetic analysis of rbcL sequences representing Saxifragaceae s.s. and other
non-Magnoliid dicots (not including autapomorphies; length = 804 steps, consistency index = 0.402, retention index = 0.541). This tree is identical
to the 50% majority rule consensus tree (including compatible groups) constructed from the four shortest trees. Nodes that did not occur in all
shortest trees are marked by black triangles. Numbers above each branch indicate the number of base substitutions. Numbers in italics below each
branch are bootstrap and decay values. The first number represents the percentage occurrence of each monophyletic group in the results of 100
bootstrap replicates, and the second number indicates the number of steps longer than the shortest trees at which the group ceases to be monophyletic.
Taxa in Saxifragaceae s.s. are shown in bold type, and well-supported groups are named and indicated by brackets (Lept. -Tan. = Leptarrhena-
Tanakaea). Members of the Darmera group, which do not form a monophyletic group in this analysis, are indicated by arrows.
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1062 AMERICAN JOURNAL OF BOTANY [Vol. 80
in four shortest trees that were 804 steps long without
autapomorphies (one of these is shown in Fig. 1). In other
shortest trees (not shown) Boykinia rotundifolia is the
closest relative of Bolandra oregona and Astilbe taquetii
is the sister to a group composed of Saxifraga integrifolia,
S. punctata, and the Heuchera group. Each of the 100
random-taxon-addition tree searches resulted in the same
four trees. All four exhibit a monophyletic group of genera
united by six base substitutions (Fig. 1). These genera are
considered core members of Saxifragaceae in all taxo-
nomic treatments of the family and represent what we
will hereafter refer to as Saxifragaceae s.s. According to
the results of this analysis, the closest relatives of Saxi-
fragaceae s.s. are Itea and Pterostemon (see also Morgan
and Soltis, 1993).
Based on these analyses of rbcL sequence data, several
strongly supported clades can be identified within Saxi-
fragaceae s.s. (e.g., Heuchera group, Leptarrhena-Tana-
kaea group, Boykinia group, Saxifraga oppositifolia-S.
cernua-S. mertensiana group, and Saxifraga punctata-S.
integrifolia group, Fig. 1). However, relationships among
these strongly supported clades, and also the relationships
of these clades to the remaining members of Saxifragaceae
s.s. analyzed, remain poorly resolved.
In the results of all analyses, three of the five species
of Saxifraga analyzed (S. mertensiana, S. cernua, and S.
oppositifolia) form the sister group to the remainder of
Saxifragaceae s.s. These three species are united by 12
base substitutions; this clade has high bootstrap and decay
values (8/7% and >5, respectively) and appears well dif-
ferentiated from the remaining members of Saxifragaceae
s.s. Sequence data also suggest that Leptarrhena and Tan-
akaea are closest relatives and that these genera are the
sister group to a well-supported clade comprising Boy-
kinia, Sullivantia, Jepsonia, Telesonix, and Bolandra,
genera united by four base substitutions (bootstrap and
decay values of 83% and 4). These five genera are all
members of the Boykinia group, a group that is also very
well supported by cpDNA restriction site data (see below).
For the most part, relationships in the remainder of the
tree are poorly resolved. However, two relatively well-
supported lineages do appear in this portion of the rbcL-
based phylogeny. One of the most strongly supported
clades in the entire tree consists of Heuchera, Tellima,
Tolmiea, and Elmera, genera representing the Heuchera
group (Heuchera, Tellima, Tolmiea, Elmera, Bensoniella,
Conimitella, Lithophragma, Mitella, and Tiarella), an al-
liance that is also well supported by cpDNA restriction
site data (see Soltis et al., 199 1, and discussion of cpDNA
restriction site data below). Members of this lineage share
seven base substitutions, and this clade is well supported
in both the bootstrap and decay analyses (99% and > 5).
The final group comprises Saxifraga integrifolia and S.
punctata, which are united by 16 base substitutions and
exhibit a bootstrap value of 99% and a decay value of
>5. These species, both of section Micranthes, differ by
46 and 49 base substitutions, respectively, from the node
uniting S. mertensiana (section Micranthes), S. opposi-
tifolia (section Porphyrion), and S. cernua (section Sax-
ifraga). The placement of species of Saxifraga in two
different portions of the rbcL-based phylogeny is an in-
triguing result that is also supported by cpDNA restriction
site data (see below).
cpDNA restriction site data-The 23 endonucleases used
recognized over 600 restriction site mutations. Using the
outgroup consisting of Ribes aureum, R. sanguineum, and
Francoa sonchifolia we polarized unambiguously 588 of
these. Of the 588 restriction site mutations, 3 1 1 are shared
by two or more taxa; the remaining 277 are autapomor-
phies (see Appendix).
Because of their restricted and/or remote geographic
distributions, only a single sample of most species was
analyzed. Some species for which conspecific populations
were included exhibited intraspecific cpDNA variation
such as Bolandra oregana, Boykinia major, and B. occi-
dentalis. Despite its restricted geographic distribution in
the Columbia River Gorge of Oregon and Washington,
Bolandra oregana maintains cpDNA variation between
two populations less than 0.5 km apart. The cpDNA vari-
ation observed within both Boykinia major and B. oc-
cidentalis has implications regarding the relationship be-
tween these two species (see discussion of Boykinia group).
Unweighted parsimony analysis resulted in 132 equally
most-parsimonious trees of 409 steps each without au-
tapomorphies (Fig. 2); each of the 100 random-taxon-
addition tree searches found the same group of 132 trees.
The large number of equally parsimonious trees may re-
flect, in part, missing characters for Saxifraga merten-
siana. When S. mertensiana is excluded from the analysis,
only 24 equally parsimonious trees are obtained, each
having 394 steps. The analysis indicates that all core mem-
bers of Saxifragaceae differ from the outgroup by a min-
imum of 43 restriction site mutations.
The results of the cpDNA restriction site analysis sug-
gest that Astilbe is the sister to the remainder of Saxifra-
gaceae s.s. (Fig. 2). The monophyly of the remaining mem-
bers of Saxifragaceae s.s. is supported by nine
synapomorphies (bootstrap value of 69%; decay value of
2). The basal phylogenetic position of Astilbe is thus only
moderately supported by restriction site data. Other basal
phylogenetic branches are more poorly resolved as illus-
trated by the few mutations and the low bootstrap and
decay values supporting the lower nodes (Fig. 2).
Chrysosplenium and Peltoboykinia appear on basal
phylogenetic branches. The remainder of the cpDNA-
based phylogenetic tree for Saxifragaceae s.s. is composed,
for the most part, of five well-supported clades, although
the relationships among these lineages remain poorly re-
solved (Fig. 2): 1) the Heuchera group of genera (Heuchera,
Tiarella, Tellima, Tolmiea, Elmera, Bensoniella, Conimi-
tella, Lithophragma, and Mitella), represented for sim-
plicity in this analysis by Heuchera micrantha, Elmera
racemosa, Tellima grandiflora, and Tolmiea menziesii;
2) the Boykinia group of genera (Jepsonia, Telesonix, Bo-
landra, Suksdorfia, Boykinia, and Sullivantia); 3) the Lep-
tarrhena- Tanakaea group; 4) the Darmera group of gen-
era (Rodgersia, Darmera, Astilboides, Mukdenia, and
Bergenia); and 5) the Saxifraga punctata group (S. lyallii,
S. arguta, S. punctata, S. ferruginea). Saxifraga merten-
siana appears as the sister taxon to the Boykinia group.
This species differs from the other species of Saxifraga
analyzed herein by over 104 restriction site mutations;
thus, these two lineages of Saxifraga species are extremely
well differentiated, a result that parallels the rbcL sequence
data.
Relationships among genera of the Heuchera group are
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September 1993] SOLTIS ET AL. - SAXIFRAGACEAE SENSU STRICTO 1063
Outgroup
25 Astilbe taquetii
100, >5 Astilbe microphylla
Rodgersia podophylla 1
8 Rodgersia sambucifolia v
99 >5 Rodgersia pinnata
Rodgersia aesculifolia e
3 10 Darmera peltata
99 ,5 1 19 Astilboides tabularis E
99, >5 2 19 2 Mukdenia rosii
48,1 __Li23~ Bergenia cordifolia j
Saxifraga lyalli 1
100, >5 3 Saxifraga arguta
, > *oo ,5 1 100, >5 Saxifraga punctata 0
52 Saxifragaferruginea I xq
1 -Saxifraga mertensiana
7 1i Jepsoniaparryi
91, 4 Telesonix heucheriformis
13 1 4 Bolandra oregana 11668
31, 1 9 99, 3 Bolandra oregana 11671
4 99, 90 2< >5 Bolandra californica
3 920,01 2 , Suksdorfla violacea
20l l Lm 11 3 -Suksdorfa ranunculifolia
100, >5 Boykinia rotundifolia
Boykinia aconitifolia
Boykinia major 11620
99, >5 4 4 Boykinia major 11450 Z
1 l | ' 1 199,4 89,3 Boykinia major 11452
9, 2 0 3 Boykinia major 2084
100, 4 Boykinia occidentalis 11386
7110 8 l,72, 2 1 Boykinia major 11398
2 99, >5 100, 3 . _ Boykinia major 2077
|21, 0 l l l l | |Boykinia occidentalis 11636
3 5 Boykinia intermedia
24,1 21 Boykinia lycoctonifolia
2 Sullivantia oregana
7
16 I Tanakaea radicans
9 9 100, >5 3 Leptarrhena pyrolifolia
69,2 5 Heuchera micrantha 1 Q
13 14005 Tellima grandiflora L
Elmera racemosa F
10 >5Tolmiea menziesii
27 Peltoboykinia tellimoides
Chrysosplenium americanum
Fig. 2. One of 132 equally most-parsimonious trees resulting from phylogenetic analysis of cpDNA restriction site data for taxa of Saxifragaceae
s.s. using an outgroup consisting of Ribes aureum, R. sanguineum, and Francoa sonchifolia (not including autapomorphies; length = 409 steps,
consistency index = 0.760, retention index = 0.931). This tree is identical to the 50% majority rule consensus tree (including compatible groupings)
constructed from the shortest trees. In those instances where multiple populations were analyzed for a species (Table 1) and identical restriction
site maps were obtained, only one entry is given (e.g., Darmera pelt ata). Nodes that did not occur in all shortest trees are marked by black triangles.
Monophyletic groupings occurring in some shortest trees but not in this one are: 1) Darmera group-Saxifraga group, 2) Rodgersia sambucifolia-R.
pinnata-R. aesculifolia, 3) Boykinia major 11450, 11452, 2084-B. occidentalis 11386, and 4) Boykinia major 11398, 2077-B. occidentalis 11636-
B. intermedia. Numbers above each branch indicate the number of restriction site mutations; bootstrap and decay values are indicated as in Fig.
1. Well-supported groups are named and indicated by brackets (Lept. -Tan = Leptarrhena- Tanakaea).
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1064 AMERICAN JOURNAL OF BOTANY [Vol. 80
Outgroup
26 2 Astilbe taquetii
2 Astilbe microphylla
Rodgersia podophylla 1
8 Rodgersia sambucifolia c
Rodgersia pinnata o
Rodgersia aesculifolia >
2 1 Darmerapeltata
l l G19 _Astilboides tabularis
I I 1 2 19 -Mukdenia rosii
12 | 4 Bergenia cordifolia |
4 _ mSaxifraga arguta
3 ~~Saxifraga lalliia
Saxifraga punctata
50 Saxifraga ferruginea c
Ir16 Saxifraga mertensiana
I 1 4 8t7 Jepsonia parryi
m I 13 4 23 Telesonix heucheriformis
2 Sullivantia oregana
4 2 Bolandra oregana 11668
30 9 Bolandra oregana 11671
2 I 5 8 Bolandra californica
3 1 Suksdorfia violacea
20 I1Suksdorfia ranunculifolia =
I I IBoykinia rotundifolia
1 Boykinia aconitifolia
9 4 Boykinia major 11620
2 ~~~~~~~~~4Boykinia major 11450
1 I F I I F = Boykinia major 11452 Q
I I 11 1 Boykinia major 2084
8 Boykinia occidentalis 11386
1 Boykinia major 11398
9 1 Boykinia major 2077
Boykinia occidentalis 11636
5 Boykinia intermedia
1 Boykinia lycoctonifolia
5 I Heuchera micrantha 1 Q
13 Tellima grandiflora - Z!
Elmera racemosa j
Tolmiea menziesii i
7 I
116 Tanakaea radicans
45 Leptarrhena pyrolifolia
t 28 Peltoboykinia tellimoides
Chrysosplenium americanum
Fig. 3. The most parsimonious tree resulting from phylogenetic analysis of cpDNA restriction site data for taxa of Saxifragaceae s.s. using
character-state weighting with gains weighted 2:1 over losses; weighted length without autapomorphies = 582. Restriction site mutations and well-
supported generic groups are indicated as in Fig. 2.
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September 1993] SOLTIS ET AL. - SAXIFRAGACEAE SENSU STRICTO 1065
described in Soltis et al. (1991). Within the Boykinia group,
a very close relationship is apparent between Telesonix
and the enigmatic Jepsonia (91%; 4). Strong support is
also provided for the monophyly of a clade comprising
Bolandra, Suksdorfia, and Boykinia (97%; 5). Species of
these three genera then form two sister groups: 1) the two
species of Bolandra and Suksdorfia violacea (90%; 2);
2) Suksdorfia ranunculifolia and all species of Boykinia
(100%; > 5). Within the Suksdorfia ranunculifolia-Boy-
kinia clade, the monophyly of Boykina is strongly sup-
ported (99%; >5). Within Boykinia, B. lycoctonifolia
emerges as the sister group to the strongly supported (99%;
4) B. rotundifolia-B. aconitifolia-B. major-B. intermedia-
B. occidentalis clade. A close relationship is also suggested
among B. major, B. intermedia, and B. occidentalis (72%;
2), although the six populations of B. major do not form
a monophyletic group separate from the two populations
of B. occidentalis. Within the Darmera group, strong sup-
port is provided for the monophyly of Rodgersia (99%;
> 5). Furthermore, a close relationship is suggested among
Rodgersia, Darmera, and Astilboides (74%; 2); more weak-
ly supported (48%; 1) is the sister-taxon status of Muk-
denia and Bergenia, as well as the sister-taxon relationship
between Rodgersia and Darmera (51%; 1). Within the
Saxifraga lyallii-S. arguta-S. punctata-S. ferruginea clade,
the former three species form a well-supported mono-
phyletic subclade (100%; > 5) distinct from S. ferruginea.
Both weighted parsimony analyses (1.5:1 and 2.0:1)
gave identical results: a single tree identical in topology
to one of the 132 trees resulting from unweighted analysis
(Fig. 3). The well-supported groups obtained using char-
acter-state weighing are identical to those revealed in the
unweighted analysis. Differences in the trees obtained
among the methods employed involve only those alliances
for which there was little support in the original, un-
weighted analysis.
Combined restriction site and rbcL sequence data -The
analysis of the combined data set resulted in six shortest
trees of 372 steps, one of which is shown in Fig. 4. The
decay analysis produced a total of 1,407 trees up to five
steps longer than the shortest trees. This analysis produced
results comparable to those obtained in the two separate
analyses (compare Fig. 4 with Figs. 1-3), although in many
respects the results are most similar to the restriction site
data. In the combined data set analysis, the major groups
of taxa are again recognized and strongly supported: Heu-
chera group (100%; >5), Boykinia group (100%; >5),
Leptarrhena Tanakaea (100%; >5), and the Darmera
group (97%; > 5). The Darmera group was present in the
results of the restriction site analysis, but not the rbcL
sequence analysis. The position of Astilbe as sister to the
remainder of Saxifragaceae s.s. is also more similar to the
results of the restriction site analysis. The combined anar-
ysis also places Leptarrhena- Tanakaea as the sister to the
Boykinia group (59%; 1), a result comparable to the short-
est rbcL trees and some (but not all) of the shortest re-
striction site trees. The results of the combined analysis
reveal that the two species of Saxifraga included do not
form a monophyletic group. Although clearly not closely
related to each other, the affinities of these two species of
Saxifraga remain uncertain. Most of the basal branches
have little support, a result in agreement with the indi-
Ribes aureum
40 Astilbe taquetii
61 Peltoboykinia tellimoides
1 1 Darmera peltata 1
6 48, 1 Rodgersia pinnata 2
15 10 194, 4 Astilboides tabularis
97, >5 2 Bergenia cordifolia
62, 1 23 Mukdenia rosii j
3m 6 Saxifraga punctata
32,0 o18 Leptarrhena pyrolifolia1
100, >5 Tanakaea radicans
20 5 9, 1 Telesonix heucheriformis 1
50 93, 19 Jepsonia parryi
25
6 100, >5 5 26 Sullivantia oregana
26,0 86,3 2 Boykinia rotundifolia
100, >5 79 Bolandra oregana j
_ Saxifraga mertensiana
46, 29 3 3 Tolmiea menziesii 1
46, 2 37 3I
100 7, >5 13 1 5 Elmera racemosa k =
100, >5 Tellima grandiflora
100, >5 Heuchera micrantha j
Fig. 4. One of six most parsimonious trees resulting from analysis
of combined restriction site and rbcL data for 20 taxa of Saxifragaceae
s.s. with Ribes aureum as an outgroup (not including autapomorphies;
length = 372 steps, consistency index = 0.624, retention index = 0.794).
This tree is identical to the 50% majority rule consensus tree (including
compatible groups) constructed from the six shortest trees. Nodes that
did not occur in all shortest trees are marked by black triangles. Numbers
above each branch indicate the total number of base substitutions and
restriction site mutations; bootstrap and decay values are indicated as
on Figs. 1, 2. Generic groupings are labeled as in Figs. 1-3.
vidual analysis of rbcL sequences and cpDNA restriction
sites.
DISCUSSION
A phylogenetic tree of character-state changes in a gene
(or any other character) does not necessarily agree com-
pletely with the actual evolutionary pathways of the spe-
cies under investigation. Discrepancies between gene trees
and species trees may result from several factors, including
genetic polymorphisms in the ancestral species and hy-
bridization (see reviews by Neigel and Avise, 1985; Pam-
ilo and Nei, 1988; Rieseberg and Soltis, 1991; Doyle,
1992).
The particularly important role that hybridization/in-
trogression can play in producing fallacious phylogenies
in plants has been recently reviewed (Rieseberg and Soltis,
1991; Rieseberg and Brunsfeld, 1992) and is especially
relevant to the present investigation. In an earlier study
of phylogenetic relationships among members of the Heu-
chera group of genera (Soltis et al., 1991), ample evidence
indicated that a gene tree and a species tree do not nec-
essarily correspond in Saxifragaceae. In the Heuchera
group, evidence of hybridization and subsequent chlo-
roplast capture was observed between closely related spe-
cies, species in different sections of the large genus Heu-
chera, and even between different genera (Tellima and
Heuchera). However, the Heuchera group is well known
for its propensity for interspecific hybridization, even at
the generic level (reviewed in Soltis et al., 1991). In con-
trast, hybridization is not known to be as frequent else-
where in Saxifragaceae, with the possible exception of
species of Saxifraga. Nonetheless, with these cautionary
notes in mind, we present below the phylogenetic/evo-
lutionary implications for Saxifragaceae s.s. based on two
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1066 AMERICAN JOURNAL OF BOTANY [Vol. 80
TABLE 5. Members of Saxifragaceae sensu lato surveyed for the pres-
ence or absence of the rpl2 intron. When more than one species of
a genus was surveyed, the number of species analyzed is given in
parentheses following the name of the genus. System of classifica-
tion follows Takhtajan (1987). Data taken from Downie et al. (1991).
Brexiaceae Saxifragaceae
Brexia madagascariensis Astilbe taquetiia
Excalloniaceae Astilboides tabularis
Escallonia sp. Bensoniella oreganaa
Francoaceae Bergenia cordifoliaa
Francoa sonchifolia Bolandra californicaa
Grossulariaceae Boykinia spp. (6)a
Ribes spp. (2) Chrysosplenium
Hydrangeaceae americanuma
Cardiandra alternifolia Conimitella williamsiia
Carpenteria californica Darmera peltataa
Hydrangea sp. Elmera racemosaa
Kirengeshoma palmata Heuchera spp. (6)a
Philadelphus lewisii Lithophragma spp. (5)a
Schizophragma hydran- Mitella spp. (5)a
geoides Mukdenia rosia
Iteaceae Peltoboykinia tellimoidesa
Itea virginica Rodgersia spp. (4)a
Pamassiaceae Saxifraga spp. (5)a
Parnassia fimbriata Suksdorfia violaceaa
Penthoraceae Sullivantia oreganaa
Penthorum sedoides Tanakaea radicansa
Telesonix jamesiia
Tellima grandifloraa
Tiarella spp. (2)a
Tolmiea menziesiia
a Designates those species for which the rpl2 intron is absent.
cytoplasmic markers: comparative sequencing ofthe chlo-
roplast gene rbcL and restriction site analysis of cpDNA.
The validity of these phylogenetic hypotheses should be
tested with critical analyses of nuclear-encoded characters
as well.
Circumscription of Saxifragaceae sensu stricto-Phy-
logenetic analysis of rbcL sequences revealed the presence
of a well-defined clade of 23 species representing 19 gen-
era, all of which have traditionally been considered core
members of Saxifragaceae (Fig. 1). Based on sequence
data, the closest relatives of these genera include Itea and
Pterostemon, with other taxa that have been thought close-
ly related to Saxifragaceae, such as Ribes, Penthorum, and
Kalanchoe (Crassulaceae) more distantly related. Thus,
this analysis of additional rbcL sequences, together with
the findings of Morgan and Soltis (1993), indicates that:
1) traditional views of Saxifragaceae (e.g., Engler, 1930;
Schulze-Menz, 1964) are much too broad; 2) a mono-
phyletic assemblage of core Saxifragaceae can be identified
that should be considered to represent Saxifragaceae s.s.
Restriction site analysis of cpDNA also indicates that
the core genera of Saxifragaceae examined are well sep-
arated from Ribes aureum and R. sanguineum, the closest
outgroup taxa. According to restriction site data, Francoa
is even more distantly removed from Saxifragaceae s.s.,
in agreement with broad phylogenetic analyses of rbcL
sequence data (Fig. 1) (for more comprehensive analyses
see Chase et al., 1993; Morgan and Soltis, 1993).
Sequence data and cpDNA restriction site data there-
fore delineate a group of herbaceous genera as a mono-
phyletic assemblage, Saxifragaceae s.s. Additional mo-
lecular support for this same narrow view of the family
comes from the loss of the intron from the chloroplast
gene rpl2. The rpl2 intron is absent from 24 herbaceous
genera (50 species) of Saxifragaceae (Downie et al., 1991;
data summarized in Table 5). In contrast, representatives
of other subfamilies of Engler's (1930) Saxifragaceae, as
well as potentially related families in Rosidae (e.g., Cras-
sulaceae, Rosaceae, Fabaceae), all possess the rpl2 intron.
These core genera also share morphological, palynolog-
ical, anatomical, and chemical features (Dandy, 1927;
Dahlgren, 1930; Bensel and Palser, 1975; Hideux and
Ferguson, 1976; Bohm, Donevan, and Bhat, 1986; Bohm,
Chalmers, and Bhat, 1 988), and most classifications main-
tain them as a distinct group at the familial, subfamilial,
or tribal level (e.g., Engler, 1930; Schulze-Menz, 1964;
Thome, 1983, 1992; Takhatajan, 1987), although some
(e.g., Cronquist, 1981) have included taxa in a narrowly
defined Saxifragaceae that are only distantly related based
on molecular data (e.g., Parnassia, Lepuropetalon, Vahlia;
see review by Morgan and Soltis, 1993). The only re-
maining potential members of Saxifragaceae s.s. are the
monotypic Oresitrophe, Zahlbrucknera, Saxifragella, and
Hieronymusia (sometimes included in Suksdorfia; see
Gornall and Bohm, 1985), which were not included in
our molecular analyses because we could not obtain ma-
terial of them.
Relationships within Saxifragaceae sensu stricto-gen-
eral considerations-Chloroplast DNA restriction site data
and rbcL sequences provide very similar pictures of phy-
logenetic relationships within Saxifragaceae s.s. Several
well-supported clades of genera are recognized by both
molecular data sets: the Heuchera group, the Boykinia
group, and the Leptarrhena- Tanakaea group (Figs. 1-3).
In addition, Leptarrhena- Tanakaea appear to be the sister
to the Boykinia group. This result appears in all of the
shortest rbcL trees and some, but not all, of the shortest
restriction site trees. The sister group status of Leptar-
rhena- Tanakaea and the Boykinia group is also sup-
ported, although weakly (59%; 1), in the analysis of the
combined data sets (Fig. 4). Both data sets also indicate
that the species of Saxifraga analyzed do not form a single
monophyletic lineage. These findings are individually dis-
cussed in more detail below. The Darmera group of genera
is supported by restriction site data but not by rbcL se-
quence data. When the restriction site and rbcL data sets
are combined (Fig. 4) all of the groups and findings noted
above, including the monophyly of the Darmera group,
are supported.
The difference between the restriction site and rbcL data
sets regarding the monophyly of the Darmera group of
genera almost certainly reflects the paucity of rbcL base
substitutions at this taxonomic level. Because of the con-
servative evolution of rbcL, it has been most useful in
resolving relationships at the family level and above, al-
though intergeneric relationships have been studied with
rbcL data in Onagraceae (Conti, Fischbach, and Systma,
1993), Geraniaceae (Price and Palmer, 1993), Ericaceae
(Kron and Chase, 1993), Cupressaceae (Gadek and Quinn,
1993), Droseraceae (Williams, Albert, and Chase, 1992),
Orchidaceae (M. Chase, Kew, unpublished data), and Ro-
saceae (D. Morgan, D. E. Soltis, and K. Robertson, un-
published data). Thus, the difference between the phy-
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September 1993] SOLTIS ET AL. -SAXIFRAGACEAE SENSU STRICTO 1067
logenies based on rbcL sequences and cpDNA restriction
site variation regarding the monophyly of the Darmera
group is probably not an actual discrepancy, but only
reflects the lower limits of resolution of rbcL sequence
data. It is of interest, for example, that our ongoing com-
parative sequence analysis of the chloroplast gene ORFK,
which evolves three times faster than does rbcL, similarly
delineates a clade corresponding to the Darmera group
of genera (L. Johnson and D. Soltis, unpublished data).
Although molecular data reveal the presence of several
strongly supported monophyletic lineages within Saxifra-
gaceae s.s. (e.g., Heuchera group, Boykinia group, Lep-
tarrhena- Tanakaea, Darmera group, and the two groups
of Saxifragas), relationships among these groups are more
poorly resolved (Figs. 1-4). This low resolution is also
observed when the restriction site data and rbcL sequences
are combined (Fig. 4). Hence, a large polychotomy is
essentially present at a basal level within Saxifragaceae
s.s. The general lack of phylogenetic resolution at basal
levels in Saxifragaceae s.s. agrees with the proposal that
this group radiated rapidly. Savile (1975, 1976), for ex-
ample, examined rust parasites (Puccinia) that use some
Saxifragaceae as hosts and concluded that following a
probable origin in eastern Asia from an ancestor that
looked much like Astilbe, Saxifragaceae s.s. radiated rap-
idly. Notably, the analysis of cpDNA restriction site data,
but not rbcL sequence data, places Astilbe in a basal po-
sition in Saxifragaceae s.s. Astilbe also appears in a basal
position in the combined analysis (Fig. 4). These results
support long-standing views (e.g., Dandy, 1927; Engler,
1930; Savile, 1975) of Astilbe as the most primitive mem-
ber of Saxifragaceae.
Traditionally, Saxifragaceae s.s. have been divided into
several groups of genera. Schulze-Menz (1964) recognized
three tribes within Saxifragoideae (Table 1): 1) Astilbeae,
comprising Astilbe, Astilboides, and Rodgersia; 2) Lep-
tarrheneae, composed of Leptarrhena and Tanakaea; and
3) Saxifrageae, consisting of the remaining genera of Sax-
ifragaceae s.s. Klopfer (1973), in contrast, recognized two
basic groups (Table 2): genera centered around Heuchera
having parietal placentation and usually polytelic inflo-
rescences (essentially the Heuchera group of genera) and
genera centered around Saxifraga, having axile placen-
tation and monotelic inflorescences (all taxa of Saxifra-
gaceae s.s. other than the Heuchera group). Chrysospleni-
um, which has parietal placentation and a monotelic
inflorescence, was considered by Klopfer (1973) a partic-
ularly enigmatic genus (Table 2).
Molecular data do not support the division of Saxifra-
gaceae into the three groups (tribes) of Schulze-Menz
(1964). One of these three groups (Leptarrhena-Tana-
kaea) is strongly supported by molecular evidence, but
there is no molecular support for the tribes Astilbeae or
Saxifrageae of Schulze-Menz (1964). Astilbe, one of the
members of tribe Astilbeae, is not closely related to either
Rodgersia or Astilboides, the two remaining members of
Astilbeae. Rodgersia and Astilboides are part of the Dar-
mera group of genera (see below) and have as their closest
relatives Darmera, Bergenia, and Mukdenia, three genera
placed in tribe Saxifrageae by Schulze-Menz (1964).
In comparing our molecular results with the division
of Saxifragaceae s.s. into the two groups proposed by
Klopfer (1973) (Table 2), it is significant that the Heuchera
group of genera (Bensoniella, Conimitella, Elmera, Heu-
chera, Lithophragma, Mitella, Tiarella, Tolmiea, and Tel-
lima) is strongly supported by both restriction site and
rbcL sequence data (discussed in more detail below). How-
ever, molecular data clearly do not identify the remaining
genera (Klopfer's large group of genera centered on Sax-
ifraga) as a monophyletic assemblage. Furthermore,
Klopfer (1973) considered Chrysosplenium potentially
closely allied with his Heuchera group of genera (compare
Tables 1 and 2). Chrysosplenium is not, however allied
with the Heuchera group in our results.
Thus, sequence data and restriction site data agree with
other lines of systematic inference in calling into question
most ofthe traditionally recognized subgroups within Sax-
ifragaceae s.s. Cytological and karyological data similarly
suggest that Astilboides and Rodgersia are not closely re-
lated to Astilbe, but rather are more closely allied with
Darmera, Bergenia, and Mukdenia (Soltis, 1986). In ad-
dition, several analyses have also noted discrepancies be-
tween alliances revealed by chemical data and the tra-
ditional subgroupings of Saxifragaceae s.s. (e.g., Jay, 1970;
Miller and Bohm, 1980). Numerous lines of evidence
therefore suggest that the subgroups of Engler (1930),
Schulze-Menz (1964), and Klopfer (1973) be abandoned.
Phylogenetic analysis of molecular data indicates the pres-
ence of several well-supported groups that differ from
those recognized in traditional taxonomic treatments: the
Heuchera group, the Boykinia group, the Darmera group,
Leptarrhena- Tanakaea, and two distinct lineages of Sax-
ifraga species.
The Boykinia group - Both chloroplast DNA restriction
site data and rbcL sequences strongly support a group of
genera referred to herein as the Boykinia group (Figs. 1-
4). This group comprises Jepsonia, Telesonix, Sullivantia,
Boykinia, Suksdorfia, and Bolandra. Morphological and
chemical evidence similarly suggests a well-defined Boy-
kina group of genera (Gomall, 1980; Gomall and Bohm,
1980, 1984, 1985; Gornall, Bohm, and Taylor, 1983).
The composition of the Boykinia group as defined by
Gomall and Bohm (1985) is similar to that suggested by
molecular evidence, with the noteworthy exception that
they did not include Jeopsonia. In contrast, both sequence
data and restriction site evidence indicate clearly that
Jepsonia is part of this alliance.
The relationships of Jepsonia have long been enigmatic.
The genus exhibits a number of unique features in Sax-
ifragaceae, including an unusual caudex and contractile
root and a distylous breeding system. In his thorough
morphological study of the genus, Omduff (1969) noted
that Jepsonia has no close relatives in Saxifragaceae. In
their flavonoid analysis of Jepsonia, Bohm and Omduff
(1978) suggested possible affinities with Darmera, Heu-
chera, and Tellima. Later, Gomall and Bohm (1985) not-
ed that, although Jepsonia shares some morphological
features with Boykinia, it also has affinities with other
genera, such as Darmera, Mukdenia, and Oresitrophe.
cpDNA restriction site data not only clearly indicate that
Jepsonia is part of the Boykinia group, but also suggest
that Telesonix and Jepsonia are sister taxa. Significantly,
Gomall and Bohm (1985) noted that Telesonix and Jep-
sonia share a number of morphological features such as
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1068 AMERICAN JOURNAL OF BoTANY [Vol. 80
colporoidate pollen, ten stamens, and smooth seeds. They
viewed these characters, however, as shared primitive
features and maintained that the two genera actually had
long, independent evolutionary histories. DNA data sug-
gest instead that these shared traits are actually indicative
of a close phylogenetic relationship.
The circumscription and relationships of Boykinia, the
core member of this group of genera, are uncertain. Gor-
nall and Bohm (1985) considered Boykinia to comprise
nine species in three sections: section Boykinia (B. aconiti-
folia, B. intermedia, B. lycoctonifolia, B. major, B. occi-
dentalis, and B. rotundifolia), section Renifolium (B. ri-
chardsonii), and section Telesonix (B. jamesii and B.
heucheriformis). The last of these sections has, however,
typically been considered a distinct genus (Telesonix) of
either one or two species (reviewed in Gomall and Bohm,
1985); we have followed the treatment of Telesonix as a
distinct genus. We attempted to sample Boykinia thor-
oughly in this study; the only species not included were
B. richardsonii and Telesonixjamesii (which is sometimes
considered conspecific with Telesonix heucheriformis,
which was sampled in this study). Chloroplast DNA re-
striction site data indicate clearly that Telesonix heu-
cheriformis is not part of Boykinia, but is instead more
closely allied with the enigmatic Jepsonia, as noted above
(Figs. 2, 3). Significantly, rbcL sequence data also clearly
separate Telesonix and Boykinia (Fig. 1). Given the con-
servative evolution of rbcL, this placement is additional
evidence that Telesonix is well differentiated from Boy-
kinia. When cpDNA restriction site data and rbcL se-
quences are combined (Fig. 4), Telesonix again appears
as the sister of Jepsonia. Thus, in contrast to the conclu-
sions of Gomall and Bohm (1985), molecular data suggest
that Telesonix be retained as a genus distinct from Boy-
kinia.
The phylogenetic position of Sullivantia remains some-
what uncertain (Figs. 2, 3). In half ofthe 132 trees resulting
from the unweighted analysis, Sullivantia appears as the
sister group to a clade comprising Boykinia, Bolandra,
and Suksdorfia (Fig. 2); in the remaining 66 trees, Sulli-
vantia is the sister to Jepsonia and Telesonix. The weight-
ed analyses show only the latter position (Fig. 3). These
results are intriguing in that Sullivantia and Boykinia have
sometimes been viewed as closest allies based on the
striking morphological similarities between the two genera
(Gomall, 1980; Soltis, 1980a). Molecular data, although
not completely resolving the placement of Sullivantia,
clearly indicate that this genus is not the closest ally of
Boykinia (Figs. 2, 3).
Restriction site data indicate a particularly close rela-
tionship among Boykinia, Bolandra, and Suksdorfia, sup-
porting the conclusions of Gomall and Bohm (1985). The
three genera share similar glandular trichomes, pollen
features, and leaf texture; although Bolandra is in many.
respects intermediate between Boykinia and Suksdorfia,
it tends to resemble the latter more closely (Gomall and
Bohm, 1985).
Although cpDNA restriction site data unite Bolandra,
Suksdorfia, and Boykinia, the two species of Suksdorfia
analyzed do not form a monophyletic group (Figs. 2, 3).
Chloroplast DNA restriction site data indicate that Suks-
dorfia ranunculifolia is more closely allied with Boykinia
than with S. violacea. Thus, our molecular data suggest
that Boykinia could be expanded to include Suksdorfia
ranunculifolia. Significantly, S. ranunculifolia was once
considered a species of Boykinia (reviewed in Gornall,
1980), and Gornall and Bohm (1985) noted many mor-
phological similarities between S. ranunculifolia and Boy-
kinia.
Our DNA data also indicate that Suksdorfia violacea
has as its sister group the two species of Bolandra (B.
oregana and B. californica). That these two Suksdorfia
species may not be closest relatives is not surprising, given
their pronounced differences in floral morphology. In fact,
the three species of Suksdorfia recognized by Gornall and
Bohm (1985) were previously placed in three monotypic
genera: Hemievia ranunculifolia, Suksdorfia violacea, and
Hieronymusia alchemilloides. The last species, a narrow
endemic of Argentina, unfortunately could not be ob-
tained for this study. Gornall and Bohm (1985) placed
these three species in one genus because of similarities in
several characters (e.g., presence of a bulbiferous rhizome
and similar trichomes and seed morphology). Our data
indicate, however, that Suksdorfia violacea is well sepa-
rated phylogenetically from S. ranunculiforlia; cpDNA
restriction site data suggest that the similarities in vege-
tative characters noted by Gornall and Bohm (1985) rep-
resent convergent evolution or the retention of primitive
characters, and that the previous treatment of these spe-
cies as separate genera may be justified.
Within Boykinia, restriction site data indicate a par-
ticularly close relationship among B. occidentalis, B. ma-
jor, and B. intermedia. Boykinia intermedia has some-
times been considered a variety ofB. major (e.g., Hitchcock
et al., 1961). However, Gornall and Bohm (1985) support
specific status for B. intermedia based in large part on
their diploid count of 2n = 14 for one population of this
species (Gornall, Bohm, and Taylor, 1983); B. major has
2n = 28 (Fedorov, 1969). A subsequent chromosome
count, however, of an individual from the same popu-
lation of B. intermedia examined by Gornall, Bohm, and
Taylor (Soltis, 1987) revealed 2n = 28; thus, B. intermedia
and B. major are unique in the genus in sharing this
tetraploid chromosome number. Further supporting a close
relationship between Boykinia major and B. intermedia
are many morphological features (Gornal and Bohm,
1985).
The close relationship among Boykinia occidentalis, B.
major, and B. intermedia suggested by our DNA data is
intriguing and raises the possibility that the tetraploids
B. intermedia and B. major have been derived at least in
part (through auto- or allopolyploidy) from B. occiden-
talis, which is only known as a diploid with 2n = 14.
Natural hybrids have been reported between B. occiden-
talis and B. major, and these two taxa, while easily dis-
tinguished, also exhibit many morphological similarities
(reviewed in Gornall and Bohm, 1985). cpDNA restric-
tion site variation is present within both B. major and B.
occidentalis but does not separate these species. Rather,
cpDNA restriction site data unite one of two analyzed
populations of B. occidentalis (11386) with four popu-
lations of B. major (11620, 11450, 11452, 2084); a par-
ticularly close relationship is suggested between popula-
tion 11386 of B. occidentalis and population 2084 of B.
major. At least four hypotheses can explain this pattern.
1) The cpDNA characters that distinguish the populations
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September 1993] SOLTIS ET AL. - SAXIFRAGACEAE SENSU STRICTO 1069
of B. occidentalis were derived twice. This explanation
seems unlikely given the low frequency of convergences
in cpDNA data sets, particularly among closely related
species. 2) The pattern of distribution of the cpDNA mu-
tations is the result of hybridization/introgression. Chlo-
roplast capture resulting from hybridization/introgression
occurs frequently in plants (reviewed in Rieseberg and
Soltis, 1991, and Rieseberg and Brunsfeld, 1992) and is
well documented in the Heuchera group of genera (Soltis
et al., 199 1). Thus, chloroplast capture must be considered
as a possibility, although it would involve hybridization
between diploid B. occidentalis and tetraploid B. major.
3) The ancestor of the B. intermedia-B. major-B. occi-
dentalis clade was polymorphic for these restriction site
mutations, and as the species diverged, the cpDNA poly-
morphisms were retained in both B. major and B. occi-
dentalis (because only one population of B. intermedia
was analyzed, an assessment cannot be made for this
species). This process has been referred to as "phyloge-
netic sorting" (Avise, 1986) and has been invoked as a
possible explanation for cpDNA diversity in other plant
species (e.g., Doebley, 1990; Doyle, Doyle, and Brown,
1990). It may not be possible, however, to differentiate
between the results of hybridization and phylogenetic
sorting in the analysis of organelle-based phylogenies. 4)
Boykinia occidentalis was polymorphic for cpDNA re-
striction sites, and there were multiple origins of the tet-
raploid B. major from cytoplasmically distinct parental
populations of B. occidentalis. This hypothesis is similar
to 3), but attempts to take the ploidal level differences
into account.
The Darmera group - Chloroplast DNA restriction site
data also provide support for a close phylogenetic rela-
tionship among Darmera, Bergenia, Astilboides, Muk-
denia, and Rodgersia, a group of five genera referred to
herein as the Darmera group (Figs. 2, 3). Within the Dar-
mera group, Astilboides, Darmera, and Mukdenia are
monotypic, whereas Bergenia and Rodgersia comprise
eight and six species, respectively. The Darmera group is
supported by cpDNA restriction site data but not by rbcL
sequence data; it does appear in the analysis of the com-
bined data sets (Fig. 4). As noted above, however, this
discrepancy between the two separate molecular analyses
probably reflects the lower limits of resolution of rbcL
sequence data at this taxonomic level. Within the Dar-
mera group, two subgroups are apparent: 1) Mukdenia-
Bergenia; and 2) Astilboides-Darmera-Rodgersia.
The five genera of the Darmera group have not tradi-
tionally been considered to be particularly closely allied.
Astilboides and Rodgersia were placed in tribe Astilbeae
by Schulze-Menz (1 9 64) and subtribe Astilbinae by Engler
(1930) with Astilbe, whereas Bergenia, Darmera, and
Mukdenia were considered members of tribe Saxifrageae
(or subtribe Saxifraginae, Engler, 1930) (Table 1). Only
Dandy (1927) considered most genera of the Darmera
group, as well as the poorly understood Oresitrophe (of
which we have so far been unable to obtain material), to
be closely allied based on general habit and similarities
in floral morphology. Cytological data also agree closely
with the relationships suggested by DNA evidence. As-
tilboides, Bergenia, Darmera, and Mukdenia possess a
chromosome number of 2n = 34 (Soltis, 1986). In con-
trast, most genera of Saxifragaceae s.s. have 2n = 14, with
2n = 34 otherwise reported only rarely for species of the
very large and cytologically diverse genus Saxifraga (Fe-
dorov, 1969; Soltis, 1986). Furthermore, these four genera
also share essentially identical karyotypes (Soltis, 1986).
Thus, cytological data parallel cpDNA data in suggesting
strongly that Astilboides, Bergenia, Darmera, and Muk-
denia are closely allied.
Several chromosome numbers have been reported for
Rodgersia, including 2n = 30, 36, 60 (Fedorov, 1969).
These are also unusual chromosome numbers for Saxi-
fragaceae s.s. and have been reported elsewhere only for
a few species of Saxifraga (Fedorov, 1969). The karyotype
of Rodgersia species is also very similar to that shared by
Astilboides, Bergenia, Darmera, and Mukdenia (D. Soltis,
unpublished data). Thus, the chromosome numbers of 2n
= 30 and 36 in Rodgersia could have been derived from
an original diploid number of 2n = 34 via aneuploid
decrease and increase, respectively.
Leptarrhena-Tanakaea-Both molecular data sets in-
dicate a very close relationship between Leptarrhena and
Tanakaea, in agreement with the traditional view based
on morphological evidence. Engler (1930) placed the two
genera in their own subtribe, Leptarrheninae (tribe Lep-
tarrheneae of Schulze-Menz, 1964), based primarily on
their unusual mode of anther dehiscence and character-
istic thick foliage. Cytological studies demonstrated that
both of these monotypic genera have 2n = 14 and dis-
tinctive karyotypes that differ not only from each other,
but also from all other taxa of Saxifragaceae s.s. so far
analyzed (Soltis, 1984, 1987, 1988).
Saxifraga-Saxifraga is by far the largest and most
taxonomically complex genus of Saxifragaceae s.s. The
genus comprises approximately 300 species grouped in
nine to 16 sections (reviewed in Spongberg, 1972). The
initial broad concept of the genus established by Linnaeus
was questioned by Haworth (1812, 1821; reviewed in
Spongberg, 1972), who divided Saxifraga into 16 genera.
Small (in Small and Rydberg, 1905) followed a similar
strategy, placing the North American species of Saxifraga
in 13 genera. More recently, however, the broad Linnaean
interpretation of Saxifraga has been widely accepted due
to the relatively uniform floral morphology found
throughout the genus (Spongberg, 1972). Species of Sax-
ifraga are characterized by five sepals, five petals, ten
stamens, and a gynoecium of typically two or three carpels
that are free or connate below the middle. However, Sax-
ifraga is extremely diverse in vegetative morphology, pal-
ynology, ecology, and cytology. For example, chromo-
some numbers for the genus range from 2n = 16 to 2n =
120, a striking contrast to the cytological uniformity (typ-
ically 2n = 14) found elsewhere in Saxifragaceae s.s.
Not only are alliances within Saxifraga enigmatic, but
the generic relationships of Saxifraga are also poorly un-
derstood. Genera considered closely allied to Saxifraga
by at least some authors include Bergenia, Leptarrhena,
Heuchera, and Saxifragella (Spongberg, 1972). The latter
genus comprises two species from Tierra del Fuego; un-
fortunately we were unable to obtain material of this genus
because of its isolated and remote distribution.
In this initial effort to elucidate the generic affinities of
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1070 AMERICAN JOURNAL OF BOTANY [Vol. 80
Saxifraga in Saxifragaceae s.s., we included five species
of Saxifraga in our rbcL sequence analysis: S. merten-
siana, S. punctata, and S. integrifolia (section Mi-
cranthes), S. oppositifolia (section Porphyrion), and S.
cernua (section Saxifraga). These taxa represent three of
the 16 sections of Saxifraga recognized by Engler and
Irmscher (1916-1919). The species included in the cpDNA
restriction site analysis (S. arguta, S. ferruginea, S. in-
tegrifolia, S. lyalli, S. mertensiana, S. punctata) all rep-
resent section Micranthes. Significantly, although this rep-
resents only a preliminary survey of Saxifraga,
phylogenetic analyses of both molecular data sets suggest
that Saxifraga may not be a monophyletic group. In the
rbcL sequence analysis, two distinct clades of Saxifraga
species are recognized (Fig. 1), with the two most divergent
species of these two clades (S. cernua and S. punctata)
differing by 82 base substitutions.
Our cpDNA restriction site analysis of Saxifraga spe-
cies provides results similar to those revealed by sequence
data. As noted above, the cpDNA restriction site analysis
of Saxifraga was restricted to those few taxa for which
we could easily obtain more than 20 g of leaf material
(see Materials and Methods for details). The species in-
vestigated in the restriction site portion of the study (S.
arguta, S. ferruginea, S. lyallii, S. mertensiana, and S.
punctata) all represent section Micranthes. Significantly,
however, cpDNA restriction site data agree with rbcL
sequence data by also indicating that S. mertensiana is
well separated from the remaining species of Saxifraga
analyzed (Figs. 1-3), a result that is also found when the
two data sets are combined (Fig. 4). Saxifraga merten-
siana appears as the sister taxon to the Boykinia group
in the results of all analyses. Restriction site data indicated
that the remaining species of Saxifraga analyzed (S. ar-
guta, S. ferruginea, S. lyallii, and S. punctata, for sim-
plicity referred to as the S. punctata clade), form a well-
supported monophyletic assemblage. Members of the S.
punctata clade are well differentiated from S. mertensiana,
as well as from other taxa of Saxifragaceae s.s. The group
is supported by 35 restriction site mutations; the node
supporting this clade differs from S. mertensiana by 104
restriction site mutations. It must be emphasized, how-
ever, that these species were sometimes so divergent from
all other Saxifragaceae s.s. that a precise interpretation of
some mutations separating them from other Saxifragaceae
could not be made. Hence, this is an underestimate of
the divergence of the S. punctata clade.
Thus, this initial molecular analysis of species of Sax-
ifraga raises the possibility that the genus is not mono-
phyletic and strongly suggests that sectional boundaries
(i.e., section Micranthes) do not accurately reflect evo-
lutionary relationships. As noted above, species of this
large, morphologically diverse genus are held together by
similar floral morphology. However, these floral features
may simply be symplesiomorphic in these well-separated
Saxifraga lineages. The enormous divergence of rbcL se-
quences and cpDNA restriction sites detected among this
small sampling of Saxifraga species parallels the tremen-
dous diversity in ecology, cytology, chemistry, palynolo-
gy, and vegetative morphology present in Saxifraga. For
example, in a preliminary flavonoid survey of species of
Saxifraga, Miller and Bohm (1980) encountered great
flavonoid diversity among the several species they studied
and noted that sectional boundaries in the genus seemed
problematic. Hideux and Ferguson (1976) and Ferguson
and Webb (1970) noted tremendous palynological diver-
sity in Saxifraga.
This preliminary molecular analysis of Saxifraga spe-
cies not only underscores the critical need for a detailed
systematic study of the entire genus Saxifraga, but con-
comitantly indicates that molecular data have great po-
tential for resolving phylogenetic relationships in this large,
complex genus. Given that 16 sections of Saxifraga typ-
ically are recognized, molecular data hold great promise
for elucidating relationships within Saxifraga, as well as
ascertaining the affinities of its component taxa to other
members of Saxifragaceae s.s.
The Heuchera group -Molecular data indicate that one
of the best-defined groups of genera in Saxifragaceae s.s.
is the Heuchera group, which comprises Heuchera, Ti-
arella, Mitella, Elmera, Conimitella, Tellima, Litho-
phragma, Bensoniella, and Tolmiea. Both cpDNA re-
striction site data and rbcL sequences strongly support
this alliance. An earlier cpDNA restriction site analysis
not only indicated that this was a monophyletic assem-
blage, but also demonstrated relationships within the
Heuchera group (Soltis et al., 1991); hence, relationships
within this group will not be considered again here.
Molecular data join other lines of evidence that simi-
larly indicate that the genera of the Heuchera group are
a well-supported alliance. These nine genera all possess
parietal placentation and an indeterminate (polytelic) in-
florescence axis (see reviews by Rosendahl, Butters, and
Lakela, 1936; Wells, 1984; Soltis, 1988; Soltis et al., 1991).
These genera are also the hosts of Puccinia rusts, which
occur only rarely elsewhere in Saxifragaceae s.s. (Savile,
1975, 1976). All nine genera of this group also have x =
7, with 2n = 14 common. Furthermore, six genera of the
group are united by chromosome morphology. Conimi-
tella, Lithophragma, most species of Mitella, Tiarella,
Tolmiea, and Heuchera have identical karyotypes not
found elsewhere in Saxifragaceae s.s. (Soltis, 1980b, 1981,
1988; Soltis and Bohm, 1984). The remaining three genera
of the Heuchera group, Elmera, Bensoniella, and Tellima,
all have distinctive karyotypes (Soltis, 1980b, 1981, 1984,
1988).
Chemical, morphological, and cytological evolution -
We reiterate the critical importance for the interpretation
of phylogenetic trees to distinguish between a character
tree (in this case, a gene or genome tree) and a species
tree; the former does not necessarily agree with the actual
evolutionary pathways of the species under investigation.
Ultimately, the phylogenies presented here based on the
chloroplast genome should be compared to phylogenies
based on nuclear data. With this in mind, we continue
with a general analysis of character evolution in Saxifra-
gaceae s.s.
The utility of restriction site analysis of chloroplast
DNA (cpDNA) and comparative rbcL sequences for re-
constructing phylogenetic relationships has been well re-
viewed (e.g., Palmer, 1985, 1987; Birky, 1988; Palmer et
al., 1988; Clegg, 1989; Crawford, 1990; Clegg and Zu-
rawski, 1992; numerous chapters in Soltis, Soltis, and
Doyle, 1992). As recently noted by several investigators
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September 1993] SOLTIS ET AL. - SAXIFRAGACEAE SENSU STRICTO 1071
Astilbe (2)
Rodgersia (4)
Darmera peltata
Astilboides tabularis
Mukdenia rosii
Bergenia cordifolia
Saxifraga (4)
Saxifraga mertensiana
Jepsonia parryi
Telesonix heucheriformis
Bolandra (2)
Suksdorfia violacea
Suksdorfia ranunculifolia
Boykinia (4)
Sullivantia oregana
Tanakaea radicans
Leptarrhena pyrolifolia
Conimitella williamsii
Mitella (2)
Tellima grandiflora
EJ Heuchera (4)
Tiarella cordifolia
Tiarella trifoliata o
Elmera racemosa
Mitella (2)
n U ~~~~~Tellima grandiflora
Heuchera (7)
Mitella (2)
Mitella (2)
Bensoniella oregana
Lithophragma (4)
Tolmiea (2) J
Peltoboykinia tellimoides
Chrysosplenium americanum
0 = 0-methylation | = 6-oxygenation f = Gallylated glycosides
Fig. 5. Distribution within Saxifragaceae s.s. ofthree flavonoid struc-
tural features: 0-methylation, 6-oxygenation, and gallylated glycosides.
The tree shown represents the results of restriction site analysis of chlo-
roplast DNA without character-state weighting (Fig. 2) and has been
modified in two ways: 1) the composition of the Heuchera group is
expanded to represent the more complete results of Soltis et al. (1991);
and 2) several groups of species have been condensed into single lineages
for simplicity (the number of species included in each of these groups
is indicated in parentheses).
(e.g., Crawford, Palmer, and Kobayashi, 1990, 1992; Syts-
ma, Smith, and Berry, 1991; Sytsma and Smith, 1992),
the value of cpDNA restriction site data and other mo-
lecular markers in phylogenetic reconstruction can be two-
fold: monophyletic lineages can be defined and questions
regarding phylogenetic relationships addressed, and a well-
supported molecular phylogeny can be used to address
questions of character evolution. Through the latter ap-
plication, molecular systematics is having a major impact
on diverse fields, including studies of breeding-system
evolution, biogeography, and the evolution of secondary
compounds and morphological characters. This two-step
process has been applied to several genera, families, and
even subclasses of angiosperms for which detailed bio-
systematic data sets were previously available (e.g., Co-
reopsis, Crawford, Palmer, and Kobayashi, 1990, 1992;
Zea, Doebley, 1990; Microseris, Wallace and Jansen, 1990;
Clarkia, Sytsma, Smith, and Berry, 1991; Sytsma and
Smith, 1992; Salix, Brunsfeld, Soltis, and Soltis, 1992;
Leguminosae, Doyle, Lavin, and Bruneau, 1992; Com-
positae, Jansen et al., 1992; Asteridae, Olmstead et al.,
1992). Below, we present insights gained into flavonoid,
morphological, and cytological evolution based on our
phylogenetic trees.
Astilbe (2)
Rodgersia (4)
Darmerapeltata
Astilboides tabularis
Mukdenia rosii
Bergenia cordifolia
Saxifraga (4)
Saxifraga mertensiana
Jepsoniaparryi
Telesonix heucheriformis
Bolandra (2)
Suksdorfia violacea
Suksdorfia ranunculifolia
Boykinia (4)
Sullivantia oregana
Tanakaea radicans
Leptarrhena pyrolifolia
Conimitella williamsii
Mitella (2)
Tellima grandiflora 1
Heuchera (4)
Tiarella cordifolia
Tiarella trifoliata Z
Elmera racemosa
Mitella (2)
Tellima grandiflora s
Heuchera (7)
Mitella (2)
Mitella (2)
Bensoniella oregana
Lithophragma (4)
Tolmiea (2)
Peltoboykinia tellimoides
| | Chrysosplentium americanum
0-methylation: | =Gain 6-oxygenation: =Gain Gallylated Ii =Gain
=Loss glycosides: 41 =Loss
Fig. 6. Most parsimonious evolutionary interpretations for the dis-
tribution in Saxifragaceae s.s. of the three chemical features 0-methyl-
ation, 6-oxygenation, and gallylated glycosides (see Fig. 4). With PAUP
version 3.0s, the tree topology of Fig. 4 was specified as a constraint
tree, then each chemical feature was analyzed separately as a two-state
(present or absent) character. The results shown for each feature represent
the fewest steps possible (gains and losses) to explain the distribution
given this topology. For both 6-oxygenation and gallylated glycosides a
second most parsimonious result was found (not shown); for both of
these features the second explanation consisted of multiple independent
gains with no losses.
Chemical evolution -Saxifragaceae s.s. may be one of
the best-studied groups of flowering plants for flavonoid
chemistry through the work of Bohm and co-workers.
The chemical data are, in fact, so extensive that it is
beyond the scope of the present investigation to discuss
these data exhaustively from a phylogenetic perspective.
Using our molecular-based phylogenetic hypotheses we
will stress one fundamental theme: the apparent multiple
origins and losses of several different classes of flavonoid
compounds in diverse lineages within Saxifragaceae s.s.
Below, we discuss several classes of flavonoids with this
theme in mind, and for three of these (0-methylation,
6-oxygenation, gallylated glycosylation) present distri-
bution (Fig. 5) and possible evolutionary interpretations
(Fig. 6). These three classes were chosen for more detailed
discussion because they illustrate well the complex evo-
lutionary history of flavonoids.
The major flavonoids of Saxifragaceae s.s. are kaemp-
ferol, quercetin, and myricetin, all of which occur as 3-0-
mono-, 3-0-di-, 3-0-tri- and 3,7-0-triglycosides based
on various combinations of glucose, galactose, arabinose,
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1072 AMERICAN JOURNAL OF BOTANY [Vol. 80
xylose, and rhamnose (e.g., Miller and Bohm, 1980; Nich-
olls and Bohm, 1984; Bohm and Bhat, 1985; Bohm, Do-
nevan, and Bhat, 1986, and references therein). Based on
our molecular phylogenies, 0-methylation is a structural
feature that has clearly evolved several times in Saxifra-
gaceae s.s. Simple 0-methylated flavonoids (e.g., isor-
hamnetin = 3'-O-methylquercetin,7-O-methylgenetin) are
absent in the closely related Itea. Within Saxifragaceae
s.s. these compounds have been reported from some
members of the Heuchera group, including Heuchera
(Bohm and Wilkins, 1978; Wells and Bohm, 1980), Tol-
miea menziesii (Soltis and Bohm, 1986), most species of
Lithophragma (Nicholls and Bohm, 1984), and Mitella
diphylla and M. nuda (Nicholls, Bohm, and Wells, 1986).
These compounds are absent, however, from most species
of Mitella, as well as from other genera of the Heuchera
group (i.e., Conimitella, Bensoniella, Elmera, Tellima,
and Tiarella). Isorhamnetin appears again in Leptarrhena
(Miller and Bohm, 1979). In Chrysosplenium, 0-meth-
ylated flavonoids are also apparent, but in this genus, large
quantities and a diverse array of these compounds are
present (Bohm, Collins, and Bose, 1977; Bohm and Col-
lins, 1979). In fact, 0-methylated flavones are the major
flavonoid constituents of most species of Chrysosplenium.
0-methylation is also common in members of the Boy-
kinia group, including species of Boykinia, Suksdorfia,
Bolandra, Sullivantia, and one of two species of Telesonix
(Gornall and Bohm, 1980; Soltis, 1980a). However,
0-methylation is not apparent in Jepsonia (Bohm and
Ornduff, 1978). 0-methylation also has been reported
from several species of Saxifraga, including S. integrifolia,
a species included in our molecular analysis. The most
parsimonious explanation for the distribution of these
compounds in Saxifragaceae s.s. necessitates seven in-
dependent gains (Fig. 6). Other, less parsimonious inter-
pretations might additionally involve losses. Whatever
the actual explanation, 0-methylation exhibits a complex
evolutionary history in Saxifragaceae s.s.
Similarly, 6-oxygenation has likely evolved multiple
times based on our DNA-based phylogenetic hypotheses.
This feature, which is absent in Itea, is well developed in
Chrysosplenium, as well as in some members of the Boy-
kinia group. Within the Boykinia group this chemical
theme is present in Telesonix heucheriformis, Bolandra,
Sullivantia, and all species of Boykinia with the exception
of B. intermedia; it is absent from Telesonixjamesii (not
analyzed herein), Jepsonia, and Suksdorfia (Gornall and
Bohm, 1980; Soltis, 1980a) (Fig. 5). 6-oxygenation also
has been reported from Saxifraga caespitosa, which was
not analyzed herein (Miller and Bohm, 1980). Thus,
6-oxygenation likely arose a minimum of two times (and
perhaps three, depending on the phylogenetic position of
S. caespitosa) based on our results, once in the Boykinia
group and once in Chrysosplenium (Fig. 6). In the Boy-
kinia group alone, multiple gains and/or losses of this
structural feature have occurred. According to one most
parsimonious explanation (Fig. 6), for example, if
6 -oxygenation were present in the ancestor of the Boykinia
group, then this pathway was subsequently lost indepen-
dently in Jepsonia, Suksdorfia violacea, and S. ranun-
culifolia.
Gallylated flavonoid glycosides are absent from Itea,
but appear in some, but not all, members of Saxifragaceae
s.s. Within the Heuchera group, these compounds have
been reported from Tellima (Collins and Bohm, 1974;
Collins, Bohm, and Wilkins, 1975), Heuchera (Wilkins
and Bohm, 1976; Bohm and Wilkins, 1978; Wells and
Bohm, 1980), Tolmiea (Bohm, 1979; Soltis and Bohm,
1986), and several species of Mitella from Japan, but none
ofthe North American species (Nicholls, Bohm, and Wells,
1986). However, these compounds are absent from other
members of the Heuchera group of genera, such as Ben-
soniella, Conimitella, Lithophragma, and Tiarella (Nich-
olls and Bohm, 1985; Soltis and Bohm, 1984; Nicholls,
Bohm, and Wells, 1986). Gallylated flavonoid glycosides
have also been reported from Jepsonia of the Boykinia
group (Bohm and Omduff, 1978) and Darmera of the
Darmera group (Bohm and Wilkins, 1976) (Fig. 5). The
most parsimonious explanations for the distribution of
these compounds require either several independent gains,
or in the case of the Heuchera group, a gain followed by
a loss and two regains (Fig. 6).
Flavones typically constitute a small proportion of the
total flavonoid chemistry of members of Saxifragaceae
s.s., whereas only flavones are present in the closely related
Itea. Small amounts and numbers of these compounds
(typically only luteolin) have been reported in several
lineages. In the Heuchera group, luteolin is present in
some, but not all, species of Heuchera (Bohm and Wilkins,
1978; Wells and Bohm, 1980), as well as one of three
species of Tiarella (Soltis and Bohm, 1984), but has not
been found elsewhere in the group. Some members of the
Boykinia group (e.g., Sullivantia, Suksdorfia, and several
species of Boykinia) also produce flavones. However, only
in Sullivantia and Chrysosplenium do flavones play a
dominant role, both in terms of number of compounds
and total concentration (Bohm, Collins, and Bose, 1977;
Bohm and Collins, 1979; Soltis, 1980a). Thus, once again
one explanation for the distribution of flavones would be
independent origins in some members of the Heuchera
group, Boykinia group, and Chrysosplenium. Alterna-
tively, given their presence in Itea, flavones were present
in the ancestor of Saxifragaceae s.s. and have been lost
several times within Saxifragaceae s.s. Under either sce-
nario, the evolutionary history may be complex, involving
multiple gains and/or losses in both the Heuchera and
Boykinia groups.
3,7-0-glycosylation also appears in several lineages of
Saxifragaceae s.s. Within the Boykinia group, this gly-
cosylation pattern is found in all species of Boykinia,
Suksdorfia, and Bolandra investigated (Gomall and Bohm,
1980), but not in Sullivantia or Jepsonia (Bohm and Orn-
duff, 1978; Soltis, 1980a). 3,7-0-glycosylation is also found
in some members of the Heuchera group: in one species
of Lithophragma (Nicholls and Bohm, 1984) and most
species of Heuchera investigated (Wilkins and Bohm, 1976;
Bohm and Wilkins, 1978; Wells and Bohm, 1980). These
compounds are lacking, however, in other members of
the Heuchera group, such as Mitella, Tiarella, Tolmiea,
Elmera, Bensoniella, and Conimitella (Bohm and Wil-
kins, 1978; Bohm, 1979; Nicholls, Bohm, and Wells, 1986;
Soltis and Bohm, 1986). 3,7-0-glycosylation is also found
in Saxifraga. These compounds were detected in S. caes-
pitosa, S. michauxii, and S. ferruginea (the latter species
was included in our cpDNA restriction site phylogenies),
but absent from several other species, including S. inte-
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September 1993] SOLTIS ET AL.-SAXIFRAGACEAE SENSU STRICTO 1073
grifolia (Miller and Bohm, 1979) (also included in mo-
lecular analyses). Again, therefore, a complex evolution-
ary history involving multiple gains and losses of this
biosynthetic pathway must be invoked.
Although most members of Saxifragaceae s.s. are char-
acterized by rich mixtures of flavonoid glycosides, some
taxa are conspicuous because of their extremely simple
flavonoid profiles. These taxa include Peltoboykinia (Gor-
nall and Bohm, 1980), Bergenia (Bohm, Donevan, and
Bhat, 1986), Conimitella (Nicholls, Bohm, and Wells,
1986), and Sullivantia (Soltis, 1 980a). Bergenia, Conimi-
tella, and Sullivantia are members of the Darmera, Heu-
chera, and Boykinia group of genera, respectively. Fla-
vonoid reduction has been considered a dominant
evolutionary trend in the angiosperms, including Saxi-
fragaceae (reviewed in Gornall and Bohm, 1978). Our
DNA phylogenies permit us to place this trend in a more
precise phylogenetic context, supporting at least four ma-
jor occurrences of this tendency in Saxifragaceae s.s. That
is, each of the genera noted above likely represents an
independent case of flavonoid reduction based on our
phylogenetic hypotheses.
Thus, this brief consideration of flavonoid evolution in
Saxifragaceae s.s. reveals a complex evolutionary pattern
involving multiple gains and/or losses of certain structural
features, as well as multiple occurrences of major flavo-
noid reduction trends. These results on a large, generic-
level scale parallel similar evolutionary trends that we
described at the species level in an earlier cpDNA inves-
tigation of Lithophragma (D. Soltis et al., 1992), a member
of the Heuchera group of genera. Within this single genus,
multiple gains and losses of certain flavonoid types were
evident, as were two separate instances of flavonoid re-
duction.
It is noteworthy that even without benefit of the generic-
level phylogenetic hypothesis provided herein, it was ap-
parent to Bohm that multiple gains and losses of certain
flavonoid structural features must have occurred in Sax-
ifragaceae. He referred, for example, to the "apparent
haphazard distribution of both individual flavonoids and
different structural types" in Saxifragaceae s.s. and con-
cluded that "other than in the few situations . . . where
genera are known to be closely related, attempts to apply
flavonoid data broadly to intergeneric relationships with-
in the Saxifragaceae [= Saxifragaceae s.s.] have failed"
(Miller and Bohm, 1980).
Morphological evolution -Several trends in floral re-
duction are apparent in light of our DNA-based phylog-
enies. For example, most species of Saxifragaceae s.s.
standardly have five sepals, as do closely related genera
(e.g., Itea, Pterostemon; Table 6). Three genera of Saxi-
fragaceae s.s. are noteworthy in being characterized by
four sepals, Chrysosplenium, Astilboides of the Darmera
group, and Tolmiea of the Heuchera group (Table 6).
Given the well-separated phylogenetic positions of these
taxa, this distribution almost certainly reflects three sep-
arate instances of floral reduction in Saxifragaceae s.s.
Similarly, five petals also characterize most members
of Saxifragaceae s.s., as well as closely related genera such
as Itea and Pterostemon. However, only four petals are
present in Astilboides and Tolmiea. In Rodgersia, one or
two petals are typical, whereas petals are completely lack-
ing in Chrysosplenium and Tanakaea. The phylogenetic
hypotheses presented herein (Figs. 1-4) suggest that these
genera each represent separate reductions in petal number,
because they clearly are not component members of a
single cohesive lineage.
Ten stamens characterize most genera of Saxifragaceae
s.s. It is hard to determine the likely ancestral state for
this character because one close relative, Itea, has five
stamens, whereas Pterostemon has five stamens and five
staminodes (Table 6). If the ancestral stamen number of
Saxifragaceae s.s. were ten, several independent reduc-
tions in stamen number apparently have occurred (Table
6). In the Boykinia group, for example, Jepsonia and Tele-
sonix have ten stamens. Because these are the sister to
the rest of the Boykinia group and all remaining genera
ofthe Boykinia group have five stamens, stamen reduction
apparently occurred a single time in this group. Stamen
reduction also may have occurred independently in the
Heuchera group. Several genera have exclusively ten sta-
mens (Lithophragma, Tiarella, Tellima). In Mitella, some
species have ten stamens, and others have five. Five sta-
mens also characterize Bensoniella, Elmera, Conimitella,
and Heuchera. Based on the cpDNA phylogeny for the
Heuchera group of genera, it is very likely that the five-
stamen condition evolved multiple times within this group
(Soltis et al., 1991); this hypothesis awaits confirmation,
however, via a nuclear-based phylogeny. Finally, only
three stamens are present in Tolmiea, which thus rep-
resents an additional reduction in stamen number. Al-
ternatively, given that other relatives of Saxifragaceae s.s.
such as Itea have five stamens, multiple instances of sta-
men increase in Saxifragaceae s.s. must be raised as a
possibility. An increase to ten stamens may have occurred
independently in Saxifraga, Peltoboykinia, and some
members of the Boykinia, Heuchera, and Darmera groups.
Under either of these two scenarios, the evolutionary his-
tory of this trait may have been complex, involving mul-
tiple gains and/or losses.
A virtual continuum of ovary positions exists in Sax-
ifragaceae s.s. from superior to inferior (Table 6). Deter-
mining the ancestral state for this character is also difficult
given that Pterostemon and Itea have inferior and superior
ovaries, respectively. If we start with a superior ovary as
the ancestral state, a trend toward hypanthium fusion has
apparently occurred multiple times within Saxifragaceae
s.s. There has been a trend toward complete hypanthium-
ovary fusion in the Boykina group, as well as in the Heu-
chera group; some species in both groups have completely
inferior ovaries (Table 6). It also seems likely that multiple
trends toward ovary fusion have occurred within each of
these groups. For example, just considering the Boykinia
group, multiple events of hypanthium-to-ovary fusion
need to be invoked to explain the distribution of ovary
conditions. A trend toward hypanthium fusion to the
ovary may have occurred separately in Telesonix, Suks-
dorfia violacea, some Sullivantia species, and the ancestor
of the Suksdorfia ranunculifolia-Boykinia clade. Alter-
natively, hypanthium-ovary fusion may have occurred
twice: once in Telesonix and once in the ancestor of the
Sullivantia-Boykinia-Suksdorfia-Bolandra clade, with a
reversal to a superior ovary in Bolandra and some species
of Sullivantia.
Still another probable instance of some hypanthium-
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1074 AMERICAN JOURNAL OF BOTANY [Vol. 80
TABLE 6. Summary of floral morphology and cytological data for genera of Saxifragaceae s.s. and the closely related Itea and Ribes. Under sepals,
petals, and stamens the common number is given first, followed by other numbers in brackets. Under ovary, ovary position is given: S =
superior, I = inferior, subS = some slight fusion of hypanthium to ovary. Haploid chromosome numbers are given for all genera but Saxifraga.
Because so many chromosome numbers are reported for species of Saxifraga, diploid numbers are given. Solid line separates three possible
close relatives (Pterostemon, Itea, and Ribes) from Saxifragaceae s.s. Dotted lines help delineate the well supported groups of genera within
Saxifragaceae s.s.
Chromosome
Taxon Sepals Petals Stamens Ovary number
Pterostemon 5 5 1 Oa I
Itea 5 5 5 5 n =ll
Ribes 5 [4] 5 [4] 5 [4] I n= 8
Astilbe 5 [7-10] 5 10 5 n = 7
Chrysosplenium 4 0 8 [4, 7, or 1 0] S-I n = 11, 12
Peltoboykinia 5 5 10 subS n = 11
Boykinia group
Boykinia 5 5 5 1/3-1/21 n = 7
Sullivantia 5 5 5 S-l1/21 n = 7
Bolandra 5 5 5 s n =7
Jepsonia 5 5 10 5 n= 7
Telesonix 5 5 10 1/21 n = 7
Suksdorfia 5 5 5 1/21-I n = 7
Darmera group
Darmera 5 5 10 SubS n = 17
Bergenia 5 5 10 subS n = 17
Aceriphyllum 5, 6 5, 6 5, 6 subS n = 17
Rodgersia 5-7 [0] 1, 2 [rarely 5] 10 [-14] subS n = 15, 18
Astilboides 4 4 8 (6) subS n = 17
Leptarrhena- Tanakaea
Tanakaea 5 [4, 7] 0 10 5 n =7
Leptarrhena 5 5 10 5 fl = 7
Heuchera group
Lit hophragma 5 5 10 S-I n = 7
Mite/la 5 5 10 [4, 5] S---- n =7
Tiarella 5 5 10 5 n =7
Tolmiea 4 4 [5] 3 5 n =7
Heuchera 5 5 5 -4 n =7
Tellima 5 5 10 1/41 n =7
Conimitella 5 5 5 1/2-1/31 n = 7
Elmera 5 5 5 5 n =7
Bensoniella 5 5 5 subS n = 7
Saxifraga punctata group
Saxifraga integrifolia 5 5 10 b 2n = 38, -56
Saxifraga punctata 5 5 10 b 2n = 28, --70, --76
Saxifraga arguta 5 5 10b
Saxifraga lyalli 5 5 10 b 2n = -58
Saxifraga ferruginea 5 5 10 b 2n = 38
Saxifraga mertensiana group
Saxifraga cernua 5 5 10 b 2n = 36, 50, 60-70,
-60, -64, -66
Saxifraga oppositifolia 5 5 10 b 2n = 26, 52
Saxifraga mertensiana 5 5 10 b 2n = --48, 36, --48-50
aPterostemon has five fertile stamens and five staminodes.
bThe ovary position for the genus Saxifraga ranges from superior to inferior; the ovary position of individual species of Saxifraga is not given.
to-ovary fusion is represented by Saxifraga. The ovary
position in this genus ranges from superior to half-inferior.
Given that Saxifraga is large, morphologically diverse,
and may not be monophyletic based on DNA data pre-
sented herein, it is likely that this trend has occurred
multiple times in this genus alone. This hypothesis awaits
testing following a detailed molecular phylogenetic anal-
ysis of Saxifraga. These results for Saxifragaceae s.s. re-
garding multiple fusion of hypanthium to ovary parallel
the results reported in our cpDNA analysis of Litho-
phragma (D. Soltis et al., 1992), a member ofthe Heuchera
group, in which at least two instances of hypanthium-
ovary fusion occurred within this genus alone.
Other morphological features similarly appear to have
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September 1993] SOLTIS ET AL. - SAXIFRAGACEAE SENSU STRICTO 1075
arisen multiple times in Saxifragaceae s.s. Two placen-
tation types occur in Saxifragaceae s.s.: parietal placen-
tation, present in Chrysosplenium and all members of the
Heuchera group, and axile placentation, found in all other
Saxifragaceae s.s. Again, it is difficult to establish the likely
ancestral state because Itea and Pterostemon possess pa-
rietal and axile placentation, respectively. If we assume
that axile placentation is ancestral in the family, our phy-
logenetic trees suggest two possible independent origins
of parietal placentation, once in Chrysosplenium and once
in the ancestor of the Heuchera group. Alternatively, if
parietal placentation is considered ancestral, several in-
dependent origins of axile placenation are suggested (Figs.
2, 3).
Cytological evolution -The DNA-based phylogenetic
trees also provide insights into cytological evolution in
Saxifragaceae s.s. Although by far most members of the
family have x = 7, x = 17 characterizes most members
of the Darmera group, x = 11 is found in Peltoboykinia,
and x = 11 and 12 are found in Chrysosplenium; in the
cytologically complex Saxifraga, x = 9-14 have been
reported. If x = 11 is primitive (this base number occurs
in Itea), then x = 7 may have evolved multiple times in
Saxifragaceae s.s. if Astilbe (with x = 7) is truly the sister
to the rest of the genus, as restriction site data suggest.
Alternatively, if we begin with x = 7 as ancestral, evo-
lutionary trends to higher base chromosome numbers must
have occurred on multiple occasions in Saxifragaceae s.s.,
although it is difficult to establish how many times due
to poorly resolved basal phylogenetic branches for the
family. For example, the relationship between Peltoboy-
kinia and Chrysosplenium is not well resolved, so the base
numbers of x = 11 and x = 1 1, 12, respectively, in these
genera may reflect a single origin, or perhaps two separate
origins.
DNA evidence for the monophyly ofthe Darmera group
suggests a single origin of x = 17, which characterizes four
of the five genera in this group (Darmera, Astilboides,
Mukdenia, and Bergenia). Because DNA evidence firmly
establishes a close relationship between Rodgersia and the
above taxa, the base chromosome numbers of x = 15 and
18 in this genus are better understood. These numbers
most likely reflect aneuploid decrease and increase, re-
spectively, from an original number of x = 17. The origin
of x = 17 in the Darmera group is still unknown. It may
represent ancient polyploidy or substantial aneuploid in-
crease from either x = 11 or x = 7.
The incredible cytological complexity of Saxifraga must
also be viewed from a new perspective given DNA evi-
dence for the polyphyly of the genus. Regardless of wheth-
er x = 7 or 11 is primitive, it is likely that multiple events
of aneuploid (and perhaps polyploid) increase have oc-
curred, given that two distinct clades of Saxifraga species
are present (Figs. 1-4); the Saxifraga species included
herein have a diverse array of chromosome numbers (Ta-
ble 6).
Conclusions-Sequence data for the chloroplast gene
rbcL and cpDNA restriction site data have circumscribed
a natural group of genera that we feel best represents
Saxifragaceae. These data agree with the taxonomic dis-
tribution of the loss of the intron for the chloroplast gene
rp/2 in delimiting a well-defined Saxifragaceae s.s. that
agrees with Takhtajan's (1987) view of the family. These
taxa also are distinguished from closely related genera
(e.g., Ribes, Itea, Pterostemon) by morphological, paly-
nological, anatomical, and chemical features.
Molecular data have also helped to resolve generic re-
lationships within Saxifragaceae s.s. Both rbcL sequence
data and cpDNA restriction site data recognize the Heu-
chera, Boykinia, and Leptarrhena-Tanakaea groups of
genera, and also suggest that Saxifraga may not be mono-
phyletic. Thus, although rbcL sequences have typically
been used at the family level and above, this study illus-
trates the potential of rbcL to elucidate phylogenetic re-
lationships among closely related genera of flowering
plants. In contrast, cpDNA restriction site data, but not
rbcL sequences, recognized the Darmera group of genera.
This apparent discrepancy probably reflects the lower lim-
its of resolution of rbcL sequence data in the phylogenetic
reconstruction of Saxifragaceae s.s.
A large number of poorly resolved basal phylogenetic
branches exists in the phylogenies obtained based on both
rbcL sequences and cpDNA restriction site data. These
results suggest that Saxifragaceae s.s. radiated rapidly in
its early evolutionary history. This finding is in agreement
with the hypothesis of Savile (1975), who, based on the
detailed examination of rust parasites (Puccinia) that use
some Saxifragaceae as hosts, concluded that following a
probable origin in Asia, Saxifragaceae s.s. radiated rap-
idly.
Molecular data have also clarified relationships within
the Boykinia group. Both rbcL sequences and cpDNA
restriction site data indicate clearly that Jepsonia is a
member of this group of genera. The enigmatic Jepsonia
was long considered to have no close allies in Saxifra-
gaceae; however, restriction site data indicate that its sister
taxon is Telesonix. Within the Boykinia group, cpDNA
restriction site data also indicate that Suksdorfia (sensu
Gornall and Bohm, 19 8 5) is not monophyletic. Suksdorfia
ranunculifolia is the closest ally of Boykinia, whereas S.
violacea is the sister to Bolandra.
The DNA-based phylogenetic hypotheses generated also
permitted us to address questions of character evolution
in Saxifragaceae s.s. A wealth of detailed flavonoid chem-
ical data are available for this group. Although we con-
sidered only a few classes of compounds, it is clear that
our phylogenetic trees suggest a complex evolutionary
history of chemical characters in the family, often in-
volving numerous gains and/or losses of particular struc-
tural features (e.g., 6-oxygenation, 0-methylation, fla-
vones, 3,7-0-glycosylation, gallylation). In addition, major
trends in flavonoid reduction (loss of compounds and
flavonoid complexity) occurred at least four times within
the family. These results on a large, generic-level scale
parallel similar chemical evolutionary trends observed at
the species level in a cpDNA investigation of Lithophrag-
ma (D. Soltis et al., 1992), a member of the Heuchera
group of genera.
Several trends in floral reduction are also apparent in
light of the DNA-based phylogenies. Our data suggest
several independent instances of reduction in stamen
number, petal loss, and hypanthium-ovary fusion in Sax-
ifragaceae s.s. Given that these sometimes represent key
taxonomic characters, the taxonomic problems at the ge-
neric level in Saxifragaceae s.s. are, in part, better un-
derstood.
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1076 AMERICAN JOURNAL OF BOTANY [Vol. 80
This initial molecular analysis of the large (approxi-
mately 300 species) and taxonomically and cytologically
complex Saxifraga also underscores the critical need for
a detailed molecular systematic study of more represen-
tatives of this genus. We found considerable rbcL se-
quence divergence and cpDNA restriction site differences
among just nine species of the genus; furthermore, both
data sets suggest that the genus is not monophyletic. Mo-
lecular data hold great promise for elucidating relation-
ships within Saxifraga, as well as ascertaining the affinities
of its component taxa to other genera of Saxifragaceae
s.s.
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APPENDIX. Matrix of restriction site data used in analysis of Saxifragaceae s.s. Included in the matrix are 311 mutations that were each present
in two or more taxa; autapomorphies (G = gains, L = losses) are indicated at the end of the restriction site data for each taxon in which they
occurred. A complete list of mutations, including locations and fragment sizes, is available from the authors.
Outgroup
0001000000000000111100000001101100000001011111000011010001011110111101110110101010
0100001000110111001110101001100010011010010101011000001100101110000000011101111001
0001000010001001100111111101001001100100100111000000010110111110o00011100000010010
10111111110010001000000100101111101010000010101101000001000111101
Astilbe taquetii
0011001000010001001110000000000100011001010111010001110001010111001101100o11100010
1100001100000111000010101101001000001010010101011001001110000110001000001111111001
0001100010001111101111110100000001110100101101000000011110101101000011000000011011
10110010110010000000101000100101101010000000101101000001000111101 OG OL
Astilbe microphylla
0011001000010001001110000000000100011001010111010001110001010111001101100111100010
1100001100000111000010101101001000001010010101011001001110000110001000001111111001
0001100010001111101111110100000001110100101101000000011110101101000011000000011011
10110010110010000000101000100101101010000000101101000001000111101 1G OL
Rodgersia podophylla
0011001100010000101110010000100100001011010111010000110001010110011111111111100011
0100001001000111010010001000000000011010o10oioioiiooiooiiioooooioooiooooioiiiiiiooi
00011000110011111001111111o010100011010010o101100000110110111011000010011000010l1
10100011110001100000101100100100011000000010101101000001000111101 OG 1L
Rodgersia sambucifolia
0011001100010000101110010000100100001011010111010000110001010110011111111111100011
0100001001000111010010001000000000011010010101011001001110000010001000010l1ll1001
00011000110011111001111111o010100011010010o101100000110110111011000010011000010l1
10100011110001100001101100100100011000000010101101000001000111101 OG OL
Rodgersia pinnata
001100110001000010111001000010010000101101011101000011000101011001i111111111100011
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September 1993] SOLTIS ET AL. - SAXIFRAGACEAE SENSU STRICTO 1079
0100001001000111010010001000000000011010010101011001001110000010001000010111111001
0001100011001111100111111101011000110100101101100000110110111011000010011000010111
10100011110001100001101100100100011000000010101101000001000111101 OG OL
Rodgersia aesculifolia
0011001100010000101110010000100100001011010111010000110001010110011111111111100011
0000001001000111010010001000000000011010010101011001001110000010001000010111111001
0001100011001111100111111101011000110100101101100000110110111011000010011000010111
10100011110001100001101100100100011000000010101101000001000111101 OG OL
Darmera peltata
0011001000010000001110010000100100001011010111010000110001010110011111111111100011
0100001000000111000010001001000000011010010101011001001110000110001000010111111001
0001100010001111100111111101011000110100101101000000010100111011000010011000010011
10100011110001100001101100100100011100000010001101000001000111101 3G 2L
Mukdenia rosii
00 1100100 00 10 0001011100 00000100100 111011010111010000110001010110011111111111100011
0100001000000111000010001001000000011010010101011001001110000110001000010111111001
0001100010001111100111111101011000110100101101001000010110111111000010001000000110
10100011110001100000101100100100011000000010101101000001000111101 12G 4L
Astilboides tabularis
0011001000010000101110010000100100001011010111010000110001010110011111111111000011
0100001000000111000010001001000000011010010101011001001110000110001000010111111001
0001100010001111100110111101010000110100101101000000010110111011000010011000010111
10100011110001100000101100100100011000000010101101000001000111101 OG 1L
Bergenia cordifolia
0011001000010000101110000000100100011011010111010000110001010110011111111111000011
0100001000000111000010001001000000011110010101011001001110000110001000010111111001
0001100010001111100110111101010000110100101101000000010110111111000110001000000111
10100011110001100000101100100100001000000010111101000001000111101 8G 8L
Saxifraga mertensiana
00??????00011000?0111000000010010000?00??????101?0?11100???????00111?11???11100??0
??000?10??100111000?1010100100000001001001??0101100000111?00011000?0000100?????011
10001000100011111101???1110100?00111010010111100100001010011???100011100??0?0?0?10
101000?1110???101000l?1110100?1?10100?000110111101000001000111101 26G 13L
Chrysosplenium americanum
0011001000010000101110000000100100001001010111010001110001010110011111110111100010
0100001000000111000010101000000000011010010101011001001110000110001000010111110001
0001100010001111100111111101001000110100101111000000010110111111000011001000010011
10110010110001101000101100100101001000000010101101000001000111101 16G 9L
Peltoboykinia tellimoides
0011001000010000101110000000100100001001010111010001110101010110011111110111100010
0100001000000111000010001001000000011010010101011001001110000110001000010111111001
0001100010001110100110111101001100110100101111000000010110111111000011001000010011
10110011110001101000101100100101001000000110101101000001000111101 28G ilL
Tanakaea radicans
0011001000010000101010000000100100001001000110010001110000010110011111110011100011
0100001000000111000010101001000100001010010101111001101110000110001110010111111001
0001100010001111100101111101100000110100101101000001010110111111000111000000010111
10100010110001101001101100100111001000000010101101000001000111101 4G 1L
Leptarrhena pyrolifolia
0011001000010000101010000000100100001001000110010001110000010110011111110011100011
0100001000000111000010101001000100001110010101111001101110000110001110010111111001
0001100010001111100101111101100000110100101101001001010110111111000111001000010111
10100010110001101000101100100111001000000010101101000001000111101 1G OL
Jepsonia parryi
0011001001011010101111001010110010001001010111011101111101110010011110010111100001
01001000000001110000110 0100000010001001011'0101011001011111000010011000110111101001
1101110010011110000011111001001100110000101110010000010110011111100101001011010111
10110011110001101000101010000111001001000110101101000001000111101 8G 4L
Bolandra oregana 11668
0011001001011010101111001010110010001001010111011101111101110010011110010111100001
0100100000000010000011101001000100010011,110000011100011111000110011000110010111010
10001100100011110000101111000011