Int. J. Plant Sci. 160(3):585–594. 1999.

q 1999 by The University of Chicago. All rights reserved.

1058-5893/99/6003-0016$03.00

PHYLOGENY OF SELAGINELLACEAE: EVALUATION OF GENERIC/SUBGENERIC

RELATIONSHIPS BASED ON rbcL GENE SEQUENCES
Petra Korall,1 Paul Kenrick,2 and James P. Therrien3
Department of Botany, Stockholm University, S-106 91 Stockholm, Sweden; Department of Palaeontology, Natural History Museum, Cromwell
Road, London SW7 5BD, United Kingdom; and Department of Botany, University of Kansas, Lawrence, Kansas 66045-2106, U.S.A.

A cladistic analysis based on rbcL gene sequences from a representative sample of 18 species yields three
most parsimonious trees that strongly support monophyly of Selaginellaceae. Within Selaginellaceae, the mor-
phologically distinctive subgenus Selaginella is resolved as sister group to a clade composed of all other species,
here termed the rhizophoric clade. In the rhizophoric clade, subgenus Stachygynandrum is paraphyletic to
subgenera Ericetorum, Tetragonostachys, and Heterostachys. Monophyly of Ericetorum and Tetragonostachys
is strongly corroborated. Results support a close relationship between “resurrection plants” in Stachygyn-
andrum and the mat-forming or tufted drought-tolerant species of Tetragonostachys, indicating a common
origin of xerophytism in these groups. A close relationship for all isophyllous species, as hypothesized in many
classifications, is not supported by the rbcL data. Leaf isophylly and reduction in Ericetorum and Tetrago-
nostachys most probably represent independent reversals of the marked anisophyllous condition in Stachy-
gynandrum. Leaf reduction is one of a suite of characters that may have evolved in response to seasonal
drought.

Keywords: systematics, Selaginellaceae, rbcL, phylogeny, xerophyte.

Introduction species diversity, in particular, the origins of the diverse xer-

Selaginellaceae are an ancient group of fern allies composed
of an estimated 750 living species. Greatest diversity occurs in
lowland to midmontane primary tropical rain forest, but this
cosmopolitan family is also widely distributed in subtropical,
temperate, montane, and rarely subarctic regions. Most species
are characterized by strongly flattened, frondlike branching
and dimorphic leaves (microphylls). These and other unique
features of the epidermis (e.g., iridescence, lens-shaped epi-
dermal cells, presence of chloroplasts; Hébant and Lee 1984)
are probably adaptations to poor light quality—a consequence
of life on the forest floor. Species may be scandent or climbing
on rocks and trees, but, in contrast to the closely related Ly-
copodiaceae, very few are obligate epiphytes. Some Selaginel-
laceae are able to survive extended periods of drought, and
several strategies for drought tolerance (xerophytism) have
evolved. Most xerophytic species have a mat-forming or tufted
habit and thick cutinized leaves that taper to a fine hair point.
Others, such as the “resurrection plants,” have the ability to
roll up leaves and stems to prevent excessive water loss and
to reverse this process on rehydration. A few are annuals that
grow and reproduce rapidly during a short wet season. Despite
a lengthy fossil record, the evolution of the family is poorly
understood. The absence of an explicit phylogenetic treatment
is a major obstacle to investigating the evolution of modern
1
Author for correspondence and reprints; fax 46-8-16-25-77; e-mail
petra.korall@botan.su.se.
2
E-mail P.Kenrick@nhm.ac.uk.
3
E-mail fernally@ukans.edu.
Manuscript received October 1998; revised manuscript received December 1998.
ophytic and humid tropical groups. Here we present a prelim-
inary phylogenetic analysis based on chloroplast rbcL gene
sequences as a first step toward developing a detailed phylo-
genetic framework for the family.
Previous systematic studies have identified several major
groups within Selaginellaceae, and our usage is based on the
classification by Jermy (1986; fig. 1), which recognizes five
subgenera in Selaginella: Selaginella, Ericetorum, Tetragonos-
tachys, Stachygynandrum, and Heterostachys. A basic di-
chotomy between isophyllous and anisophyllous species is a
feature of some early taxonomies (e.g., Homeophyllum/Het-
erophyllum; Spring 1850; Hieronymus 1901), and this char-
acteristic is still an important element of more recent classi-
fications (Walton and Alston 1938; Tryon and Tryon 1981;
Jermy 1986). The importance of leaf dimorphism as a diag-
nostic character is further reinforced by its early (Moscovian;
Late Carboniferous) occurrence in the fossil record (Thomas
and Quansah 1991). Among isophyllous species, S. selagino-
ides and the very similar Hawaiian endemic S. deflexa are
consistently recognized as distinct from other species, based
on helical phyllotaxy, similarity of leaf and sporophyll, and
the erect growth habit with stems emerging from a basal node
(Selaginella sensu Jermy 1986; fig. 1). Approximately 50 spe-
cies of mat-forming or tufted xerophytes that inhabit regions
characterized by prolonged seasonal drought are grouped in
Tetragonostachys (sensu Jermy 1986; fig. 1), morphologically
distinguished by tetrastichous sporophylls. Ericetorum (sensu
Jermy 1986; fig. 1) is a small group that contains three pro-
teaceous heathland species from Australia, Tasmania, and
South Africa. The species are characterized by decussately ar-
ranged leaves and tetrastichous sporophylls. Most species of

585
586 INTERNATIONAL JOURNAL OF PLANT SCIENCES
Fig. 1 A comparison of three widely cited classifications of Selaginellaceae. Our usage is based on Jermy’s classification, which recognizes
one genus (Selaginella) and five subgenera: Selaginella, Ericetorum, Tetragonostachys, Stachygynandrum, and Heterostachys. The rank of these
taxa referred to in the text is subgeneric, unless otherwise stated.
Selaginellaceae are anisophyllous, and there is little agreement
over taxonomic division within this group (e.g., Heterophyl-
lum: Hieronymus 1901; fig. 1; Stachygynandrum, Homos-
tachys, Heterostachys: Walton and Alston 1938). Jermy (1986)
recognizes two subgroups, Stachygynandrum and Heteros-
tachys. Subgroups have been established on strobilus mor-
phology, general plant habit, and stem anatomy (Spring 1850;
Braun 1865; Baker 1887; Hieronymus 1901; Walton and Al-
ston 1938; Jermy 1986). Stachygynandrum (sensu Jermy 1986)
is the largest group (ca. 600 species) and is characterized by
isomorphic sporophylls. Approximately 60 species with di-
morphic sporophylls are classified in Heterostachys (Walton
and Alston 1938; Jermy 1986). Within Stachygynandrum, one
of the most widely recognized and distinctive groups is Arti-
culatae with ca. 50 species (Spring 1850). This group contains
species with both conspicuous nodes below stem dichotomies
and rhizophores that emerge from the upper surface of the
stem and loop downward. Most Articulatae are Neotropical,
but the group also contains the widely grown S. kraussiana
(southern Africa) as well as at least one Southeast Asian spe-
cies, S. remotifolia.
The aim of this article is to develop an outline phylogenetic
framework for Selaginellaceae based on sequence data from
the chloroplast rbcL gene. These data are used to investigate
the relationships of major taxonomic units and to begin to
address questions on the origins of xerophytic, humid tropical,
and temperate woodland species. A secondary aim is to eval-
uate the utility of rbcL sequences as a phylogenetic tool within
this ancient family.
Material and Methods
Choice of Taxa
Ingroup. In this preliminary study, 18 species were se-
lected to represent the major taxonomic units recognized in
recent classifications (fig. 1; table 1). Choice of taxa was also
influenced by growth environment and included xerophytic,
humid tropical, and temperate woodland species. Prior to this
study, the rbcL gene had been sequenced for two species of
Selaginellaceae: S. kraussiana (S. apoda, Manhart 1994) and
S. selaginoides (Wikström and Kenrick 1997). Isophyllous spe-
cies included representatives of Selaginella, Ericetorum, and
Tetragonostachys (sensu Jermy 1986). Subgenus Selaginella
contains two species, the widespread Northern Hemisphere S.
selaginoides and the Hawaiian endemic S. deflexa, both of
which were sequenced for the analysis. Two (S. uliginosa, S.
gracillima) of the three known species of Ericetorum were
included. Selaginella gracillima is also interesting because it is
one of the few annuals in Selaginellaceae. Tetragonostachys
was represented by three North American xerophytes: S. ru-
pestris, S. arizonica, and S. rupincola. Anisophyllous species
included representatives of Stachygynandrum and Heteros-
tachys (sensu Jermy 1986). Stachygynandrum is here repre-
 KORALL ET AL.—PHYLOGENY OF SELAGINELLACEAE 587

Table 1
Sources of Plant Material, Previously Published Sequences, and Accession Numbers in the EMBL Sequence Database
for the Species Included in This Article
Species DNA source/voucher Accession number/reference
Selaginella apoda (L.) Fern.a .................... Z. E. Murrell 6484 (S) AJ010854
S. arizonica Maxonb ............................. T. Eriksson 13/10 1996 (S) AJ010851
S. bombycina Springb ............................ Korall 1997:31 (S) AJ010848
S. deflexa Brackenridgeb ........................ D. Palmer 2651 (KANU) AF093253
S. denticulata (L.) Springa ....................... Korall & Eriksson TE 715 (S) AJ010853
S. diffusa (Presl) Springb ........................ Korall 1997:33 (S) AJ010852
S. exaltata (Kunze) Springa ..................... Korall 1996:1 (S) AJ010849
S. gracillima (Kunze) Springc ................... Nordenstam & Anderberg 1124 (S) AJ010844
S. haematodes (Kunze) Springa ................. Korall 1996:15b (S) AJ010846
S. kraussiana (Kunze) A. Br.b ................... Korall 1997:30 (S) AJ010845
S. lepidophylla (Hook. & Grev.) Springb ...... Therrien 1996:s.n. (KANU) AF093254
S. moellendorfii Hieron.b ........................ Renzaglia s.n. (KANU) AF093256
S. moritziana Spring ex Klotzscha .............. Korall 1996:12 (S) AJ010856
S. pulcherrima Liebm. ex Fourn.b .............. Korall 1997:32 (S) AJ010847
S. rupestris (L.) Springb ......................... Therrien 1996:356 (KANU) AF093255
S. rupincola Underw.b ........................... T. Eriksson 14/10 1996 (S) AJ010850
S. selaginoides (L.) Link ........................ T. Eriksson 704 (S) Y07940
S. uliginosa (Labill.) Springa .................... Gray, Holmgren & Wanntorp 523 (S) AJ010843
Andreaea rupestris Hedw. ...................... Renzaglia 864 (ETSU) L13473 (Manhart 1994)
Angiopteris evecta (G. Forst) Hoffm. .......... Nagata 12/20/88 (TAMU) L11052 (Manhart 1994)
Ephedra tweediana C. A. Mey ................. ) L12677 (Chase et al. 1993)
Equisetum arvense L. ........................... Manhart 05/29/87-1 (TAMU) L11053 (Manhart 1994)
Huperzia campiana B. Øllg. .................... B. Øllgaard 100612 (AAU) X98282 (Wikström and Kenrick 1997)
H. cumingii (Nessel) Holub .................... B. Øllgaard 100836 (AAU) Y07930 (Wikström and Kenrick 1997)
H. hippuridea (Christ) Holub .................. B. Øllgaard 100619 (AAU) Y07931 (Wikström and Kenrick 1997)
H. linifolia (L.) Trevisan ........................ B. Øllgaard 100594 (AAU) Y07932 (Wikström and Kenrick 1997)
H. selago (L.) C. Martius & Schrank ......... N. Wikström 36 (S) Y07934 (Wikström and Kenrick 1997)
H. wilsonii (L.) Underw. & F. Lloyd .......... B. Øllgaard 100611 (AAU) Y07933 (Wikström and Kenrick 1997)
Isoetes lacustris L.b .............................. ) AJ010855
I. melanopoda (Gay & Durieu)
ex. Durieu ..................................... Manhart 03/10/88-1 (TAMU) L11054 (Manhart 1994)
Lycopodiella alopecuroides (L.) Cranfill ...... B. Øllgaard 100822 (AAU) Y07937 (Wikström and Kenrick 1997)
L. inundata (L.) Holub .......................... H.-E. Wanntorp & N. Wikström 19/7 1995 (S) Y07938 (Wikström and Kenrick 1997)
Lycopodium clavatum L. ....................... N. Wikström 164 (S) Y07936 (Wikström and Kenrick 1997)
L. digitatum A. Braun .......................... Manhart 06/12/88-1 (TAMU) L11055 (Manhart 1994)
L. obscurum L. .................................. T. Eriksson 666 (GH) Y07935 (Wikström and Kenrick 1997)
Marchantia polymorpha L. ..................... ) X04465 (Ohyama et al. 1986)
Phylloglossum drummondii Kunze ............. ) Y07939 (Wikström and Kenrick 1997)
Pinus edulis Engelm. ............................ ) X58137
Pseudotsuga menziesii (Mirb.) Franco ......... ) X52937 (Hipkins et al. 1990)
Psilotum nudum (L.) P. Beauv. ................. Manhart 04/06/88-1 (TAMU) L11059 (Manhart 1994)
Sphagnum palustre L. ........................... Renzaglia 752 (ETSU) L13485 (Manhart 1994)
Zamia inermis Vovides, Rees &
Vázquez-Torres ................................ ) L12683 (Chase et al. 1993)
Note. Voucher location abbreviations follow Index Herbariorum (Holmgren et al. 1990). Ellipses indicate that information on voucher
specimens is missing.
a
DNA extracted from silica gel–dried material.
b
DNA extracted from fresh material.
c
DNA extracted from herbarium material.

sented by nine species, three of which are in subgroup Arti-
culatae. Taxa were chosen from the humid tropics (S.
bombycina, S. diffusa, S. exaltata, S. haematodes, S. morit-
ziana, S. pulcherrima), subtropics (S. kraussiana, S. moellen-
dorfii), temperate woodland (S. apoda, S. denticulata), and
seasonally arid areas (S. lepidophylla: resurrection plant). The
North American species S. apoda was resequenced because the
source for the previously published sequence (Manhart 1994)
was a misidentified specimen of S. kraussiana (voucher
examined).
Outgroup. Twenty-four outgroup species were chosen to
represent other lycopsids and major land-plant groups. Het-
erosporous lycopsids were represented by two species of Is-
oetes (I. melanopoda, I. lacustris). Twelve species of homo-
sporous lycopsid were included to represent the major groups
of Lycopodiaceae (Phylloglossum drummondii, Huperzia, Ly-
588 INTERNATIONAL JOURNAL OF PLANT SCIENCES

copodiella, Lycopodium). Ferns and sphenopsids were repre-
sented by Angiopteris evecta (Marattiaceae) and Equisetum
arvense (Equisetaceae). The enigmatic Psilotum nudum (Psi-
lotaceae) was included because of its putative basal position
within ferns (Stevenson and Loconte 1996). Seed plants were
represented by Pseudotsuga menziesii (Pinaceae), Pinus edulis
(Pinaceae), Zamia inermis (Cycadales), and Ephedra tweedi-
ana (Gnetaceae). Three bryophytes were included: the liver-
wort Marchantia polymorpha (Marchantiales) and the mosses
Andreaea rupestris (Andreaeales) and Sphagnum palustre
(Sphagnales). Trees were rooted using M. polymorpha.
DNA Extraction, Amplification, and Sequencing
Total DNA was extracted from 18 specimens (17 species of
Selaginellaceae and I. lacustris), following a modified version
of the protocol outlined by Doyle and Doyle (1987). DNA
extracts were made from fresh material, specimens dried in
silica gel, and also from some herbarium specimens (table 1).
Leaves taken from herbarium specimens were rehydrated in
CTAB-buffer for 2 h–1 d. Total DNA of S. selaginoides was
kindly provided by N. Wikström. The species included in the
analysis, details of voucher specimens, accession numbers, and
references to sequences taken from the literature are given in
table 1. Fragments corresponding to bases 18–1383 of the rbcL
gene of Marchantia polymorpha (Ohyama et al. 1986) were
amplified using the polymerase chain reaction (PCR) and two
primers (rbcL 1F corresponding to bases 1–17 and rbcL 1409R
corresponding to bases 1384–1409). Primer sequences are
given in table 2. The PCR used the following method, with
minor variations for some species. PCR was done in 50-mL
aliquots using Taq-polymerase from Promega. The reactions
were run in a Perkin-Elmer Thermal Cycler with one cycle of
977C for 2 min and 30 cycles of 947C for 30 s, 507C for 30
s, and 727C for 2 min. A second PCR, using product from the
first PCR as template, was occasionally necessary to obtain
sufficient DNA for sequencing. The Thermo Sequenase Fluo-
rescent Sequencing kit from Amersham was used to sequence
double-stranded PCR products for the rbcL gene. Samples were
electrophoresed on 6% Pharmacia Long Ranger acrylamide
gels on the Pharmacia automated “ALF-express” sequencer.
All species were sequenced in both directions using six different
primers (table 2). Sequences were assembled and edited using
the Staden Package (Staden 1996) on a PC running Linux. All
sequences have been deposited in the EMBL sequence database
(http://www.embl-heidelberg.de/srs5) (table 1).
Phylogenetic Analysis
Visual alignment of the rbcL sequences was unproblematic
because of the absence of insertions and deletions. Parsimony
analyses of the data were performed using PAUP 3.1.1 (Swof-
ford 1991). Analyses used the heuristic search option with
collapse of zero-length branches and MULPARS selected. Ran-
dom sequence addition used 100 replications and TBR branch
swapping. An equal weighting scheme was employed with no
transition-transversion bias (Albert and Mishler 1992). In all
analyses, trees were rooted using the liverwort Marchantia
polymorpha, which represents a basal group in land plants
(Mishler et al. 1994; Qiu et al. 1998).
Support for individual clades was assessed using the decay
index (Bremer 1988; Donoghue et al. 1992) and bootstrap
values (Felsenstein 1985). Decay indices were calculated using
programs AutoDecay 3.0.2 (Eriksson and Wikström 1995) and
PAUP 3.1.1 (Swofford 1991). PAUP 3.1.1 settings were used
to find the tree length of the reversely constrained trees: 10
replicates of random addition sequence, TBR branch swap-
ping, and MULPARS on. Bootstrap values were calculated us-
ing PAUP 3.1.1 by performing 1000 replicates with the fol-
lowing options selected: (1) heuristic search, (2) collapse of
zero-length branches, (3) MULPARS, (4) random sequence ad-
dition with 10 replications, and (5) TBR branch swapping.
Results
The heuristic search yielded three most parsimonious trees
of 2663 steps, excluding uninformative characters (fig. 2;
CI 5 0.35; RI 5 0.67). All trees obtained came from the same
island of trees. Of 1366 characters in the matrix, 700 were
variable and 576 were phylogenetically informative. The rbcL
data provide strong support for monophyly of Selaginellaceae
(decay index, 30; bootstrap, 100%), Selaginella (decay index,

Table 2
List of Primers Used for Amplification and Sequencing
Name Sequence
Forward primers:
rbcL 1F ........... 50-ATGTCACCACAAACGGA-30
rbcL 35F ......... 50-GATTCAAGGCTGGCGTTAAAGAT-30
rbcL 406F ........ 50-GAAGATCTGCGAATTCCCCCCGCTTATTC-30
rbcL 880F ........ 50-CACCGCGCGATGCATGC-30
Reverse primers:
rbcL 770R ....... 50-GCGAATTCTGCCCTTTTCATCATTTCCTCGCA-30
rbcL 1192R ...... 50-AATCATCTCCAAATATTTCAGTCAAAGCGGGCA-30
rbcL 1402R ...... 50-CAAACTTGATTTCTTTCCATACC-30
rbcL 1409R ...... 50-TCAAATTCAAACTTGATTTCTTTCCA-30
Note. Primers rbcL 1F and rbcL 1409R were used for amplification, and the others,
for sequencing. The primer names indicate the 50 position on the Marchantia polymorpha
rbcL gene (Ohyama et al. 1986). Primers were constructed using known sequences of
Lycopodiaceae, Isoetaceae, and Selaginellaceae (except for primers rbcL 880F, rbcL 1F, and
rbcL 1409R constructed by N. Wikström [Wikström and Kenrick 1997]).
 KORALL ET AL.—PHYLOGENY OF SELAGINELLACEAE 589

Fig. 2 Strict consensus tree based on the three most parsimonious trees (tree length 5 2663 steps), depicting relationships among living
lycopsids on the basis of rbcL sequences. Branches with a bootstrap value !50% are collapsed. Except for one collapsed branch (S. denticulata
(S. pulcherrima to S. moritziana)), the ingroup relations are identical with the strict consensus tree. Numbers above branches denote decay values
and numbers below branches, bootstrap values.
590 INTERNATIONAL JOURNAL OF PLANT SCIENCES
72; bootstrap, 100%), Ericetorum (decay index, 46; bootstrap,
100%), and Tetragonostachys (decay index, 38; bootstrap,
100%). Subgenus Selaginella is resolved as a sister group to a
clade composed of all other species of Selaginellaceae. Results
clearly show that the large Stachygynandrum group is para-
phyletic to Ericetorum, Heterostachys, and Tetragonostachys.
The basal dichotomy in this clade has strong support. Some
species of Stachygynandrum (S. apoda to S. bombycina) form
a clade with Heterostachys (decay index, 15; bootstrap,
100%), whereas others (S. kraussiana to S. lepidophylla) are
closely related to Ericetorum and Tetragonostachys (decay in-
dex, 11; bootstrap, 92%). Monophyly of Articulatae is not
supported in any of the alternative trees. A sister-group rela-
tionship between the resurrection plant, S. lepidophylla, and
other xerophytes in the Tetragonostachys clade has moderate
support (decay index, 5; bootstrap, 70%). Moderate support
is also found for monophyly of lycopsids (decay index, 2;
bootstrap, 80%) and a sister-group relationship between Is-
oetaceae and Selaginellaceae. Lycopsids are resolved as a sister
group to seed plants, but bootstrap support (53%) for this
grouping is comparatively weak.
Discussion
Phylogenetic Relationships
Our analysis included a very broad selection of outgroups
because we wanted to examine the effects of additional species
of Selaginellaceae on relationships within lycopsids and the
placement of lycopsids within land plants. Previous rbcL anal-
yses have grouped lycopsids with seed plants (Manhart 1994),
whereas phylogenies based on other molecular (Raubeson and
Jansen 1992; Kolukisaoglu et al. 1995; Kranz and Huss 1996)
and morphological data (Mishler et al. 1994; Stevenson and
Loconte 1996; Kenrick and Crane 1997) group ferns and
horsetails with seed plants, placing lycopsids in a basal position
within vascular plants. The rbcL data are controversial in this
respect, and Manhart (1994) suggests that the lycopsid-seed
plant clade might be a consequence of inadequate taxon sam-
pling and long-branch attraction. Our inclusion of 16 species
of Selaginellaceae, representing all major groups within the
family, in addition to the 12 recently sequenced species of
Lycopodiaceae (Wikström and Kenrick 1997) and two species
of Isoetaceae, had no effect on the decay index (3) and boot-
strap support (53%) for the lycopsid-seed plant clade, which
remained low.
Although the circumscription of Selaginellaceae has re-
mained unchallenged for more than 100 years, recent cladistic
studies have shown that monophyly of the family is poorly
corroborated because clear morphological synapomorphies are
absent (Bateman 1992; Kenrick and Crane 1997). Further-
more, two characteristics of subgenus Selaginella (determinate
growth and rootstock) have been interpreted as support for a
closer relationship to Isoetales than to other species of Sela-
ginellaceae (Bateman 1992; see alternative interpretation in
Kenrick and Crane 1997, pp. 212–213). One potential unique
characteristic of Selaginellaceae is the number of megaspores
per megasporangium. Notwithstanding within-family varia-
tion (Duerden 1929), a single megaspore tetrad is probably
the plesiomorphic state and a potential family-level synapo-
morphy. The inclusion of palaeobotanical data renders this
characteristic equivocal because similar megaspore numbers
are known in closely related fossils that are most likely not
members of Selaginellaceae (Bateman 1992). In other words,
sporangia containing a single megaspore tetrad is probably a
more general feature of heterosporous lycopsids. A second
unique feature of living Selaginellaceae is the presence of a
natural cavity surrounding the stele. As an identifier of the
family in the fossil record, this characteristic is problematic
because tissue decay can result in similar cavities. It is, there-
fore, difficult to know whether the absence of extrastelar tis-
sues in fossils is a real feature of the living plant or simply a
consequence of taphonomic changes. Although these potential
morphological synapomorphies remain equivocal, there is
strong support for monophyly of Selaginellaceae in the rbcL
sequences.
Results support the phylogenetically isolated position of sub-
genus Selaginella (two living species) within Selaginellaceae.
The rbcL data resolve a basal dichotomy between Selaginella
and a large clade, here termed the rhizophoric clade (fig. 3),
composed of all other species (S. uliginosa to S. moritziana)
and including Stachygynandrum, Tetragonostachys, Ericeto-
rum, and Heterostachys. One of the most conspicuous mor-
phological characteristics of the rhizophoric clade is the root-
like structures that develop along the lower surface of leafy
stems, usually in an axillary position (rhizophores). Other
characteristics include dimorphic leaves in four ranks and pla-
nar branching (lost in Tetragonostachys and Ericetorum). The
rhizophoric clade is divided into two strongly supported mono-
phyletic groups (figs. 2, 3). One group (S. apoda to S. mor-
itziana) is composed of part of Stachygynandrum and also
includes the single sampled species of Heterostachys. The phy-
logenetic relationships show clearly that sporophyll dimor-
phism, one of the distinguishing features of Heterostachys, is
derived from the isomorphic condition prevalent within Stach-
ygynandrum. The second well-supported group within the rhi-
zophoric clade (S. uliginosa to S. lepidophylla) is composed of
elements of Stachygynandrum (Articulatae, resurrection
plants) as well as the Tetragonostachys and Ericetorum groups.
Surprisingly, monophyly of Articulatae, represented by three
species, was not supported in this analysis. In all three most
parsimonious trees, the Articulatae is paraphyletic to Erice-
torum, and in one tree, S. exaltata is resolved as sister group
to an Ericetorum, Tetragonostachys, Articulatae clade. Para-
phyly is, however, very weakly supported, and relationships
collapse to a polytomy in the consensus tree. Notwithstanding
the uncertainty in the rbcL data, the Articulatae possess several
unique and distinctive morphological characteristics that
strongly indicate that the group is monophyletic (stem artic-
ulations or swellings below dichotomies, rhizophores devel-
oping from the upper surface of the stem, microsporangial
dehiscence; Somers 1978). Intriguingly, the resurrection plant
S. lepidophylla is resolved as a sister group to the mat-forming
Tetragonostachys, pointing to a common origin of these xer-
ophytes. However, bootstrap support for this relationship is
not strong. Both S. lepidophylla and Tetragonostachys share
similarities in rhizophore development (i.e., rhizophores de-
velop without relation to branch axils; Harvey-Gibson 1902),
but our results indicate that a more detailed comparative study
of rhizophore morphology in these taxa would be appropriate.
 KORALL ET AL.—PHYLOGENY OF SELAGINELLACEAE 591

Fig. 3 Phylogram showing branch lengths of one of the three most parsimonious trees depicting relationships among living lycopsids on
the basis of rbcL sequences. Taxa in bold are isophyllous. Accelerated transformation (ACCTRAN) was used for finding branch lengths. Tree
length is 2663 steps (uninformative characters excluded), CI 5 0.35, RI 5 0.67.
592 INTERNATIONAL JOURNAL OF PLANT SCIENCES
The results of this study indicate that further investigation
of major phylogenetic patterns within Selaginellaceae should
focus on Stachygynandrum (ca. 600 species) and Hetero-
stachys (ca. 60 species). Sampling of Stachygynandrum should
include a broader representation of the species groups recog-
nized in previous classifications (e.g., Hieronymus 1901; Wal-
ton and Alston 1938). Clearly, further sampling of species di-
versity within Heterostachys (60 species) is desirable to
investigate monophyly of this subgroup. Sampling also needs
to reflect a wider geographic area as well as a broader eco-
logical diversity including temperate, tropical rain forest, and
xerophytic species. Further sampling of the predominantly
South American Articulatae (ca. 40 species) should also include
the single Asian species, S. remotifolia. Inclusion of more res-
urrection plants is desirable to explore the possibility of a
common ancestry with other xerophytes in Tetragonostachys.
One striking property of the rbcL gene highlighted in this
study is the heterogeneity of the Selaginellaceae sequences com-
pared with other land plants (fig. 3). The branch lengths of
rbcL in Selaginellaceae exceed that separating the most basal
and distal outgroups used in this analysis, spanning an enor-
mous range of land plant diversity (liverworts to derived gym-
nosperms). The molecular basis for this unusual degree of het-
erogeneity remains unexplored and requires further
investigation, including analyses of the evolutionary rate of
other genes. Although the rbcL gene is a highly informative
phylogenetic marker within Selaginellaceae, there does also
appear to be rate heterogeneity within the family, leading to
relatively short branch lengths and low signal in some clades
(S. denticulata to S. moritziana clade). More rapidly evolving
loci will be necessary to resolve relationships within these
groups.
The Fossil Record and Calibration of the
Phylogenetic Tree
Selaginellaceae have a lengthy fossil record extending back
into the Late Paleozoic. The earliest fossil generally attributed
to the family is Selaginellites resimus (Rowe 1988), a herba-
ceous, isophyllous plant of Visean epoch (Early Carboniferous,
Mississippian, ca. 345 Ma). The group may, however, have an
earlier origin, because heterosporous fossils related to Isoetales
(Selaginellaceae sister group) appear in the Upper Devonian
(Bateman and DiMichele 1994; Kenrick and Crane 1997),
which implies that Selaginellaceae had evolved by this time.
We estimate, therefore, that the divergence of the Selaginel-
laceae and Isoetaceae lineages may have already occurred by
the Upper Devonian (370 Ma).
Unequivocal Selaginellaceae typical of the rhizophoric clade
are well documented by the Late Carboniferous (Moscovian
epoch; ca. 310 Ma), first appearing in the Saar-Lorraine Basin,
Germany (Thomas 1997). Fossils such as S. gutbieri (Rößler
and Buschmann 1994; Thomas 1997) have well-developed,
possibly planar branching and dimorphic leaves in four ranks.
Leaves are typically ovate-lanceolate with acute apices and
denticulate margins. These fossils bear a strong resemblance
to living anisophyllous forms in branch and leaf morphology,
and they indicate that the rhizophoric clade (fig. 3) was well
established in tropical wetlands by the Carboniferous (ca. 310
Ma). An ancient origin of this group is also consistent with
the diverse geographic distributions of living species in the two
major subclades identified in our analysis (fig. 3).
Several morphological characteristics of subgroups within
Selaginellaceae are potentially recognizable but poorly docu-
mented in the fossil record. Rhizophores, one of the most char-
acteristic features of the rhizophoric clade, have not been doc-
umented in any Paleozoic or Mesozoic fossils, even from rare,
comparatively complete specimens (e.g., S. harrisiana: Town-
row 1968). The distribution of rhizophores is quite variable
in living Selaginellaceae. In many species with flattened, frond-
like branching systems, rhizophores are confined to prostrate
stems, but in others they are also borne on upright stems.
Whereas in most species the rhizophore is axillary, in Tetra-
gonostachys they have a more extended distribution, occurring
between branches in many places along the stem. The rela-
tionships of Tetragonostachys indicate that the extended dis-
tribution of rhizophores is a derived characteristic of this sub-
group (figs. 2, 3). The polarity of the rhizophore is reversed
in the Articulatae, where it develops from the upper side of
the stem, looping downward. One possible reason for the ab-
sence of rhizophores in Paleozoic and Mesozoic fossils is that
these structures were confined to rhizomatous axes—parts of
the plant that are seldom fossilized. This hypothesis implies
that extended rhizophore distributions are a comparatively re-
cent feature of the family, an idea that requires further inves-
tigation. The earliest documented rhizophore-like structures
occur in Limnothetis gobiensis (Krassilov 1982) from the Early
Cretaceous. Adventitious roots are borne along the stem in
this plant, but, unlike in Selaginellaceae, the roots develop in
leaf axils. Axillary leaves, another characteristic of the rhi-
zophoric clade, are first recognized in the Late Triassic S. an-
asazia (Ash 1972), but it is very probable that this feature has
been overlooked in earlier fossils. Axillary leaves often resem-
ble the larger of the dimorphic leaf types, but they occur singly
in the axillary position at all stem dichotomies. Many Sela-
ginellaceae possess two or more distinct steles. The earliest
record of a bistelic form is also the Late Triassic S. anasazia
(Ash 1972), where two vascular strands are visible in stem
compressions with preserved cuticle. Other potentially rec-
ognizable morphological characteristics in fossil plants include
swellings of the stem (Articulatae: Somers 1978), dimorphic
sporophylls (Heterostachys: Jermy 1990), and the distribution
of mega- and microsporangia within the strobilus (Horner and
Arnott 1963; Somers 1978). It is the identification in the fossil
record of characters such as these, combined with a better
understanding of phylogenetic relationships among living spe-
cies, that is necessary to the development of a well-calibrated
phylogenetic tree.
Palynological data are also of potential use in dating clades
within Selaginellaceae. Certain species possess a unique form
of megaspore that is characterized by a regular, compact, grid-
like arrangement of sporopollenin wall elements. This struc-
ture gives the spore a distinctive opalescent iridescence, a phe-
nomenon that has also been observed in fossil spores from
sediments as old as the Jurassic (Collinson 1991). Megaspores
of this type are known to occur in Stachygynandrum (Tryon
and Lugardon 1991; Hemsley et al. 1992), but a better un-
derstanding of the relationships of species with iridescent meg-
aspores and the taxonomic distribution of this spore type
within Stachygynandrum is required. Future phylogenetic
 KORALL ET AL.—PHYLOGENY OF SELAGINELLACEAE 593

work should include species of Stachygynandrum with irides- 1991). This characteristic was used as a primary criterion for
cent megaspore walls (e.g., S. myosurus, S. galeottii, S. lyallii, splitting the family in the early classifications of Spring (1850)
S. willdenovii, S. marginata). The ease of recognition of this and Hieronymus (1901), and it is still important in more recent
spore type in the fossil record makes it a potentially valuable taxonomies (Walton and Alston 1938; Tryon and Tryon 1981;
marker for calibrating clades. Jermy 1990; Thomas and Quansah 1991). It has been argued
that the early appearance of species with dimorphic leaves in
Origin of Tropical Rain Forest and Drought- the fossil record (Late Carboniferous, Pennsylvanian; Mos-
Adapted Species covian epoch; ca. 310 Ma; Thomas 1997) justifies the use of
this characteristic at a high systematic level within the family
Fossil evidence is consistent with a long ecological associ-
(Thomas and Quansah 1991; Thomas 1992). The results of
ation between Selaginellaceae and the humid tropics. The ear-
our study indicate that, whereas two occurrences of isophylly
liest Selaginellaceae were a component of the coal-forming
are clearly plesiomorphic (subgenus Selaginella), at least two
tropical wetland vegetation of the Carboniferous (Rößler and
other instances are probably independent reversals from het-
Buschmann 1994; Thomas 1997), and it seems likely that el-
erophyllous ancestors. Mapping leaf dimorphism on our pre-
ements of the family have persisted in similar environments
ferred, most parsimonious tree supports a reversal to the is-
throughout the Mesozoic and Cenozoic. Fossil evidence would
ophyllous condition in Tetragonostachys and Ericetorum (fig.
therefore indicate that xerophytes and temperate woodland
3). It should be noted, however, that this pattern is only mar-
species originated from humid tropical ancestors. In other
ginally less parsimonious than independent gains of leaf di-
words, it seems likely that the humid tropics have been a source
morphism in various dimorphic groups. Clearly a larger sam-
of species diversity for temperate and arid tropical to sub-
ple of Stachygynandrum species is required, but, with this
tropical regions. We suspect that the drought-tolerant Tetra-
caveat in mind, we interpret isophylly in Tetragonostachys and
gonostachys group has ancient origins, perhaps evolving dur-
Ericetorum as reversals. Leaves in both groups are generally
ing the Triassic in parallel with the widespread development
small, and this reduction of leaf size is probably related to
of xerophytic features in other groups, such as ferns and seed
drought tolerance.
plants. Our phylogenetic analysis does not provide a rigorous
test of these hypotheses because our sample of ecological, ge-
ographic, and taxonomic diversity within Stachygynandrum is
Acknowledgments
relatively small. If these hypotheses are correct, we would ex-
pect rain forest species to form a basal grade within, for ex-
We acknowledge the help and support of our institutions
ample, the rhizophoric clade. This is clearly not a pattern,
and financial support from the Swedish Natural Science Re-
however, that emerges from our preliminary data. In the S.
search Council (NFR research grant to Paul Kenrick: B-AA/
apoda to S. moritziana clade (fig. 2), both S. apoda and S.
BU 10728-301), Helge Ax:son Johnsons Stiftelse (grant to Pe-
denticulata are temperate species. These temperate species are
tra Korall), the Royal Swedish Academy of Sciences (KVA
paraphyletic to a humid tropical group (S. pulcherrima to S.
grants to Petra Korall from the Hierta-Retzius fund and En-
moritziana) from South America and Australia. However, this
anderska fonden), Wallenbergstiftelsens Jubileumsfond (grant
relationship is poorly supported. In the S. uliginosa to S. lep-
to Petra Korall), and a General Research Fund Allocation,
idophylla clade, the xerophytic (S. rupincola, S. arizonica, S.
University of Kansas, to James P. Therrien. We thank M. Käl-
rupestris, S. lepidophylla), temperate (S. uliginosa, S. gracil-
lersjö (Molecular Systematics Laboratory, Swedish Museum of
lima), and humid tropical species (S. kraussiana, S. diffusa, S.
Natural History) for guidance and access to sequencing facil-
exaltata) form a polytomy (fig. 2). We anticipate that a broader
ities and Bonnie Liscek (University of Kansas) for assistance
sample of Stachygynandrum species, in particular tropical
in automated sequencing. We are very grateful to the following
groups, will improve phylogenetic resolution and provide a
persons for providing specimens: Torsten Eriksson (S. selagi-
clearer picture of the origin of drought-tolerant and temperate
noides, S. arizonica, S. rupestris), Mark Fishbein (S. arizonica,
woodland species.
S. rupestris), Alan Gray, Hans-Erik Wanntorp, and Livia
Wanntorp (S. uliginosa), Zack E. Murrell (S. apoda), Dan Pal-
Leaf Dimorphism
mer and Ken Wilson (S. deflexa), and Karen Renzaglia (S.
The systematic significance of leaf dimorphism in Selagi- moellendorffii). Finally, we thank the two reviewers for con-
nellaceae has been widely discussed (Thomas and Quansah structive criticism of the manuscript.

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