Korall Et Al2===Phylogeny of Selaginellaceae===Rbcl

585

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/SUBGENERICRELATIONSHIPS 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, CromwellRoad, 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 threemost 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 tosubgenera Ericetorum, Tetragonostachys, and Heterostachys. Monophyly of Ericetorum and Tetragonostachysis 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 commonorigin of xerophytism in these groups. A close relationship for all isophyllous species, as hypothesized in manyclassifications, 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 seasonaldrought.

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

Introduction

Selaginellaceae are an ancient group of fern allies composedof an estimated 750 living species. Greatest diversity occurs inlowland to midmontane primary tropical rain forest, but thiscosmopolitan family is also widely distributed in subtropical,temperate, montane, and rarely subarctic regions. Most speciesare characterized by strongly flattened, frondlike branchingand dimorphic leaves (microphylls). These and other uniquefeatures of the epidermis (e.g., iridescence, lens-shaped epi-dermal cells, presence of chloroplasts; Hebant and Lee 1984)are probably adaptations to poor light quality—a consequenceof life on the forest floor. Species may be scandent or climbingon 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, andseveral strategies for drought tolerance (xerophytism) haveevolved. Most xerophytic species have a mat-forming or tuftedhabit and thick cutinized leaves that taper to a fine hair point.Others, such as the “resurrection plants,” have the ability toroll up leaves and stems to prevent excessive water loss andto reverse this process on rehydration. A few are annuals thatgrow and reproduce rapidly during a short wet season. Despitea lengthy fossil record, the evolution of the family is poorlyunderstood. The absence of an explicit phylogenetic treatmentis a major obstacle to investigating the evolution of modern

1 Author for correspondence and reprints; fax 46-8-16-25-77; [email protected].

2 E-mail [email protected] E-mail [email protected].

Manuscript received October 1998; revised manuscript received December 1998.

species diversity, in particular, the origins of the diverse xer-ophytic and humid tropical groups. Here we present a prelim-inary phylogenetic analysis based on chloroplast rbcL genesequences as a first step toward developing a detailed phylo-genetic framework for the family.

Previous systematic studies have identified several majorgroups within Selaginellaceae, and our usage is based on theclassification by Jermy (1986; fig. 1), which recognizes fivesubgenera in Selaginella: Selaginella, Ericetorum, Tetragonos-tachys, Stachygynandrum, and Heterostachys. A basic di-chotomy between isophyllous and anisophyllous species is afeature 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 (Thomasand Quansah 1991). Among isophyllous species, S. selagino-ides and the very similar Hawaiian endemic S. deflexa areconsistently recognized as distinct from other species, basedon helical phyllotaxy, similarity of leaf and sporophyll, andthe 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 regionscharacterized by prolonged seasonal drought are grouped inTetragonostachys (sensu Jermy 1986; fig. 1), morphologicallydistinguished by tetrastichous sporophylls. Ericetorum (sensuJermy 1986; fig. 1) is a small group that contains three pro-teaceous heathland species from Australia, Tasmania, andSouth Africa. The species are characterized by decussately ar-ranged leaves and tetrastichous sporophylls. Most species of

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 recognizesone genus (Selaginella) and five subgenera: Selaginella, Ericetorum, Tetragonostachys, Stachygynandrum, and Heterostachys. The rank of thesetaxa referred to in the text is subgeneric, unless otherwise stated.

Selaginellaceae are anisophyllous, and there is little agreementover 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 byisomorphic sporophylls. Approximately 60 species with di-morphic sporophylls are classified in Heterostachys (Waltonand Alston 1938; Jermy 1986). Within Stachygynandrum, oneof the most widely recognized and distinctive groups is Arti-culatae with ca. 50 species (Spring 1850). This group containsspecies with both conspicuous nodes below stem dichotomiesand rhizophores that emerge from the upper surface of thestem 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 phylogeneticframework for Selaginellaceae based on sequence data fromthe chloroplast rbcL gene. These data are used to investigatethe relationships of major taxonomic units and to begin toaddress 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 withinthis 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 inrecent classifications (fig. 1; table 1). Choice of taxa was alsoinfluenced by growth environment and included xerophytic,humid tropical, and temperate woodland species. Prior to thisstudy, the rbcL gene had been sequenced for two species ofSelaginellaceae: S. kraussiana (S. apoda, Manhart 1994) andS. selaginoides (Wikstrom and Kenrick 1997). Isophyllous spe-cies included representatives of Selaginella, Ericetorum, andTetragonostachys (sensu Jermy 1986). Subgenus Selaginellacontains two species, the widespread Northern Hemisphere S.selaginoides and the Hawaiian endemic S. deflexa, both ofwhich were sequenced for the analysis. Two (S. uliginosa, S.gracillima) of the three known species of Ericetorum wereincluded. Selaginella gracillima is also interesting because it isone of the few annuals in Selaginellaceae. Tetragonostachyswas represented by three North American xerophytes: S. ru-pestris, S. arizonica, and S. rupincola. Anisophyllous speciesincluded 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 Databasefor the Species Included in This Article

Species DNA source/voucher Accession number/reference

Selaginella apoda (L.) Fern.a . . . . . . . . . . . . . . . . . . . . Z. E. Murrell 6484 (S) AJ010854S. arizonica Maxonb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T. Eriksson 13/10 1996 (S) AJ010851S. bombycina Springb . . . . . . . . . . . . . . . . . . . . . . . . . . . . Korall 1997:31 (S) AJ010848S. deflexa Brackenridgeb . . . . . . . . . . . . . . . . . . . . . . . . D. Palmer 2651 (KANU) AF093253S. denticulata (L.) Springa . . . . . . . . . . . . . . . . . . . . . . . Korall & Eriksson TE 715 (S) AJ010853S. diffusa (Presl) Springb . . . . . . . . . . . . . . . . . . . . . . . . Korall 1997:33 (S) AJ010852S. exaltata (Kunze) Springa . . . . . . . . . . . . . . . . . . . . . Korall 1996:1 (S) AJ010849S. gracillima (Kunze) Springc . . . . . . . . . . . . . . . . . . . Nordenstam & Anderberg 1124 (S) AJ010844S. haematodes (Kunze) Springa . . . . . . . . . . . . . . . . . Korall 1996:15b (S) AJ010846S. kraussiana (Kunze) A. Br.b . . . . . . . . . . . . . . . . . . . Korall 1997:30 (S) AJ010845S. lepidophylla (Hook. & Grev.) Springb . . . . . . Therrien 1996:s.n. (KANU) AF093254S. moellendorfii Hieron.b . . . . . . . . . . . . . . . . . . . . . . . . Renzaglia s.n. (KANU) AF093256S. moritziana Spring ex Klotzscha . . . . . . . . . . . . . . Korall 1996:12 (S) AJ010856S. pulcherrima Liebm. ex Fourn.b . . . . . . . . . . . . . . Korall 1997:32 (S) AJ010847S. rupestris (L.) Springb . . . . . . . . . . . . . . . . . . . . . . . . . Therrien 1996:356 (KANU) AF093255S. rupincola Underw.b . . . . . . . . . . . . . . . . . . . . . . . . . . . T. Eriksson 14/10 1996 (S) AJ010850S. selaginoides (L.) Link .. . . . . . . . . . . . . . . . . . . . . . . T. Eriksson 704 (S) Y07940S. uliginosa (Labill.) Springa . . . . . . . . . . . . . . . . . . . . Gray, Holmgren & Wanntorp 523 (S) AJ010843Andreaea 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 (Wikstrom and Kenrick 1997)H. cumingii (Nessel) Holub .. . . . . . . . . . . . . . . . . . . B. Øllgaard 100836 (AAU) Y07930 (Wikstrom and Kenrick 1997)H. hippuridea (Christ) Holub .. . . . . . . . . . . . . . . . . B. Øllgaard 100619 (AAU) Y07931 (Wikstrom and Kenrick 1997)H. linifolia (L.) Trevisan . . . . . . . . . . . . . . . . . . . . . . . . B. Øllgaard 100594 (AAU) Y07932 (Wikstrom and Kenrick 1997)H. selago (L.) C. Martius & Schrank .. . . . . . . . N. Wikstrom 36 (S) Y07934 (Wikstrom and Kenrick 1997)H. wilsonii (L.) Underw. & F. Lloyd .. . . . . . . . . B. Øllgaard 100611 (AAU) Y07933 (Wikstrom and Kenrick 1997)Isoetes lacustris L.b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ) AJ010855I. melanopoda (Gay & Durieu)

ex. Durieu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manhart 03/10/88-1 (TAMU) L11054 (Manhart 1994)Lycopodiella alopecuroides (L.) Cranfill . . . . . . B. Øllgaard 100822 (AAU) Y07937 (Wikstrom and Kenrick 1997)L. inundata (L.) Holub .. . . . . . . . . . . . . . . . . . . . . . . . . H.-E. Wanntorp & N. Wikstrom 19/7 1995 (S) Y07938 (Wikstrom and Kenrick 1997)Lycopodium clavatum L. . . . . . . . . . . . . . . . . . . . . . . . N. Wikstrom 164 (S) Y07936 (Wikstrom and Kenrick 1997)L. digitatum A. Braun .. . . . . . . . . . . . . . . . . . . . . . . . . Manhart 06/12/88-1 (TAMU) L11055 (Manhart 1994)L. obscurum L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T. Eriksson 666 (GH) Y07935 (Wikstrom and Kenrick 1997)Marchantia polymorpha L. . . . . . . . . . . . . . . . . . . . . . ) X04465 (Ohyama et al. 1986)Phylloglossum drummondii Kunze . . . . . . . . . . . . . ) Y07939 (Wikstrom and Kenrick 1997)Pinus edulis Engelm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ) X58137Pseudotsuga 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 &

Vazquez-Torres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ) L12683 (Chase et al. 1993)

Note. Voucher location abbreviations follow Index Herbariorum (Holmgren et al. 1990). Ellipses indicate that information on voucherspecimens 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), andseasonally arid areas (S. lepidophylla: resurrection plant). TheNorth American species S. apoda was resequenced because thesource for the previously published sequence (Manhart 1994)

was a misidentified specimen of S. kraussiana (voucherexamined).

Outgroup. Twenty-four outgroup species were chosen torepresent 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 groupsof Lycopodiaceae (Phylloglossum drummondii, Huperzia, Ly-


Table 2

List of Primers Used for Amplification and Sequencing

Name Sequence

Forward primers:rbcL 1F .. . . . . . . . . . 5′-ATGTCACCACAAACGGA-3′

rbcL 35F .. . . . . . . . 5′-GATTCAAGGCTGGCGTTAAAGAT-3′

rbcL 406F .. . . . . . . 5′-GAAGATCTGCGAATTCCCCCCGCTTATTC-3′

rbcL 880F .. . . . . . . 5′-CACCGCGCGATGCATGC-3′

Reverse primers:rbcL 770R .. . . . . . 5′-GCGAATTCTGCCCTTTTCATCATTTCCTCGCA-3′

rbcL 1192R .. . . . . 5′-AATCATCTCCAAATATTTCAGTCAAAGCGGGCA-3′

rbcL 1402R .. . . . . 5′-CAAACTTGATTTCTTTCCATACC-3′

rbcL 1409R .. . . . . 5′-TCAAATTCAAACTTGATTTCTTTCCA-3′

Note. Primers rbcL 1F and rbcL 1409R were used for amplification, and the others,for sequencing. The primer names indicate the 5′ position on the Marchantia polymorpharbcL gene (Ohyama et al. 1986). Primers were constructed using known sequences ofLycopodiaceae, Isoetaceae, and Selaginellaceae (except for primers rbcL 880F, rbcL 1F, andrbcL 1409R constructed by N. Wikstrom [Wikstrom and Kenrick 1997]).

copodiella, Lycopodium). Ferns and sphenopsids were repre-sented by Angiopteris evecta (Marattiaceae) and Equisetumarvense (Equisetaceae). The enigmatic Psilotum nudum (Psi-lotaceae) was included because of its putative basal positionwithin ferns (Stevenson and Loconte 1996). Seed plants wererepresented 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 mossesAndreaea 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 ofSelaginellaceae and I. lacustris), following a modified versionof the protocol outlined by Doyle and Doyle (1987). DNAextracts were made from fresh material, specimens dried insilica gel, and also from some herbarium specimens (table 1).Leaves taken from herbarium specimens were rehydrated inCTAB-buffer for 2 h–1 d. Total DNA of S. selaginoides waskindly provided by N. Wikstrom. The species included in theanalysis, details of voucher specimens, accession numbers, andreferences to sequences taken from the literature are given intable 1. Fragments corresponding to bases 18–1383 of the rbcLgene of Marchantia polymorpha (Ohyama et al. 1986) wereamplified using the polymerase chain reaction (PCR) and twoprimers (rbcL 1F corresponding to bases 1–17 and rbcL 1409Rcorresponding to bases 1384–1409). Primer sequences aregiven in table 2. The PCR used the following method, withminor variations for some species. PCR was done in 50-mLaliquots using Taq-polymerase from Promega. The reactionswere run in a Perkin-Elmer Thermal Cycler with one cycle of977C for 2 min and 30 cycles of 947C for 30 s, 507C for 30s, and 727C for 2 min. A second PCR, using product from thefirst PCR as template, was occasionally necessary to obtainsufficient DNA for sequencing. The Thermo Sequenase Fluo-rescent Sequencing kit from Amersham was used to sequencedouble-stranded PCR products for the rbcL gene. Samples wereelectrophoresed on 6% Pharmacia Long Ranger acrylamidegels on the Pharmacia automated “ALF-express” sequencer.

All species were sequenced in both directions using six differentprimers (table 2). Sequences were assembled and edited usingthe Staden Package (Staden 1996) on a PC running Linux. Allsequences 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 unproblematicbecause of the absence of insertions and deletions. Parsimonyanalyses of the data were performed using PAUP 3.1.1 (Swof-ford 1991). Analyses used the heuristic search option withcollapse of zero-length branches and MULPARS selected. Ran-dom sequence addition used 100 replications and TBR branchswapping. An equal weighting scheme was employed with notransition-transversion bias (Albert and Mishler 1992). In allanalyses, trees were rooted using the liverwort Marchantiapolymorpha, which represents a basal group in land plants(Mishler et al. 1994; Qiu et al. 1998).

Support for individual clades was assessed using the decayindex (Bremer 1988; Donoghue et al. 1992) and bootstrapvalues (Felsenstein 1985). Decay indices were calculated usingprograms AutoDecay 3.0.2 (Eriksson and Wikstrom 1995) andPAUP 3.1.1 (Swofford 1991). PAUP 3.1.1 settings were usedto find the tree length of the reversely constrained trees: 10replicates 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 ofzero-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 treesof 2663 steps, excluding uninformative characters (fig. 2;

; ). All trees obtained came from the sameCI 5 0.35 RI 5 0.67island of trees. Of 1366 characters in the matrix, 700 werevariable and 576 were phylogenetically informative. The rbcLdata provide strong support for monophyly of Selaginellaceae(decay index, 30; bootstrap, 100%), Selaginella (decay index,


Fig. 2 Strict consensus tree based on the three most parsimonious trees (tree steps), depicting relationships among livinglength 5 2663lycopsids 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 valuesand numbers below branches, bootstrap values.


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 aclade composed of all other species of Selaginellaceae. Resultsclearly show that the large Stachygynandrum group is para-phyletic to Ericetorum, Heterostachys, and Tetragonostachys.The basal dichotomy in this clade has strong support. Somespecies of Stachygynandrum (S. apoda to S. bombycina) forma clade with Heterostachys (decay index, 15; bootstrap,100%), whereas others (S. kraussiana to S. lepidophylla) areclosely related to Ericetorum and Tetragonostachys (decay in-dex, 11; bootstrap, 92%). Monophyly of Articulatae is notsupported in any of the alternative trees. A sister-group rela-tionship between the resurrection plant, S. lepidophylla, andother xerophytes in the Tetragonostachys clade has moderatesupport (decay index, 5; bootstrap, 70%). Moderate supportis 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 sistergroup to seed plants, but bootstrap support (53%) for thisgrouping is comparatively weak.

Discussion

Phylogenetic Relationships

Our analysis included a very broad selection of outgroupsbecause we wanted to examine the effects of additional speciesof Selaginellaceae on relationships within lycopsids and theplacement of lycopsids within land plants. Previous rbcL anal-yses have grouped lycopsids with seed plants (Manhart 1994),whereas phylogenies based on other molecular (Raubeson andJansen 1992; Kolukisaoglu et al. 1995; Kranz and Huss 1996)and morphological data (Mishler et al. 1994; Stevenson andLoconte 1996; Kenrick and Crane 1997) group ferns andhorsetails with seed plants, placing lycopsids in a basal positionwithin vascular plants. The rbcL data are controversial in thisrespect, and Manhart (1994) suggests that the lycopsid-seedplant clade might be a consequence of inadequate taxon sam-pling and long-branch attraction. Our inclusion of 16 speciesof Selaginellaceae, representing all major groups within thefamily, in addition to the 12 recently sequenced species ofLycopodiaceae (Wikstrom and Kenrick 1997) and two speciesof Isoetaceae, had no effect on the decay index (3) and boot-strap support (53%) for the lycopsid-seed plant clade, whichremained low.

Although the circumscription of Selaginellaceae has re-mained unchallenged for more than 100 years, recent cladisticstudies have shown that monophyly of the family is poorlycorroborated because clear morphological synapomorphies areabsent (Bateman 1992; Kenrick and Crane 1997). Further-more, two characteristics of subgenus Selaginella (determinategrowth and rootstock) have been interpreted as support for acloser relationship to Isoetales than to other species of Sela-ginellaceae (Bateman 1992; see alternative interpretation inKenrick and Crane 1997, pp. 212–213). One potential uniquecharacteristic of Selaginellaceae is the number of megasporesper megasporangium. Notwithstanding within-family varia-tion (Duerden 1929), a single megaspore tetrad is probablythe plesiomorphic state and a potential family-level synapo-

morphy. The inclusion of palaeobotanical data renders thischaracteristic equivocal because similar megaspore numbersare known in closely related fossils that are most likely notmembers of Selaginellaceae (Bateman 1992). In other words,sporangia containing a single megaspore tetrad is probably amore general feature of heterosporous lycopsids. A secondunique feature of living Selaginellaceae is the presence of anatural cavity surrounding the stele. As an identifier of thefamily in the fossil record, this characteristic is problematicbecause 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 aconsequence of taphonomic changes. Although these potentialmorphological synapomorphies remain equivocal, there isstrong support for monophyly of Selaginellaceae in the rbcLsequences.

Results support the phylogenetically isolated position of sub-genus Selaginella (two living species) within Selaginellaceae.The rbcL data resolve a basal dichotomy between Selaginellaand 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 leafystems, usually in an axillary position (rhizophores). Othercharacteristics include dimorphic leaves in four ranks and pla-nar branching (lost in Tetragonostachys and Ericetorum). Therhizophoric 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 alsoincludes the single sampled species of Heterostachys. The phy-logenetic relationships show clearly that sporophyll dimor-phism, one of the distinguishing features of Heterostachys, isderived 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 ofelements of Stachygynandrum (Articulatae, resurrectionplants) as well as the Tetragonostachys and Ericetorum groups.Surprisingly, monophyly of Articulatae, represented by threespecies, was not supported in this analysis. In all three mostparsimonious trees, the Articulatae is paraphyletic to Erice-torum, and in one tree, S. exaltata is resolved as sister groupto an Ericetorum, Tetragonostachys, Articulatae clade. Para-phyly is, however, very weakly supported, and relationshipscollapse to a polytomy in the consensus tree. Notwithstandingthe uncertainty in the rbcL data, the Articulatae possess severalunique and distinctive morphological characteristics thatstrongly indicate that the group is monophyletic (stem artic-ulations or swellings below dichotomies, rhizophores devel-oping from the upper surface of the stem, microsporangialdehiscence; Somers 1978). Intriguingly, the resurrection plantS. lepidophylla is resolved as a sister group to the mat-formingTetragonostachys, pointing to a common origin of these xer-ophytes. However, bootstrap support for this relationship isnot strong. Both S. lepidophylla and Tetragonostachys sharesimilarities 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 studyof rhizophore morphology in these taxa would be appropriate.


Fig. 3 Phylogram showing branch lengths of one of the three most parsimonious trees depicting relationships among living lycopsids onthe basis of rbcL sequences. Taxa in bold are isophyllous. Accelerated transformation (ACCTRAN) was used for finding branch lengths. Treelength is 2663 steps (uninformative characters excluded), , .CI 5 0.35 RI 5 0.67


The results of this study indicate that further investigationof major phylogenetic patterns within Selaginellaceae shouldfocus on Stachygynandrum (ca. 600 species) and Hetero-stachys (ca. 60 species). Sampling of Stachygynandrum shouldinclude 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 toinvestigate monophyly of this subgroup. Sampling also needsto reflect a wider geographic area as well as a broader eco-logical diversity including temperate, tropical rain forest, andxerophytic species. Further sampling of the predominantlySouth American Articulatae (ca. 40 species) should also includethe single Asian species, S. remotifolia. Inclusion of more res-urrection plants is desirable to explore the possibility of acommon ancestry with other xerophytes in Tetragonostachys.

One striking property of the rbcL gene highlighted in thisstudy is the heterogeneity of the Selaginellaceae sequences com-pared with other land plants (fig. 3). The branch lengths ofrbcL in Selaginellaceae exceed that separating the most basaland 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 furtherinvestigation, including analyses of the evolutionary rate ofother genes. Although the rbcL gene is a highly informativephylogenetic marker within Selaginellaceae, there does alsoappear to be rate heterogeneity within the family, leading torelatively short branch lengths and low signal in some clades(S. denticulata to S. moritziana clade). More rapidly evolvingloci will be necessary to resolve relationships within thesegroups.

The Fossil Record and Calibration of thePhylogenetic Tree

Selaginellaceae have a lengthy fossil record extending backinto the Late Paleozoic. The earliest fossil generally attributedto 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 anearlier 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 bythe Upper Devonian (370 Ma).

Unequivocal Selaginellaceae typical of the rhizophoric cladeare well documented by the Late Carboniferous (Moscovianepoch; ca. 310 Ma), first appearing in the Saar-Lorraine Basin,Germany (Thomas 1997). Fossils such as S. gutbieri (Roßlerand Buschmann 1994; Thomas 1997) have well-developed,possibly planar branching and dimorphic leaves in four ranks.Leaves are typically ovate-lanceolate with acute apices anddenticulate margins. These fossils bear a strong resemblanceto living anisophyllous forms in branch and leaf morphology,and they indicate that the rhizophoric clade (fig. 3) was wellestablished in tropical wetlands by the Carboniferous (ca. 310Ma). An ancient origin of this group is also consistent with

the diverse geographic distributions of living species in the twomajor subclades identified in our analysis (fig. 3).

Several morphological characteristics of subgroups withinSelaginellaceae 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 variablein living Selaginellaceae. In many species with flattened, frond-like branching systems, rhizophores are confined to prostratestems, 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, occurringbetween 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 reversedin the Articulatae, where it develops from the upper side ofthe stem, looping downward. One possible reason for the ab-sence of rhizophores in Paleozoic and Mesozoic fossils is thatthese structures were confined to rhizomatous axes—parts ofthe plant that are seldom fossilized. This hypothesis impliesthat extended rhizophore distributions are a comparatively re-cent feature of the family, an idea that requires further inves-tigation. The earliest documented rhizophore-like structuresoccur in Limnothetis gobiensis (Krassilov 1982) from the EarlyCretaceous. Adventitious roots are borne along the stem inthis plant, but, unlike in Selaginellaceae, the roots develop inleaf 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 hasbeen overlooked in earlier fossils. Axillary leaves often resem-ble the larger of the dimorphic leaf types, but they occur singlyin the axillary position at all stem dichotomies. Many Sela-ginellaceae possess two or more distinct steles. The earliestrecord of a bistelic form is also the Late Triassic S. anasazia(Ash 1972), where two vascular strands are visible in stemcompressions with preserved cuticle. Other potentially rec-ognizable morphological characteristics in fossil plants includeswellings of the stem (Articulatae: Somers 1978), dimorphicsporophylls (Heterostachys: Jermy 1990), and the distributionof mega- and microsporangia within the strobilus (Horner andArnott 1963; Somers 1978). It is the identification in the fossilrecord of characters such as these, combined with a betterunderstanding of phylogenetic relationships among living spe-cies, that is necessary to the development of a well-calibratedphylogenetic tree.

Palynological data are also of potential use in dating cladeswithin Selaginellaceae. Certain species possess a unique formof 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 fromsediments as old as the Jurassic (Collinson 1991). Megasporesof this type are known to occur in Stachygynandrum (Tryonand 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 typewithin Stachygynandrum is required. Future phylogenetic


work should include species of Stachygynandrum with irides-cent megaspore walls (e.g., S. myosurus, S. galeottii, S. lyallii,S. willdenovii, S. marginata). The ease of recognition of thisspore type in the fossil record makes it a potentially valuablemarker for calibrating clades.

Origin of Tropical Rain Forest and Drought-Adapted Species

Fossil evidence is consistent with a long ecological associ-ation between Selaginellaceae and the humid tropics. The ear-liest Selaginellaceae were a component of the coal-formingtropical wetland vegetation of the Carboniferous (Roßler andBuschmann 1994; Thomas 1997), and it seems likely that el-ements of the family have persisted in similar environmentsthroughout the Mesozoic and Cenozoic. Fossil evidence wouldtherefore indicate that xerophytes and temperate woodlandspecies originated from humid tropical ancestors. In otherwords, it seems likely that the humid tropics have been a sourceof species diversity for temperate and arid tropical to sub-tropical regions. We suspect that the drought-tolerant Tetra-gonostachys group has ancient origins, perhaps evolving dur-ing the Triassic in parallel with the widespread developmentof xerophytic features in other groups, such as ferns and seedplants. Our phylogenetic analysis does not provide a rigoroustest of these hypotheses because our sample of ecological, ge-ographic, and taxonomic diversity within Stachygynandrum isrelatively small. If these hypotheses are correct, we would ex-pect rain forest species to form a basal grade within, for ex-ample, the rhizophoric clade. This is clearly not a pattern,however, that emerges from our preliminary data. In the S.apoda to S. moritziana clade (fig. 2), both S. apoda and S.denticulata are temperate species. These temperate species areparaphyletic to a humid tropical group (S. pulcherrima to S.moritziana) from South America and Australia. However, thisrelationship is poorly supported. In the S. uliginosa to S. lep-idophylla clade, the xerophytic (S. rupincola, S. arizonica, S.rupestris, S. lepidophylla), temperate (S. uliginosa, S. gracil-lima), and humid tropical species (S. kraussiana, S. diffusa, S.exaltata) form a polytomy (fig. 2). We anticipate that a broadersample of Stachygynandrum species, in particular tropicalgroups, will improve phylogenetic resolution and provide aclearer picture of the origin of drought-tolerant and temperatewoodland species.

Leaf Dimorphism

The systematic significance of leaf dimorphism in Selagi-nellaceae has been widely discussed (Thomas and Quansah

1991). This characteristic was used as a primary criterion forsplitting the family in the early classifications of Spring (1850)and Hieronymus (1901), and it is still important in more recenttaxonomies (Walton and Alston 1938; Tryon and Tryon 1981;Jermy 1990; Thomas and Quansah 1991). It has been arguedthat the early appearance of species with dimorphic leaves inthe fossil record (Late Carboniferous, Pennsylvanian; Mos-covian epoch; ca. 310 Ma; Thomas 1997) justifies the use ofthis characteristic at a high systematic level within the family(Thomas and Quansah 1991; Thomas 1992). The results ofour study indicate that, whereas two occurrences of isophyllyare clearly plesiomorphic (subgenus Selaginella), at least twoother instances are probably independent reversals from het-erophyllous ancestors. Mapping leaf dimorphism on our pre-ferred, most parsimonious tree supports a reversal to the is-ophyllous condition in Tetragonostachys and Ericetorum (fig.3). It should be noted, however, that this pattern is only mar-ginally less parsimonious than independent gains of leaf di-morphism in various dimorphic groups. Clearly a larger sam-ple of Stachygynandrum species is required, but, with thiscaveat in mind, we interpret isophylly in Tetragonostachys andEricetorum as reversals. Leaves in both groups are generallysmall, and this reduction of leaf size is probably related todrought tolerance.

Acknowledgments

We acknowledge the help and support of our institutionsand financial support from the Swedish Natural Science Re-search Council (NFR research grant to Paul Kenrick: B-AA/BU 10728-301), Helge Ax:son Johnsons Stiftelse (grant to Pe-tra Korall), the Royal Swedish Academy of Sciences (KVAgrants to Petra Korall from the Hierta-Retzius fund and En-anderska fonden), Wallenbergstiftelsens Jubileumsfond (grantto Petra Korall), and a General Research Fund Allocation,University of Kansas, to James P. Therrien. We thank M. Kal-lersjo (Molecular Systematics Laboratory, Swedish Museum ofNatural History) for guidance and access to sequencing facil-ities and Bonnie Liscek (University of Kansas) for assistancein automated sequencing. We are very grateful to the followingpersons for providing specimens: Torsten Eriksson (S. selagi-noides, S. arizonica, S. rupestris), Mark Fishbein (S. arizonica,S. rupestris), Alan Gray, Hans-Erik Wanntorp, and LiviaWanntorp (S. uliginosa), Zack E. Murrell (S. apoda), Dan Pal-mer and Ken Wilson (S. deflexa), and Karen Renzaglia (S.moellendorffii). Finally, we thank the two reviewers for con-structive criticism of the manuscript.

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