506===phylogenetic relationships in selaginellaceae rbcl===+

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American Journal of Botany 89(3): 506517. 2002.

PHYLOGENETIC RELATIONSHIPS IN SELAGINELLACEAE

BASED ON RBCL SEQUENCES1

PETRA KORALL2,4 AND PAUL KENRICK3

2Department of Botany, Stockholm University, SE-106 91 Stockholm, Sweden, and Molecular Systematics Laboratory,

Swedish Museum of Natural History, Box 50007, SE-104 05 Stockholm, Sweden; and3Department of Palaeontology, The Natural History Museum, Cromwell Road, London, SW7 5BD, UK

A phylogenetic framework is developed for the clubmoss family Selaginellaceae based on maximum parsimony analyses of molecular

data. The chloroplast gene rbcL was sequenced for 62 species, which represent nearly 10% of living species diversity in the family.

Taxa were chosen to reflect morphological, geographical, and ecological diversity. The analyses provide support for monophyly of

subgenera Selaginella and Tetragonostachys. Stachygynandrum and Heterostachys are polyphyletic. Monophyly of Ericetorum is

uncertain. Results also indicate a large number of new groupings not previously recognized on morphological grounds. Some of these

new groups seem to have corresponding morphological synapomorphies, such as the presence of rhizophores (distinctive root-like

structures), aspects of rhizophore development, and leaf and stem morphology. Others share distinctive ecological traits (e.g., xero-

phytism). For many groups, however, no morphological, ecological, or physiological markers are known. This could reflect patchy

sampling and a lack of detailed knowledge about many species. Despite a lengthy fossil record dating from the Carboniferous Period,

cladogram topology indicates that most of the living tropical species are probably the products of more recent diversifications. Res-

urrection plants, extreme xerophytes characterized by aridity-driven inrolling of branches and rapid revival on rehydration, have evolved

at least three times in quite different clades.

Key words: lycopod; phylogeny; rbcL; resurrection plant; rubisco; Selaginellaceae; xerophyte.

Selaginellaceae Willk. are an ancient group of lycopodscomprising some 700 living species. Most are easily recog-nizable by their delicate dichotomously branching stems thatbear ranks of minute leaves in two distinct sizes (Jermy, 1990).In these characteristics, the morphology of Selaginellaceae haschanged little since the group was first encountered in the fos-sil record in the tropical wetland floras of the Carboniferous

Period (Thomas, 1992, 1997). Selaginellaceae are a cosmo-politan family with species capable of growing under a widerange of climate, soil, and light regimes. The group containsfrost-tolerant, arctic-alpine species, delicate terrestrial rainfo-rest species, and physiologically robust, drought-adapted xe-rophytes of desert scrub and heathland. Greatest diversity oc-curs in primary tropical rainforest, and it is conceivable thatsome elements of the group might have persisted in similarenvironments since the Late Paleozoic. Recently, we devel-oped an outline phylogeny for living Selaginellaceae based onrbcL gene sequences (Korall, Kenrick, and Therrien, 1999).Here, we build on this previous molecular work to develop amore detailed phylogenetic framework for the family.

Previous systematic studies divided Selaginellaceae into nu-

merous groups (Spring, 1850; Braun, 1857; Baker, 1883; Hi-eronymus, 1901; Walton and Alston, 1938; Jermy, 1986; So-jak, 1992), and we follow the classification of Jermy (1986).Jermy recognized one genus (Selaginella Pal. Beauv.) con-taining five subgenera: Selaginella (2 species), Ericetorum Jer-

1 Manuscript received 3 May 2001; revision accepted 11 October 2001.The authors thank S. Stefanovic (University of Washington, Seattle) for

providing some important DNA extracts for this analysis, J. Therrien (Uni-versity of Kansas, Lawrence) for providing an rbcL sequence, the many otherpeople who have contributed material to this study, and Catarina Rydin andTorsten Eriksson for comments on the manuscript. This work was financiallysupported by the Swedish Natural Science Research Council (NFR researchgrants to Paul Kenrick: B-AA/BU 10728-301, and to Paul Kenrick and POKaris: B 1393/1999), and Helge Ax:son Johnsons Stiftelse (grant to Petra

Korall).4 Author for reprint requests (e-mail: [email protected]).

my (3 species), Tetragonostachys Jermy (50 species), Stach-ygynandrum (Pal. Beauv.) Baker (600 species), and Heter-ostachys Baker (60 species). Our preliminary molecular phy-logeny based on a representative sample of 18 species revealedthat Selaginellaceae constitutes a monophyletic group and thatthe morphologically distinctive subgenus Selaginella is sistergroup to a clade comprising all other species (Korall, Kenrick,

and Therrien, 1999). In addition, the large subgenus Stachy-gynandrum was shown to be polyphyletic with some membersparaphyletic to Tetragonostachys, Ericetorum, and Heteros-tachys. Tetragonostachys and Ericetorum are monophyletic,but monophyly of Heterostachys was untested.

Here we extend our phylogenetic data set of rbcL gene se-quences from 18 to 62 species. Our sample includes a broadand representative selection of diversity, adding up to almost10% of living species. In particular, we have focused on im-proving our sample of Stachygynandrum, choosing speciesfrom all continents and a wide range of environments. Taxonchoice has been tailored to investigate the relationships of xe-rophytic species, and we have included four resurrection plantsas well as other drought-tolerant forms. Taxa have also been

chosen to reflect the morphological diversity within the group,focusing on those features that have been used by previousauthors to define groups. In addition to characters of leaf,branch, and root, we have paid particular attention to stelemorphology, sporangial arrangement, and megaspore wallstructure. We have been careful to select characteristics thatare observable in fossils, with the long-term aim of calibratingour phylogenetic tree.

MATERIALS AND METHODS

NomenclatureTaxonomy and nomenclature at the species level follow

Alston, Jermy, and Rankin (1981) and Stefanovic, Rakotondrainibe, and Badre

(1997) where applicable; otherwise Reed (19651966) is followed.

Choice of speciesIngroupChoice of ingroup was based on previoustaxonomic work (Baker, 1883; Hieronymus, 1901; Walton and Alston, 1938;


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March 2002] 507KORALL AND KENRICKPHYLOGENY OF SELAGINELLACEAE

Jermy, 1986), comparative morphology (emphasizing growth form, sporangial

arrangement, and leaf, stele, and megaspore morphology), geographical dis-

tribution, and chromosome number. A total of 62 species were chosen and 44

of the sequences analysed here were not previously published (Table 1). Spe-

cies recognition in Selaginellaceae is difficult and much effort was devoted

to accurate identification. Where necessary and whenever possible, specimens

were compared to type material, as indicated in Table 1. Most information on

sporangial arrangement (Horner and Arnott, 1963; Fraile and Riba, 1981;

Quansah, 1988), chromosome numbers (Manton, 1950; Tchermak-Woes and

Dolezal-Janish, 1959; Kuriachan, 1963; Jermy, Jones, and Colden, 1967; Love

and Love, 1976; Takamiya, 1993), and some data on stele arrangement (Hi-

eronymus, 1901; Mickel and Hellwig, 1969; Jermy, 1990) were taken from

the literature. Because species determination in Selaginellaceae is difficult and

herbarium and cultivated specimens often are labelled incorrectly, data culled

from the literature should be used with caution. Sporangial arrangement, rhi-

zophore development, and stele form have, as far as possible, been verified

through studies of herbarium material.

Isophyllous species were represented by the two species in subg. Selagi-

nella (S. selaginoides, S. deflexa), the three Ericetorum species (S. gracillima,

S. uliginosa, S. pygmaea), and four species of the drought-adapted Tetrago-

nostachys group (S. arizonica, S. rupestris, S. rupincola, S. sellowii).

Fifty-three anisophyllous species were chosen from Stachygynandrum (in-cluding ten members of the series Articulatae Spring) and Heterostachys. All

growth forms (e.g., creeping, erect, twining, rosettes) and a wide range of

geographical distributions are covered (Table 1). Monostelic, bistelic, and po-

lystelic species were included (Table 1). The steles of bistelic forms are pre-

dominantly terete, whereas those of monostelic forms are elliptical to strap-

shaped. Selaginella exaltata has a unique actino-plectostele (Mickel and

Hellwig, 1969). Selaginella novae-hollandiae is quite variable in general mor-

phology and widespread in Central and South America (Alston, Jermy, and

Rankin, 1981), and we suspect that it may represent a group of closely related

species. Two morphotypes were included, one from Venezuela and one from

Ecuador. Similarly, S. pallescens has two different growth forms, rosette (a

so-called resurrection plant) and erect, and both forms were included in the

analysis.

In addition to the ten articulate species, five possibly closely related non-articulate species were chosen (S. australiensis, S. lyalli, S. myosurus, S. po-

lymorpha, and S. sinensis). These possess at least one of the characteristics

of articulate species (i.e., single, rarely few, basal megasporangium subtended

by sterile sporophylls). Chromosome numbers are known for 24 of the species

chosen for the analysis (Table 1), x 8, 9, and 10 were represented, as well

as 2n 5060 (S. martensii).

OutgroupTwo species of Isoetaceae, Isoetes melanopoda and I. lacustris,

were included as outgroups. The sister-group relationship between Selaginel-

laceae and Isoetaceae has been confirmed in several studies, both morpholog-

ical (Kenrick and Crane, 1997) and molecular (Kranz and Huss, 1996; Wiks-

trom and Kenrick, 1997; Korall, Kenrick, and Therrien, 1999).

DNA extraction, amplification, and sequencingTotal DNA was extracted

from 34 specimens using the DNeasy Plant Mini Kit from Qiagen (Santa

Clarita, California, USA). Total DNA from nine species, mainly Madagascan,

was most kindly provided by S. Stefanovic (Department of Botany, University

of Washington, Seattle, Washington, USA). The rbcL sequence of the rosette

form of S. pallescens was kindly donated by James Therrien (Department of

Botany, University of Kansas, Lawrence, Kansas, USA). DNA extracts were

made from fresh material, from specimens dried in silica gel, or from her-

barium specimens. The species included in the analysis and references to

sequences taken from the literature are given in Table 1. Fragments corre-

sponding to bases 181383 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 117 and rbcL 1409R

corresponding to bases 13841409) (for primer sequences see Korall, Ken-

rick, and Therrien, 1999). Polymerase chain reaction was performed in 25-

L aliquots using Ready-To-Go PCR beads from Amersham Pharmacia Bio-tech (Uppsala, Sweden). The reactions were run in a Perkin-Elmer Thermal

Cycler with one cycle of 95C for 5 min and 30 cycles of 94C for 30 sec,

50C for 30 sec, and 72C for 2 min. A second amplification, using product

from the first PCR as template was occasionally necessary to obtain sufficient

DNA for sequencing. Two different amplification strategies were used: high

amounts of DNA template (15 L) and only 15 PCR cycles, or nested PCR,

where one internal primer was used in combination with one of the amplifi-

cation primers. Nested PCR was the most successful method, and allowed us

to obtain sequences from poor-quality or old material, such as S. pilifera

collected in 1907. The Thermo Sequenase Fluorescent Sequencing Kit from

Amersham Pharmacia Biotech (Uppsala, Sweden) was used to sequence dou-

ble-stranded PCR products for the rbcL gene. Samples were electrophoresed

on 6% Pharmacia Long Ranger acrylamide gels on an Amersham Pharmacia

Biotech (Uppsala, Sweden) automated ALF-express sequencer. All species

were sequenced in both directions using six different primers (Korall, Kenrick,

and Therrien, 1999). Sequences were assembled and edited using the Staden

Package (Staden, 1996). All sequences are deposited in EMBL.

A smaller partial PCR product was obtained for four species (S. willdenovii,

S. myosurus, S. plana, and S. helvetica). In these cases an internal primer was

used in combination with one of the amplification primers. The length of the

resulting sequences varied between 677 and 985 base pairs.

Phylogenetic analysisVisual alignment of the rbcL sequences was un-problematic because of the absence of insertions and deletions. The data ma-

trix contained 1299 characters corresponding to bases 831382 of the rbcL

gene of Marchantia polymorpha (Ohyama et al., 1986). Parsimony analyses

of the data were performed using PAUP* 4.0 (Swofford, 2000). Analyses

used the heuristic search option and the settings were random-sequence ad-

dition with 500 replicates, tree bisection-reconnection (TBR) branch swap-

ping, collapse of zero length branches, and MULTREES on. An equal weight-

ing scheme was employed with no transition-transversion bias (Albert and

Mishler, 1992). In all analyses, trees were rooted using both Isoetes species.

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 AutoDecay 4.0.2 (Eriksson, 1999) and PAUP*

4.0 (Swofford, 2000). PAUP* 4.0 settings used during decay analyses to find

the tree length of constrained trees were: heuristic search with 200 replicatesof random addition sequence, TBR branch swapping, collapse of zero length

branches, random sequence addition with one replicate, and MULTREES off.

Bootstrap values were calculated using PAUP* 4.0 by performing 30 000 rep-

licates with the following options selected: heuristic search, TBR branch

swapping, collapse of zero length branches, random sequence addition with

one replicate, and MULTREES off.

RESULTS

Of 1299 characters, 566 were phylogenetically informativefor parsimony. An heuristic search yielded 1355 equally mostparsimonious trees of 2403 steps, consistency index (CI) 0.375, retention index (RI) 0.758, uninformative characters

excluded. The strict consensus tree is shown in Fig. 1. Cladesare referred to throughout the text by the outermost (top andbottom) species of the clade as they are found in Figs. 1 and2. Note that the circumscriptions are dependent on how thetree is drawn and are only relevant when compared to Figs. 1and 2.

Results are consistent with monophyly of Selaginellaceae(Fig. 1). Within this clade, subg. Selaginella is monophyletic(decay index 54; bootstrap 100%) and sister group to a poorlysupported clade comprising all other species, which we termthe rhizophoric clade (decay index 2; bootstrap 50%).The rhizophoric clade (Fig. 1) is divided into two subclades,here termed clades A (S. sellowii/S. australiensis) and B (S.tamariscina/S. denticulata). Clade A contains both isophyllous

and anisophyllous species and has low support (decay index2; bootstrap 50%). Several of the basal nodes within clade


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TAB

LE1.

Continued.

Taxa

(!)

comparedtotypematerial

Subgenus/series

Geographicaldistribution

Stelearrangementa

Chromosome

numbera

S.

rupestris(L.)Springb

S.

rupincolaUnderw.b

S.

selaginoides(L.)Linkb

S.

sellowiiHieron.

S.

sericeaA.Braun

S.

simplexBaker!

Tetragonostachys

Tetragonostachys

Selaginella

Tetragonostachys

Stachygynandrum/Articulatae

Heterostachys

N.America

S.USA

Circumboreal

Cuba,Mexico,S.America

S.America

S.America

monostelic,terete

monostelic,elliptical

monostelic,terete

monostelic

bistelic,terete

unknown

2n

18

unknown

2n

18

unknown

unknown

unknown

S.

sinensis(Desv.)Spring

S.

stauntonianaSpring!

S.

suavis(Spring)Spring

S.

sulcata(Desv.)Spring

Stachygynandrum

Stachygynandrum



China

E.Asia

S.America

S.America

monostelic


bistelic,terete

bistelic

unknown

unknown

unknown

2n

20

S.

tamariscina(Beauvais)Spring

S.

uliginosa(Labill.)Springb

S.

umbrosaLemaireexHieron.

S.

willdenovii(Desv.)Baker

Stachygynandrum

Ericetorum

Stachygynandrum

Stachygynandrum

E.Asia,northernpartsofSEAsia

Australia,Tasmania

S.America

SEAsia,introducedinNewWorldtropics


solenostelic


polystelic,elliptical

2n

20

2n

18

2n

20

2n

18

Outgroup

IsoeteslacustrisL.b

Isoetesmelanopoda(Gay&Durieu)ex.D

urieuh

Europe

USA

unknown

unknown

2n

110g

2n

22g

a

Seetextforreferences.

b

Previouslypublishedsequences:Korall,

Kenrick,andThierren(1999).

c

CollectedinVenezuela.

d

CollectedinEcuador.

e

Rosetteform.

f

Erectform.

g

MusselmanandHeafner(1997).

h

Previouslypublishedsequences:Manhart(1994).


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510 [Vol. 89AMERICAN JOURNAL OF BOTANY

Fig. 1. Strict consensus of 1355 equally most parsimonious trees, consistency index 0.375, retention index 0.758. Support values above branchesdenote decay indices and below branches bootstrap values (a dash indicates a bootstrap value less than 50%). Branches with bootstrap values 80% are thicker.Support values with arrows pointing to nodes denote values on these nodes when S. sinensis and S. australiensis are excluded from the analysis. All species

not labeled with subgenus (first column) are classified in subg. Stachygynandrum. Note that series Articulatae is found within Stachygynandrum.1

The rosetteform of S. pallescens. 2The erect form of S. pallescens. 3Specimen collected in Venezuela. 4Specimen collected in Ecuador.


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Fig. 2. Phylogram showing one of the most parsimonious trees. Taxa in boldface are isophyllous. Stele arrangement is shown as: one circle monostelic;two circles bistelic; three ellipses polystelic; * actino-plectostele; ? stele type unknown.


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A are also weakly supported, leaving three major well-sup-ported groups: (1) the isophyllous subg. Tetragonostachys,which is monophyletic with very strong support (decay index44; bootstrap 100%); (2) a group of articulate species (all Ar-ticulatae except S. exaltata) (decay index 12; bootstrap 96%);and (3) a very strongly supported clade comprising the three

species in Ericetorum and three Madagascan species (S. lyallii,S. moratii, and S. polymorpha) (decay index 21; bootstrap100%). However, monophyly of Ericetorum is not supported.Also of interest is the sister-group relationship between S. lep-idophylla (resurrection plant) and the moss-like xerophytes inTetragonostachys, which has moderate support (decay index4; bootstrap 70%). Other notable groupings in clade A includespecies with rhizophores emerging from the upper surface ofbranches, here termed the dorsal rhizophoric clade. Thisgroup includes Articulatae, Tetragonostachys, and their re-spective sister taxa: S. myosurus and S. lepidophylla (decayindex 2; bootstrap 50%). Also, there is weak support formonophyly of Articulatae (decay index 2; bootstrap 50%).

Clade B comprises only anisophyllous species (decay index

3; bootstrap 76%), and it contains several well-supported sub-clades (Fig. 1). There is strong support for a cosmopolitanclade (Asia, North America, Africa/Madagascar) of drought-adapted species (S. tamariscina/S. imbricata, decay index 43;bootstrap 100%). This predominantly xerophytic group is sis-ter to a clade containing all other species: S. apoda/S. denti-culata. Some of the subsequent nodes are weakly or moder-ately supported, leaving two strongly supported major nodes:A clade of southeast Asian and Australian species, S. longi-

pinna/S. alopecuroides (decay index 4; bootstrap 97%), and aclade of European, Madagascan, and Asian species, S. plana/S. denticulata (decay index 17; bootstrap 100%). In addition,a poorly supported clade of Central and South American hu-mid tropical species is found, S. pallescens/S. novae-hollan-

diae (decay index 1; bootstrap 50%). Species with dimorphicsporophylls (Heterostachys sensu Jermy, 1986) form a poly-phyletic assemblage. Neither the two geographic variants of S.novae-hollandiae nor the two morphologically different formsof S. pallescens were resolved as monophyletic.

DISCUSSION

SystematicsDespite its large size (700 species), only ahandful of taxonomic groups are widely recognized within Se-laginellaceae. Of the groupings that have been establishedbased on morphological criteria, rbcL data corroborate the is-ophyllous subg. Selaginella and Tetragonostachys. Likewise,

the anisophyllous series Articulatae (subg. Stachygynandrum),excluding S. exaltata, has strong support. Monophyly of thedrought-adapted, mainly North American Tetragonostachyshas been supported previously by two molecular studies thatsampled most species of the group (based on rbcL and thenuclear ribosomal internal transcribed spacer region [ITS];Therrien, Haufler, and Korall, 1999; Therrien and Haufler,2000). In previous systematic treatments, major divisions ofthe family have often been based on one or more morpholog-ical features, such as leaf dimorphism (isophylly/anisophylly),phyllotaxy, and growth form. The rbcL data suggest that sev-eral of these key characters have evolved iteratively, implyingthat these groups are polyphyletic or paraphyletic. It should benoted, however, that earlier classifications are in most cases

not phylogenetically based systems and were thus not neces-sarily intended to reflect evolutionary relationships (exceptions

include Sojak, 1992). Of the groups recognized by Jermy(1986), the rbcL tree indicates that Stachygynandrum and Het-erostachys are polyphyletic. Whether Ericetorum is monophy-letic or not cannot be deduced from this study. Of the groupsrecognized by Sojak (1992) the genera Selaginella ( subg.Selaginella of Jermy, 1986) and Bryodesma ( subg. Tetra-

gonostachys of Jermy, 1986) are monophyletic, but the largegenus Lycopodioides Boehm ( subgenera Ericetorum, Stach-ygynandrum, and Heterostachys of Jermy, 1986) is paraphy-letic to Bryodesma.

Perhaps the most surprising aspect of our analysis is thelarge number of new and well-supported groupings not pre-viously recognized on morphological grounds. These cladesrange enormously in size from those containing perhaps ahandful of species to others with many hundreds. Examplesof these include S. remotifolia/S. fragilis, S. pygmaea/S. gra-cillima, S. sinensis/S. australiensis, S. tamariscina/S. imbri-cata, S. apoda/S. denticulata, S. longipinna/S. alopecuroides,and S. plana/S. denticulata. One of these groupings shares thedistinctive ecological trait of being xerophytic (S. tamariscina/

S. imbricata), but the other clades do not appear to be sup-ported by any obvious characters. This may reflect a poor un-derstanding of the comparative morphology, ecology or phys-iology of species in this large and diverse family. It may alsobe an artifact of limited sampling because the rbcL data sample10% of living species diversity. There is certainly a need toreevaluate these species groups from other perspectives. Thetaxonomic status of various groups is discussed in more detailbelow.

New groupsOur previous molecular analysis (Korall, Ken-rick, and Therrien, 1999) identified a clade with a conspicuousmorphological marker, which we have called the rhizophoricclade (Fig. 1). All species in this group bear a distinctive and

unique root-like structure termed the rhizophore (Fig. 3C andF), which is lacking in the two species of subg. Selaginella.Another morphological character supporting the rhizophoricclade is the decussate arrangement of sporophylls. Bootstrapsupport for this clade is very low. Within the rhizophore bear-ing group, a basal dichotomy gives rise to two major clades:clade A (poorly supported) with both isophyllous and aniso-phyllous species and clade B (moderate support) with onlyanisophyllous species (Fig. 1). The exclusion of two specieswith exceptionally long branches (S. sinensis and S. austral-iensis), strengthens support for these three clades (see belowbranch lengths, pseudogenes, and morphotypes).

All species in the poorly supported dorsal rhizophoric clade

(Fig. 1) also possess a conspicuous and unique morphologicalmarker. Instead of developing from the lower surface of thestems, the rhizophore develops from the upper surface (surfacebearing small leaves) and loops over the branch to grow down-wards (Fig. 3C). This distinctive form of development is welldocumented in Articulatae (e.g., S. kraussiana), but has notbeen recognized previously as a synapomorphy of a largergroup. Dorsal rhizophores have been reported for two speciesof Tetragonostachys (S. densa Rydb. in Webster and Steeves[1963], S. wallacei Hieron. in Webster and Steeves [1964]),and we have observed this developmental pattern in an addi-tional ten out of50 species in this group. Observations ofherbarium and living material show that dorsal rhizophoresalso occur in the resurrection plant S. lepidophylla and in S.

myosurus.The well-supported S. remotifolia/S. fragilis clade contains


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Fig. 3. Morphology of Selaginella. (AC) Selaginella arizonica (subg. Tetragonostachys). (A) Habit, note the incurling vegetative branch tips. (B) Close-up of vegetative branch showing uniform spirally arranged leaves. (C) Close-up of branch dichotomy showing rhizophore originating on the dorsal side of thebranch. (DF) Selaginella martensii (subg. Stachygynandrum). (D) Habit. (E) Close-up of upper side of vegetative branch showing dimorphic leaves, decussatelyarranged in four rows. (F) Close-up of branch dichotomy showing rhizophore originating on the ventral side of the branch. Drawing by Pollyanna von Knorring.

all of the articulate species sampled, except S. exaltata. In1850, Spring incorporated all species with articulations into awidely recognized and predominantly South American groupcalled Articulatae (Braun, 1865; Hieronymus, 1901; Waltonand Alston, 1938). Articulations are small swellings of thestem that occur below dichotomies. Somers (1978) listed sev-eral additional morphological features found in this group.Distinguishing features include (1) dorsal rhizophores, (2) asingle, basal megasporangium (rarely two) associated withseveral enlarged sterile sporophylls, (3) large megaspores, and(4) unique microsporangia. Monophyly of Articulatae is con-

sistent with the strict consensus tree (Fig. 1), but has very lowsupport. Although the articulations seem to be a synapomor-

phy of the group, many of the characteristics documented bySomers (1978) have also been reported for nonarticulate spe-cies (e.g., the dorsal rhizophores). It seems likely, therefore,that a more detailed study of Articulatae and close relativeswill show that some of the putative characteristics of the groupare in fact synapomorphies of a series of more inclusiveclades.

The South American articulate species (S. sericea/S. fragil-is) form a well-supported subclade that is sister to a cladecontaining the only two Old World articulate species sampled(i.e., S. kraussiana, S. remotifolia; Somers, 1978). These two

species are very similar in morphology and growth form andhave been considered geographic variants of a single species


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(S. kraussiana; Baker, 1884). Differences in stele and leafshape (P. Korall, personal observations; Hieronymus, 1901;Somers, 1978) together with 91 base-pair differences in therbcL sequences support their treatment as distinct species.Whereas S. remotifolia is strictly confined to the Old World(parts of east and southeast Asia), the distribution ofS. kraus-

siana spans both New World and Old World (parts of Africa,South America). Alston, Jermy, and Rankin (1981) point outthat the general assumption is that S. kraussiana has been in-troduced into South America by humans. In addition to S.kraussiana there is at least one other Articulatae species inAfrica. Selaginella grallipes Alston differs markedly in grossmorphology from the creeping S. kraussiana and S. remotifoliaand more closely resembles the scandent South American S.exaltata (Moran and Smith, 2001). Including S. grallipes inthe analysis might help elucidate the weakly supported rela-tionships between basal Articulatae species and close relatives.

The strongly supported S. pygmaea/S. gracillima clade doesnot possess any obvious unique morphological synapomor-phies. Morphologically, the three Madagascan species (S. lyal-lii, S. moratii, S. polymorpha) and Ericetorum (S. gracillima,S. uliginosa, S. pygmaea) are quite dissimilar. Ericetorum con-tains small, isophyllous plants that inhabit temperate heathlandin Australia and South Africa. Selaginella pygmaea and S.gracillima are annuals. In contrast, S. lyallii, S. polymorpha,and S. moratii are tall plants (up to 80 cm height; Stefanovic,Rakotondrainibe, and Badre, 1997) with frondose branches inthe upper part, anisophyllous, and tropical. The rbcL sequenc-es of the three Madagascan species differ in only one or twobase pairs, and although the species are very similar in mor-phology, Stefanovic, Rakotondrainibe, and Badre (1997) con-sider them to be different species.

The very large pantropical S. apoda/S. denticulata clade

contains almost half of the 62 species included in the analysis.All have dimorphic leaves and are members of either Stach-

ygynandrum or Heterostachys. Most species are confined tothe humid tropics. One exception is the sister species to therest of the group, S. apoda, which grows in moist, temperateenvironments in North America. It is likely that this cladeincludes many unsampled species from the humid tropics ofsoutheast Asia and South America. The restricted geographicdistribution and the relatively small differences in branchlengths (Fig. 2) among species within some subclades is con-sistent with comparatively recent speciation events.

Morphological characters previously used as diagnostic

features in classificationsThe presence or absence of leavesof two distinctive sizes (leaf dimorphism or anisophylly) hasbeen used as a criterion for subdividing the family (see Spring,1850; Baker, 1883; Hieronymus, 1901; Thomas and Quansah,1991), but our study shows that isophylly has a complex evo-lutionary history. Anisophylly is characteristic of the subgen-era Heterostachys and Stachygynandrum (Fig. 3DF), whereasTetragonostachys (Fig. 3AC), Selaginella, and Ericetorumare isophyllous (Fig. 2). Outgroup comparison with Isoetaceaeindicates that leaves of one size is the plesiomorphic state forthe family. The isophyllous states of Tetragonostachys and

Ericetorum are most parsimoniously interpreted as reversals,but because most basal nodes have low support the possibility

that isophylly is a retained plesiomorphic state and that ani-sophylly has originated several times cannot be ruled out.

A resupinate strobilus, one that exhibits sporophyll dimor-phism with the smaller sporophylls borne in the same planeas the larger vegetative leaves, was used to characterize thepolyphyletic subg. Heterostachys. Five resupinate species wereincluded in the analysis (Fig. 1). Although they all appear inclade B, the rbcL data indicate that the evolution of resupinate

strobili involved parallelisms and/or reversals. In a futurestudy, it would be interesting to include S. bemarahensis S.Stefanovic and Rakotondrainibe and S. marinii S. Stefanovicand Rakotondrainibe. These species have resupinate strobili,but the rhizophores are dorsal (Stefanovic and Rakotondrain-ibe, 1996), which implies a phylogenetic position closer to

Articulatae and Tetragonostachys.Stele morphology has been used as a basis for recognizing

groups within Selaginellaceae (Hieronymus, 1901), and thischaracteristic is documented for most species (Fig. 2, Table1). It is clear from Fig. 2 that the bistelic condition has evolvedat least once among the articulate species and that the polys-telic condition has arisen independently at least four times, inS. lyallii and S. polymorpha, in S. articulata, in S. acanthos-

tachys and in S. willdenovii and S. plana. A solenostele isreported as characteristic of Ericetorum (Jermy, 1986).

Several studies have shown that distinctive patterns of spo-rangial arrangement exist in Selaginella (e.g., Horner and Ar-nott, 1963; Fraile and Riba, 1981; Quansah, 1988). Majortrends that have been recognized include the organization ofsporangia into rows (two rows of microsporangia and tworows of megasporangia) and the restriction of megasporangiato a zone at the base of the strobilus. The only pattern thatseems always to be consistent within species is the presenceof a single (rarely two) basal megasporangium (Fraile andRiba, 1981; Quansah, 1988). This arrangement has been usedas a feature unifying the articulate species (Somers, 1978), butis also found in other taxa. This was recognized by Hierony-

mus (1901) who included sporangial arrangement as a diag-nostic character in his classification by dividing the large sub-genus Heterophyllum (corresponding to Stachygynandrum and

Heterostachys together) into Oligomacrosporangiatae (fewmegasporangia per strobilus) and Pleiomacrosporangiatae(many megasporangia per strobilus). Included in our analysis,in addition to the articulate species, are S. myosurus, S. aus-traliensis, S. lyallii, and S. polymorpha and S. sinensis. Ourresults show that species with a single basal megasporangiumare all found in clade A, but that this characteristic does notuniquely circumscribe a distinct clade.

Branch lengths, pseudogenes, and morphotypesWith

566 phylogenetically informative characters for 62 taxa, therbcL gene has a very high level of variation in Selaginellaceae,and this is distributed unequally within the family (Fig. 2).The branches in clade A are longer (more substitutions) thanthose in clade B, and this phenomenon has the potential ofcausing analytical problems. Long branches can cause spuri-ous groupings (Felsenstein, 1978), but this is mainly a problemwhen they are connected by short internodes. This pattern isnot seen in our tree (Fig. 2) in which internodes are of asimilar length to terminal branches for the most part. Thebranches leading to S. sinensis and S. australiensis are excep-tional even for Selaginellaceae and have led to unstable to-pologies. Under some ingroup/outgroup combinations, thesetwo species have even grouped with seed plants in the Gne-

tales, also a group with an exceptionally long branch in rbcLdata sets. Because of this problem we acknowledge the pos-


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sibility of spurious groupings, but we suspect that this is nota major problem because many groupings within the tree arecomparatively stable and well supported, both in terms ofbootstrap support and decay index. Furthermore, if S. sinensisand S. australiensis are excluded from the analyses, the sametopology is found and support for the basal nodes increases.

The support for the rhizophoric clade, clade A, and clade B,rise from bootstrap values/decay indices of50%/2 to 94%/12,50%/2 to 85%/7, and 50%/3 to 91%/4, respectively. Inview of the branch lengths, it is particularly important to testthe rbcL gene tree through the development of a parallel dataset based on additional genes.

Our laboratory data indicate the possible existence withinSelaginellaceae of a pseudogene that is shorter than the func-tional rbcL gene. Double PCR products were amplified in sixof the included species (S. acanthostachys, S. arizonica, S.novae-hollandiae [from Venezuela], S. remotifolia, S. rupin-cola, S. sellowii), and multiple products were observed in onespecies (S. plana). Pseudogenes of rbcL have previously beenfound in both chlorophyllous and parasitic angiosperms and in

red algae (see Sennblad, Endress, and Bremer, 1998, and ref-erences therein). Because no attempts to sequence the shorterfragments have been made, we cannot exclude the possibilitythat the fragments are amplifications of unrelated parts of thegenome.

The rbcL sequences revealed problems in the species delim-itation of S. novae-hollandiae. Two morphotypes of S. novae-hollandiae were included in the analysis. These specimens col-lected from Venezuela and Ecuador were not resolved as amonophyletic group (Fig. 1) and the rbcL sequences differ in27 base pairs. Analyzing the data set with a constraint thatforced the two taxa together produced trees five steps longerthan the most parsimonious trees. These results are consistentwith the presence of more than one species in S. novae-hol-

landiae. Similarly, the rbcL data show that the rosette anderect forms of S. pallescens differ in nine base pairs and thatthey are closely related but not a monophyletic group (Fig. 1).In this case, a constraint yields trees only two steps longerthan the most parsimonious trees. This might also constitutegrounds for considering the separation of rosette and erectforms at the species level. Interspecific variation in the rbcLgene of Selaginellaceae has not previously been examined, butstudies on ferns show that within-species variation is low: only02 base-pair differences are commonly found between indi-viduals considered to belong to the same species (Hauk, 1995;Yatabe, Takamiya, and Murakami, 1998). Our results indicatethat species diversity may be underestimated in the family and

reflect the need of a thorough revision of the family at thespecies level.

Origins of SelaginellaceaeThe earliest fossil evidence ofSelaginellaceae comes from the tropical wetland floras of theCarboniferous Period (Visean Epoch, 333350 million yearsago [Ma]; Rowe, 1988). By the Late Carboniferous (290323Ma) branching stems that bore minute leaves of two distinctsizes were widespread in coal measure floras (Thomas, 1992,1997). These observations demonstrate that the characteristicof leaf dimorphism appeared early on and that the family wasan important component of the humid tropical floras of theLate Paleozoic. Selaginellaceae are predominantly forest-floor-dwelling species, and the possession of leaves of two distinct

sizes is most probably an adaptation to poor light quality (He-bant and Lee, 1984). The evolution of this characteristic broad-

ly coincides with earliest evidence for closed-canopy forest,which was caused by the rise of tree ferns during the West-phalian, some 303320 Ma (DiMichele et al., 1992). The eco-logical association between Selaginellaceae and coal measureforests and the similarity of modern species to fossils raisesthe possibility that some living tropical clades may be relicts

of these ancient Carboniferous groups (Korall, Kenrick, andTherrien, 1999).If elements of Selaginellaceae have persisted in tropical wet-

lands since the Carboniferous, we would expect to resolve spe-cies from the humid tropics as a basal grade or clade withinthe family, which would be paraphyletic or basal to xerophyticand temperate clades. The results of our molecular analysis donot, however, support this interpretation. Results documenttwo major clades of predominantly humid tropical species, theS. pallescens/S. denticulata clade and a clade containing Ar-ticulatae and its sister species, S. myosurus. The S. pallescens/S. denticulata clade is further divided into strongly supportedSoutheast AsianAustralian (S. longipinna/S. alopecuroides)and weakly supported neotropical elements (S. pallescens/S.

novae-hollandiae). Several of the basal clades in Selaginella-ceae are either temperate, xerophytic, or contain significantproportions of species that fit these categories. Subgenus Se-laginella contains the temperate/arctic alpine S. selaginoides.In clade A, the S. pygmaea/S. australiensis clade is probablytemperate, and the Tetragonostachys plus S. lepidophylla cladeis xerophytic. Likewise, in clade B, the S. tamariscina/S. im-bricata clade contains drought-adapted species, and the nextbranch up is S. apoda, a temperate North American species.These data show a comparatively derived position for bothmajor clades of humid tropical species. The cladogram topol-ogy therefore indicates that species and species groups of themodern humid tropics are unlikely to be relicts of Carbonif-erous cladogenesis, but are probably of more recent origin.

How can this interpretation be reconciled with the early ap-pearance of species with dimorphic leaves in the tropical wet-lands of the Carboniferous Period? The relationships of theearly fossil species are currently very poorly understood. Thisis mainly because many are compression fossils, and they donot contain much information on anatomical or reproductivestructures. They are therefore relatively information poor. It ispossible that some of these early species would branch fromthe stem group of the rhizophoric clade or perhaps even fromthe Selaginellaceae stem group. In either case, these specieswould not be expected to possess the distinctive rhizophore,and in this context it is interesting to note that rhizophoreshave not been observed in any Paleozoic fossils.

Multiple origins of resurrection plantsAlthough mostspecies diversity in Selaginellaceae occurs in primary tropicalrainforest, a substantial number of species are able to with-stand seasonal drought or even live in very arid parts of theworld. The moss-like xerophytes (Fig. 3AC) in Tetragonos-tachys have been shown to be a monophyletic group (Korall,Kenrick, and Therrien, 1999; Therrien, Haufler, and Korall,1999; Therrien and Haufler, 2000). Physiologically, species inthis group are able to withstand prolonged desiccation, andmorphologically they exhibit adaptations to reducing waterloss by possessing small leaves with thick cuticles. In Stach-

ygynandrum, at least ten species have a rosette of branchesand during drought the branches curl inwards, forming a ball.

These are called resurrection plants and the name alludesto their ability to revive rapidly by uncurling their branches to


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reform the rosette habit under more favorable moisture con-ditions. We included four of these ten species in our analysisin order to investigate their relationships to each other and toTetragonostachys. Results show that resurrection plants haveevolved at least three times in Selaginellaceae. The resurrec-tion plant S. lepidophylla is sister to the moss-like xerophytes

of the Tetragonostachys clade. Selaginella lepidophylla alsohas the distinctive rhizophore development that characterizesthe more inclusive dorsal rhizophoric clade. The resurrectionplants S. tamariscina (eastern Asia) and S. pilifera (southernUSA, Mexico) are found in a strongly supported clade withother non-resurrection Asian (S. stauntoniana) and Madagas-can species (S. digitata, S. helioclada, S. imbricata). All spe-cies in this clade grow in seasonally dry areas. In contrast toother xerophytes, the rosette form of the resurrection plant S.

pallescens is nested in a humid tropical clade. The rbcL datatherefore support diverse relationships for resurrection plants,indicating that this response to arid conditions has evolvediteratively in Selaginellaceae. Incurling of branches in a lesspronounced fashion is also found in other drought-adapted

species such as some species of Tetragonostachys (e.g., S. ar-izonica; Tryon, 1955; see Fig. 3AC), and the non-rosette-forming species of the S. tamariscina/S. imbricata clade (S.stauntoniana and S. imbricata; P. Korall, personal observa-tions; S. digitata and S. helioclada; Stefanovic, Rakotondrain-ibe, and Badre, 1997). There is evidence therefore in the mor-phology of other species that the resurrection plants adapta-tions to aridity may just be an extreme example of a moregeneral but less dramatic trend of branch incurling that is com-mon in the family.

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506===phylogenetic relationships in selaginellaceae rbcl===+

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