Molecular Phylogenetics of Anogramma Species and Related

490

Systematic Botany (2003), 28(3): pp. 490–502q Copyright 2003 by the American Society of Plant Taxonomists

Molecular Phylogenetics of Anogramma Species and Related Genera(Pteridaceae: Taenitidoideae)

TAKUYA NAKAZATO and GERALD J. GASTONY1

Department of Biology, Indiana University, Jordan Hall, 1001 East Third Street,Bloomington, Indiana 47405-3700

1Author for correspondence ([email protected])

Communicating Editor: Alan T. Whittemore

ABSTRACT. Anogramma is a genus of eight putative species with small annual sporophytes and potentially perennatinggametophytes. Phylogenetic relationships within the genus as well as its relationships with other putatively taenitidoid generaand with traditionally cheilanthoid Cosentinia vellea have been poorly resolved and are investigated here. Maximum parsi-mony, maximum likelihood, and Bayesian inference analyses of rbcL sequences were used to test 1) the monophyly ofAnogramma, 2) support for the proposed specific distinctness of A. guatemalensis and A. caespitosa from A. leptophylla, and 3)the asserted close (sister) relationship between Anogramma and Pityrogramma, as well as to infer the phylogenetic relationshipsamong these genera. Results reveal that Anogramma in the traditional sense is polyphyletic. Anogramma guatemalensis and A.caespitosa nest within A. leptophylla. Pityrogramma is not sister to Anogramma as a whole but only to A. chaerophylla and A.novogaliciana. Anogramma osteniana is deeply separated from its traditional congeners and the new combination Jamesoniaosteniana (Dutra) Gastony is made. Cosentinia vellea should not be subsumed within Cheilanthes but instead should beconsidered a taenitidoid genus closely related to A. leptophylla and A. lorentzii. Neighbor joining analysis of Amplified Frag-ment Length Polymorphism data sets inferred relationships among the A. leptophylla accessions, embedding A. guatemalensisand A. caespitosa in respective New World and Old World clades of A. leptophylla.

Anogramma is a homosporous fern genus in whicheight species have been proposed: A. ascensionis, A.caespitosa, A. chaerophylla, A. guatemalensis, A. leptophyl-la, A. lorentzii, A. novogaliciana, and A. osteniana. Thesespecies are found mainly in the tropics of Mexico andCentral and South America. Anogramma leptophylla,however, has an exceptionally wide range of distribu-tion, in the cited regions of the New World and in theOld World where it occurs in Africa, southern Europeto northern India, Australia, and New Zealand (Tryonet al. 1990), and A. caespitosa is reported only from Mt.Kilimanjaro, Tanzania. Sporophytes of these speciesare typically small (1–10 cm), and often grow in en-vironments with alternating wet and dry seasons(Tryon and Tryon 1982).

The taxonomic treatment of species of Anogrammaand its generic relatives based on non-molecular char-acters remains uncertain and intuitive. Although soralmorphology has traditionally suggested that Anogram-ma is closely related to Pityrogramma and Eriosorus, itis generally considered to be a distinctive lineage(Christensen 1938; Copeland 1947; R. Tryon 1962). Thisis primarily because species of Anogramma, except A.osteniana, are unique among ferns in having gameto-phytes whose archegonial cushion areas form pea-liketubercles ca. 1–3 mm in diameter. Tubercles can be-come dormant during environmentally stressful sea-sons and generate new gametophytic lobes or supportthe outgrowth of a dormant embryo when favorableenvironmental conditions resume (Baroutsis 1976). Be-cause of its tubercles, Anogramma gametophytes areconsidered perennial although the sporophytes func-tion as annuals (Mehra and Sandhu 1976). Anogramma

osteniana differs from its congeners in lacking game-tophytic tubercles and having glandular trichomes onthe leaf and distinctive spore morphology (Tryon andTryon 1982). It has been suggested that A. ostenianamay be closely related to Eriosorus based on similari-ties of leaf and spore morphology, although the dimin-utive sporophyte habit of A. osteniana is like that ofother Anogramma species and unlike that of Eriosorusspecies. Anogramma guatemalensis (Domin 1928) fromGuatemala and Mexico, and A. caespitosa (Pichi Ser-molli 1972) from Mt. Kilimanjaro of Tanzania, arethought to be closely related to A. leptophylla based ontheir overall morphological resemblance, despite someminor distinctive characteristics. Whether or not theyare genetically distinctive enough from A. leptophylla tobe treated as separate species has been controversial(Baroutsis 1976). Similarly, A. ascensionis (Hooker1854), endemic to Ascension Island, and A. novogalici-ana (Mickel 1992), endemic to Jalisco and Michoacan,Mexico are clearly related to A. chaerophylla based ontheir overall morphology, but the relationships amongthese species have not been investigated.

Cosentinia is an Old World genus of a single species(C. vellea) that is widely distributed in regions border-ing the Mediterranean Sea and in Macaronesia andsouthwest Asia eastward to the western Himalayas(Badre and Reichstein 1983; Vida et al. 1983; Rivas-Martınez and Salvo 1984; Zimmer 1991). The taxonom-ic treatment of this species has long been unsettled(Fee 1852; Todaro 1866; Copeland 1947; Pichi Sermolli1977; Saenz de Rivas and Rivas-Martınez 1979; Rivas-Martınez et al. 1981; Badre et al. 1982; Tryon andTryon 1982; Tryon et al. 1990). Most of these authors

2003] 491NAKAZATO & GASTONY: PHYLOGENETICS OF ANOGRAMMA

FIGS. 1–3. Sporophyte morphologies of Anogramma lepto-phylla and Cosentinia vellea. 1. Pressed and dried whole maturesporophyte of A. leptophylla at left (Sheehan s.n. 7 April 1973,Greece [IND]) and mature leaf of C. vellea (Gastony 90-1, Spain[IND]) at right with some lower pinnae partly or entirely bro-ken away. Scale bar 5 20 mm. 2. Close-up of glabrous centralright pinna of A. leptophylla from Fig. 1, bearing dehisced spo-rangia and dispersed spores. Scale bar 5 5 mm. 3. Close-upof densely wooly central left pinna of C. vellea of Fig. 1, spo-rangia partially obscured by tomentum. Scale bar 5 5 mm.

have considered C. vellea a species of Notholaena (N.lanuginosa) or Cheilanthes (C. vellea) of Pteridaceae sub-family Cheilanthoideae, because of its habit and woolyindument of trichomes and perhaps because of the sea-sonally xeric habitat it shares with these cheilanthoidgenera. Pichi Sermolli (1985), however, concluded thatCosentinia vellea is closely related to Anogramma of Pter-idaceae subfamily Taenitidoideae, primarily because itsspore morphology is nearly identical to that of Ano-gramma leptophylla (Lugardon 1963; Badre et al. 1982;Pichi Sermolli 1985). Probably because the sporophytemorphologies of C. vellea and A. leptophylla are strik-ingly different (Figs. 1–3), most notably in the woolyindument of C. vellea versus the glabrous, delicate lam-inas of A. leptophylla, C. vellea has most recently beenmaintained as a species of Cheilanthes (Tryon et al.1990) or at least as a member of subfamily Cheilan-thoideae (Zimmer 1991). However, if the close relation-ship between Anogramma and C. vellea is true, the lattershould be removed from Pteridaceae subfamily Chei-lanthoideae to subfamily Taenitidoideae. Molecularphylogenetic analysis should help to resolve the ge-neric relationships and placement of this species.

This study uses rbcL nucleotide sequences from thechloroplast genome to investigate phylogenetic rela-tionships among the species of Anogramma and be-tween Anogramma and traditionally related genera andto determine the generic relationships and subfamilialplacement of Cosentinia vellea. It also investigates ge-netic relationships among geographically diverse ac-cessions of widely distributed A. leptophylla using Am-plified Fragment Length Polymorphisms (AFLPs; Voset al. 1995) of total genomic DNA.

MATERIALS AND METHODS

Study Samples. Sixteen accessions representing all Anogrammaspecies, except A. ascensionis were included in the phylogeneticanalysis. Anogramma ascensionis has not been recorded from As-cension Island since 1958—searches for it by Quentin Cronk in1976, 1986, and 1995 were unsuccessful (Antonia Eastwood, pers.comm.). It is suspected to be extinct and was not included in thisstudy because neither suitable specimens nor viable spores wereavailable. Spores of available Anogramma accessions except A. nov-ogaliciana were sown on Metro-Mixt (Scotts, Marysville, OH) inour laboratory, and total genomic DNA was extracted from re-sulting gametophytes or sporophytes, following the protocol inDNeasy Plant Mini Kit (Qiagen, Inc., Valencia, CA). The sameDNeasy kit was used to extract DNA from herbarium specimensof Anogramma novogaliciana and Eriosorus flexuosus and from freshleaf tissue of Platyzoma microphyllum kindly provided by Peter Bos-tock. DNA of Cosentinia vellea was obtained by CTAB extraction(Doyle and Doyle 1987) of a field-collected sporophyte. The rbcLgene sequence from Jamesonia canescens was provided by RaymondCranfill. Sequences of rbcL from species of 21 related genera in-cluded in the analyses of Hasebe et al. (1995), as well as sequencesof Pteris cretica, Onychium lucidum, and two species of Pityrogrammawere obtained from GenBank.

Accessions included in this study are listed in Table 1, with thefollowing exception. To minimize Table 1, rbcL sequences used inthis study but taken from previously published sources are listedalphabetically with GenBank accession number in paragraph for-

mat here. Superscript 1 indicates data in Hasebe et al. (1995); su-perscript 2 indicates data in Gastony and Johnson (2001): 1Acro-stichum aureum L. (U05601); 2Actiniopteris radiata (Sw.) Link(AF336100); 1Adiantum pedatum L. (U05602); 1Ananthacorus angus-tifolius Underw. & Maxon (U20932); 2Anogramma lorentzii (Hieron.)Diels (AF336102); 1Antrophyum reticulatum (G. Forst.) Kaulf.(U05604); 1Argyrochosma delicatula (Maxon & Weath.) Windham(U19500); 1Bommeria ehrenbergiana (Klotzsch) Underw. (U19497);1Ceratopteris thalictroides (L.) Brongn. (U05609); 1Cheilanthes allosu-roides Mett. (U27239); 1Cheilanthes lanosa (Michx.) D. C. Eaton(U27205); 1Coniogramme japonica (Thunb.) Diels (U05611); 1Doryop-teris concolor (Langsd. & Fisch.) Kuhn (U05621); 1Hemionitis levyi E.

492 [Volume 28SYSTEMATIC BOTANY

TABLE 1. Sources of material for rbcL sequence analyses and AFLP genotyping in this study. GenBank accession numbers are givenfor all new rbcL sequences in this study. The AFLP data matrix used to generate Table 3 and Fig. 6 is available from the authors. Taxaare listed alphabetically by genus and species. To minimize table space, species for which data were previously published are presentedin paragraph format under MATERIALS AND METHODS. All collections are vouchered at IND except when noted otherwise.

Anogramma caespitosa Pic. Serm. Tanzania, Mt. Kilimanjaro, Hemp 2079, AY168718 (fig. 5, 6)Anogramma chaerophylla (Desv.) Link. Brazil, Rio Grande do Sul, Sehnem s.n., 10/12/72, AY168712 (fig. 5); Costa Rica, Prov.

San Jose, Gomez 5432, AY168713 (fig. 5)Anogramma guatemalensis (Domin) C. Chr. Guatemala, Chimaltenango, Gastony 1037D, AY168716 (fig. 5, 6)Anogramma leptophylla (L.) Link. Mexico, Vera Cruz, Conant 846, AY168715 (fig. 5, 6); Peru, Cuzco, Vargas s.n., 4/18/73, AFLP

data only (fig. 6); UK, Channel Islands, Guernsey, Schollick s.n., 1972, AFLP data only (fig. 6); France, Sumene, Mandil s.n.,6/71, AFLP data only (fig. 6); Greece, Nauplion, Sheehan s.n., 4/7/73, AFLP data only (fig. 6); Turkey, Izmir, Fraser-Jenkins2653, AY168719 (fig. 5, 6); Canary Islands, La Talura, Kunkel s.n., 1/13/73, AFLP data only (fig. 6); Morocco, Cap Serrat,Ornduff 7923, AFLP data only (fig. 6); New Zealand, Canterbury, Given & Stemmer 8163, AY168717 (fig. 5, 6)

Anogramma novogaliciana Mickel. Mexico, Jalisco, Gonzalez & Palafox 45 (MICH), AY168714 (fig. 5)Anogramma osteniana Dutra. Brazil, Rio Grande do Sul, Sehnem s.n., 11/5/72, AY168711 (fig. 5)Cosentinia vellea (Aiton) Tod. Spain, Malaga, Gastony 90-1, AY168720 (fig. 4, 5)Eriosorus flexuosus (Humb., Bonpl., et Kunth) Copel. Dominican Republic, Gastony 737, AY168709 (fig. 4, 5)Jamesonia canescens Kunze. Venezuela, Merida, Sanchez-Baracaldo 332 (UC), AY168710 (fig. 4, 5)Platyzoma microphyllum R. Br. Australia, Queensland, Bostock, s.n., AY168721 (fig. 4, 5)

TABLE 2. AFLP primers used for selective amplifications. All 12 combinations of EcoRI and MseI primers were used in the analyses.Symbols in parentheses indicate fluorescent dye molecules.

EcoRI primers: Eacg (6-FAM): 59GACTGCGTACCAATTC ACG39; Eaac (NED): 59GACTGCGTACCAATTC AAC39MseI primers: Macaaa: 59GATGAGTCCTGAGTAA ACAAA39; Macaag: 59GATGAGTCCTGAGTAA ACAAG39; Macaat: 59GATGAGTCCT-GAGTAA ACAAT39; Mactg: 59GATGAGTCCTGAGTAA ACTG39; Matagc: 59GATGAGTCCTGAGTAA ATAGC39; Matagg: 59GAT-GAGTCCTGAGTAA ATAGG39

Fourn. (U27725); 1Microlepia strigosa (Thunb.) C. Presl (U05931);1Monachosorum arakii Tagawa (U05636); 1Notholaena rosei Maxon(U27728); 1Onychium japonicum (Thunb.) Kunze (U05641); 2Ony-chium lucidum Spreng. (AF360359); 1Pellaea andromedifolia (Kaulf.)Fee (U19501); 2Pityrogramma calomelanos (L.) Link (AF336103); 2Pi-tyrogramma trifoliata (L.) R. M. Tryon (AF336104); 1Polytaenium li-neatum (Sw.) J. Sm. (U20935); 2Pteris cretica L. (AF360360); 1Pterisfauriei Hieron. (U05647); 1Taenitis blechnoides (Willd.) Sw. (U05654);1Vittaria flexuosa Fee (U05656). The data sets are available onTreeBASE (study accession number 5 S897; matrix accession num-bers 5 M1471, M1472, M1473, and M1474).

Amplification and Sequencing of the rbcL Gene. Amplificationof the rbcL gene was carried out in a reaction mixture containing30 mM Tricine, 50 mM KCl, 2 mM MgCl2, 5% acetamide, 10 mMof each dNTP, 0.2 mM of each primer, 22 ng DNA template, and0.25 ml of Taq polymerase in a 10 ml reaction. Primers 1F and1351R (Table 2 of Gastony and Rollo 1995) were used to amplifya 1325 bp fragment via PCR reactions carried out in an MJ Re-search Thermal Cycler (MJ Research, Inc., Watertown, MA) withthe following program: initial denaturation for 60 sec at 948C fol-lowed by 35 cycles of 30 sec at 948C, 30 sec at 508C, and 30 sec at728C, with a final extension of 5 min at 728C. PCR products werepurified prior to sequencing reactions following the protocol in theElu-Quickt DNA purification Kit (Schleicher & Schuell, Keene,NH) or in the QIAquicky PCR Purification Kit (Qiagen, Inc., Va-lencia, CA). Sequencing reactions were carried out following theprotocols in the DNA Sequencing Kit (Applied Biosystems, FosterCity, CA) using an MJ Research Thermal Cycler. Suitable primerslisted in Table 2 of Gastony and Rollo (1995) were used to se-quence the gene in both directions. Sequencing products were pu-rified following the protocols in the DyeExy Spin Kit (Qiagen,Inc., Valencia, CA) or by precipitation with 0.1 volume of 7.5 Mammonium acetate and 2.5 volume of 95% ethanol followed by awash with 5 volumes of 70% ethanol and resuspension with 3 mlof formamide loading dye. Automated sequencing was carried outwith the 377 DNA Sequencing System or a 3700 DNA Analyzer(Applied Biosystems, Foster City, CA). Sequences were assembled

and contigs were constructed using the software Sequenchery3.1.1 (Gene Codes Corporation, 1994).

AFLP Genotyping. AFLP analysis was carried out followingprotocols described in Kim and Rieseberg (1999), except that prim-ers were labeled with 6-FAM and NED fluorescent dye. Primerpairs used in selective amplifications are listed in Table 2. Ampli-fied fragments were detected with the 3700 DNA Analyzer. AFLPelectropherograms were generated using Genotypert software(Applied Biosystems, Foster City, CA). Each AFLP locus was treat-ed as independent of others and scored as a dominant marker,either present (1) or absent (0) across all loci. All loci from 50 bpto 500 bp in size, including autapomorphic loci were scored, unlesspresence or absence could not be reliably discriminated.

Phylogenetic Analysis: rbcL. Phylogenetic analyses were con-ducted based on 1325 bp rbcL sequences. In the global analysis(see Approaches to Phylogenetic Analyses of Anogramma Species be-low and Fig. 4), phylogeny was inferred using maximum parsi-mony (MP) and maximum likelihood (ML) methods. PAUP*(Swofford 1998) version 4.0b8 was used for both MP (1,000 rep-licates of a heuristic search using random taxon addition, TBRbranch swapping, and steepest descent) and ML (100 replicates ofa heuristic search using random taxon addition and steepest de-scent). The best-fit models for the global analysis under ML wereestimated using the software MODELTEST (Posada and Crandall1998) version 3.06, and the TrN1I1G and GTR1I1G models wereselected under the hLRT and AIC criteria, respectively. TheTrN1I1G model was used. To estimate support for the rbcL to-pology, 1,000 bootstrap replicates were conducted under MP and100 bootstrap replicates under ML, with 10 random taxon additionsequences each in both cases. In the more focused analysis (Fig.5), PAUP* (Swofford 1998) version 4.0b10 was used for both MP(Branch and Bound search, guaranteed to find the shortest tree)and ML (200 replicates of a heuristic search using random taxonaddition and steepest descent, and based on the TrN1I1G best-fit model found by MODELTEST under both the hLRT and AICcriteria for the focused data set). In addition to MP and ML anal-yses, the more focused data set was also analyzed by recently


FIG. 4. ML phylogram resulting from the initial rbcL analysis intended to place Anogramma, Cosentinia, Actiniopteris, Erio-sorus, Jamesonia, and Pityrogramma within the context of Fig. 3 of Hasebe et al. (1995), specifying Monachosorum and Microlepiaas outgroups. See key for ML phylogram branch length. Numbers above lines are the branch lengths obtained under the MPcriterion. Numbers below lines are bootstrap values based on 1,000 replicates under MP (upper) and 100 replicates under ML(lower).

developed Bayesian Inference (BI; Mau et al. 1999) carried out us-ing the software MRBAYES (Huelsenbeck and Ronquist 2001) for500,000 generations, setting other parameters as defaults. Five in-dependent runs were carried out to ensure chain convergence. Onetree per 10 generations was retained, yielding 50,000 trees, ofwhich the first 10,000 were eliminated because they correspondedto the first 100,000-generation burn-in period, given that the chain

converged at ;50,000 generations. 50% majority rule consensustrees were constructed for the 40,000 trees of each of the five runs.All had the same topology, and one was selected for use in thispaper. To estimate support for clades resulting from the focusedanalyses, 10,000 heuristic bootstrap replicates with 10 random tax-on addition sequences each were carried out under MP, and 925bootstrap replicates with 10 random taxon addition sequences


FIG. 5. ML phylogram based on rbcL sequences of species of Anogramma and Cosentinia (boldface) and related genera. Seekey for ML phylogram branch lengths. Numbers above lines are branch lengths obtained under the MP criterion. Numbersbelow lines are bootstrap values based on 10,000 replicates under MP (upper), 925 replicates under ML (center), and theBayesian posterior probability in percentage (bottom; e.g., 98 represents a posterior probability of 0.98). To facilitate discussion,the three major clades containing Anogramma species are lettered A–C and provenances of Anogramma accessions are indicatedin parentheses.

each were run under ML through Indiana University ResearchComputing services. MRBAYES generated posterior probabilityvalues for the BI consensus tree. In all analyses, all characters andcharacter states were weighted equally.

Phylogenetic Analysis: AFLP. Phylogenetic analyses were con-ducted using 348 loci for A. guatemalensis and the two New Worldpopulations of A. leptophylla and 388 loci for A. caespitosa and theseven Old World populations of A. leptophylla, with all loci derivedfrom the same 12 AFLP primer pairs (Table 2). Genetic distancematrices were generated following Nei and Li (1979) using allavailable AFLP data. Based on these matrices, phylogenetic rela-

tionships were inferred using the Neighbor-joining (NJ) method(Saitou and Nei 1987) of PAUP*4.0b8. Support for the resultingphylogenies was determined with 1,000 bootstrap replicates withten random addition sequence replicates each.

Approaches to Phylogenetic Analyses of Anogramma Species.Anogramma has generally been placed within tribe Taenitideae orsubfamily Taenitidoideae of fern family Pteridaceae (Tryon andTryon 1982; Tryon et al. 1990), but it was not included in the globalanalysis of fern phylogeny by Hasebe et al. (1995) based on rbcLnucleotide sequences. To assess appropriate ingroup context andoutgroup selection for an rbcL cladistic analysis of Anogramma, the


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placement of A. lorentzii (Gastony and Johnson 2001) within theclade of Pteridaceae plus Vittariaceae (Hasebe et al. 1995) was firstdetermined in the context of the clade from Platyzoma to Conio-gramme in Fig. 3 of Hasebe et al. (1995). Also included in thisbroad-context, initial global analysis were putative taenitidoids Er-iosorus flexuosus, Jamesonia canescens, Pityrogramma calomelanos, Ac-tiniopteris radiata, and taxonomically controversial Cosentinia vellea.The rbcL sequence of Platyzoma microphyllum used in Hasebe et al.(1995) was replaced with a more complete and accurate sequencefrom a newly obtained accession of this species (Table 1) becausethe Platyzoma sequence in Hasebe et al. (1995) is 59 bases shorterthan sequences generated in our lab. Monachosorum and Microlepiafrom the sister clade in Fig. 3 of Hasebe et al. (1995) were usedas outgroups for this initial analysis.

Anogramma lorentzii and Cosentinia vellea nested within the tra-ditional (Tryon et al. 1990) core of taenitidoid ferns from Taenitisthrough Onychium in the preceding initial analysis (Fig. 4). Basedon those results, rbcL sequences from accessions of all availableAnogramma species were subjected to a more focused analysiswithin the context of the clade from Platyzoma microphyllum to Pter-is fauriei of Fig. 4. Sequences of Pityrogramma trifoliata, Pteris cretica,and Onychium lucidum, from Gastony and Johnson (2001), werealso included in this analysis to decrease branch lengths associatedwith those genera in Fig. 4. Platyzoma microphyllum (Pteridaceaesubfamily Platyzomatoideae of Tryon et al. 1990) and Pteris faurei(Pteridaceae subfamily Pteridoideae of Tryon et al. 1990) are un-convincingly grouped with genera of the taenitidoid clades (MLbootstrap values of 23% and 54%, respectively in Fig. 4), callinginto question their suitability as outgroups for the taenitidoids theybracket in Fig. 4. Therefore Ceratopteris thalictroides and Acrostichumaureum from the clade sister to the Platyzoma through Pteris com-plex in Fig. 4 were chosen as the outgroup for this more focusedanalysis whose results are presented in Fig. 5.

Because of the conservative nature of the rbcL gene, phylogeneticrelationships within the clade from Anogramma leptophylla (Mexico)through A. leptophylla (Turkey) of Fig. 5, consisting of populationsof A. leptophylla, A. guatemalensis, and A. caespitosa (A. leptophyllaclade hereafter) was poorly resolved (see RESULTS). Analyses ofrelatively rapidly evolving AFLP loci were therefore used to im-prove phylogenetic resolution within the A. leptophylla clade. Giventhat rbcL sequence data clearly divided the A. leptophylla clade intotwo groups, New World and Old World populations (Fig. 5), theseaccessions and all available additional accessions of taxa withinthe A. leptophylla clade, were partitioned into New and Old Worldgroups, and AFLP data were analyzed separately for each group.In addition to the high bootstrap and BI values supporting thedivergence of the New and Old World clades (98% MP bootstrap,96% ML bootstrap, and 100% BI; Fig. 5), distinctive AFLP bandingpatterns of each group (data not shown) supported partitioningthese accessions in this way. Analyses combining accessions fromboth New and Old World populations were not performed because1) the analysis based on rbcL sequences established the relation-ship between these groups, 2) the New and Old World populationswere genetically too diverged to reliably assess homologous AFLPloci across them, and 3) the appropriate outgroup (A. lorentzii) forthe clade of these two groups was too diverged from members ofthe A. leptophylla clade to assess its AFLP homologies with suchan ingroup. For the AFLP analysis of New World accessions of A.leptophylla the Old World New Zealand accession was used as out-group, and for the AFLP analysis of the Old World accessions ofA. leptophylla the New World Peruvian accession was used as out-group. Genetic distances among accessions within the New andOld World sets (Table 3) were estimated from the D-values (Neiand Li 1979). Similarly, genetic distances between the New andOld World taxa were estimated from D-values between ingroupsand outgroups in each set (Table 3).

As stated under DISCUSSION below, some rbcL sequences ob-tained from GenBank are truncated by 76–119 nucleotides at their59 and 39 ends relative to the 1,325 bp sequence reads generatedin our lab. DNAs of Eriosorus flexuosus and Anogramma novogalici-ana were derived from herbarium material (Table 1), and respec-tively yielded 18 and 30 bases of uncertainty, including 20 posi-


tions at amplification primers 868–887 bases from the beginningof the gene in the case of Anogramma novogaliciana. As averagedacross all accessions, 3.85% and 1.59% of 1,325 rbcL bp in the glob-al and the more focused analysis, respectively, were treated asmissing or uncertain data coded as N, R, Y, or K. The proportionof missing AFLP data was only 0.22% of 348 loci and 0.46% of 388loci in the analysis of New and Old World A. leptophylla groups,respectively.

RESULTS

ML analysis of rbcL sequences from Anogramma lor-entzii plus other putative taenitidoids and Cosentiniavellea in the context of the Pteridaceae plus Vittariaceaeclade of Fig. 3 of Hasebe et al. (1995) positioned Ano-gramma and Cosentinia within the taenitidoid cladefrom Taenitis through Pityrogramma (Fig. 4). Analysisunder the MP criterion found a single most parsimo-nious tree with a length of 1,609 steps, a consistencyindex of 0.469, and a retention index of 0.517. The MPtopology was congruent with that under ML in Fig. 4,except that under MP, Pteris fauriei was sister to thetaxa from Platyzoma microphyllum through Pityrogram-ma calomelanos, and P. calomelanos was sister to Ano-gramma lorentzii and Cosentinia vellea. Thus in bothanalyses Anogramma lorentzii was placed in a cladewith traditionally related genera Taenitis, Eriosorus, Ja-mesonia, and Pityrogramma of subfamily Taenitidoideae.In both analyses Actiniopteris and Onychium (which aretaenitidoids sensu Tryon et al. 1990) were placed basalto this central core of taenitidoids. In both analysesCosentinia vellea was deeply separated from cheilan-thoid genera Cheilanthes and Notholaena in which it isoften placed, nesting instead among taenitidoid generawhere its position sister to Anogramma is supported by18 MP synapomorphies and 100% MP and ML boot-strap values, in agreement with its placement by PichiSermolli (1985). MP and ML bootstrap values (Fig. 4)for the clades uniting Platyzoma with the taenitidoids(39%, 23%), Pityrogramma with taxa from Taenitisthrough Cosentinia (71%, 58%), and Pteris with Acti-niopteris1Onychium (27%, 54%) were relatively weak.

Results of the more focused analysis of Anogrammaspecies within the context of all available rbcL sequenc-es from taenitidoid ferns are presented in Fig. 5 asdetermined by ML. Analysis under the MP criteriongenerated a single most parsimonious tree with alength of 769 steps, a consistency index of 0.615, andretention index of 0.694. This MP tree is topologicallyidentical to the ML tree shown in Fig. 5. As in theinitial analysis (Fig. 4), bootstrap values supportingsome of the basal branches are relatively low. However,bootstrap values supporting clades A, B, and C them-selves in Fig. 5 are moderate to high, and those sup-porting relationships within each of these three cladesare very high. The topology from BI analysis was iden-tical to the ML topology, and support for each node,indicated by percent posterior probability, was higher

than the bootstrap values under ML when the latterwere not 100% (Fig. 5). Anogramma itself is highlypolyphyletic (Fig. 5). Anogramma chaerophylla is moreclosely related to species of Pityrogramma, and A. os-teniana to species of Jamesonia, Eriosorus, and Taenitisthan to other species of Anogramma. Cosentinia vellea isvery strongly supported as sister to the clade of A.leptophylla plus A. lorentzii. Anogramma novogaliciana dif-fers from accessions of A. chaerophylla by only seven tonine apomorphies, whereas the two accessions of A.chaerophylla itself differ from each other by six apo-morphies. Anogramma guatemalensis and A. caespitosawere sister to and only slightly diverged from acces-sions of A. leptophylla. The A. leptophylla clade (from A.leptophylla Mexico through A. leptophylla Turkey) con-sists of the New and Old World populations that arehighly diverged from each other, although there areinsufficient apomorphies to resolve relationships with-in the Old World subclade itself. This entire A. lepto-phylla clade is highly supported by the bootstrap anal-yses and BI percent posterior probability and stronglydistinct from its sister species, A. lorentzii.

The 348 and 388 polymorphic AFLP loci scorablewithin the respective New and Old World clades of A.guatemalensis, A. leptophylla, and A. caespitosa yield thegenetic distance matrices shown in Table 3 for the Newand Old World populations. Relatively large but con-sistent average genetic distances were obtained be-tween New and Old World taxa (0.3273 and 0.3210,estimated from the New and the Old World popula-tions, respectively). Genetic distance ranges from0.0875 to 0.1346 among the New World populations,and from 0.0236 to 0.1824 among the Old World pop-ulations. The genetic distances among the three NewWorld accessions are relatively large (average 50.1178). The Mexican accession of A. leptophylla sharesa higher genetic similarity with A. guatemalensis thanwith the Peruvian accession of the same species. With-in the Old World group, the New Zealand accession isthe most diverged, as indicated by the large geneticdistances (average 5 0.1423) between it and the otherOld World populations, and 20.7% of the polymor-phisms within the Old World populations occur be-tween the New Zealand accession and the others. Ge-netic distances among the European accessions (UK,France, Greece, and Turkey: 0.0236–0.0343) are muchsmaller than those among the African accessions (Mo-rocco, Canary Islands, and Tanzanian A. caespitosa:0.1065–0.1407). The NJ networks of relationships gen-erated from the respective New and Old World geneticdistance matrices of Table 3 were joined to produce theunrooted phenogram of populations seen in Fig. 6. In-ferred relationships among accessions were relativelywell-supported, as indicated by moderate to high boot-strap values, except among A. leptophylla accessionsfrom France, UK, and Turkey, whose relative positions


FIG. 6. Unrooted NJ phenogram based on AFLP loci obtained from 12 primer pairs, resulting from joining the New Worldand Old World A. leptophylla clades. Lengths of branches correspond to genetic distances calculated following Nei and Li (1979).The branch length joining the two clades is the mean value of genetic distances between the outgroups (New Zealand andPeruvian A. leptophylla) and their respective ingroups as computed by the NJ algorithm of PAUP*. Numbers on branches arebootstrap values based on 1,000 replicates. Provenances are indicated in parentheses.

cannot be resolved because of insufficient synapomor-phies. As in Fig. 5, Anogramma guatemalensis and A.caespitosa nest deeply among accessions of A. lepto-phylla.

DISCUSSION

General Considerations. The MP, ML, and BI anal-yses all produced a single, identical tree topology (Fig.

5), but positions of the Pteris and Actiniopter-is1Onychium clades in that tree have relatively lowbootstrap support. This may be partly because the rbcLsequences of Acrostichum aureum, Ceratopteris thalictro-ides, Onychium japonicum, Pteris faurei, and Taenitisblechnoides obtained from GenBank are truncated by atotal of 76–119 nucleotides at their 59 and 39 ends rel-ative to the sequence reads generated in our lab. These


shortened sequences may have introduced some un-certainty into the analyses since missing data areknown to influence recovery of true relationships(Huelsenbeck 1991; Platnick et al. 1991; Novacek 1992;Wiens and Reeder 1995; Wilkinson 1995; Wiens 1998).In addition, the limited taxon sampling available forthis analysis may have resulted in unstable topologies(Derijk et al. 1995; Poe 1998; Omland et al. 1999; Soltiset al. 1999; Yoder and Irwin 1999; Saunders and Ed-wards 2000; Johnson 2001; Murphy et al. 2001; but seeRosenberg and Kumar 2001). Inclusion of additionaltaxa in this alliance (Tryon et al. 1990) might improverecovery of true phylogenetic relationships and stabil-ity of clades. The placements of clades A, B, and C ofFig. 5 relative to each other also have relatively lowbootstrap support ranging from 50% under MP to 71%under ML. However, relationships within each of thesethree clades, the central focus of this study, were rel-atively well resolved, and it is apparent that Anogram-ma is highly polyphyletic and that Cosentinia is closelyrelated to part of traditional Anogramma.

The Occurrence of Gametophytic Tubercles. Thedevelopment of gametophytic tubercles is a synapo-morphy of all traditional Anogramma species examinedfor this character with the exception of A. osteniana(Baroutsis 1976). With that exception, ‘‘perennating’’gametophytic tubercles are often cited as a definingcharacter of Anogramma (Copeland 1947; R. Tryon1962; Tryon and Tryon 1982; Tryon et al. 1990); theyare not known to occur in Pityrogramma. Gametophytictubercles are an elaboration of the thickened archego-nial region or ‘‘cushion’’ of an Anogramma gameto-phyte and arise when ecological factors prevent fertil-ization and/or sporophyte development (see cover il-lustration, this journal issue). They are able to surviveseasons of hot, dry weather and subsequently regen-erate gametophytic lobes or permit the rapid devel-opment of an embedded dormant embryo upon res-toration of favorable environmental conditions, butthey cannot produce a sporophyte and simultaneouslycontinue to develop as gametophytes (Baroutsis 1976).Thus gametophytic tubercles perennate only in a qual-ified sense.

In terms of the tree topology in Fig. 5, the occur-rence of gametophytic tubercles can be explained ei-ther by parallel gains at the bases of the Anogrammasubclades in clades B and C or by the gain of thischaracter state in the common ancestor of clades B andC with subsequent parallel losses in Pityrogramma andCosentinia. It is likely that Cosentinia has never beendeliberately investigated for development of gameto-phytic tubercles, but they could not be found in cul-tures of Cosentinia gametophytes grown for sevenmonths on MetroMix in our laboratory, whether or notthe gametophytes produced sporophytes during thattime.

The Relationship of Anogramma to Pityrogramma.The rbcL analysis supports the close relationship be-tween Anogramma and Pityrogramma indicated by R.Tryon (1962) on the basis of architecture of the lamina,grooving of the leaf axes, soral similarity, and partic-ularly spore morphology. As seen in Fig. 5, however,this relationship is particularly true with regard to A.chaerophylla and A. novogaliciana, which are more close-ly related to P. calomelanos and P. trifoliata than to otherAnogramma species. Based on morphological similarity(Baroutsis 1976, p. 44), it is likely that rbcL wouldgroup A. ascensionis with A. chaerophylla and A. novo-galiciana, but materials of A. ascensionis were unavail-able for this study as noted above. Presence of scaleson mature rhizomes in both Pityrogramma (R. Tryon1962) and A. chaerophylla (Baroutsis 1976), but not inother Anogramma species, supports the rbcL relation-ship between Pityrogramma and A. chaerophylla in cladeB of Fig. 5. Mickel (1992) cited the lack of rhizomescales in A. novogaliciana as the character distinguish-ing it from A. chaerophylla. In terms of the phylogenyin clade B of Fig. 5, loss of rhizome scales is an auta-pomorphy of A. novogaliciana. Based on what is ob-served here, A. novogaliciana is only weakly distinctfrom A. chaerophylla. Pityrogramma sulphurea and sev-eral other species of that genus, unavailable for thisstudy, were specifically cited by R. Tryon (1962) asmorphologically closest to Anogramma. Where rbcLwould place those Pityrogramma species relative to theAnogramma clades of Fig. 5 remains to be determined.

Relationships of Anogramma osteniana. Twenty-three MP synapomorphies and 100% MP, 99% ML, and100% BI confidence values support placing Anogrammaosteniana in the subclade with Eriosorus and Jamesoniain Fig. 5 clade A, deeply separating A. osteniana fromother Anogramma species. Baroutsis (1976) and Tryonand Tryon (1982) questioned maintaining this speciesin Anogramma because it differs from others in thatgenus in lacking gametophytic tubercles and in havingglandular trichomes on its leaves and spores with asingle equatorial flange lacking parallel ridges, unlikethe equatorial flange plus two parallel ridges charac-teristic of Anogramma. Glandular trichomes occur onthe leaves of a few species of Jamesonia and about halfthe species of Eriosorus (A. Tryon 1962, 1970). Withregard to its single equatorial ridge or flange, thespores of A. osteniana (Baroutsis 1976) are rather likethose of Taenitis blechnoides illustrated by Tryon andLugardon (1991). They are also more similar to thoseof Eriosorus and Jamesonia, where a single triangulardistal ridge is more or less parallel to the equatorialflange (A. Tryon 1962, 1970; Tryon and Lugardon1991), than they are to the equatorially three-ridgedspores of other Anogramma species (Baroutsis 1976). Inview of its gametophyte and sporophyte morphologyand its molecular phylogenetic placement in this study,


it is now quite clear that A. osteniana should be exclud-ed from Anogramma and that its affinities are withAmerican taenitidoid genera Eriosorus and Jamesoniaand perhaps with Nephopteris and Pterozonium whoserbcL sequences were not available for this study. Ge-neric distinction between Jamesonia and Eriosorus wasquestioned by A. Tryon (1962, e.g., pp. 116, 129; 1970,e.g., p. 60), who reported intergradation in leaf mor-phology and several instances of intergeneric hybridsand concluded that Jamesonia has been derived frommore than one element within Eriosorus. Broader taxonsampling in these genera than was available here isneeded to specify the precise relationships of A. osten-iana within this complex, but if these genera are com-bined, Jamesonia has priority as the generic name (A.Tryon 1962, p. 116). Because A. osteniana clearly cannotbe maintained within Anogramma, a new combinationis made here to facilitate reference to this species in itsmore appropriate genus.

Jamesonia osteniana (Dutra) Gastony, comb. nov.Anogramma osteniana Dutra, Ostenia (Montevideo): 5–6, Figs. 1–2. 1933.—TYPE: BRASIL. Rio Grande do Sul:Sao Leopoldo, Morro das pedras, 1 Sep 1932, Dutra 48(holotype: ICN).

Abandoning Anogramma guatemalensis. Thisstudy firmly embeds A. guatemalensis within A. lepto-phylla on the basis of both rbcL (Fig. 5) and AFLP (Fig.6) data. Recognizing A. guatemalensis as specificallydistinct from A. leptophylla would render the latter par-aphyletic (Fig. 5). Baroutsis (1976) concluded that A.leptophylla and A. guatemalensis have similar sporo-phyte morphology, identical spore morphology, a sim-ilar gametophyte developmental pattern, and a sharedpreference for cool growing conditions. Gastony andBaroutsis (1975) regarded A. guatemalensis, whose re-ported geographic range from Mexico to Costa Rica iswithin that of A. leptophylla, as questionably distinctfrom A. leptophyllya at the species level. Tryon andTryon (1982) implicitly subsumed A. guatemalensis intoA. leptophylla, and Mickel and Beitel (1988) explicitlydid so. Stolze (1981) maintained A. guatemalensis as theonly species reported from Guatemala but noted thatit differs from A. leptophylla only in its somewhat moredissected lamina and relatively narrower segmentsand that in some areas the two species have been col-lected together with evidence of transitional states.Smith (1981) regarded A. guatemalensis as conspecificwith A. leptophylla, noting that both the frond type typ-ical of A. leptophylla and the finely dissected frondscharacteristic of A. guatemalensis can be found on thesame rhizome. Abandoning any taxonomic distinctionbetween A. guatemalensis and A. leptophylla would beconsistent with the rbcL and AFLP results of thisstudy.

Abandoning Anogramma caespitosa. Anogrammacaespitosa was distinguished from A. leptophylla by Pichi

Sermolli (1972) based on the habit of the whole plantwith a large number of homomorphic leaves crowdedon the rhizome. The number of leaves per rhizome andrhizome development itself, however, must be a func-tion of the age and longevity of the sporophyte. Per-haps its mid altitude locality on virtually equatorialMt. Kilimanjaro in Tanzania provides what would oth-erwise be a normal A. leptophylla population with asuitable moist habitat for prolonged sporophytegrowth resulting in the robust habit of the sporophyte.Our material of A. caespitosa was collected by AndreasHemp from the same locality as Pichi Sermolli’s (1972)type specimen (Andreas Hemp, pers. comm.), and itsrbcL sequence firmly embeds it within the Old Worldsubclade of A. leptophylla in clade C of Fig. 5. AFLPdata were used to analyze the relationship between theTanzanian material and an increased number of OldWorld accessions of A. leptophylla (Fig. 6), placing itsister to the European accessions and robustly main-taining it within the clade of Old World A. leptophylla.The average AFLP divergence of A. caespitosa from oth-er Old World populations (0.0967; Table 3) is very closeto the average AFLP divergence among all Old WorldA. leptophylla accessions (0.0947) and is less than theaverage AFLP divergences of the accessions from Mo-rocco, Canary Islands, and New Zealand. Purely interms of the AFLP data, if species are to be monophy-letic and not paraphyletic, the New Zealand, Moroc-can, and Canary Islands populations of Fig. 6 wouldeach have to be accepted as independent species beforethis would be justified for A. caespitosa, potentiallyleaving the accessions from Turkey through Greece asanother species. No meaningful morphological distinc-tions are presently known to correlate with such a tax-onomy. Our results support the view of Tryon andTryon (1982) that A. caespitosa is merely a robustgrowth form of A. leptophylla.

Generic Disposition of A. chaerophylla and A.novogaliciana. MP, ML, and BI analyses group A.chaerophylla and A. novogaliciana with Pityrogramma inclade B (Fig. 5), well separated from very robustly sup-ported clade C of A. leptophylla, A. lorentzii, and Cos-entinia. This placement indicates that A. chaerophyllaand A. novogaliciana should be removed from Anogram-ma (typified by A. leptophylla in clade C) and that theirprecise relationship to Pityrogramma should be exam-ined in the context of additional species of that genus,particularly P. sulphurea and others noted by R. Tryon(1962) as morphologically closest to traditional Ano-gramma. Results of that expanded study will help de-termine whether A. chaerophylla and A. novogalicianashould be considered congeneric with Pityrogramma ortreated as a coordinate independent genus. If they arecongeneric with Pityrogramma, Domin (1928) alreadyprovided the name Pityrogramma chaerophylla (Desv.)Domin for one of them.


Generic segregation of A. chaerophylla and A. novo-galiciana from A. leptophylla and A. lorentzii is consistentwith their rbcL divergence values relative to those ofother fern taxa. Interspecific rbcL divergence valueswithin fern genera have been estimated at 0.2%–1.8%(Wolf et al. 1994), 0.3%–3.0% (Haufler and Ranker1995), and 0.4%–2.8% (Gastony and Ungerer 1997). IfA. guatemalensis and A. caespitosa are subsumed into A.leptophylla as indicated by Fig. 5 clade C and discussedabove, interspecific rbcL divergence values between A.leptophylla and A. lorentzii of Fig. 5 clade C (2.42–2.65%)are comparable to the higher interspecific values in theforegoing citations. Maintaining A. chaerophylla and A.novoglaciana in Anogramma, however, would increasethe interspecific divergence value of Anogramma spe-cies up to 6.73%, which is exorbitantly high amongfern genera and higher than all but two of the inter-generic rbcL divergence values reported for cheilan-thoid and onocleoid genera (Gastony and Ungerer1997, Tables 3 and 5). How much significance shouldbe accorded these sequence divergence values is un-clear because rates of evolution for a given gene mayvary from taxon to taxon (particularly at the level offamily differentiation in angiosperms: Bousquet et al.1992; Gaut et al. 1992), but branch lengths in Fig. 5clades B and C do not indicate rate heterogeneity with-in traditional Anogramma.

Phylogenetic Position of Cosentinia vellea. Cos-entinia was established by Todaro (1866) for the singlespecies C. vellea. As summarized by Vida et al. (1983),this genus was not widely accepted, and C. vellea wastreated as a species of Notholaena (N. lanuginosa) innearly all European floras before 1964, after which Eu-ropean species of Notholaena were commonly regardedas species of Cheilanthes. Its spore morphology, atypicalof cheilanthoids, led Saenz de Rivas and Rivas-Martı-nez (1979) to segregate C. vellea into Cheilanthes sub-genus Cosentinia. Copeland (1947) considered C. velleaan extremely pubescent Cheilanthes, and it was main-tained as a species of Cheilanthes in recent general treat-ments of ferns (Tryon and Tryon 1982; Tryon et al.1990), even though its spores were regarded as ‘‘ex-ceptional in Cheilanthes, but similar to those of generaallied with Anogramma in the subfamily Taenitidoi-deae’’ (Tryon and Lugardon 1991). Indeed, its sporesare so exceptional in Cheilanthes, so like those of Ano-gramma (particularly A. leptophylla), and so similar tothe spores of other taenitidoid genera as to single-handedly make its placement in Cheilanthes untenable.Vida et al. (1983) examined leaf development in liveplants of C. vellea (as N. lanuginosa) and determinedthat it does not produce a pseudo-indusium (5 re-flexed, modified leaf margin generally thought to char-acterize Cheilanthes) at any stage ‘‘neither when youngnor mature.’’ Pichi Sermolli (1985) summarized thesoral, indusial, palynological, and chromosomal data

relating to C. vellea, and concluded that this speciesshould be returned to Todaro’s genus Cosentinia, whichTodaro had classified with Anogramma in tribe Lepto-grammeae. Pichi Sermolli noted that in Cosentinia thesporangia occur along the ultimate veinlets or a littlebelow their forking as in Anogramma, Eriosorus, andsome species of Pityrogramma. We have confirmed thisAnogramma-like soral disposition in leaf clearings ofour C. vellea voucher from Spain. Spores of C. vellea arevirtually identical to those of A. leptophylla (Pichi Ser-molli 1985; Ferrarini et al. 1986; Tryon and Lugardon1991). Cosentinia vellea is known to occur as two cyto-types, a diploid with n 5 29, and a tetraploid with n5 58 (Vida et al. 1970; Badre and Reichstein 1983; Vidaet al. 1983). Chromosome numbers of Notholaena sensustricto are all based on x 5 30 (Windham 1987), as arethose of Cheilanthes except for species of the C. alaba-mensis group (Benham and Windham 1992). Chromo-some numbers of taenitidoid genera including Ano-gramma are based on x 5 29 (Lovis et al. 1993). Thus,even without our rbcL data, C. vellea appears wellplaced among the taenitidoid ferns and misplacedamong the cheilanthoids.

Results of the initial rbcL analysis (Fig. 4) very deep-ly separate Cosentinia vellea (sister to Anogramma lor-entzii among the taenitidoid ferns) from the cheilan-thoid ferns (Argyrochosma delicatula through Bommeriaehrenbergiana). The more focused rbcL analysis concen-trating on taenitidoid ferns (Fig. 5) very robustly main-tains the relationship between C. vellea and Anogram-ma. Twenty MP synapomorphies, 100% MP and MLbootstrap confidence, and 100% BI posterior probabil-ity place C. vellea in clade C sister to the subclade fromA. leptophylla (Mexico) through A. lorentzii. Brazilian A.lorentzii is in turn robustly sister to the subclade of A.leptophylla sensu lato, including both New and OldWorld accessions and including A. guatemalensis andA. caespitosa. This placement of Cosentinia in clade C isconsistent with spore and soral morphology, eventhough the dense pubescence of the leaves of C. velleastrongly contrasts with the glabrous leaves of its sistertaxa (Figs. 1–3). The plesiomorphic taenitidoid basechromosome number of x 5 29 of C. vellea persists inA. lorentzii. Determining chromosome numbers for A.leptophylla has been exceptionally difficult, with con-flicting reports in the literature (Gastony and Baroutsis1975; Baroutsis and Gastony 1978; Rasbach and Reich-stein 1990; Lovis et al. 1993). According to the reviewand reinterpretation by Lovis et al. (1993), A. leptophyllais uniquely and uniformly characterized by the de-rived, reduced number of n 5 26. If the reinterpreta-tion by Lovis et al. (1993) is correct, this apomorphicreduction occurred in the common ancestor of theclade from A. leptophylla (Mexico) through A. leptophylla(Turkey).

Anogramma leptophylla is much more closely related


to C. vellea than it is to A. chaerophylla. Divergence val-ues based on rbcL between Cosentinia and Anogramma(A. leptophylla and A. lorentzii) range from 3.02 to3.93%, which is comparable with the intergeneric di-vergence values between Polypodium and Pleopeltis (2.8–4.5%; Haufler and Ranker 1995) and with the range ofintergeneric rbcL divergence values computed by Gas-tony and Ungerer (1997) for cheilanthoids (3.0–7.4) andonocleoids (2.0–4.7). If the ML, MP, and BI topologyseen in Fig. 5 is correct, the taenitidoid genus Cosen-tinia can be maintained for C. vellea as can Anogrammafor A. lorentzii and A. leptophylla sensu lato, but A. chaer-ophylla (including A. novogaliciana) cannot be main-tained in a monophyletic Anogramma and certainly nei-ther can A. osteniana. Maintaining Cosentinia as gener-ically distinct from Anogramma is consistent with thephylogeny of Fig. 5 and with the fact that it is notknown to produce gametophytic tubercles. This gener-ic disposition also acknowledges the divergent sporo-phyte morphology of C. vellea (Figs. 1–3) that led somany to place it in a separate subfamily from Ano-gramma for so long.

ACKNOWLEDGEMENTS. We are grateful to Andrea Schwarzbachand Leslie Goertzen for helpful advice, to Andreas Hemp and Pe-ter Bostock for providing plant materials of Anogramma caespitosaand Platyzoma microphyllum, respectively, to William Anderson forproviding MICH material of A. novogaliciana, to Raymond Cranfillfor the rbcL sequence of Jamesonia canescens, to Antonia Eastwoodfor information about the probable extinction of Anogramma ascen-sionis, to Paulo Brack for help with the type of A. osteniana, toJudith Gordon for use of her figures in the cover illustration, andto Robbin Moran and Thelma Barbara. We are also grateful to thefollowing who supplied materials of Anogramma species to GJG in1972–74 for the doctoral studies of Judith Baroutsis. Spores fromrefrigerated leaves of these specimens generated gametophytesfrom which DNAs were extracted for this study: Frederic Badre,David Conant, David Given, Luis Gomez, A. Clive Jermy, GuntherKunkel, Ramon Riba, Aloysio Sehnem, Mark Sheehan, and CesarVargas. GJG acknowledges research support from National ScienceFoundation grants DEB-9307068 and DEB-0128926.

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