Utility of a large, multigene plastid data set in inferring higher-order relationships in ferns and relatives (monilophytes)

1444

American Journal of Botany 97(9): 1444–1456. 2010.

American Journal of Botany 97(9): 1444–1456, 2010; http://www.amjbot.org/ © 2010 Botanical Society of America

Considerable progress has been made in inferring phyloge-netic relationships of the three major branches of vascular-plant phylogeny: the lycophytes, monilophytes, and seed plants (e.g., Doyle, 1998 ; Pryer et al., 2001 ). For example, the lycophytes are the sister group of a clade that comprises seed plants and monilophytes, and the latter two lineages comprise the extant eu-phyllophytes (see Pryer et al., 2001 ). The extant monilophytes (a name based on a “ moniliform ” or necklace-like stele thought to be ancestral in the group; Kenrick and Crane, 1997 ; Pryer et al., 2004 ) are sometimes referred to simply as “ ferns ” ( Pryer et al., 2004 ). They comprise Equisetaceae (horsetails), Ophio-glossaceae (ophioglossoid ferns), Psilotaceae (whisk ferns), Marattiaceae (marattioid ferns), and leptosporangiate ferns. Suggestions that Equisetaceae are sister to seed plants among

extant taxa ( Rothwell, 1999 ; Rothwell and Nixon, 2006 ) have been rejected with moderate support in an analysis of morpho-logical data from living taxa and have also consistently been strongly rejected by molecular data, which fi rmly place them as part of the monilophyte clade (see Schneider et al., 2009 ). How-ever, extinct moniliform ferns may represent stem relatives of euphyllophytes as a whole ( Rothwell and Stockey, 2008 ; cf. Pryer et al., 2004 ), which would mean that monilophytes (or ferns as a whole) are a grade, considering extant and extinct taxa. We refer to the extant clade here.

The early branches of monilophyte phylogeny may have split from each other relatively rapidly in the late Devonian ( Pryer and Schuettpelz, 2009 ). Therefore, one might expect that recon-structing their phylogenetic history would be as problematic as resolving that of the other ancient plant radiations that have left only a handful of major extant lineages (e.g., seed plants; Mathews, 2009 ). Nonetheless, recent exemplar-based multi-gene approaches have provided considerable insights into the broad circumscription of major monilophyte clades, including the composition and arrangement of their constituent orders and families (e.g., Pryer et al., 2001 , 2004 ; Schuettpelz and Pryer, 2007 ), allowing their classifi cation to be updated ( Smith et al., 2006 ).

Despite these substantial advances, several key unresolved problems persist in monilophyte phylogeny. These unresolved relationships include the arrangement of the major monilophyte lineages in relation to each other. For example, Doyle (1998: fi g. 1 ), citing morphological and fossil evidence presented in

1 Manuscript received 1 October 2009; revision accepted 25 June 2010. The authors thank K. M. Pryer, P. G. Wolf, E. Schuettpelz, J. Zgurski,

J. Bain, A. Murdock, C. La Farge, N. Wikstr ö m, the Royal Botanic Gardens, Kew, and the Missouri Botanical Garden for plant material or access to data. We also thank M. Berbee, W. Maddison, K. Ritland, J. Whitton, and anonymous reviewers for helpful comments on the manuscript. This research was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) postgraduate scholarship to H.S.R. and an NSERC Discovery grant to S.W.G.

2 Corresponding author (e-mail: [email protected]); present address: Department of Wildland Resources, 5230 Old Main Hill, Utah State University, Logan, Utah 84322, USA.

doi:10.3732/ajb.0900305

UTILITY OF A LARGE, MULTIGENE PLASTID DATA SET IN INFERRING HIGHER-ORDER RELATIONSHIPS IN FERNS AND

RELATIVES (MONILOPHYTES) 1

Hardeep S. Rai 2 and Sean W. Graham

UBC Botanical Garden & Centre for Plant Research (Faculty of Land & Food Systems), 2357 Main Mall, and Department of Botany, 6270 University Boulevard, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada

• Premise of the Study: The monilophytes (ferns and relatives) — the third largest group of land plants — exhibit a diverse array of vegetative and reproductive morphologies. Investigations into their early ecological and life-history diversifi cation require ac-curate, well-corroborated phylogenetic estimates. We examined the utility of a large plastid-based data set in inferring back-bone relationships for monilophytes.

• Methods: We recovered 17 plastid genes for exemplar taxa using published and new primers. We compared results from maximum-likelihood and parsimony analyses, assessed the effects of removing rapidly evolving characters, and examined the extent to which our data corroborate or contradict the results of other studies, or resolve current ambiguities.

• Key Results: Considering multifamily clades, we found bootstrap support comparable to or better than that in published studies that used fewer genes from fewer or more taxa. We fi rmly establish fi lmy ferns (Hymenophyllales) as the sister group of all leptosporangiates except Osmundaceae, resolving the second deepest split in leptosporangiate-fern phylogeny. A clade com-prising Ophioglossaceae and Psilotaceae is currently accepted as the sister group of other monilophytes, but we recover Equi-setum in this position. We also recover marattioid and leptosporangiate ferns as sister groups. Maximum-likelihood rate-class estimates are somewhat skewed when a long-branch lineage ( Selaginella ) is included, negatively affecting bootstrap support for early branches.

• Conclusions: Our fi ndings support the utility of this gene set in corroborating relationships found in previous studies, improv-ing support, and resolving uncertainties in monilophyte phylogeny. Despite these advances, our results also underline the need for continued work on resolving the very earliest splits in monilophyte phylogeny.

Key words: deep phylogeny; Equisetaceae; euphyllophytes; Hymenophyllaceae; long-branch attraction; Marattiaceae; Selaginellaceae; tracheophytes; vascular-plant phylogeny.

1445September 2010] Rai and Graham — Monilophyte Phylogeny

and Pryer ’ s (2007) study , but the relative arrangement of these threes lineages to each other is poorly supported by bootstrap analysis.

Monilophyte phylogeny has not been examined as exten-sively as the phylogeny of the monilophytes ’ sister group, the seed plants, considering the variety of taxon and gene samplings used ( Table 1 ). For example, fi ve major studies that were fo-cused in large part on the monilophyte phylogenetic backbone ( Pryer et al., 2001 , 2004 ; Wikstr ö m and Pryer, 2005 ; Schuettpelz et al., 2006 ; Schneider et al., 2009 ) considered similar sets of monilophytes (either 21 or 52 – 53 taxa), and they consistently included relatively few seed plants (six exemplar species total; Table 1 ). Dense taxon sampling helps to minimize long-branch attraction artifacts and generally improves accuracy (e.g., Hillis, 1998 , Zwickl and Hillis, 2002 , Hillis et al., 2003 , Hedtke et al., 2006 ), and it may be particularly important to sample outgroups densely when long branches connect the ingroup to its nearest relatives (e.g., Graham and Iles, 2009), as is the case here. Pryer and colleagues employed substantially overlapping sets of genes for four molecular studies of monilophyte phylogeny (variously, three or four plastid genes, one nuclear and one mito-chondrial gene, but all including a core set of four genes; Table 1 ). Qiu et al. (2006 , 2007 ) included substantially more seed-plants (47 total) in two studies that have largely overlapping sets of genes (i.e., four plastid genes, one or two mitochondrial genes, and a nuclear gene); three of the genes they examined also over-lap with the molecular studies of Pryer et al. ( Table 1 ).

Further studies would therefore be valuable for addressing this major branch of the land-plant tree of life. We explored relationships among the major monilophyte clades and the broad backbone of the leptosporangiate ferns using new data from 17 conserved plastid genes ( Graham and Olmstead, 2000a , b ; Rai et al., 2003 , 2008 ). These markers and their associated noncoding regions have provided a powerful means for infer-ring a broad variety of deep phylogenetic questions in other major land-plant clades (i.e., “ early diverging ” angiosperms, monocots, conifers, cycads, and bryophytes; see Graham and Olmstead, 2000a , b ; Rai et al., 2003 , 2008 ; Graham et al., 2006; Saarela et al., 2007 ; Zgurski et al., 2008 ; Graham and Iles, 2009; Y. Chang and S. W. Graham unpublished data). The util-ity of this sampling of genes for inferring monilophyte relation-ships has not been considered before (only two of the genes have been considered; Table 1 ). We infer monilophyte phylog-eny on the basis of a large-scale survey of these genes across a phylogenetically diverse and representative sampling of monilo-phyte taxa. We also include a substantial number of outgroups from the sister taxon of monilophytes: 22 seed plants in total.

We use this new data set to address three main goals. The fi rst is to determine whether using a large set of plastid genes corroborates monilophyte relationships with equal or better branch support based on likelihood or parsimony bootstrap analysis. If our current gene sampling proves to be at least as effective as that used in other recent studies ( Table 1 ), this sup-ports its utility for future additional studies of monilophyte higher-order phylogeny. To facilitate denser sampling efforts in the future, we also present new “ universal ” primers designed to complement existing seed-plant primers ( Graham and Olmstead, 2000b ) for recovering these plastid genes in ferns and relatives. Our second goal is to reexamine poorly understood or confl ict-ing aspects of monilophyte phylogeny in recent studies — specifi cally, the earliest splits of monilophyte, leptosporangiate fern, and polypod fern phylogeny — to determine whether these problematic nodes can be resolved into well-supported clades

Skog and Banks (1973) and Stein et al. (1984) , suggested that horsetails (Equisetaceae) may be sister to all other living monilophytes. In a morphological analysis of living taxa only, Schneider et al. (2009) recovered Marattiaceae as the sister group of all other monilophytes with moderate support from parsimony and Bayesian analysis. In contrast, Schuettpelz and Pryer (2008) accepted Ophioglossaceae-Psilotaceae (= Psilo-topsida in Smith et al., 2006 ) as the sister group of all other monilophytes, a result that is strongly supported in a Bayesian analysis by Wikstr ö m and Pryer (2005) , but only moderately supported by parsimony or likelihood bootstrap analysis ( Pryer et al., 2001 , 2004 ). This result is now included in infl uential textbooks ( Judd et al., 2007 ) and has been used as the basis for reconstructing early evolutionary transitions in monilophyte biology ( Johnson and Renzaglia, 2009 ). However, we believe this result should be treated with caution, as it is well known that Bayesian inference can yield infl ated or skewed clade-support values for ancient radiations in situations where maximum-likelihood or parsimony analysis may be more conservative (e.g., Suzuki et al., 2002 ; Cummings et al., 2003 ; Simmons et al., 2004 ; Susko, 2008 ; Kolaczkowski and Thornton, 2007 ). Finally, the largest studies to date in terms of genes and taxa examined ( Qiu et al., 2006 , 2007 ) do not provide a well-supported resolution to the deepest divi-sions in monilophyte phylogeny based on likelihood and par-simony bootstrap analyses.

The leptosporangiate clade of ferns (Filicales of Doyle, 1998; Polypodiopsida of Smith et al., 2006; Leptosporangiatae of Cantino et al., 2007) represents a signifi cant component of ter-restrial ecosystems (e.g., Schnieder et al., 2004), with about 9 000 – 12 000 extant species in 33 families ( Smith et al., 2006 ; Moran, 2008 ). This makes it the most species-rich branch of monilophyte phylogeny, and the third richest land-plant clade after angiosperms and mosses. The major aspects of the phylo-genetic backbone of leptosporangiate ferns have been success-fully inferred and corroborated in several studies (e.g., Hasebe et al., 1995 ; Smith et al., 2006 ; Schuettpelz and Pryer, 2007 ), but multiple deep nodes along its phylogenetic backbone re-main unclear. For example, while Osmundaceae are consis-tently and strongly supported as the sister group of all other leptosporangiate ferns, the next split in leptosporangiate phy-logeny is not clear, as noted by Schuettpelz and Pryer (2008) , as the relative positions of the fi lmy ferns (the monofamilial Hy-menophyllales) and the gleichenioid ferns (Gleicheniales) to each other and to the remaining leptosporangiate orders have not been adequately resolved. Different arrangements of these early branches have been found that confl ict among multiple studies or that are only weakly supported by likelihood and par-simony analyses (see Pryer et al., 2001 ; Qiu et al., 2006 , 2007 ; Schuettpelz et al., 2006 ; Schuettpelz and Pryer, 2007 ; Schneider et al., 2009 ).

The largest group of leptosporangiate ferns is the polypod ferns (Polypodiales of Smith et al., 2006, with 15 families). The relationships of four polypod families that emerge near the base of this clade (i.e., Dennstaedtiaceae, Lindsaeaceae, Pteridaceae, and Saccolomataceae) are also uncertain. Their overall relation-ships have been examined only rarely using multigene data ( Table 1 ). Lindsaeaceae and Saccolomataceae may together compose a clade that is the sister-group of all other polypod ferns, but this relationship is only moderately supported in the three-gene study of Schuettpelz and Pryer (2007) . Similarly, a clade comprising Dennstaedtiaceae, Pteridaceae, and the re-maining “ eupolypod ” lineages is well supported in Schuettpelz

1446 American Journal of Botany [Vol. 97

We surveyed 17 genes and associated noncoding regions considered by Graham and Olmstead (2000b) and Rai et al. (2003 , 2008 ). However, we ex-cluded an intergenic spacer between rps 7 and ndh B from consideration. This region is present in all seed plants surveyed for our study but is apparently not present as a contiguous region in most leptosporangiate ferns, owing to a large inversion that involves a substantial portion of the inverted repeat ( Raubeson and Stein, 1995 ; Wolf et al., 2003 ).

DNA extraction, amplifi cation, and sequencing — We extracted genomic DNA using the protocol of Doyle and Doyle (1987) , as modifi ed in Rai et al. (2003) , from fresh, silica-dried, and herbarium specimens. Amplifi cation and sequencing follows Graham and Olmstead (2000b) and Rai et al. (2003 , 2008 ). We designed a set of 16 new fern-specifi c primers to facilitate amplifi cation and sequencing (Appendix 2). With a few minor exceptions, all products were com-pletely sequenced in both forward and reverse directions. The new data were added to a previously generated alignment ( Rai et al., 2008 ) using criteria out-lined in Graham et al. (2000) and Rai et al. (2008) . Regions in some taxa that we could not amplify or sequence were coded as missing data in the fi nal matrix (see Appendix 1).

The aligned regions considered for analysis included all of the coding re-gions ( Table 1 ) in addition to unambiguously aligned noncoding regions from two introns ( rpl 2 and ndh B) and seven intergenic spacer regions (i.e., 3 ′ - rps 12- rps 7, and three each in the psb E- psb F- psb L- psb J and psb B- psb T- psb N- psb H clusters). We dealt with hard-to-align regions, often limited to individual taxa, by staggering them in the alignment (e.g., Saarela and Graham, 2010 ), coding the gap cells as missing data. Offset unique regions are effectively ignored in parsimony analysis and should have only minor effects in maximum-likelihood analysis (e.g., on the estimation of base frequencies). This approach contributes signifi cantly to the large length of the full concatenated alignment (36 139 bp, based on ~10 kb per taxon in monilophytes; Table 1 ). For the full alignment, 2573 bp are variable and uninformative across vascular plants and 7479 bp are parsimony informative.

Phylogenetic analyses — We performed a heuristic maximum likelihood (ML) search using PhyML 2.4.4 ( Guindon and Gascuel, 2003 ) with a BIONJ starting tree, NNI (nearest-neighbor-interchange) branch swapping, and model parameters estimated from the data in each case. We chose a DNA

using this approach. Our third goal is to assess whether remov-ing the fastest-evolving characters — and therefore potentially the most misleading ones in phylogenetic inference ( Felsen-stein, 1978 , 1983 ) — has a substantial infl uence on phylogenetic inference here. We use a character-fi ltering approach based on a maximum-likelihood classifi cation of site rates, used previ-ously in seed-plant phylogenetic inference (e.g., Burleigh and Mathews, 2004 , 2007 ; Rai et al., 2008 ; Graham and Iles, 2009).

MATERIALS AND METHODS

Taxonomic and genomic sampling — The fi nal matrix considered here in-cludes 64 representatives of the major land-plant lineages (Appendix S1; see Supplemental Data at http://www.amjbot.org/cgi/content/full/ajb.0900305/DC1; and see Appendix 1 for a list of newly generated sequences and associated GenBank accession numbers). Of these, four represent the major lineages of lycophytes ( Selaginella uncinata and Huperzia lucidula sequences were ob-tained from GenBank; accession numbers AB197035 and NC_006861, re-spectively). We included 16 gymnosperms and 6 angiosperms to represent the deepest splits in seed-plant phylogeny ( Rai et al., 2008 ; Graham and Iles, 2009). We also included four bryophyte outgroups (GenBank accession num-bers for Anthoceros formosae , Marchantia polymorpha , and Physcomitrella patens are NC_004543, NC_001319, and NC_005087, respectively; see Ap-pendix 1 for Sphagnum sp.). The remaining 34 taxa sample the major clades of monilophytes according to Pryer et al. (2001 , 2004 ) and Schuettpelz and Pryer (2007) and were chosen, as far as possible, to sample the crown-clade root nodes (= most recent common ancestors, MRCAs) of relevant clades. They include eight eusporangiate monilophytes and 26 leptosporangiate ferns ( Adiantum capillus-veneris , Angiopteris evecta , and Psilotum nudum sequences were obtained from GenBank, accession numbers NC_004766, NC_008829, and NC_003386, respectively; see Appendix 1 for the remain-der). We sampled all seven orders and 21 of the 33 families of leptosporan-giate ferns recognized by Smith et al. (2006) ; unsampled families are mainly small ones in Cyatheales and Polypodiales (largely comprising one to fi ve genera; Smith et al., 2006 ).

Table 1. Taxonomic scope, analysis method (MP = parsimony; ML = likelihood; BI = Bayesian inference), and character samplings used in recent multigene analyses that included a substantial sampling of monilophytes (pt = plastid, nu = nuclear, and mt = mitochondrial), compared with the present study.

ReferencePrimary sampling focus

(number of taxa)Other taxa sampled

(number of taxa)Method of analysis

No. of genes Aligned characters Genes

Present study Monilophytes (34) Bryophytes (4), Lycophytes (4), Seed plants (22)

ML, MP 17 36 139 bp a All pt: atp B, ndh B, ndh F, psb B, psb C, psb D, psb E, psb F, psb J, psb L, psb N, psb H, psb T, rpl 2, rps 7, 3 ′ - rps 12, rbc L

Schneider et al., 2009

Monilophytes (21) Bryophytes (5), Lycophytes (3), Seed plants (6),

MP, BI – 136 morph. –

Qiu et al., 2007 Land plants (181) Green algae (9) ML, MP b 7 14 533 bp Pt- atp B, pt- rbc L, pt-LSU, pt-SSU, nu-18S rDNA, mt- atp 1, mt-LSU

Qiu et al., 2006 Land plants (184) Green algae (9) ML, MP 6 13 631 bp Pt- atp B, pt- rbc L, pt-LSU, pt-SSU, nu-18S rDNA, mt-LSU

Schuettpelz and Pryer, 2007

Leptosporangiate ferns (400)

Other monilophytes (5) ML 3 4092 bp Pt- atp B, pt- rbc L, pt- atp A

Schuettpelz et al., 2006

Monilophytes (52) Seed plants (6) ML, BI 5 6432 bp Pt- atp B, pt- rbc L, pt- rps 4, pt- atp A, nu-18S rDNA

Wikstr ö m and Pryer, 2005

Monilophytes (21) Bryophytes (5), Lycophytes (3), Seed plants (6)

BI 5 5258 bp, + 138 morph.

Pt- atp B, pt- rbc L, pt- rps 4, nu-18S rDNA, mt- atp 1

Pryer et al., 2004 Monilophyes (53) Lycophytes (3), Seed plants (6)

ML, MP, BI

4 5 049 bp Pt- atp B, pt- rbc L, pt- rps 4, nu-18S rDNA

Pryer et al., 2001 Monilophytes (21) Bryophytes (5), Lycophytes (3), Seed plants (6)

ML, MP 4 5072 +, 136 morph. (MP)

Pt- atp B, pt- rbc L, pt- rps 4, nu-18S rDNA

a Aligned length (ignoring excluded regions for the taxon subset considered in the present study). Median unaligned length for monilophytes in which we recovered all 17 genes = 9851 bp (range: 8181 – 13 116 bp).

b MP values not reported for all nodes of interest here.


but were moderately well supported by MP (clade aa, and clade e = monilophytes excluding Equisetaceae). The remaining clades recovered for the shortest MP or ML trees confl icted be-tween phylogenetic inference methods but were also poorly supported (i.e., clades u and x, seen in the ML tree [ Fig. 1] , and clades ab, ac, and ae, observed in the single MP tree but not in the best ML tree [ Table 2] ). Only one substantial confl ict was observed in total (clade c vs. ad, mentioned above).

Rate-class analyses — When we fi ltered the most rapidly evolving rate classes (RC78), with Selaginella included or ex-cluded during site-rate assignment, and reran the ML analyses with the remaining more conservative characters (RC0 – RC6), 15 clades had approximately the same ML support values be-fore and after removal of rapidly evolving characters, consider-ing both fi ltering methods. The ML bootstrap values changed (fell) by 10% or more bootstrap support for both fi ltering meth-ods for fi ve clades total in the best ML tree (i.e., clades k, l, m, o, aa; see Table 2 ). We considered > 10% change in bootstrap support as noteworthy, following Schuettpelz et al. (2006 : fi g. 2). Considering the two fi ltering cases here (fast sites re-moved, with Selaginella included or excluded during the rate-class assignments), distinctly different patterns of change in ML bootstrap support were observed in six cases, for com-parisons of bootstrap support values before and after fi ltering of fast sites ( Table 2 ). In four cases (clades b, c, d, and h), the ML bootstrap support fell by 10% or more compared with the unfi ltered analysis when Selaginella was included during rate-class assignment but was essentially static for the same clades when Selaginella was excluded. All four clades are early branches of monilophyte or vascular-plant phylogeny (i.e., lycophytes as a whole, euphyllophytes, monilophytes as a whole, and leptosporangiate ferns as a whole). Support for clade a (Iso ë taceae+Selaginellaceae) fell by more than 10% when Selaginella was included in rate-class inference, with no corresponding value available for when Selaginella was excluded (by defi nition).

A possible sister-group relationship between Dennstaedti-aceae and Pteridaceae (clade x) was poorly supported in the unfi ltered ML analysis ( < 50% support), but this increased to moderate support when Selaginella was excluded from rate assignments (71% ML bootstrap support); a similar pattern was observed for the fi rst branch in monilophyte phylogeny: Equisetaceae was inferred to be the sister to all other monilo-phytes with weak support in the unfi ltered ML analysis (clade e, 58% support) but moderate support with fast sites removed and Selaginella excluded from rate assignments (71% support; Table 2 ).

We compared how the ML assignments of individual sites to rate classes differed when Selaginella was included or de-leted. We observed substantial overlap in rate-class assign-ments with or without this problematic taxon ( Table 3 ) , and most differences in how rate classes were assigned involved neighboring rate classes (either one rate class higher or lower). Thus, rate-class assignments were in general similar whether Selaginella was present or deleted. However, with either fi ltering approach ( Selaginella included vs. deleted), the alternative case tended to place more characters in the next-highest class, rather than the next-lowest class, particu-larly for the lower rate classes (considering displacements off the diagonal in Table 3 ; note the relative size and direc-tion of change in rate assignments moving out from the di-agonal). On the whole, the subset of characters that were

substitution model for ML analysis using the hierarchical likelihood ratio test (hLRT) and the Akaike information criterion (AIC) in Modeltest 3.7 ( Posada and Crandall, 1998 ). The optimal model chosen in each case was GTR + Γ + I (general-time-reversible [GTR] rate matrix with proportion of invariable sites [I] considered, and among-site rate variation accounted for using the gamma [ Γ ] distribution as described by the shape parameter alpha ( α )]. We also performed a heuristic maximum-parsimony (MP) search using PAUP* 4.0b10 ( Swofford, 2003 ), with all characters and character-state changes equally weighted, and using TBR (tree-bisection-reconnection) branch swap-ping, with 100 random-addition replicates, and otherwise using default set-tings. We performed nonparametric bootstrap analysis ( Felsenstein, 1985 ) using the same search criteria, with 100 bootstrap replicates (and a single random-addition replicate per parsimony bootstrap replicate). We use “ weak, ” “ moderate, ” and “ strong ” in reference to clades that have bootstrap support values < 70%, 70 – 89%, and ≥ 90%, respectively (e.g., Graham et al., 1998 ; Rai et al., 2003 , 2008 ).

Inference of nucleotide rate classes and exploration of the effect of long branches — We partitioned the data into nine rate classes using HyPhy ( Pond et al., 2004 ; and see Burleigh and Mathews, 2004 ; Rai et al., 2008 ). We used the best MP tree topology to assign each of the aligned sites to its most likely indi-vidual rate category (using the GTR model and eight discrete rate classes). We then excluded the two fastest rate classes (RC7 and RC8) and performed a maximum-likelihood search and bootstrap analysis on the remaining characters (RC0 – RC6), using the search criteria outlined above. The sequence for Selagi-nella is highly divergent compared with the rest of the vascular plants (see Re-sults; also evident from a visual examination of the alignment). There is evidence of elevated plastid genome evolution for the family, reported for rbc L by Korall and Kenrick (2002 ; 2004 ), which may be a consequence of the exten-sive RNA editing observed in Selaginella ( Tsuji et al., 2007 ). We therefore re-calculated rate classes with Selaginella removed from consideration and repeated the ML search (again for RC0 – RC6). Overlaps in the nucleotides esti-mated to be in each rate class, with Selaginella either included or deleted, were determined by including or excluding the relevant rate-class character sets in PAUP*.

RESULTS

Phylogeny of ferns and relatives — We have reported on re-sults for seed plants elsewhere, and so we focus here on phylo-genetic relationships involving monilophytes and lycophytes. The majority of the vascular-plant clades inferred here were strongly supported by both ML and MP analysis. These include all seven monilophyte families sampled for two or more taxa and 18 of the 27 multifamily clades recovered in the best ML tree (which are labeled as clades a – aa; Fig. 1 , Table 2 ). The latter clades comprise lycophytes, monilophytes, Psilotopsida (= Ophioglossaceae-Psilotaceae), all fi ve multifamily orders in Smith et al. (2006 ; i.e., Cyatheales, Gleicheniales, Polypodi-ales, Salviniales, Schizaeales), leptosporangiate ferns, leptospo-rangiate ferns excluding Osmundaceae, core leptosporangiate ferns (Salviniales+Cyatheales+Polypodiales), eupolypods, eupolypods I, and several others of interest (clades labeled as: a, j, m, r, and v in Fig. 1 and Table 2 ). The eupolypods II clade (= clade z) was strongly supported by ML and moderately well supported by MP, and two clades were moderately strongly supported by both ML and MP (clades l and p). Thus, 78% of clades (= 21 of 27 taxon partitions) seen in the ML tree that in-volved two or more families of ferns or lycophytes simultane-ously had moderate to strong support from ML and MP bootstrap analysis.

Two clades were moderately strongly supported by ML anal-ysis but had < 50% support from MP (clade g, with Marattiaceae sister to leptosporangiate ferns, and clade c = euphyllophytes). The latter clade, the euphyllophytes, was also contradicted by MP (clade ad = lycophytes+seed plants, which had 94% sup-port from parsimony). Two clades had weak support from ML


DISCUSSION

Congruence and discordance with other phylogenetic studies of ferns and relatives — Phylogenetic congruence, the ability to corroborate phylogenetic relationships using differ-ent gene and taxon sets, is a key pillar of systematic biology (e.g., Penny et al., 1982 ; Hillis, 1995 ; Graham et al., 1998 ; Pryer et al., 2001 ; Schneider et al., 2009 ) because it builds

assigned differently by the two methods tended to be placed in faster rate classes when Selaginella was included in rate assignments. This effect was most pronounced for the zero-rate class. When Selaginella was included, 349 characters were assigned to RC2 – RC8 that were assigned to RC0 with Selaginella excluded. By contrast, no characters assigned to RC0 with Selaginella excluded were assigned to higher rate classes when this taxon was included ( Table 3 ).

Fig. 1. Maximum-likelihood tree ( – ln L = 235,448.293) found using 17 plastid genes and associated noncoding regions (including all nine rate classes, RC0 – RC8). Maximum likelihood bootstrap values are indicated beside branches (asterisk indicates 100% ML bootstrap support); clades comprising mul-tifamily collections of ferns or lycophytes are also indicated beside branches (see Table 2 ). Inverted arrowheads indicate alternative placements of Equise-tum found using different samplings of bryophytes in the analysis (left, including only the hornwort Anthoceros ; right, excluding all bryophytes). Monilophyte families (following Smith et al., 2006 ) are noted in circles: As = Aspleniaceae, Bl = Blechnaceae, Cy = Cyatheaceae, De = Dennstaedtiaceae, Eq = Equisetaceae, Dk = Dicksoniaceae, Dp = Dipteridaceae, Dr = Dryopteridaceae, Gl = Gleicheniaceae, Hy = Hymenophyllaceae, Li = Lindsaeaceae, Ly = Lygodiaceae, Mr = Marattiaceae, Ms = Marsileaceae, Mt = Matoniaceae, Op = Ophioglossaceae, Os = Osmundaceae, Pl = Plagiogyriaceae, Po = Polypodiaceae, Ps = Psilotaceae, Pt = Pteridaceae, Sa = Saccolomataceae, Sc = Schizaeaceae, Sl = Salviniaceae, and Th = Thelypteridaceae.


Tab

le 2

. Su

ppor

t fo

r th

e m

ultif

amily

mon

iloph

yte

and

lyco

phyt

e cl

ades

as

dete

rmin

ed b

y pa

rsim

ony

boot

stra

p (M

P),

likel

ihoo

d bo

otst

rap

(ML

), o

r po

ster

ior

prob

abili

ty (

PP,

as p

erce

ntag

e;

italic

s). L

abel

s (fi

rst

col

umn)

cor

resp

ond

to c

lade

s (s

ee F

ig. 1

for

cla

des

a – aa

). F

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moderately supported in Schuettpelz and Pryer, 2007 ). The primers described here (Appendix 2) would facilitate expanded taxon sampling to test this hypothesis for the current gene set.

Early phylogenetic splits in leptosporangiate ferns — We re-solved a key issue in leptosporangiate fern phylogeny that has resisted satisfactory resolution in all current studies, with rea-sonably strong support here from ML and MP bootstrap analy-sis. Although the deepest split is clearly between Osmundaceae and all other taxa, a result that is well supported in all current studies (i.e., clade i; Table 2 ), the next deepest split in the lep-tosporangiate-fern phylogenetic tree has been unclear. Current multigene or morphological analyses fi nd various confl icting relationships involving Hymenophyllaceae; these have only poor support from MP or ML analysis, and while Bayesian sup-port values tend to be much stronger, these confl ict moderately to strongly considering three possible resolutions (see clades viii – x in Table 4 ). Qiu et al. (2006) found Hymenophyllaceae (= Hymenophyllales) to be the sister group of the remaining leptosporangiate ferns (excluding Osmundaceae), with 70% MP bootstrap support (vs. 54% for ML). A later, larger sam-pling also had similar support for this arrangement (i.e., 59% ML bootstrap support in Qiu et al., 2007 ; see clade l in Table 2 ). We fi nd this same relationship here, but for the fi rst time with moderately strong support from ML and MP bootstrap analysis (88% ML, 83% MP; Fig. 1 ; clade l in Table 2 ).

Early splits in the monilophytes — We recovered Equiseta-ceae as the sister group of all other monilophytes, a result that is moderately well supported by MP bootstrap analysis, and also by a rate-fi ltered ML analysis that excluded Selaginella in rate-class assignments (see clade e in Table 2 ). We also fi nd Marattiaceae as the sister group of leptosporangiate ferns (moderately supported in the main ML analysis; clade g in Table 2 ). However, at present perhaps the best that can be said about all relationships among the major lineages of monilo-phytes in current studies is that we do not understand them very well, excepting a well-corroborated relationship between Ophioglossaceae and Psilotaceae (also well supported here; clade f in Table 2 ). This stance is supported by the fact that when we reduced the sampling of bryophyte outgroups here, this tended to disrupt early monilophyte relationships (note the effect this has on the position of Equisetaceae in Fig. 1 ).

Therefore, concerning all other relationships among these taxa, all current phylogenetic estimates of the arrangement of the basal splits in monilophyte phylogeny are questionable, as they have at best relatively moderate ML or MP support ( Table 2 ), and they confl ict among studies (ignoring even stronger Bayesian

confi dence that the recovered topologies refl ect evolutionary history rather than arbitrary groupings due to stochastic error. Conversely, discordance among studies may also allow us to fl ag and investigate possible instances of systematic error, such as long-branch attraction ( Felsenstein, 1978 ). Our analy-sis of vascular-plant relationships using a large multigene plastid data set is, in general, highly congruent with earlier multigene studies, corroborating clades in common across studies ( Fig. 1 , Table 2 ). In addition, our ML bootstrap support values for major multifamily clades either are within a few percentage points of other studies or are equal to them or better ( Table 2 ). Even at the currently relatively limited taxon den-sity, we found moderate to strong ML bootstrap support for ~85% (23 of 27) multifamily clades in the best ML tree ( Fig. 1 ), including some of the most problematic nodes. This sup-ports the general utility of our approach for the current and future investigations of monilophyte phylogeny. However, several major branches of monilophyte phylogeny are poorly supported or in confl ict in all published studies. Here, we focus on three important areas of monilophyte phylogeny that are currently poorly understood: the branching order of the earliest splits in monilophytes, leptosporangiate ferns, and polypod fern phylogeny, respectively.

Early phylogenetic splits in polypod ferns — Few multigene studies have suffi cient taxon sampling to adequately address the earliest splits of polypod fern phylogeny ( Table 1, 2 ), and so this remains a relatively unresolved question. Our ML and MP analyses disagreed weakly about whether Saccolomataceae or Lindsaeaceae are the respective sister taxon to other polypods, and we did not recover the Lindsaeaceae-Saccolomataceae clade observed in Schuettpelz and Pryer (2007) . We corrobo-rated the well-supported Dennstaedtiaceae-Pteridaceae-eupolypod clade observed in Schuettpelz and Pryer (2007) , also with strong ML support here (clade v; Table 2 ). However, a novel sister-group relationship found here between Dennstaedtiaceae and Pteridaceae was only poorly supported (i.e., clade x; Table 2 ). While it is possible that many more data per taxon will be needed to resolve these relationships, denser taxon sampling for gene sets as large as ours may also help. This has been a pro-ductive approach in comparable situations (e.g., Graham et al., 2006). Denser taxon sampling may also be useful for address-ing other relationships among the remaining polypod ferns, in-cluding the possibility that the deepest splits in eupolypods I and II are each defi ned by small isolated groups containing a few genera that were previously considered to belong to other families (i.e., fragments of Dryopteridaceae for eupolypods I, and Woodsiaceae for eupolypods II, arrangements that are only

Table 3. Overlap in ML site-rate class assignments with Selaginella included or excluded prior to estimating the most likely rate class for each site. Columns: rate assignments with Selaginella included (number in parentheses at column head is total characters in that class). Rows: rate assignments with Selaginella excluded (number in parentheses at row start is total characters in that class). RC = rate class, from lowest (RC0, no change predicted) to highest (RC8); RC1 is an empty set.

RC0 (26 087) RC2 (292) RC3 (1031) RC4 (1127) RC5 (1224) RC6 (1806) RC7 (2497) RC8 (2075)

RC0 (26 436) 26 087 10 261 61 5 5 1 6RC2 (255) 0 244 11 0 0 0 0 0RC3 (850) 0 38 681 131 0 0 0 0RC4 (946) 0 0 78 790 76 2 0 0RC5 (1236) 0 0 0 145 1013 76 2 0RC6 (1818) 0 0 0 0 130 1600 88 0RC7 (2560) 0 0 0 0 0 121 2373 66RC8 (2038) 0 0 0 0 0 2 33 2003


(i.e., lycophytes + seed plants). These reductions in support values may be consistent with expectations of increased sam-pling error in smaller data sets. However, we observed an ex-ceptionally long branch for Selaginella ( Fig. 1 ), also noted by Korall and Kenrick (2002 , 2004 ) for rbc L. When we removed this taxon prior to rate classifi cation, and repeated ML analysis on the fi ltered data set, four of these clades no longer experi-enced a reduction in support (and support for two clades sur-passed what was seen for the unfi ltered ML analysis; clades e and x). These clades include some of the earliest splits in eu-phyllophyte phylogeny. Examination of how nucleotides were assigned to different rate classes with or without this taxon indicates that some sites behave quite differently when Selag-inella is included in the rate assignments ( Table 3 ). This may be at the root of how this taxon may reduce some ML support values in ferns and relatives. However, until this effect is bet-ter characterized, it may be useful to exclude Selaginella as an outgroup in future studies of monilophyte phylogeny, at least for plastid-derived sequence data.

One strong confl ict between ML and MP involves a well-supported (but likely incorrect) sister-group relationship in-ferred here between lycophytes and seed plants using MP (with corresponding poor MP support for euphyllophytes; Table 2 ). Our ML analysis did not recover this relationship, but instead moderately supported euphyllophyte monophyly (81% sup-port). Schneider et al. (2009) also recovered the euphyllophyte clade in their morphological study, with strong MP bootstrap support ( Table 2 ). However, in other molecular studies a simi-lar pattern of good ML versus poor MP bootstrap support for euphyllophytes has been found, for example by Pryer et al. (2001) , who used a comparable sampling of bryophyte out-groups ( Table 1 ). Subsequent molecular studies by Pryer and colleagues either did not report parsimony bootstrap values for euphyllophytes ( Wikstr ö m and Pryer, 2005 ) or did not include bryophytes in the analysis ( Pryer et al., 2004 ), precluding full assessment of euphyllophyte monophyly ( Table 2 ). The best

confl icts; see clades i – vii in Table 4 ). Although we do not satis-factorily resolve the question here, our fi ndings clearly demon-strate that it is too early to accept Psilotopsida as the sister group of all other monilophytes (cf. Schuettpelz and Pryer, 2008 ). Our fi nding of Equisetaceae as the sister-group of all other monilo-phytes may be on equally shaky ground but is possibly more consistent with fossil evidence (see Doyle, 1998 ) and should clearly remain in contention as a possible solution. More data (including more genes) are needed to resolve this satisfactorily. However, simply improving taxon sampling in this part of monilophyte phylogeny may not help much here, as the exem-plar taxon sampling that we used spanned the deepest nodes of most of the earliest lineages, including Marattiaceae (see Mur-dock, 2008a , b ), Ophioglossaceae (see Hauk et al., 2003 ), lep-tosporangiate ferns, and likely also Psilotaceae (we sampled both genera). Nonetheless, it would still be worth including ad-ditional taxa to sample a greater phylogenetic diversity of Equi-setaceae (see Des Marais et al., 2003 ; Guillon, 2004 ; 2007 ).

Effect of long branches and rapidly evolving sites on in-ference of deep fern phylogeny — Maximum likelihood is ex-pected to be less sensitive to long-branch attraction than parsimony (e.g., Chang, 1996; Huelsenbeck, 1997 , 1998 ; Sullivan and Swofford, 2001 ; Swofford et al., 2001 ). Strong disagree-ment between ML and MP analysis may therefore be a fl ag for long-branch attraction in phylogenetic inference. How-ever, considering the multifamily clades of monilophytes and lycophytes, our parsimony and likelihood estimates were generally similar, which we view as a reassuring result. Rapidly evolving sites should be accommodated in phyloge-netic inference using maximum likelihood, so long as the sub-stitution model is a reasonable fi t. When we fi ltered out high-rate sites (RC78), this generally had little effect on phy-logenetic inference beyond reducing some ML bootstrap support values ( Table 2 ), even though the model MP tree used for rate classifi cation likely had an incorrect relationship

Table 4. Summary of selected confl icts concerning early branches of monilophyte and leptosporangiate-fern phylogeny (P01 = Pryer et al., 2001 ; P04 = Pryer et al., 2004 ; WP05 = Wikstr ö m and Pryer, 2005 ; S06 = Schuettpelz et al., 2006 ; Q06 = Qiu et al., 2006 ; and Sch = Schneider et al., 2009). MP = parsimony bootstrap, ML = likelihood bootstrap, and BI = Bayesian inference (with posterior probability expressed as a percentage). Only instances with ≥ 70% support from at least one analysis method are reported.

Confl ict summary

Multifamily clade

Clade label on Figure 1 and Table 2 Study Criterion Support (%)

Support here: ML (MP)

Confl icts with clade:

i Monilophytes excluding Equisetaceae e Here MP → 58 (74) ii – iii, v – viiii Monilophytes excluding Psilotopsida – P01 ML 87 < 50 ( < 50) i, iii, vi

P04 ML/MP/BI 88, 87, 100WP05 BI 100

iii Monilophytes excluding Marattiaceae Sch09 MP/BI 71, 77 < 50 ( < 50) i, ii, iv – v, viiiv [Marattiaceae+leptosporangiate ferns] – Here ML → 82 ( < 50) iii, vv [Equisetaceae+Marattiaceae] – P04 MP/BI 76, 82 < 50 ( < 50) i, iii, iv, vi

WP05 BI 93vi [Equisetaceae+Psilotaceae] – Sch09 MP/BI 75, 99 < 50 ( < 50) i, ii, v, viivii [Equisetaceae+Marattiaceae+ leptosporangiate

ferns] – WP05 BI 100 < 50 ( < 50) i, iii, vi

viii Leptosporangiate ferns excluding Osmundaceae & Hymenophyllaceae

1 Here ML/MP → 88 (83) ix, xWP05 BI 99Q06 MP 70S06 BI 80

ix Leptosporangiate ferns excluding Osmundaceae & Gleicheniales

– Sch09 BI 72 < 50 ( < 50) viii, x

x [Hymenophyllaceae+Gleicheniales] – P04 BI 96 < 50 ( < 50) viii, ix


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I)E

U35

2281

EU

3496

02E

U55

2829

EU

3282

43E

U55

8412

EU

3523

08E

U55

8443

EU

5584

96n/

a

Poly

podi

acea

e Po

lypo

dium

hes

peri

um M

axon

(H

. S. R

ai &

J.

M. Z

gurs

ki 1

4-09

-02-

2, A

LTA

)E

U35

2282

EU

3496

03E

U55

2830

EU

3282

44E

U55

8413

EU

3523

09E

U55

8444

EU

5584

97E

U55

8467

Pter

idac

eae

Cer

atop

teri

s ri

char

dii

EU

3522

69E

U34

9590

EU

5528

14E

U32

8228

EU

5583

97E

U35

2297

EU

5584

30E

U55

8481

EU

5584

59B

rong

n.(P

. Kill

ip 4

4595

, GH

)Pt

erid

acea

e Vi

ttar

ia v

olke

nsii

Hie

ron.

(E

. T. A

fric

a,

Che

rang

ani T

wee

dia

2708

, KE

W)

EU

3522

87n/

aE

U55

2836

EU

3282

50E

U55

8419

EU

3523

15E

U55

8448

EU

5585

03n/

a

Sacc

olom

atac

eae

Sacc

olom

a in

aequ

ale

(Kun

ze)

Met

t. (3

7207

6,

DU

KE

)E

U35

2283

EU

3496

04E

U55

2831

EU

3282

45E

U55

8414

EU

3523

10n/

aE

U55

8498

n/a

Salv

inia

ceae

Salv

inia

S é g

. sp.

(H

. S. R

ai 1

023,

UB

C)

EU

3522

84n/

aE

U55

2832

EU

3282

46E

U55

8415

EU

3523

11E

U55

8445

EU

5584

99E

U55

8468

Schi

zaea

ceae

Schi

zaea

dic

hoto

ma

(L.)

J. S

m. (

S. W

. Gra

ham

02

-03-

36B

s.n

.)E

U35

2285

EU

3496

05E

U55

2833

EU

3282

47E

U55

8416

EU

3523

12n/

aE

U55

8500

n/a

The

lypt

erid

acea

e Th

elyp

teri

s re

ticu

lata

(L

.) P

roct

or (

J. S

. Mill

er

& M

. C. M

erel

lo 8

864,

MO

)E

U35

2286

n/a

EU

5528

34E

U32

8248

EU

5584

17E

U35

2313

EU

5584

46E

U55

8501

n/a

a Pre

viou

sly

publ

ishe

d se

quen

ces.

Acc

essi

ons

in b

rack

ets

wer

e pr

oduc

ed b

y ot

her

wor

kers

; see

Gra

ham

and

Olm

stea

d (2

000a

, b ),

Rai

et a

l. (2

003 ,

200

8 ), a

nd S

aare

la e

t al.

(200

7) f

or a

com

plet

e lis

t of

taxa

and

acc

essi

on n

umbe

rs f

or o

ther

seq

uenc

es e

mpl

oyed

her

e. b

Her

bari

um a

bbre

viat

ions

: ALT

A =

Uni

vers

ity o

f A

lber

ta;

DU

KE

= D

uke

Uni

vers

ity;

GH

= H

arva

rd U

nive

rsity

; K

EW

= R

oyal

Bot

anic

Gar

dens

, K

EW

; M

O =

Mis

sour

i B

otan

ical

Gar

den;

N

YB

G =

New

Yor

k B

otan

ical

Gar

den;

TI

= U

nive

rsity

of

Toky

o; U

BC

= U

nive

rsity

of

Bri

tish

Col

umbi

a; U

C =

Uni

vers

ity o

f C

alif

orni

a, B

erke

ley;

and

UT

C =

Uta

h St

ate

Uni

vers

ity, L

ogan

.

App

endi

x 1.

Con

tinue

d

1456 American Journal of Botany

Appendix 2. New primers designed for this study.

Primer name and sequence (5 ′ – 3 ′ ) a,b Gene/region

F1F: CCATAATTTRCARGAACATTC 3 ′ - rps 12L1F: GAGRTAACRGCTTACATAC 3 ′ - rps 12L2F: AAACAACTTGGTGTCYAAGG 3 ′ - rps 12L2R: CTTAGACACCAAGTTGTTTC 3 ′ -rps 12L4F: TGGAAAGCTGTATTCGATG 3 ′ -rps 12- rps 7 IGSL4R: TCATCGAATACAGCTTTCC 3 ′ -rps 12L5F: GATCCAATTTATCGTAATCG rps 7L5R: GATTACGATAAATTGGATC 3 ′ -rps 12- rps 7 IGSF9F: TTATGGGTGGARCAAGTTC ndh BF9R: TAGAAGAACTTGYTCCACC ndh BF13F: GAAACGTATGCTTGCATATTC ndh BF13R: GAATATGCAAGCATACGTTTC ndh BF20F: ATATCGTSAAATWGATTTTCG rpl 2F24R: ATCTCTTCCCRAACTGTAC rpl 2F41F: GGTCCTGARGCACARGG psb DF45R: CATTAAAGAGCGTTTCCAC psbD

a The prefi x ‘ F ’ indicates a primer designed to work across all ferns. b The prefi x ‘ L ’ indicates a primer designed to work specifi cally in

leptosporangiate ferns.

Utility of a large, multigene plastid data set in inferring higher-order relationships in ferns and relatives (monilophytes)

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