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Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev 5 6

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Horsetails are the sister group to all other monilophytes and Marattiales are sister to leptosporangiate ferns q

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Nils Knie, Simon Fischer 1, Felix Grewe 2, Monika Polsakiewicz, Volker Knoop ⇑

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IZMB – Institut für Zelluläre und Molekulare Botanik, Abteilung Molekulare Evolution, Kirschallee 1, D-53115 Bonn, Germany

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a r t i c l e

i n f o

Article history: Received 4 March 2015 Revised 9 May 2015 Accepted 11 May 2015 Available online xxxx Keywords: Ferns Monilophyte phylogeny Equisetales Mitochondrial DNA Chloroplast DNA Group II introns

a b s t r a c t The ‘‘Monilophyte’’ clade comprising ferns, horsetails, and whisk ferns receives unequivocal support from molecular data as the sister clade to seed plants. However, the branching order of its earliest emerging lineages, the Equisetales (horsetails), the Marattiales, the Ophioglossales/Psilotales and the large group of leptosporangiate ferns has remained dubious. We investigated the mitochondrial nad2 and rpl2 genes as two new, intron-containing loci for a wide sampling of taxa. We found that both group II introns – nad2i542g2 and rpl2i846g2 – are universally present among monilophytes. Both introns have orthologues in seed plants where nad2i542g2 has evolved into a trans-arrangement. In contrast, and despite substantial size extensions to more than 5 kb in Psilotum, nad2i542g2 remains cis-arranged in the monilophytes. For phylogenetic analyses, we filled taxonomic gaps in previously investigated mitochondrial (atp1, nad5) and chloroplast (atpA, atpB, matK, rbcL, rps4) loci and created a 9-gene matrix that also included the new mitochondrial nad2 and rpl2 loci. We extended the taxon sampling with two taxa each for all land plant outgroups (liverworts, mosses, hornworts, lycophytes and seed plants) to minimize the risk of phylogenetic artefacts. We ultimately obtained a well-supported molecular phylogeny placing Marattiales as sister to leptosporangiate ferns and horsetails as sister to all remaining monilophytes. In addition, an indel in an exon of the here introduced rpl2 locus independently supports the placement of horsetails. We conclude that under dense taxon sampling, phylogenetic information from a prudent choice of loci is currently superior to character-rich phylogenomic approaches at low taxon sampling. As here shown the selective choice of loci and taxa enabled us to resolve the long-enigmatic diversifications of the earliest monilophyte lineages. Ó 2015 Published by Elsevier Inc.

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1. Introduction

52

Ferns, ‘‘fern allies’’, and seed plants are vascular plants (tracheophytes) that share the ability to transport water and nutrients over long distances within the plant body. This evolutionary innovation gave rise to one of the most dramatic changes of life on earth. The classic term ‘‘fern allies’’ describes taxa representing the most ancient lineages of tracheophytes that have puzzled plant biologists for long owing to their unique, unorthodox anatomies: the whisk ferns (Psilotales), the horsetails (Equisetales) and the lycophytes (the latter comprising quillworts, club mosses and

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q

This paper has been recommended for acceptance by Elizabeth Zimmer. ⇑ Corresponding author. Fax: +49 228 73 6467. E-mail address: [email protected] (V. Knoop). 1 Present address: Innere Medizin III, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 669, D-69120 Heidelberg, Germany. 2 Present address: Field Museum of Natural History, Integrative Research Center, Department of Science and Education, 1400 South Lake Shore Drive, Chicago, IL 60605, USA.

spike mosses). The common names of the lycophyte groups are particularly misleading. Club mosses (Lycopodiales) and spike mosses (Selaginellales) are not mosses, but true vascular plants. Molecular data have ultimately helped to resolve many of the phylogenetic enigmas. A first key insight set the lycophytes apart from all other vascular plants, which share a characteristic chloroplast genome inversion (Raubeson and Jansen, 1992). A second significant insight came from molecular phylogenetic analyses clearly confirming the previously suggested clade of monilophytes (‘‘Mo niliformopses’’, Kenrick and Crane, 1997) to include the whisk ferns (Psilotales) and the horsetails (Equisetales) together with all eusporangiate and leptosporangiate ferns (Pryer et al., 2001). Moreover, a sister group relationship of the Psilotales and the Ophioglossales (adders’ tongues and moonworts, respectively) was early identified based on molecular data (Pryer et al., 2001; Vangerow et al., 1999; Wolf et al., 1998) suggesting the suspicious lack of elaborate fronds to be a secondary morphological reduction in the former. Accordingly, recent taxonomic treatments have suggested to classify Ophioglossales and Psilotales jointly on higher

http://dx.doi.org/10.1016/j.ympev.2015.05.008 1055-7903/Ó 2015 Published by Elsevier Inc.

Please cite this article in press as: Knie, N., et al. Horsetails are the sister group to all other monilophytes and Marattiales are sister to leptosporangiate ferns. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.05.008

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taxonomic ranks, for example as a class Psilotopsida (Smith et al., 2006) or a subclass Ophioglossidae (Christenhusz and Chase, 2014), respectively. Monilophytes are the sister clade of seed plants and together these two large vascular plant clades make up the clade of ‘‘Euphyllophytes’’ separating them from the microphyllous lycophytes. These insights have been confirmed with independent molecular studies on the backbone phylogeny of land plants (e.g. Qiu et al., 2006). However, the radiation of the early monilophyte lineages has remained unclear and left the branching order of the four prime monilophyte lineages Psilotopsida (or Ophioglossidae), Equisetales, Marattiales and the clade of leptosporangiate ferns as an unresolved polytomy. Obviously, we need further and more informative molecular data sets to resolve this crucial issue in understanding the diversification of the most ancient lineages of extant euphyllophytes.

Chloroplast loci have been the prime choices for molecular phylogenetic studies among land plants for obvious reasons. Chloroplast genomes are highly conserved and stoichiometrically dominating in total plant DNA preparations allowing for straightforward primer design and PCR amplification approaches covering wide taxon ranges. Plant mitochondrial genomes in contrast are structurally much more variable. On the other hand, exactly this mtDNA variability may contain interesting cladistic information. The now widely accepted placement of hornworts as the sister clade to tracheophytes has been suggested early on the basis of a characteristic shared mitochondrial intron in the nad5 gene (Groth-Malonek et al., 2005). In contrast to chloroplasts, mitochondrial intron patterns diverge strongly between early plant clades and contribute phylogenetic information (Pruchner et al., 2002, 2001; Qiu et al., 1998). Similarly, plant mitochondrial intron sequences (Volkmar and Knoop, 2010; Volkmar et al., 2012;

A – nad2 gene i830g2 i156g2

i542g2

i709g2

i1282g2

100 bp

B – nad2i542g2

EBS1

III

A CU AA A C C C 8 UU G CAA CGC GU CC GC UG UA U A UA A AU U C CC CGC A CG AU AU GU AA G U C C CU U A GA C C IV 8 C GGCC* UC A UG A C* A II 1622 CG GC CG EBS2 ACU A AGG U C A CA CG CC A A A G AU A A CAC ACUU CC U C GU GC A CU UACG A G C G 8 GUG UGAGGC G A A U C CC CG A A G AA G G CGUU G A G A U A GGCU A GCGUGGGCUC AA C A C A G G G G γ G G G G C C G G U G C A U AG G C A U A GG AUGC C C A CG CC *C A G A C C* 7 A G U G G UUAU G G AA GCCAG AGCCGUAU C UAUCG *CG CGGCC UUGGCAUG CA AUAGC CA A U *A Ic 41 7 A G UC C G C C A C C G C UA UG AGU A GA G C C G U G C G G γ´ UAGG A 16 G A UC U A G U C A *CA C G G UA U A A CA A C GGG A C * C U G C GA U UA U C Ia * G C U G CC 37 A G CUC U G U U G C C UA C U C Ib AU U G U IBS1 U A A VI G U G C 3`Exon U G U 5`- Exon G C C* 79 IBS2 C* A U

V

Fig. 1. (A) The mitochondrial nad2 gene in seed plants contains four group II introns (nad2i156g2, nad2i542g2, nad2i709g2 and nad2i1282g2), which are differentially conserved among bryophytes and lycophytes with the latter additionally featuring intron nad2i830g2 (Pruchner et al., 2002). Owing to genomic disruption of intron nad2i542g2, trans-splicing is necessary for the maturation of the nad2 mRNA in seed plants. Targeting a PCR amplicon encompassing nad2i542g2 now identifies this intron as universally conserved in the cis-splicing state in monilophytes. Arrows indicate the positions of the PCR primers used in this study. The scale bar indicates 100 base pairs (bp). (B) Secondary structure modeling of the monilophyte nad2i542g2 intron of Gleichenia dicarpa identifies all features typically conserved in group II introns. The six intron domains are labelled with Roman numerals (I–VI) and tertiary interaction sites are named with Greek letters (Michel and Ferat, 1995). EBS: exon binding site, IBS: intron binding site. The branch point adenosines for lariat formation is encircled. Numbers indicate unpaired nucleotides. Potential sites of RNA editing are marked with asterisks.

Please cite this article in press as: Knie, N., et al. Horsetails are the sister group to all other monilophytes and Marattiales are sister to leptosporangiate ferns. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.05.008

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Wahrmund et al., 2010), mtDNA rearrangements (Wahrmund et al., 2008), or mitochondrial gene migrations to the nucleus (Groth-Malonek et al., 2007) have already proven to carry interesting phylogenetic information for bryophyte evolution. As of today, however, the sampling of mitochondrial loci is low for monilophytes, simply related to the fact that not a single complete monilophyte mitochondrial DNA genome sequence is currently available. Work on the backbone phylogeny of ferns is essentially restricted to an early study on the nad5 gene (Vangerow et al., 1999) and to a study on the atp1 gene

(Wikström and Pryer, 2005). Interestingly, both studies reported on novel introns (nad5i1242g2 and atp1i361g2, respectively) lacking counterparts in bryophytes or seed plants and both studies reported likely losses of these introns among individual monilophyte lineages. We here report on the investigation of two other mitochondrial loci, nad2 and rpl2, both chosen for the presence of introns conserved among seed plants: nad2i542g2 and rpl2i846g2. Our interest in the mitochondrial nad2 gene originated from the fact that its continuity is disrupted not only in angiosperms (Binder et al.,

A – rpl2 gene i28g2

i846g2

100 bp

B – rpl2i846g2

EBS1

(26) G AG U G UAUG EBS2 GU (15) U A UC CG G Azolla:+28 AU U GC GU GU UA CG GC C UAGCGGGA (19-23) C C GU G G A G A GCA G GCGA G C G G G U C G U CUGGG A GCCAA G GC U G CGGUU A A ACACGA C III A A II C (16-19) A GUGCU A A C CG U C UG UU (28-32) (33-38) G C G GC IV GA GC AU A C C (130-920) GC 13 UA Psilotales: 13 GC +18 (17-19) UG Ophioglossum: 15 GC 126 C GU GC AU G G AU +90 11 A C° A G C GC U A G C G GG CG (23) °C A GG C AUG AUG CC A A C U G AU G GG G A °C A A UU GC CG AG UG C U U GC U C G AU UAG UGA C C UA U (57-64) G G CG C UA GC γ C C A G Ic U G 58 A CG A C A G UA A C G C (57-61) A AAGGCCCGGG GCGGAG AGCGGA GAGCCGUAU V C ° A UUGCCC* CGCUUU CTTGGCACGUA G A GUUCCGGGCCC (34-40) C U U G ° A U GU 16 C U G CU A A CGC (16-20) A γ´ GAG U C AC A GC G G U C GG GA U U U G C U * G G U U C** CG A C AA C C UA C G UAA A C C G U AG A G CAAGCC C A* AGCG A CA G A C G C U U GC A UCCGG 16 U C A G GU G AG IBS1 CG ° C U U UAG A CG U A GCU Ib CG CG C U (19) U U U (18-21) Ia VI G C A A C 3`- Exon (25-54) A C A AC C U (34-35) C A IBS2 A U 5`- Exon G C Fig. 2. (A) The plant mitochondrial rpl2 gene features a conserved group II intron rpl2i846g2 in tracheophytes approximately in the middle of the reading frame. Intron rpl2i846g2 is between ca. 1400 and 1900 bp in seed plants. An unrelated upstream intron rpl2i28g2 is present in liverworts. The mitochondrial rpl2 gene is absent in hornworts and in the lycophytes Isoetes engelmannii and Selaginella moellendorffii, likely owing to EGT to the nucleus (Knoop, 2013). The rpl2 amplicon investigated in this study covers the largest part of the reading frame and identified several indel regions in the flanking exons (light shading, only Indels > 3 amino acids are shown). The position of the phylogenetically interesting indel described in Section 3.4 is shown with a black arrowhead. The scale bar indicates 100 base pairs (bp). (B) A secondary structure model for rpl2i846g2 is shown for the Gleichenia dicarpa rpl2i846g2 analogous to Fig. 1B. The intron is highly conserved in all monilophytes with differences essentially restricted to unpaired (loop) structures. Numbers in brackets represent the total lengths of the respective homologous structures in all investigated monilophyte introns. All Insertions greater than four nucleotides are indicated. A–C mismatches in stem structures of the Gleichenia dicarpa intron labelled with the degree sign (°) may be subject to RNA editing and correspond to A–U or G–C base pairs in other ferns.

Please cite this article in press as: Knie, N., et al. Horsetails are the sister group to all other monilophytes and Marattiales are sister to leptosporangiate ferns. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.05.008

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Table 4). This essentially affects sequence insertions in the most variable loop regions (especially in domain IV) of the mitochondrial introns shared by only few taxa. Given the general absence of intron atp1i361g2 in the basal monilophyte groups (Equisetales, Ophioglossales, and Psilotales) that were in the focus of our study, only the atp1 coding regions were included in the phylogenetic analysis of the concatenated alignment. Phylogenetic trees were obtained by the maximum likelihood method using the GTR + C + I substitution model (Rodríguez et al., 1990), which was proposed after model tests both using the Bayesian Information Criterion (BIC, Schwarz, 1978) and the corrected Akaike Information Criterion (AICc, Burnham and Anderson, 2002). Model testing was performed using the model test function implemented in MEGA 5.05. Tree searches were run in parallel (see Table 2) with MEGA, RaxML (Stamatakis, 2014), PhyML (Dereeper et al., 2008; Guindon et al., 2010), and IQ-Tree (Nguyen et al., 2014). Node support was determined with bootstrapping using 1000 alignment pseudoreplicates. The same settings were used to determine phylogenetic trees from all nine individual loci. The individual bootstrap support results are documented in Table 2.

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1992) but also in gymnosperms (Chaw et al., 2008), likely owing to a recombination event disrupting its group II intron nad2i542g2 into a trans-splicing status early in the spermatophyte stem lineage (Fig. 1). Cis-splicing orthologues of nad2i542g2 were identified in the ferns Asplenium nidus and jk (Malek and Knoop, 1998; Malek et al., 1997) and subsequently also in the lycophytes Isoetes engelmannii (Grewe et al., 2009) and Selaginella moellendorffii (Hecht et al., 2011) clearly demonstrating the cis-arrangement of nad2i542g2 to be the evolutionary ancestral, plesiomorphic state as expected. Like nad2, the plant mitochondrial rpl2 gene (Fig. 2) has gained a group II intron, rpl2i846g2, in the very early tracheophyte evolution as documented by its presence in the lycophyte Huperzia squarrosa (Liu et al., 2012) and in seed plants. A loss of rpl2i846g2 has been reported for monkeyflower, Mimulus guttatus (Mower et al., 2012). Several angiosperms, the lycophytes Isoetes engelmannii and Selaginella moellendorffii, and hornworts have lost the rpl2 gene from their mtDNAs altogether, likely owing to endosymbiotic gene transfer (EGT) to the nucleus, as frequently observed for genes encoding ribosomal proteins (Adams et al., 2002; Adams and Palmer, 2003). Both introns, nad2i542g2 and rpl2i846g2, are present in a wide sampling of fern taxa chosen to address the open issue of early monilophyte phylogeny. An indel in the downstream rpl2 exon supports a molecular phylogeny obtained from a 9-gene organellar data set integrating the two novel loci investigated here.

3. Results

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2. Materials and methods

3.1. The nad2 gene

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158

2.1. Plant material and molecular work

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159

3.2. The rpl2 gene

232

179

Monilophyte material was from the Botanical Garden of the University of Ulm (see Vangerow et al., 1999) or from the University Bonn Botanical Garden (Suppl. Table 1). We sampled at least two representatives for each monilophyte order (see Table 1, Suppl. Table 1 and Fig. 4). Nucleic acids were isolated using the CTAB method (Doyle and Doyle, 1990) followed by RNA digestion with RNase A (Thermo Scientific/Fermentas). The organellar loci of interest were amplified by Touchdown-PCR. Initial annealing temperatures started at 55 °C or 45 °C and were lowered to 45 °C or 42 °C, respectively. Elongation time varied between 2 min 30 s and 3 min 30 s. PCR primer sequences are listed in supplementary Table 2. PCR-products were separated with a gel electrophoresis using 0.8% agarose gels, recovered with the NucleoSpin Extract II Kit (Macherey Nagel) and cloned into the pGEM-T Easy Vector (Promega). Sequencing of plasmids was performed by Macrogen Europe (Amsterdam, Netherlands) or GATC Biotech AG (Konstanz, Germany). Completely new data sets were created for nad2 and rpl2 and several taxon sampling gaps were filled for previously investigated loci atp1 (21 new accessions), nad5 (13 new accessions), and matK (6 new accessions). All new sequences obtained for this study were submitted to GenBank (Table 1).

We investigated a wide sampling of monilophyte taxa (Table 1 and Suppl. Table 1) with an amplicon targeting the nad2i542g2 insertion site and obtained PCR amplification products of highly variable sizes. The PCR products were cloned and sequenced and consistently revealed correct targeting of the expected nad2 amplicon and conservation of nad2i542g2 in its ancestral cis-arranged status in the monilophyte taxa investigated. All essential structural elements contributing to the canonical six-domain core group II intron structure (Michel et al., 2009) were conserved (Fig. 1), but high variability of the non-conserved looped-out regions (mainly of domain IV) caused significant overall intron length variability. The nad2i542g2 homologues of monilophytes ranged in length variability from 1347 bp in Marsilea hirsuta to more than 5 kbp in Psilotum nudum. In contrast, the flanking nad2 exon sequences were highly conserved except for the expected requirement of pyrimidine exchanging RNA editing known to exist in fern organellar transcriptomes (Vangerow et al., 1999; Wolf et al., 2004).

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2.2. Sequence handling and phylogenetic analyses

181

Sequence handling, alignment and alignment inference were done using the alignment feature of MEGA 5.05 (Tamura et al., 2011). Automatic sequence alignment was done using the MUSCLE algorithm as implemented in MEGA with additional manual adjustment. The concatenated full alignment and the resulting phylogenetic tree (Fig. 4) were submitted to TreeBase under accession number S17521 (http://purl.org/phylo/treebase/phylows/ study/TB2:S17521). Gaps and missing data in the alignment were treated with the partial deletion option and the site coverage cut-off was set to 12%. With these settings every character that was not present in at least five taxa was excluded (Suppl.

In monilophytes, rpl2 including intron rpl2i846g2 turned out to be near-universally conserved with sizes ranging from 724 to 788 bp except for size extensions in the Ophioglossaceae (e.g. up to 1655 bp in Ophioglossum petiolatum). A unique exception within monilophytes is the loss of rpl2i846g2 in Equisetum bogotense (Suppl. Table 3). The secondary structure of rpl2i846g2 folds into the conserved structural stems branching off the central group II intron core and looped-out regions with high variability across different taxa (Fig. 2B). In contrast to core respiratory chain subunits like nad2, the rpl2 reading frame features several indels in the coding region. Most notably, rpl2 includes a variability in the coding region with an insert of nearly 200 amino acids in the upstream exon in the Equisetales and a phylogenetically informative indel in the downstream exon (Fig. 2A), as we will discuss below.

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Please cite this article in press as: Knie, N., et al. Horsetails are the sister group to all other monilophytes and Marattiales are sister to leptosporangiate ferns. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.05.008

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Table 1 Monilophyte and outgroup taxon sampling. Database accessions are given for the sequences of the nine organellar loci investigated for phylogenetic tree constructions. Accession numbers in bold indicate new sequences obtained in this study, ‘‘n.d.’’ indicates that no data are available. The respective epithet is indicated below the accession number when originating from a different species of the same genus. For the jungermanniid liverwort outgroup taxon, the chloroplast sequences of Pellia endiviifolia (JX827163) and the mitochondrial sequences of Pleurozia purpurea (FJ999996) were combined. Monilophytes

atpA

atpB

matK

rbcL

rps4

atp1

nad2

nad5

rpl2

Adiantum capillusveneris Anemia mexicana Anemia phyllitidis Angiopteris madagascariensis Asplenium nidus

AY178864

AY178864

AY178864

AY178864

AY178864

DQ110142

n.d. AY612687 EF463485 A. evecta AY612688

U05603 AJ303391 EF463239 A. evecta U05907

AY612649 n.d. DQ821119 A. evecta JQ724305 A. normale AY612651

KJ944570 KJ944571 KJ944577

KJ944516 KJ944517 KJ944524

KJ944566

Y07910

AJ130738

KJ944513

AY612689

n.d. JF303905 DQ821119 A. evecta JF303923 A. tripteropus n.d.

KJ944536 A. peruvianum n.d. KJ944537 KJ944542

KP757852

n.d. AM176474 DQ390544 A. evecta DQ390545 A. theciferum DQ390547 A. pinnata EF452080 B. occidentale DQ390549

KP757849 A. caudatum n.d. n.d. KJ944555

KJ944561

KJ944544

AJ130739

KJ944507

JF832157 B. spicant U93826

JF303938 B. orientale KP757848

KP757842

KP757850

KP757853

KJ944559

KJ944530

AF313553 C. poeppigii U93829 JN968380

JF303907 C. podophylla HM021802 JN968380

KJ944548

KJ944533

KJ944511

KJ944547 n.d.

Equisetum bogotense n.d. Equisetum hyemale KC117177 Gleichenia dicarpa DQ390562

KC893871 KC117177 AF313550

AJ583678 KC117177 AF313599

n.d. KJ944560 KJ944550

AJ130745 AJ130749 E. telmateia KP757858 AJ130748 KJ944538

KJ944510 KJ944532 E. hyem. jap. KP757855 KJ944531 KJ944518

Helminthostachys zeylanica Hymenophyllum hirsutum Leptopteris superba

n.d.

DQ646095

KP757846 AY226139 KC117177 KC117177 HM021798 G. AF313584 japonica KP757847 EU352293

DQ110159 B. oneidense KP757843 C. contaminans AJ548853 KJ944581 E. giganteum KP757844 DQ646213 KJ944572

AJ130740 B. brasiliense AJ130742

AY870436

DQ110149

n.d.

KP757856

KJ944529

DQ390565

AF313538

DQ390566 L. hymenophyll. KF225593

AY612699 L. wilkesiana KF225593

JF303898 H. polyanthos KM925081

AB496599 H. sp.Schuettpelz EF588758 L. fraseri KF225593

DQ646226 H. sp.Qiu KJ944576

KJ944552 H. triangulare KJ944554

KJ944539 H. triangulare KJ944541

KJ944521 H. trichomanoides KJ944523

KP757845

n.d.

n.d.

DQ390567 M. alata EF463786

AF313546 M. KM925080 attenuata AF313551 HM021801 M. mutica AY612704 JF303903 U93825 O. HF585134 reticulatum HM535629 HM535629 AP004638 AP004638 AF313552 JF303906

AF313581 M. attenuata EU352306

EU439107

KJ944578

KJ944556

AJ130750 L. circinatum KJ944543

AF313608 M. mutica AY612666 AF313594 O. reticulatum HM535629 AP004638 AF313600

KJ944563

AJ131136

KJ944573 KJ944580

KJ944546 M. hirsuta KJ944551 KJ944558

AJ548852 AJ548873 KJ944562

KP757851 KJ944557 KJ944545

AY612709

JF303899

AY612683

n.d.

n.d.

AJ131137 AJ131139 O. vulgatum KP757857 AJ012794 AJ131147 S. auriculata n.d.

KJ944509 M. hirsuta KJ944519 KJ944528 O. pedunculosum KP757854 KJ944526 KJ944508

U93823 T. tannensis AY612714 AY612715

KJ569699

EU352294

KM925082 JF303901

Azolla filiculoides Blechnum gibbum Botrychium lunaria Cyathea dealbata Dicksonia antarctica Equisetum arvense

Lygodium japonicum Marattia laevis Marsileadrummondii Matonia pectinata Ophioglossum petiolatum Pteridium aquilinum Psilotum nudum Salvinia molesta Schizaeadichotoma Tmesipteris elongata

EF463640 C. poeppigii AM176442 JN968380

DQ390568 DQ390571 O. reticulatum HM535629 AP004638 DQ390576 S. cucullata EF463861 DQ390579 T. obliqua DQ390580 DQ390581

KF225593

U24185 A. caroliniana AB040545 B. brasiliense L13474 B. biternatum AF313585 C. poeppigii U05919 JN968380

AF275645 AY612678 L. wilkesiana KF225593

EU352307 AF313582 O. reticulatum HM535629 AP004638 EU269668

JN168071 B. eburneum AY870429 AF313601 C. poeppigii AF313596 JN968380

KJ944525

KJ944579

n.d.

n.d.

KJ944527

AY612686 AF275650

DQ390583 S. laevigata AF313593 T. obliqua EF588776 AF537123

n.d.

KJ944575 KJ944574

KJ944553 n.d.

KJ944540 AJ131145

KJ944522 KJ944520

Todea barbara Vandenboschia radicans Woodwardia radicans EF463623 W. virginica

EF463359 W. JF303937 W. virginica japonica

AY137667

AF533865

KJ944567

KJ944549

KJ944535

KJ944514

Outgroups Huperzia lucidula

AY660566

AY660566

AY660566

AY660566

AY660566

Isoetes flaccida

GU191333

GU191333

GU191333

GU191333

GU191333

KC285889

KC285889

KC285889

KC285889

KC285889

JQ002659 H. squarrosa FJ536259 I. engelmannii EU660574

JQ002659 H. squarrosa FJ536259 I. engelmannii EU660574

JQ002659 H. squarrosa Not present

Nothoceros aenigmaticus Anthoceros formosae

JQ002659 H. squarrosa FJ536259 I. engelmannii EU660574

AB086179

AB086179

AB086179

AB086179

AB086179

n.d.

AP005672 KJ817846 AY522330 AP009339 X04465

AP005672 KJ817846 AY522330 AP009339 X04465

AP005672 KJ817846 AY522330 AP009339 X04465

AP005672 KJ817846 AY522330 AP009339 X04465

AB251495 KJ817845 BA000029 AP009381 M68929

DQ845731 A. agrestis AB251495 KJ817845 BA000029 AP009381 M68929

n.d.

AP005672 KJ817846 AY522330 AP009339 X04465

AJ409117 A. agrestis AB251495 KJ817845 BA000029 AP009381 M68929

JX827163

JX827163

JX827163

JX827163

JX827163

FJ999996

FJ999996

FJ999996

FJ999996

Physcomitrella patens Tetraphis pellucida Oryzasativa Cycas taitungensis Marchantia polymorpha Pellia endiviifolia/ Pleurozia purpurea

Not present

AB251495 KJ817845 BA000029 AP009381 M68929

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Table 2 Comparison of contributions from 9 organellar loci for key nodes of monilophyte phylogeny. Bootstrap values are indicated and given in bold where at least 90% for the individual loci and for the fused data determined with MEGA (ME5), PhyML (PML) and IQ-Tree (IQ), respectively. Clade acronyms are SCP (Salviniales, Cyatheales, Polypodiales), SSCP (Schizaeales, Salviniales, Cyatheales, Poly po di ales), GMH (Gleicheniales (including Matoniaceae), Hymenophyllales), GHSSCP (Gleicheniales, Hymenophyllales, Schizaeales, Salviniales, Cyatheales, Polypodiales), OP (Ophioglossales, Psilotales) and HT (Hornworts, Tracheophytes), respectively. Lack of data to support a clade is indicated by ‘‘n.d.’’. Two hyphens indicate that the respective clade is not identified in the respective single gene analysis, but without bootstrap support (>70%) for alternative topologies. Exceptions are: A Gleichenia-Hymenophyllales clade with 87% support in the nad5 analysisa, a spermatophyte-lycophyte linkage with 90% support in the atpB analysisb and supports of 80% and 94% linking Ophioglossum instead of Botrychium to Helminthostachys in the atpB and matK analysesc, respectively. Node

Single loci analyses

Fused 9-gene matrix

atpA

atpB

matK

rbcL

rps4

atp1

nad2

nad5

rpl2

ME5

PML

IQ

(Blechnum, Woodwardia) (Asplenium, Blechnum, Woodwardia) Polypodiales without Adiantum Polypodiales Cyatheales (Cyatheales, Polypodiales) (Azolla, Salvinia) Salviniales SCP clade (‘‘core leptosporangiates’’) Schizaeales (Anemia, Schizaea) SSCP clade Gleicheniales incl. Matoniaceae (GM) Hymenophyllales GMH clade GHSSCP clade Osmundales Leptosporangiates Marattiales (Marattiales, Leptosporangiates) Psilotales Ophioglossales (Botrychium, Helminthostachys) OP clade (OP, Marattiales, Leptosporangiates) Monilophytes Euphyllophytes Tracheophytes HT clade Hornworts Mosses Liverworts

98 99 83 99 93 89 100 89 98 99 99 94 42 100 – 91 100 100 100 48 100 100 82 88 44 81 70 80 76 100 99 98

92 99 99 100 93 64 100 98 99 95 99 71 87 99 – 99 100 99 100 36 100 100 –c 91 37 77 –b 82 68 100 88 97

99 100 52 100 77 85 n.d. 78 100 99 100 96 47 100 42 100 100 100 100 68 100 100 –c 100 96 94 95 79 92 100 100 100

92 84 77 99 97 69 100 99 99 99 100 88 89 99 67 96 100 84 100 – 100 100 60 89 – 50 – 50 73 100 68 38

62 90 – 97 76 – 100 92 100 70 98 35 – 99 – 92 99 99 100 91 100 94 41 96 – 99 94 – 52 100 94 100

– – 83 – – – 100 – 98 99 n.d. 51 – 98 – 80 99 87 100 – 100 99 92 62 – 85 82 – – n.d. 100 100

98 99 – 84 – – 94 – 83 n.d. n.d. n.d. – n.d. 12 37 99 30 81 – n.d. 85 n.d. – – 13 – – – 78 77 49

99 94 65 89 – – 100 59 100 100 n.d. 30 –a 94 78 34 100 82 100 – n.d. 99 82 95 15 83 22 – 80 100 100 100

– – – 98 79 72 100 99 99 100 n.d. 96 45 95 – 100 99 99 100 – 100 99 74 90 – 100 99 99 n.d. n.d. 100 100

100 100 100 100 100 92 100 99 100 100 100 100 83 100 – 100 100 100 100 89 100 100 99 100 99 100 96 96 99 100 100 100

100 100 97 100 100 96 100 100 100 100 100 100 – 100 – 100 100 100 100 100 100 100 55 100 95 100 100 100 97 100 100 100

100 100 96 100 100 98 100 100 100 100 100 100 – 100 – 100 100 100 100 98 100 100 62 100 95 100 100 100 97 100 100 100

Number of nodes scoring > 90

19

21

22

15

20

11

4

13

19

29

29

29

248

3.3. Further organellar loci investigated

249

We incorporated the new monilophyte nad2 and rpl2 data sets into a nine-gene matrix together with previously established monilophyte data for the chloroplast loci atpA, atpB, matK, rbcL and rps4 and for the mitochondrial atp1 and nad5 locus (Table 1 and Suppl. Table 1). To that end, we also filled taxon sampling gaps in atp1 (21 new sequence accessions), nad5 (13 new sequence accessions), and matK (6 new sequence accessions). Whereas the five chloroplast protein coding regions are devoid of introns, both nad5 and atp1 feature monilophyte-specific introns (Fig. 3 and Suppl. Table 3). The nad5 amplicon contains group II intron nad5i1242g2 conserved in the lycophyte mtDNAs and in most monilophytes, but this is absent in Anemia phyllitidis, Equisetum hyemale, E. telmateia, E. bogotense, Ophioglossum vulgatum and O. petiolatum (Vangerow et al., 1999 and this study). However, nad5i1242g2 is present in Botrychium, Helminthostachys and Psilotum, strongly suggesting independent losses in Anemia, Equisetum and Ophioglossum (Suppl. Table 3). The atp1 gene carries an intron (atp1i361g2) in most ferns without orthologues outside of monilophytes and an as yet unclear history of gain(s) and losses (Rothfels and Schuettpelz, 2014; Wikström and Pryer, 2005). The 21 new atp1 accessions obtained in the course of our study (Table 1 and Suppl. Table 3) confirm that intron atp1i361g2 is absent in Equisetales and Ophioglossidae and,

250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272

in the light of the phylogeny presented here (Fig. 4), are congruent with its gain in the common ancestor of Marattiales and leptosporangiate ferns. The absence of atpi361g2 in a subset of Pteridaceae (Rothfels and Schuettpelz, 2014) and Danaea elliptica (Wikström and Pryer, 2005) likely reflect independent losses of this intron.

273

3.4. Phylogenetic studies

278

We used the nine organellar loci jointly for phylogenetic tree reconstruction (Fig. 4 and Suppl. Table 4) but also individually to evaluate their individual support for critical nodes and to check for potential conflict between them (Table 2). We here only consider node supports of at least 70% in maximum likelihood bootstrapping analyses to be of sufficient significance for further consideration. Individually, the nine loci show weaker or stronger support for certain nodes but only very few cases for potentially conflicting topologies at this level of significance (Table 2). The extended nine-locus analysis results in a well-resolved phylogeny (Fig. 4) without conflict to the individual single gene analyses. In our phylogenetic study, the horsetails (Equisetales) are the sister group to all other extant monilophyte lineages, followed by a well-supported joint Ophioglossales/Psilotales clade, itself sister to a clade with Marattiales as sister clade to the leptosporangiate ferns (Fig. 4). Within the latter, the Osmundales branch off first as sister to all other leptosporangiates. Still unclear is the following split of Hymenophyllales, which may or may not

279

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i1050g2 i1019g2 i805g2

i361g2

i989g2

i1127g2

atp1 100 bp

i1477g2 i1455g2

i230g2

i753g1

i1242g2

i1872g2

nad5 100 bp Fig. 3. The two mitochondrial loci previously investigated among monilophytes (Rothfels and Schuettpelz, 2014; Vangerow et al., 1999; Wikström and Pryer, 2005), atp1 and nad5, revealed monilophyte-specific group II intron atp1i361g2 and group II intron nad5i1242g2, which is shared with lycophytes (bold). The atp1 gene is devoid of introns in spermatophytes and lycophytes but shows different patterns of introns in bryophytes (italics): atp1i805g2 and atp1i1019g2 in hornworts, atp1i989g2 in liverworts, atp1i1050g2 in liverworts and hornworts and atp1i1127g2 in mosses. The nad5 gene carries four group II introns in seed plants, two of which are trans-splicing (stippled lines) with differential conservation among bryophytes. Mosses and liverworts feature a highly conserved group I intron, nad5i753g1. The scale bars indicate 100 base pairs (bp).

297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324

be included in a joint assembly together with Gleicheniales (includingMatoniales/-aceae) in a ‘‘GMH clade’’ that has been suggested in previous studies (Knoop, 2005; Pryer et al., 2004). Despite exceptionally long branches and as in previous studies, the Schizaeales are clearly identified as the sister clade to the joint assembly of the water ferns (Salviniales), the tree ferns (Cyatheales) and the Polypodiales comprising most of the extant fern diversity (Fig. 4). The individual loci support monophyly of Cyatheales and Salviniales to different degrees (Table 2). The evolutionary scenario with tree ferns as a sister clade to the Polypodiales, as reflected in our multigene tree (Fig. 4), is the most likely one, mainly supported by chloroplast loci atpA and matK and the here investigated mitochondrial rpl2 locus plus minor support from atpB and rbcL while the other four loci do not offer any significant support here (Table 2). The deepest dichotomy in the phylogeny of monilophytes, now identified as a sister clade relationship of horsetails to all ferns, is independently supported by an indel in the rpl2 coding region (Fig. 5). The Equisetum spp. rpl2 downstream exon sequences clearly reflect a plesiomorphic state shared with bryophytes, lycophytes and spermatophytes whereas the Ophioglossales, Psilotales, Marattiales, and the leptosporangiate fern rpl2 sequences feature a derived synapomorphic state with a sequence deletion. Hence, as in the case of rps4 for the monilophyte clade as a whole (Pryer et al., 2001; Schneider et al., 2004), a coding sequence indel as a molecular synapomorphy is an attractive independent support, in this case for a clade of monilophytes excluding Equisetales.

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4. Discussion

326

Early molecular phylogenetic studies with low taxon sampling have already indicated the inclusion of horsetails and whisk ferns in one clade together with ‘‘true’’ ferns (Duff and Nickrent, 1999; Nickrent et al., 2000), even before the seminal molecular phylogenetic study (Pryer et al., 2001) confirming the

327 328 329 330

Monilophyte/Moniliformopses concept (Kenrick and Crane, 1997). However, taxonomic treatments of monilophytes have hitherto avoided a clear systematic assignment of horsetails to any other fern group, reflecting their ambiguous or even apparently contradictory placement in alternative phylogenies. This is also summarized in recent schematic phylogenetic trees leaving the lineages of Ophioglossopsida, Equisetales, Marattiales and leptosporangiate ferns as an unresolved tetratomy (Sessa et al., 2014; Wolf et al., 2011, 2010). For example, Psilotopsida/Ophioglossidae were considered as the sister lineage to all other ferns with an unclear relationship of Equisetales, Marattiales or both jointly as sister to the leptosporangiate taxa (Lehtonen, 2011; Pryer et al., 2009; Schuettpelz et al., 2006; Smith et al., 2006). Essentially the same result was obtained from a multigene analysis of chloroplast loci (Pryer et al., 2004, 2001). In contrast, a study relying on complete plastid genomes found horsetails to be sister of Psilotopsida (Grewe et al., 2013), similar to other studies using only moderate monilophyte taxon sampling (Gao et al., 2013; Ruhfel et al., 2014; Zhong et al., 2014). Interestingly, while still relying on a comparative small taxon sampling, the very recent addition of further fern cp genomes from key leptosporangiate orders Osmundales, Gleicheniales and Schizaeales resulted in a phylogenetic tree without any contradiction to ours (Kim et al., 2014). This is similarly true for a matK-based monilophyte study (Kuo et al., 2011), in full agreement with our extended and comparative phylogenies and previous observations (Rothfels et al., 2012) assigning matK a top position as a phylogenetically informative locus (Table 2). Moreover, although lacking convincing support, the results of an earlier chloroplast multigene study (Rai and Graham, 2010) are also without conflict to our observations. Likewise, an early study exploring the nuclear phytochrome gene family suggested that ‘‘Equisetum arose prior to Psilotum’’ (Kolukisaoglu et al., 1995). A recent pioneering phylotranscriptomic study also found Equisetum basal among monilophytes (Wickett et al., 2014). However, it must be noted that this study, at low monilophyte taxon sampling, also identified Marattiales as sister to Ophioglossidae. In addition,

Please cite this article in press as: Knie, N., et al. Horsetails are the sister group to all other monilophytes and Marattiales are sister to leptosporangiate ferns. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.05.008

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Fig. 4. Maximum likelihood phylogenetic tree of the nine-gene (atpA-atpB-matK-rbcL-rps4-atp1-nad2-nad5-rpl2) data set. Bootstrap values from 1000 pseudoreplicates are shown where exceeding 70. The tree shown is obtained with MEGA5 but independently confirmed with PhyML and IQ-TREE (see Table 2).

Fig. 5. A coding sequence indel in rpl2 comes as independent support for a clade of monilophytes excluding horsetails (gray shading). For clarity, only selected representative taxa are shown. Equisetales share a plesiomorphic state with seed plants, lycophytes and bryophytes, a sequence motif with the majority consensus sequence YHQPAQxxxxHTDxLRFQDHxVR, which is absent in Ophioglossales, Psilotales, Marattiales and leptosporangiate ferns.

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depending on the type of analysis, that study identified bryophytes as monophyletic or hornworts as sister to all other land plants in contradiction to other studies recently converging on a hornworts-tracheophyte (HT) clade.

Like in previous studies, the monophyly of leptosporangiate ferns with Osmundales branching off first and the sister grouping of Ophioglossales and Psilotales are found well-supported in our study. Other open issues of monilophyte phylogeny concern the

Please cite this article in press as: Knie, N., et al. Horsetails are the sister group to all other monilophytes and Marattiales are sister to leptosporangiate ferns. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.05.008

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splits separating (i) Polypodiales, Cyatheales and Salviniales and (ii) the Gleicheniales, Hymenophyllales and the remaining leptosporangiates including Schizaeales. The branching of Cyatheales and Salviniales in the fern crown group of ‘‘core leptosporangiates’’ (Pryer et al., 2004) is particularly affected by unequal rates of evolution (very likely mainly resulting from highly divergent generation times) with extremely short branches in the tree ferns (Korall et al., 2010; Zhong et al., 2014) as opposed to extremely long branches in the water ferns. We here find the sister group relationship of Polypodiales and Cyatheales well confirmed. In contrast, we conclude that the issue of a possible ‘‘GMH clade’’ comprising Gleicheniales, Matoniaceae and Hymenophyllales needs further attention and denser taxon sampling (e. g. including Dipteridaceae) in the future. While molecular phylogenies, especially when encompassing numerous loci may be ultimately hard to grasp in a cladistic sense, certain molecular features may serve well as convincing morphological synapomorphies to define a clade. A case in point is the 3-codon insertion in the chloroplast rps4 gene supporting the monilophyte clade as a whole (Pryer et al., 2004, 2001). In other cases, significant changes in the monilophyte organellar genomes such as gene or intron losses are inconclusive (Kim et al., 2014; Wikström and Pryer, 2005). The isolated position of Equisetum bogotense among Equisetales in an as yet unresolved trichotomy with the subgenera Hippochaete and Equisetum both based on molecular phylogenetic (Des Marais et al., 2003) and biochemical (Xue and Fry, 2012) characters and the here observed loss of rpl2i846g2 only in E. bogotense (Suppl. Table 3) are remarkable in that sense. In contrast, we here find that the mitochondrial rpl2 gene supplies such a molecular synapomorphy separating the Equisetales from all other monilophytes, in full accordance with our multigene phylogeny. Part of the rpl2 coding region retains a plesiomorphy in Equisetales shared both with seed plants and the lycophyte outgroup but lacking in all other monilophytes. The placement of horsetails as the remaining extant representative of the large clade of mostly extinct sphenophytes is fully compatible with the fossil record (Kenrick and Crane, 1997; Taylor et al., 2009).

414

5. Conclusions

415

423

We here resolve the long enigmatic early radiation of the most ancient monilophyte lineages (Equisetales, Ophioglossidae, Marattiales and leptosporangiate ferns). A new multigene data set and an indel in the mitochondrial rpl2 locus strongly support Equisetales as the sister group to all other monilophytes. We conclude that, for the time being, the combination of adequate taxon sampling combined with careful, and often serendipitous, choice of loci is superior to phylogenomic approaches accumulating massive amounts of data for (too) few taxa in molecular phylogeny.

424

Acknowledgments

425

429

We are extremely grateful for the expert help of Bernhard Reinken managing the Bonn Botanical Garden fern collection and to Dr. Wolfram Lobin, curator of the Bonn Botanical Garden. Research reported here relied on basic funds of the University of Bonn.

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Appendix A. Supplementary material

431

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2015.05. 008.

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Please cite this article in press as: Knie, N., et al. Horsetails are the sister group to all other monilophytes and Marattiales are sister to leptosporangiate ferns. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.05.008

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