Chloroplast Phylogenomics Resolves Key Relationships in Ferns

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Chloroplast phylogenomics resolves key relationships in ferns

ARTICLE in JOURNAL OF SYSTEMATICS AND EVOLUTION · SEPTEMBER 2015 Impact Factor: 1.49 · DOI: 10.1111/jse.12180

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Chloroplast phylogenomics resolves key relationships in ferns

† † Jin-Mei Lu1 , Ning Zhang2 , Xin-Yu Du1, Jun Wen2*, and De-Zhu Li1*

1Plant Germplasm and Genomics Center, Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China 2Department of Botany, National Museum of Natural History, Smithsonian Institution, Washington DC 20013-7012, USA † These authors contributed equally to this work. *Authors for correspondence. Jun Wen. E-mail: [email protected]. Tel.: 1-202-633-4881. Fax: 1-202-786-2563. De-Zhu LI. E-mail: [email protected]. Tel./Fax: 86-871-65223503. Received 1 July 2015; Accepted 27 August 2015; Article first published online 1 September 2015

Abstract Studies on chloroplast genomes of ferns and lycophytes are relatively few in comparison with those on seed plants. Although a basic phylogenetic framework of extant ferns is available, relationships among a few key nodes remain unresolved or poorly supported. The primary objective of this study is to explore the phylogenetic utility of large chloroplast gene data in resolving difﬁcult deep nodes in ferns. We sequenced the chloroplast genomes from Cyrtomium devexiscapulae (Koidz.) Ching (eupolypod I) and Woodwardia unigemmata (Makino) Nakai (eupolypod II), and constructed the phylogeny of ferns based on both 48 genes and 64 genes. The trees based on 48 genes and 64 genes are identical in topology, differing only in support values for four nodes, three of which showed higher support values for the 48-gene dataset. Equisetum L. was resolved as the sister to the Psilotales–Ophioglossales clade, and Equisetales–Psilotales–Ophioglossales clade was sister to the clade of the leptosporangiate and marattioid ferns. The sister relationship between the tree fern clade and polypods was supported by 82% and 100% bootstrap values in the 64-gene and 48-gene trees, respectively. Within polypod ferns, Pteridaceae was sister to the clade of Dennstaedtiaceae and eupolypods with a high support value, and the relationship of Dennstaedtiaceae–eupolypods was strongly supported. With recent parallel advances in the phylogenetics of ferns using nuclear data, chloroplast phylogenomics shows great potential in providing a framework for testing the impact of reticulate evolution in the early evolution of ferns. Key words: chloroplast genome, chloroplast phylogenomics, ferns, phylogenetics, polypods.

The plant chloroplast genome (or plastome) is of a moderate 1998; Wolf et al., 2003; Roper et al., 2007; Gao et al., 2009). size and can be easily sequenced. In general, there is good co- With the advent of next-generation sequencing, 15 complete linearity among chloroplast genomes of different plant taxa, chloroplast genomes have been sequenced since 2011 (Table 1; facilitating comparative analyses (Jansen et al., 2007). The also see Banks et al., 2011; Wolf et al., 2011; Gao et al., 2013; molecular evolutionary rates of the coding and non-coding Grewe et al., 2013; Kim & Kim, 2014; Kim et al., 2014; Zhong regions of chloroplast genomes are of significant difference, et al., 2014). Moreover, five partial chloroplast genomes were and thus can provide a large degree of genetic variation for recently submitted to GenBank, and these included: Ceratop- comparative phylogenetic studies at different taxonomic teris richardii Brongn. (KM052729), Cystopteris protrusa ranks (Clegg et al., 1994). These advantages have allowed the (Weath.) Blasdell (KP136830), Dipteris conjugata Reinw. wide use of chloroplast DNA sequences in phylogenetic (KP136829), Plagiogyria formosana Nakai (KP136831), and studies of plants (Jansen et al., 2007; Moore et al., 2007, 2010; Polypodium glycyrrhiza D. C. Eaton (KP136832). Gao et al., 2010). The chloroplast genome size is generally approximately Previously, studies on whole chloroplast genomes of ferns 131–168 kb in ferns (Grewe et al., 2013; Zhong et al., 2014; and lycophytes have been relatively few in comparison with Table 1), and the genome size variation is mostly due to length those on seed plants and those studies largely focused on variation in the inverted repeat (IR) and the small single copy chloroplast genome sequencing of a single species (Wakasugi (SSC) section (Grewe et al., 2013). Although the number of et al., 1998; Wolf et al., 2003; Roper et al., 2007; Tsuji et al., genes (117–124 coding genes) and gene order are relatively 2007). However, as of June 2015, the National Center for conserved in chloroplast genomes of ferns, there are some Biotechnology Information’s database included 26 chloroplast differences among taxa (Table 1). A few studies on the genomes of ferns and five of lycophytes (Table 1; Fig. 1). Early chloroplast evolutionary genomics of ferns have been production of chloroplast genomes for ferns and lycophytes published in recent years (Gao et al., 2010, 2011, 2013; Karol involved more tedious Sanger sequencing (Wakasugi et al., et al., 2010; Wolf et al., 2011; Grewe et al., 2013; Kim et al., 2014;

Table 1 General characteristics of chloroplast genomes of ferns and selected outgroups † Taxon Reference Accession Size, GC % LSC IR SSC Coding, Coding, Non- Non- Genes Protein rRNA tRNA Intron no. bp bp % coding, coding, bp % Ferns Equisetum Karol et al., 2010 NC_014699 133 309 33.40 93 542 10 149 19 469 84 365 63.29 48 944 36.71 121 84 4 33 18 arvense L. Equisetum Kim et al., 2014 JN968380 132 726 34.00 92 961 10 144 19 477 84 465 63.64 48 261 36.36 121 84 4 33 18 arvense L. Equisetum hyemale Grewe et al., 2013 NC_020146 131 760 33.74 92 580 10 093 18 994 84 612 64.22 47 148 35.78 122 85 4 33 17 Mankyua Kim & Kim, unpublished data NC_017006 146 221 37.97 106 096 9756 20 613 81 447 55.70 64 774 44.30 123 87 4 32 18 ‡ chejuense Mankyua chejuense Kim et al., 2014 KP205433 146 225 38.00 106 099 9756 20 614 82 056 56.12 64 169 43.88 124 88 4 32 19 Ophioglossum Grewe et al., 2013 NC_020147 138 270 42.20 99 058 9775 19 662 83 529 60.41 54 741 39.59 120 84 4 32 19 californicum hools hlgnmc nferns in phylogenomics Chloroplast Psilotum nudum Wakasugi et al., 1998 NC_003386 138 829 36.03 84 617 18 954 16 304 90 274 65.03 48 555 34.97 119 84 43219 Psilotum nudum Grewe et al., 2013 KC117179 138 909 36.00 84 674 18 953 16 329 85 345 61.44 53 564 38.56 119 82 4 33 20 Tmesipteris Zhong et al., 2014 KJ569699 139 736 36.10 85 629 18 914 16 279 79 507 56.90 60 229 43.10 116 81 4 31 17 elongata P. A. Dangeard Angiopteris Zhu & Mower, unpublished NC_026300 153 596 35.50 89 708 21 676 20 536 87 132 56.73 66 464 43.27 122 86 4 32 19 angustifolia data Angiopteris evecta Roper et al., 2007 NC_008829 153 901 49.50 89 709 21 053 22 086 82 600 53.67 71 301 46.33 121 86 4 31 18 Osmundastrum Kim et al., 2014 NC_024157 142 812 40.20 100 294 10 109 22 300 80 239 56.19 62 573 43.81 121 84 4 33 21 cinnamomeum Diplopterygium Kim et al., 2014 NC_024158 151 007 40.20 99 857 14 584 21 982 84 623 56.04 66 384 43.96 119 85 4 30 20 glaucum Lygodium Gao et al., 2013 NC_022136 157 260 40.64 85 448 25 080 21 652 92 740 58.97 64 520 41.03 117 86 4 27 20 japonicum Lygodium Kim et al., 2014 KF225593 157 142 41.00 85 432 25 038 21 634 91 810 58.42 65 332 41.58 117 86 4 27 19 japonicum Marsilea crenata C. Gao et al., 2013 NC_022137 151 628 42.22 87 828 20 795 22 210 91 301 60.21 60 327 39.79 117 86 4 27 21 Presl Dicksonia Zhong et al., 2014 KJ569698 168 244 41.50 85 817 30 201 22 025 96 942 57.62 71 302 42.38 118 85 4 29 15 .Ss.Evol. Syst. J. squarrosa (Forst. f.) Sw. Alsophila spinulosa Gao et al., 2009 NC_012818 156 661 40.40 86 308 24 365 21 623 91 947 58.69 64 714 41.31 118 86 4 28 21 (Wall. ex Hook.) R. M. Tryon

3() 448 (5): 53 Pteridium Der, 2010 NC_014348 152 362 41.50 84 335 23 384 21 259 92 311 60.59 60 051 39.41 117 84 4 29 21 aquilinum subsp. aquilinum Adiantum capillus- Wolf et al., 2003 NC_004766 150 568 42.00 82 282 23 447 21 392 91 562 60.81 59 006 39.19 117 84 4 29 20 veneris –

5,2015 457, Cheilanthes Wolf et al., 2011 NC_014592 155 770 42.70 83 059 25 694 21 323 92 140 59.15 63 630 40.85 118 85 4 29 22 lindheimeri

Continued 449 .Ss.Evol. Syst. J. 450 Table 1 Continued † Taxon Reference Accession Size, GC % LSC IR SSC Coding, Coding, Non- Non- Genes Protein rRNA tRNA Intron no. bp bp % coding, coding, bp % 3() 448 (5): 53 Ceratopteris Marchant et al., KM052729 148 444 36.80 83 178 22 020 21 226 91 072 61.35 57 372 38.65 117 84 4 29 20 § richardii unpublished data Cystopteris Marchant et al., KP136830 131 837 42.70 83 429 26 671 21 708 77 547 58.82 54 290 41.18 120 84 4 32 15 § protrusa unpublished data § –

5,2015 457, Dipteris conjugata Marchant et al., KP136829 123 674 45.80 81 224 10 683 20 984 66 293 53.60 57 381 46.40 82 54 4 24 11 unpublished data Plagiogyria Marchant et al., KP136831 127 710 43.00 83 747 22 396 21 563 73 717 57.72 53 993 42.28 112 81 4 27 15 § formosana unpublished data Polypodium Marchant et al., KP136832 129 223 40.10 82 565 23 759 21 486 76 916 59.52 52 307 40.48 115 82 4 29 15 § glycyrrhiza unpublished data Woodwardia This study KT599101 153 717 43.21 82 387 24 887 21 556 92 873 60.42 60 844 39.58 117 84 4 29 20 unigemmata Cyrtomium This study KT599100 151 684 42.33 82 453 23 803 21 625 92 847 61.21 58 837 38.79 117 84 4 29 20 devexiscapulae Lycophytes Huperzia lucidula Wolf et al., 2005 NC_006861 154 373 36.20 104 088 15 314 19 657 85 456 55.36 68 917 44.64 120 87 4 29 18 (Michx.) Trevis. Isoetes flaccida A. Karol et al., 2010 NC_014675 145 303 37.90 91 862 13 118 27 205 82 986 57.11 62 317 42.89 118 82 4 32 20 Braun al. et Lu Selaginella Smith, 2009 FJ755183 143 780 51.00 83 671 12 114 35 881 79 276 55.14 64 504 44.86 94 77 4 13 10 moellendorffii Hieron. Selaginella Banks et al., 2011 HM173080 143 775 51.00 83 665 12 114 35 882 80 903 56.27 62 872 43.73 98 79 4 15 11 moellendorffii Hieron. Selaginella uncinata Tsuji et al., 2007 AB197035 144 170 54.80 77 706 12 789 40 886 79 731 55.30 64 439 44.70 99 82 4 13 11 (Desv. ex Poir.) Spring Seed Plants Ginkgo biloba L. Lin et al., 2012 AB684440 156 945 39.60 99 223 17 734 22 254 85 862 54.71 71 083 45.29 122 83 4 35 22 Arabidopsis Sato et al., 1999 NC_000932 154 478 36.30 84 170 26 264 17 780 91 622 59.31 62 856 40.69 112 79 3 30 6 thaliana (L.) Heynh. Oryza sativa L. Hiratsuka et al., 1989 NC_001320 134 525 39.00 80 592 20 799 12 335 78 296 58.20 56 229 41.80 126 92 4 30 16 (Japonica group) †The gene number was adjusted by the latest National Center for Biotechnology Information sequences with slight manual adjustment in Geneious. ‡This chloroplast genome sequence was not included in Fig. 2. §Partial chloroplast genome sequence. IR, inverted repeat; LSC, large single-copy region; SSC, small single-copy region. www.jse.ac.cn Chloroplast phylogenomics in ferns 451

et al., 2006). The phylogenetic relationships among the four eusporangiate fern orders were uncertain even though recent data (e.g., Pryer et al., 2001, 2004; Gao et al., 2013; Grewe et al., 2013; Kim et al., 2014; Zhong et al., 2014) indicated that they constitute a paraphyletic assemblage. The sister relationship between Ophioglossum L. and Psilotum Sw. was revealed by analyses based on several genes (Pryer et al., 2001, 2004), and supported by some morphological characters (including heterotrophic gametophytes with multicellular rhizoids, and the reduction of the root systems) (Schneider et al., 2002). Smith et al. (2006) placed Psilotaceae and Ophioglossaceae at Fig. 1. Number of chloroplast genomes in ferns and the basalmost position of ferns, but the phylogenetic lycophytes published in the last two decades. relationships among the four eusporangiate fern groups and the leptosporangiate ferns were uncertain. The phylogenetic analyses based on chloroplast genomes confirmed the Zhong et al., 2014). These studies revealed that the paraphyly of the eusporangiate ferns, and the sister relation- evolutionary dynamics of chloroplast genomes in ferns are ship between Marattiales and the leptosporangiate ferns more complicated than previously assumed. (Grewe et al., 2013; Kim et al., 2014; Zhong et al., 2014). Chloroplast genome structural changes (e.g., multigene The primary objective of this study is to explore the inversions) generally show less homoplasy than sequence phylogenetic utility of whole chloroplast genomic data in data, and can be informative in resolving certain intractable resolving difficult deep nodes in ferns. Along with the phylogenetic issues (Kelch et al., 2004; Philippe et al., 2005; enthusiasm generated at the recent Next Generation Raubeson & Jansen, 2005; Jansen et al., 2007). The inversion Pteridology Symposium held at the Smithsonian Institution of a 3-kb region (involving trnG-GCC to trnT-GGU) (Wolf et al., (Washington, D.C., USA) for adding nuclear gene data from 2005; Gao et al., 2009; Karol et al., 2010) and the trnD-GUC next-generation sequencing in resolving relationships in ferns, inversion (Roper et al., 2007; Gao et al., 2009) can be detected the recent availability of a large number of chloroplast in ferns. Three chlorophyll biosynthesis genes (chlB, chlL, and genomes in GenBank provides a good opportunity to further chlN) were reported to be independently lost from the whisk explore the congruence of phylogenies from the two kinds of fern Psilotum nudum (L.) P. Beauv (Grewe et al., 2013). The genomes in ferns, a group with documented frequent trnK gene is absent in the leptosporangiate ferns (Wolf et al., occurrences of allopolyploidy and reticulate evolution (see 2003, 2011; Gao et al., 2009). The rpoB-psbZ (BZ) region of fern many references cited in Xiang et al., 2015). plastomes shows considerable variation in size, gene order, and repeat content (Gao et al., 2011). Two pathways have been proposed for the complex gene order change in the BZ region Material and methods in ferns (Roper et al., 2007; Gao et al., 2011). Nearly all core Taxon sampling and DNA extraction (higher) leptosporangiates have the same gene arrangement pattern as that observed in Adiantum capillus-veneris L. (the Young leaves of Cyrtomium devexiscapulae (Koidz.) Ching Adiantum type) in the BZ region, while the basal fern lineages and Woodwardia unigemmata (Makino) Nakai were collected share the same gene order found in Angiopteris evecta (G. from the plants growing in Kunming Botanical Garden, the Forst.) Hoffm. (the Angiopteris type) (Wolf et al., 2003; Roper Chinese Academy of Sciences (Kunming, China). The voucher et al., 2007; Gao et al., 2009, 2011). The major evolutionary herbarium specimens were deposited in the herbarium of changes of chloroplast genomes among fern lineages are Kunming Institute of Botany, the Chinese Academy of Sciences shown in Table 1. (KUN). These two species were selected because they Higher-level relationships of ferns have been reconstructed represent the two lineages of eupolypods, for which the primarily based on molecular phylogenetics using genes and complete chloroplast genome sequences had previously been intergenic regions from chloroplast and nuclear genomes unavailable. (Hasebe et al., 1993, 1994, 1995; Pryer et al., 1995, 2001, 2004; Total genomic DNA was extracted from 100 mg fresh leaves fi Schuettpelz & Pryer, 2007; Rothfels et al., 2012a; Liu et al., using a modi ed CTAB method (Doyle & Doyle, 1987), in 2013; Zhang & Zhang, 2015). Additionally, these gene markers which 4% CTAB was used instead of 2% CTAB, along with the and morphological characteristics supported the monophyly addition of approximately 1% polyvinyl polypyrrolidone and of monilophytes (including ferns, horsetails, and whisk ferns). 0.2% DL-dithiothreitol. Karol et al. (2010) also identified one new indel (atpA) and an inversion of a block of genes that support the monophyly of Genome sequencing, assembly, and annotation monilophytes. The complete chloroplast genomes for the two species were Although a basic phylogenetic framework of extant ferns is sequenced using an Illumina HiSeq 2000 Sequencing System available (Schuettpelz & Pryer, 2007; Rothfels et al., 2012a, (BGI-Shenzhen, Shenzhen, China), with standard Illumina 2012b; Liu et al., 2013; Zhang & Zhang, 2015), relationships sequencing protocols (Shendure & Ji, 2008). among a few key nodes remain unresolved or poorly The raw sequence reads were assembled with SOAPdenovo supported. Monilophytes consist of four orders of eusporan- version 1.04 (Li et al., 2010). Small gaps in the assemblies were giate ferns (Psilotales, Ophioglossales, Equisetales, and bridged by designing custom primers based on their flanking Marattiales) and a clade of leptosporangiate ferns (Smith sequences using polymerase chain reaction and conventional www.jse.ac.cn J. Syst. Evol. 53 (5): 448–457, 2015 452 Lu et al.

Sanger sequencing. The Mafft version 1.3 plugin of Geneious annotated plastomes have been deposited in GenBank Pro version 6.0.6 (Kearse et al., 2012) was used to generate a (Table 1). whole plastome alignment of the two new sequences against The alignment of the 48-gene dataset of 35 taxa was 32 324 three available basal polypods: Adiantum capillus-veneris, nt in length. The matrix contained 18 150 variable sites (56.2%), Cheilanthes lindheimeri Hook., and Pteridium aquilinum (L.) of which 15 975 were phylogenetically informative (88%). The Kuhn subsp. aquilinum. This alignment was used to annotate alignment of the 64-gene dataset was 44 737 nt in length. The the new sequences, coupled with manual adjustment of the matrix had 27 337 variable sites (61.1%), of which 24 057 were positions of start and stop codons and boundaries between phylogenetically informative (88%). introns and exons. The trees based on 48 genes and 64 genes are identical in topology, differing only in support values for four nodes, three Phylogenetic analyses of which showed higher support values for the 48-gene Thirty-three chloroplast genome sequences of vascular plants dataset (Fig. 2). The 48-gene dataset is considered more (25 ferns, including five taxa with incomplete chloroplast reliable because the 64-gene data included genes with lower genomes, five lycophytes, and three seed plants) were identities (<25%) and thus were likely more homoplasious. obtained from GenBank (Table 1; Fig. 1). Only KP205433 was The phylogenetic analysis strongly supported the mono- included for Mankyua chejuense B. Y. Sun, M. H. Kim & C. H. Kim phyly of ferns with bootstrap support (BS) ¼ 100 and Bayesian in the analyses because both KP205433 and NC_017006 posterior probability (PP) ¼ 1.0, and the leptosporangiate represent the same species and were submitted by the same ferns (BS ¼ 100, PP ¼ 1.0) (Fig. 2). Four Eusporangiate fern authors. As noted above, two chloroplast genomes were clades were identified in our analyses (Fig. 2). The clade of the newly sequenced in this study. The chloroplast genome marattioid ferns was sister to the leptosporangiate ferns. sequences have been deposited into GenBank under acces- Equisetum L. was resolved as sister to the clade of Psilotales– sion number KT599100 and KT599101 (Table 1). The taxa of Ophioglossales (BS ¼ 96/100 (48 genes/64 genes), PP ¼ 1.0). lycophytes and seed plants were used to root the trees in the Then the clade of Equisetales–Psilotales–Ophioglossales was phylogenetic analyses. Muscle (Edgar, 2004) was used to sister to the clade of the leptosporangiates and the marattioid obtain multisequence alignments. All alignments were ferns (BS ¼ 100, PP ¼ 1.0). concatenated for phylogenetic analyses using maximum In the core leptosporangiate ferns, the sister relationship likelihood (ML) and Bayesian inference, as implemented in between the tree ferns and polypods was strongly supported RAxML (Stamatakis, 2006) and MrBayes (Huelsenbeck & by the 48-gene tree (BS ¼ 100, PP ¼ 1.0); however, the BS for Ronquist, 2001), respectively. For the ML analysis, the ML tree this relationship was lower in the 64-gene tree (BS ¼ 82, was calculated with a GTR þ CAT model of sequence PP ¼ 1.0). Pteridaceae was sister to the clade of Dennstaed- evolution. Robustness of inference was assessed by running tiaceae and eupolypods with high support (BS ¼ 100, PP 100 fast bootstrap replicates (Stamatakis et al., 2008). The ¼ 1.0), and the sister relationship between Dennstaedtiaceae Bayesian analysis was carried out with the GTR þ G model and and eupolypods was strongly supported (BS ¼ 99/98, 1 000 000 generations with trees being sampled per 1000 PP ¼ 1.0). generations. For each analysis, two runs with four chains were performed in parallel, and the first 25% of all sampled trees were discarded as the burn-in. Discussion Two phylogenetic trees were constructed based on 64 genes and 48 genes (Tables S1 and S2). Rather than using the Our analyses support the monophyly of the leptosporangiate data from all genes, we used the identity values of the genes ferns and the sister relationship between the leptosporan- to screen out highly homoplasious data. Using 15% identity giate ferns and the marattioid ferns (Fig. 2). The latter value as the threshold, we obtained a dataset of 64 genes relationship is also supported by a single gain of an intron in (identities ranging from 15% to 61.7%, Table S1). Using 25% the mitochondrial atp1 gene in the common ancestor of the identity value as the threshold, we obtained a dataset of 48 leptosporangiates and the marattioid ferns. That intron is genes (identities ranging from 25.1% to 61.7%, Table S2). Our absent in the ophioglossoid ferns, whisk ferns, and horsetails data have been made available from the Dryad Digital (Grewe et al., 2013). Repository (http://dx.doi.org/10.5061/dryad.v23n2). Two clades of the eupolypods (eupolypod I and eupolypod II) were also recovered in our analyses (both BS ¼ 100, PP ¼ 1.0), and the sister relationship between the two clades Results was strongly supported (BS ¼ 100, PP ¼ 1.0). Our analyses also supported three key nodes, which were controversial The complete plastome of Cyrtomium devexiscapulae is in previous studies, and below we discuss the three key 151 684 bp long and includes an 82 453-bp large single-copy relationships in more detail. region, a 21 625-bp SSC region, and two 23 803-bp IRs. The complete plastome of Woodwardia unigemmata is 153 717 bp Horsetails as sister to the Psilotales–Ophioglossales clade long and includes an 82 387-bp large single-copy region, a Equisetum was resolved as sister to the Psilotales–Ophio- 21 556 bp SSC region, and two 24 887-bp IRs. The overall GC glossales clade (BS ¼ 96/100, PP ¼ 1.0; Fig. 2). Then the content is 42.33% for Cyrtomium devexiscapulae and 43.21% for Equisetales–Psilotales–Ophioglossales clade was sister to Woodwardia unigemmata. A total of 117 genes were identified the clade of the leptosporangiates and the marattioid ferns. in each genome, and each consisted of 84 protein-coding Equisetopsida (horsetails) is an ancient lineage dating genes, four rRNA genes, and 29 tRNA genes (Table 1). The two back to approximately 300 Ma (the Permian period) (Zhong

J. Syst. Evol. 53 (5): 448–457, 2015 www.jse.ac.cn Chloroplast phylogenomics in ferns 453

Fig. 2. Phylogeny of ferns bases on plastome sequence data. The topology is consistent using different genes or different methods. Bootstrap values obtained using 48 genes and 64 genes, respectively, are shown above the branch, and posterior possibilities using 48 genes and 64 genes, respectively, are shown below the branch. The diamond indicates that the bootstrap values are 100% and the posterior possibilities are 1.0 in both analyses. et al., 2014), but the extant members all belong to 2004; Des Marais et al., 2003; Wikstrom€ & Pryer, 2005; Qiu Equisetum, which diversified only within the last 50 million et al., 2006, 2007), whereas other analyses have suggested a years (Des Marais et al., 2003). Des Marais et al. (2003) position sister to the leptosporangiate ferns (Nickrent et al., provided molecular-based age estimates using the penalized 2000), or sister to the marattioid–leptosporangiate ferns likelihood approach (Sanderson, 2002), and suggested that (Schuettpelz et al., 2006), or sister to Ophioglossum–Psilotum the Equisetum crown group appears to have diversified in (Gao et al., 2013; Grewe et al., 2013), or as the sister group to all the early Cenozoic (36.3 or 48.9 Ma), whereas the extant monilophytes (Rai & Graham, 2010). Schneider (2007) Equisetales was estimated to have a Paleozoic origin integrated fossils with morphology and molecular data to (266.1 Ma, or older than 381 Ma). Stanich et al. (2009) show the sister relationship of horsetails to the clade of other pointed out that the crown group Equisetum probably ferns (fig. 2 in Schneider, 2007). However, the placement of originated during the Mesozoic, as all of the synapomor- Equisetum in the various analyses did not have strong phies of living Equisetum have been discovered in the lower statistical support. When the chloroplast genome of Tmesip- Cretaceous deposits (approximately 136 Ma). teris Bernh. (another genus of Psilotaceae) was added, Horsetails have been a particularly enigmatic group until Equisetum was sister to Psilotaceae (Tmesipteris þ Psilotum) recently with the molecular and morphological evidence in the phylogenetic trees with strong BS (Zhong et al., 2014; supporting their inclusion in ferns (Pryer et al., 2001). Several Marrattiaceae not sampled). early molecular phylogenetic analyses have suggested that Enhanced sampling of more complete chloroplast genome horsetails are sister to the marattioid ferns (Pryer et al., 2001, sequences from the eusporangiate ferns and basal www.jse.ac.cn J. Syst. Evol. 53 (5): 448–457, 2015 454 Lu et al. leptosporangiate ferns should help resolve the different Tree fern clade Cyatheales as sister to polypods phylogenetic placement. Grewe et al. (2013) sequenced the The tree fern clade Cyatheales was strongly supported in the plastid genomes of three early diverging fern taxa: Equisetum early phylogenetic studies based on the single rbcL gene hyemale L., Ophioglossum californicum Prantl, and Psilotum (Hasebe et al., 1994, 1995; Pryer et al., 1995), but the sister nudum. In their phylogenetic analyses using 49 genes, the relationship between this clade and eupolypods was sup- Equisetum species formed a monophyletic group sister to the ported by only low BS values (BS < 70). As multiple genes clade Ophioglossum–Psilotum with weak BS (BS ¼ 52). Kim were added into the analyses, a sister relationship between et al. (2014) sequenced three additional chloroplast genomes tree ferns and eupolypods was strongly supported (Pryer from the early diverged leptosporangiate ferns (Osmundas- et al., 2001; Schuettpelz et al., 2006; Schuettpelz & Pryer, trum cinnamomeum (L.) C. Presl, Diplopterygium glaucum 2007), although the support value was not high for this sister (Thunb. ex Houtt.) Nakai, and Lygodium japonicum (Thunb.) relationship in other studies (Pryer et al., 2004; Qiu et al., Sw.), and constructed phylogenetic trees based on 89 genes 2006). The tree fern clade (Alsophila R. Br. and Dicksonia from 35 taxa. The result placed Equisetales either as the sister L’Her.) was sister to polypods in the ML tree based on group of the Psilotales–Ophioglossales clade, or at the chloroplast genome data with 100% BS (Zhong et al., 2014). basalmost position of the monilophytes (Kim et al., 2014). When we added the sequences of Plagiogyria (Kunze) Mett. Kim et al. (2014) chose the tree topology of the basalmost (Plagiogyriaceae, Cyatheales) in the present analyses, the position of Equisetales in monilophytes to further discuss the sister relationship between the tree ferns and polypods was relationships even though this tree did not show strong only moderately supported (BS ¼ 82) in the 64-gene tree, support for the relationship (BS ¼ 33). This position of while the support value was high (BS ¼ 100) in the 48-gene Equisetales sister to the remaining monilophytes is supported tree. in the recent transciptome analyses on land plants (Wickett Three major lineages of the core leptosporangiates – et al., 2014), a dataset of nine organellar loci and an indel in the heterosporous ferns, tree ferns, and polypod ferns – mitochondrial rpl2 locus (Knie et al., 2015), and multiple low- originated in the Late Triassic, and the earliest divergences copy nuclear genes (Rothfels et al., 2015). within each of these lineages occurred in the Jurassic (Pryer The present study of chloroplast phylogenomic data, et al., 2004; Schuettpelz & Pryer, 2009). It is possible that however, supports the relationship of Equisetales sister to conserved genes can more reliably construct phylogenetic the Psilotales–Ophioglossales clade (Fig. 2), as in Grewe et al. relationships in the groups with a very deep evolutionary (2013). In our study, this relationship was strongly supported history. Certainly, more chloroplast genomes of tree ferns and by a 100% bootstrap value in the 64-gene tree, and a 96% polypods need to be sequenced to test this deep relationship. bootstrap value in the 48-gene tree. The sister relationship between the clade of Equisetales–Psilotales–Ophioglossales Early divergences in polypods: Pteridaceae as sister to the and the clade of leptosporangiate and marattioid ferns was clade of Dennstaedtiaceae and eupolypods also strongly supported (BS ¼ 100, PP ¼ 1.0). Pteridaceae was sister to the clade of Dennstaedtiaceae and The substitution rate in the plastid genome was reported eupolypods in our phylogenetic analyses with a high support to be elevated in horsetails compared with other early value (BS ¼ 100, PP ¼ 1.0), and the sister relationship of diverging ferns (Pryer et al., 2001, 2004; Rai & Graham, Dennstaedtiaceae and eupolypods was strongly supported 2010), and all molecular phylogenetic analyses showed a (BS ¼ 99/98, PP ¼ 1.0) (Fig. 2). long branch leading to Equisetum. The topological differ- Lonchitis L., Saccoloma Kaulf., Cystodium J. Sm., and ences may be due to the taxon sampling for the long Lindsaeaceae constituted a moderately supported clade branched taxa, and the relationships need to be further (BS ¼ 74%) within the early polypods in the analysis of tested with a broader sampling in early diverged ferns. Schuettpelz & Pryer (2007). This clade was divided into two Schneider et al. (2009) used a morphological dataset families, Lindsaeaceae and Saccolomataceae in Smith et al. (including 135 morphological, anatomical, biochemical char- (2006), whereas taxa of this clade were recognized as four acters, and one DNA structural character) to infer the families, Lindsaeaceae, Lonchitidaceae, Saccolomataceae, divergences of early ferns. The clade of Psilotales–Equise- and Cystodiaceae, in Christenhusz et al. (2011) and Christen- tales (PP ¼ 0.99, BS ¼ 75) was sister to the leptosporangiate husz & Chase (2014). fern (but with weak support, PP ¼ 0.53); Ophioglossales was The analyses of Rai & Graham (2010) using 17 plastid genes sister to an aforementioned assemblage (PP ¼ 0.77, BS ¼ 71), showed that Lindsaea Dryand. ex Sm., Lonchitis and Saccoloma whereas Marattiales was the basalmost group in ferns in were the basalmost groups in polypods. However, their result their analyses (PP ¼ 0.53, BS ¼ 73). They pointed out that the did not recover the Lindsaeaceae–Saccolomataceae clade placement of Psilotales as sister to Equisetales might be the observed in Schuettpelz & Pryer (2007). The dataset of result of shared homoplastic character states and thus could Lehtonen et al. (2010) included almost all genera associated be interpreted as a consequence of long-branch attraction with Lindsaeaceae (Cystodium, Lonchitis, Saccoloma, and (Schneider et al., 2009). Evidence from more conserved Sphenomeris Maxon). Their analyses placed Cystodium close nuclear genes or phylogenomic data such as transcriptomes to the lindsaeoids, with Lonchitis branching off from the with a broad taxon sampling scheme of diverse fern groups lindsaeoid lineage even before Saccoloma did (Lehtonen et al., should be sought to further test this highly controversial 2010). deep relationship. Our study suggests that the relationship In our study, the remaining polypods were members of of Equisetales as sister to the Psilotales–Ophioglossales three highly supported clades (BS ¼ 100%): the dennstaedtioid clade should not be dismissed, as it is strongly supported by clade, the pteroid clade, and the eupolypod clade. However, theplastomedata. the relationships among these three lineages were previously

J. Syst. Evol. 53 (5): 448–457, 2015 www.jse.ac.cn Chloroplast phylogenomics in ferns 455 unclear because BS values were lower than 50% (Schuettpelz Natural Science Foundation of China (Grant No. 31129001), the et al., 2006; Schuettpelz & Pryer, 2007). In the analyses of the Applied Fundamental Research Foundation of Yunnan Prov- multigene supermatrix (six to seven genes from chloroplast, ince (Grant Nos. 2014GA003, 2014FB168) and the Laboratories mitochondrial, and nuclear genomes) of deepest divergences of Analytical Biology of the Smithsonian Institution. N. Z. was in land plants, Pteridium Gled. ex Scop. was sister to the clade supported by the Buck Fellowship program of the National of eupolypods with moderate support (BS > 70), then this Museum of Natural History, the Smithsonian Institution. We aggregate was sister to the pteroid clade with strongly thank F. H. Wang for her assistance with the experiments, and support (BS ¼ 100) (Qiu et al., 2006, 2007). The Dennstaedtia- Dr. Elizabeth A. Zimmer and Dr. Harald Schneider for their ceae–Pteridaceae–eupolypod clade also had strong ML helpful comments on earlier versions of the manuscript. support in the analysis of Rai & Graham (2010). However, the sister-group relationships between Dennstaedtiaceae and Pteridaceae, and between the Dennstaedtiaceae–Pteridaceae References clade and eupolypods, were only poorly supported in their analysis (Rai & Graham, 2010). Banks JA, Nishiyama T, Hasebe M, Bowman JL, Gribskov M, Several molecular phylogenetic analyses were imple- dePamphilis C, Albert VA, Aono N, Aoyama T, Ambrose BA, Ashton NW, Axtell MJ, Barker E, Barker MS, Bennetzen JL, mented to clarify the relationships among eupolypods in Bonawitz ND, Chapple C, Cheng C, Correa LG, Dacre M, DeBarry J, the last few years (Rothfels et al., 2012a; Liu et al., 2013; Zhang Dreyer I, Elias M, Engstrom EM, Estelle M, Feng L, Finet C, Floyd & Zhang, 2015). No studies have aimed at the relationships SK, Frommer WB, Fujita T, Gramzow L, Gutensohn M, Harholt J, among the basal groups of polypods. Chloroplast phyloge- Hattori M, Heyl A, Hirai T, Hiwatashi Y, Ishikawa M, Iwata M, Karol nomics based on large-scale gene data or whole chloroplast KG, Koehler B, Kolukisaoglu U, Kubo M, Kurata T, Lalonde S, Li K, genome data may shed light on resolving the phylogeny of Li Y, Litt A, Lyons E, Manning G, Maruyama T, Michael TP, Mikami early polypods. The present study supports the relationship of K, Miyazaki S, Morinaga S, Murata T, Mueller-Roeber B, Nelson Pteridaceae sister to the clade of Dennstaedtiaceae and DR, Obara M, Oguri Y, Olmstead RG, Onodera N, Petersen BL, Pils ~ eupolypods as in Qiu et al. (2006, 2007) with high BS/PP B, Prigge M, Rensing SA, Riano-Pachon DM, Roberts AW, Sato Y, values. 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