1. Introduction

Lycophytes and ferns are two distinct evolutionary lineages of free-sporing vascular plants, encompassing approximately 12, 000 species (PPG I, 2016). Of these plants, the shield-fern family Dryopteridaceae is the largest fern family, containing slightly more than 2100 species (Zhang et al., 2013a; PPG I, 2016). With a nearly worldwide distribution, Dryopteridaceae exhibit their highest diversity in temperate regions and tropical mountainous areas, particularly in East Asia (Dryopteris Adans., Polystichum Roth) and the New World (Elaphoglossum Schott ex J. Sm.) (Zhang et al., 2013a). Dryopteridaceae can be found from coastal areas to alpine tree line. Most species are terrestrial or rupestral, although some tropical taxa are root climbers (e.g., Maxonia C. Chr., Polybotrya Humb. & Bonpl. ex Willd., and Cyclodium C. Presl; Moran et al., 2010a, b; Moran and Labiak, 2015) or hemiepiphytes (e.g., one species of Elaphoglossum, a few species of Bolbitis Schott, and almost all species in Arthrobotrya J. Sm., Mickelia R.C. Moran, Labiak & Sundue, Lomagramma J. Sm., and Teratophyllum Mett. ex Kuhn) (Moran et al., 2010a; Lagomarsino et al., 2012; Nie et al., 2023).

Throughout the taxonomic history of ferns, the circumscriptions of families and genera have been highly dynamic. Over the past two centuries, numerous classifications have been proposed, reflecting different interpretations of the available evidence. Although Dryopteridaceae share some common morphological features (stipes with three or more vascular bundles arranged in a semicircle or circle, scales on rhizome and stipe, and chromosome numbers x = 41) with most families of the Eupolypod I (Polypodiineae), the family has been defined differently by different pteridologists due to the large difference in habit, shape of rhizome and frond, venation, and soral arrangement among different genera (Tryon and Tryon, 1982; Kramer et al., 1990; Zhang et al., 2013a) (Fig. 1). Because of extremely diverse morphology, the taxonomic position of dryopteridoid ferns has changed several times (Table 1). Most dryopteridoid ferns were historically placed in Polypodiaceae (Presl, 1836; Hooker and Baker, 1868), Aspidiaceae (nom. illeg. = Tectariaceae) (Christensen, 1906; Ching, 1940; Copeland, 1947; Alston, 1956; Pichi-Sermolli, 1958, 1977), or Dennstaedtiaceae (Holttum, 1947). Until the mid-19th century, Dryopteridaceae were gradually recognized and accepted (Herter, 1949; Ching, 1965a; Nayar, 1970; Pichi-Sermolli, 1970).

The circumscription of Dryopteridaceae has also changed over time. Pteridologists proposed multiple classifications of Dryopteridaceae, with varying numbers of genera accepted (Pichi-Sermolli, 1977; Ching, 1978; Tryon and Tryon, 1982; Kramer et al., 1990; Wu and Ching, 1991) (Table 1). Dryopteridaceae were divided into two subfamilies (Dryopteridoideae and Athyrioideae), five tribes, and 45 genera in the most widely accepted morphology-based classification (Kramer et al., 1990), of which three tribes (Tectarieae, Physematieae, and Onocleeae) were dealt as separate families based on molecular and morphological evidence in subsequent classifications (e.g., PPG I, 2016). Smith et al. (2006), largely based on available molecular evidence at the time, included 40–45 genera within Dryopteridaceae without recognition of any subfamilies. The three genera tentatively placed in Dryopteridaceae by Smith et al. (2006) were later assigned to the other two families, respectively, i.e., Didymochlaena Desv. in Didymochlaenaceae, Hypodematium A. Rich. and Leucostegia C. Presl in Hypodematiaceae (Zhang and Zhang, 2015). Christenhusz et al. (2011) included 34 genera in Dryopteridaceae and divided this family into two subfamilies—Dryopteridoideae B.K. Nayar and Elaphoglossoideae (Pic. Serm.) Crabbe, Jermy & Mickel, and then Zhang et al. (2013b) included 25 genera in two subfamilies of Dryopteridaceae. The circumscription of Dryopteridaceae had been confusing until recent phylogenetic studies which substantially revised it (Table 2).

The monophyly of several global large genera (more than 100 species, e.g., Ctenitis (C. Chr.) C. Chr., Dryopteris, Elaphoglossum, and Polystichum) or medium genera (about 50–90 species, e.g., Bolbitis and Megalastrum Holttum) has been identified by molecular phylogenetic studies (Moran et al., 2010a; Zhang et al., 2012; Le Péchon et al., 2016a, 2016b; Hennequin et al., 2017; Nie et al., 2023). Most of the small segregate genera were found to be nested within some larger genera which are closely related and are nowadays synonyms of these genera. For example, Leptorumohra H. Itô, Lithostegia Ching, and Phanerophlebiopsis Ching were nested in Arachniodes Blume (Li and Lu, 2006a; Liu et al., 2007c; He et al., 2013; Lu et al., 2019); Ataxipteris Holttum and Pseudotectaria Tardieu were nested in Ctenitis (Dong and Christenhusz, 2013; Wang et al., 2014; Hennequin et al., 2017); Acrophorus C. Presl, Acrorumohra (H. Itô) H. Itô, Diacalpe Blume, Dryopsis Holttum & P.J. Edwards, Nothoperanema (Tagawa) Ching, Peranema D. Don, Revwattsia D.L. Jones, and Stenolepia Alderw. were nested in Dryopteris (Li and Lu, 2006a, 2006b; Liu et al., 2007a; Ebihara, 2011; McKeown et al., 2012; Zhang, 2012; Zhang and Zhang 2012; Zhang et al., 2012; Kuo et al., 2016); Cyrtogonellum Ching, Cyrtomidictyum Ching, and Sorolepidium Christ were nested in Polystichum (Little and Barrington, 2003; Liu et al., 2007b; Lu et al., 2007; Li et al., 2008; Zhang et al., 2013a; Le Péchon et al., 2016a, 2016b), Coveniella Tindale was nested in Lastreopsis Ching (Labiak et al., 2014, 2015); and Adenoderris J. Sm. was found to be polyphyletic and its taxa were then treated in either Dryopteris or Polystichum (McHenry et al., 2013). On the other hand, several genera, e.g., Mickelia, Polystichopsis (J. Sm.) C. Chr., Parapolystichum (Keyserl.) Ching, and Trichoneuron Ching were newly recognized or reinstated (Moran and Prado, 2010; Moran et al., 2010b; Labiak et al., 2014, 2015; Liu et al., 2016).

Numerous phylogenetic studies have attempted to elucidate the infra-familial relationships of Dryopteridaceae (Li and Lu, 2006a; Liu et al., 2007c, 2016) as well as the phylogenetic relationships of different lineages (Moran et al., 2010a; Zhang et al., 2012; Labiak et al., 2014; Moran and Labiak, 2015; Le Péchon et al., 2016b; Hennequin et al., 2017). Among these studies, Liu et al. (2016) implemented the comprehensive phylogenetic study of Dryopteridaceae with the most extensive generic representatives, recognized three main clades and two subclades within the family, and proposed a three-subfamily (Dryopteridoideae, Elaphoglossoideae, and Polybotryoideae Hong M. Liu & X.C. Zhang) classification. However, there are still several remaining issues that need to be further resolved. Firstly, the phylogenetic relationships of the three subfamilies of Dryopteridaceae were not fully resolved; secondly, the placement of Ctenitis and Stigmatopteris C. Chr. was only tentatively settled; and thirdly, several small genera were not sampled in their study (Liu et al., 2016).

Twenty-six genera were recognized in Dryopteridaceae in the currently most widely accepted PPG I (2016) classification. In PPG I (2016) classification, Bolbitidaceae, Elaphoglossaceae, and Peranemataceae were treated as synonyms of Dryopteridaceae, and some genera assigned earlier in Lomariopsidaceae (e.g., Lomagramma) and Tectariaceae (e.g., Ctenitis and Pleocnemia C. Presl) were moved in Dryopteridaceae (PPG I, 2016). PPG I (2016) recognized three subfamilies in Dryopteridaceae, primarily followed Liu et al. (2016) (Table 2). Given the unresolved relationships of Ctenitis and Stigmatopteris, PPG I (2016) tentatively placed these two genera in Dryopteridoideae and Polybotryoideae, respectively. Furthermore, the relationships between Arthrobotrya and Teratophyllum were unsettled (Moran et al., 2010a; Liu et al., 2016). Aenigmopteris Holttum and Dryopolystichum Copel., not placed in any subfamilies of Dryopteridaceae by PPG I (2016), were later shown to be members of Tectariaceae and Lomariopsidaceae, respectively (Chen et al., 2017a, 2017b).

The three-subfamily classification of Dryopteridaceae is only based on molecular phylogeny. The heterogeneous morphology of the current members of Dryopteridaceae makes the family and its subfamilies difficult even to define by morphological character combinations. Various states of morphological characters generally used to identify different fern lineages (families), such as habit, rhizome shape, frond morphology, rachis-costa architecture, and sorus shape, can be observed in Dryopteridaceae (Smith et al., 2006). For example, terrestrial/epiphytic habit, erect/long-creeping rhizome, monomorphic/dimorphic fronds (with different sterile and fertile fronds), simple/1- or more pinnate lamina division, and discrete/acrostichoid sori can be found in Elaphoglossoideae and Polybotryoideae. More comprehensive morphological examination is needed to better understand and circumscribe the subfamilies and genera of Dryopteridaceae.

To determine the phylogenetic placement of Ctenitis and Stigmatopteris and establish an "practical" infra-familial classification of Dryopteridaceae supported by a robust phylogenetic framework and morphological evidence, a phylogeny of Dryopteridaceae was reconstructed using complete chloroplast genomes and morphological characteristics were examined and coded for each genus. Based on phylogenetic relationships and optimizations of morphological characters on the resulting trees, we updated a more informative infra-familial classification for Dryopteridaceae here.

2. Material and methods 2.1. Taxon sampling, DNA extraction, sequencing, and plastome assembly

A total of 94 plastomes from 86 species, representing all 24 recognized genera of Dryopteridaceae (Liu et al., 2016; PPG I, 2016), and three outgroups were gathered (Du et al., 2021), among which 53 plastomes were newly generated and 41 plastomes accessed via GenBank (Table S1). Total genomic DNA of silica-gel-dried leaf materials was extracted using a modified CTAB procedure (Doyle and Doyle, 1990), and DNA extraction of specimens was followed the protocol of Zeng et al. (2018). Library preparation was conducted at the Germplasm Bank of Wild Species, Kunming Institute of Botany (CAS, China), and Illumina sequencing was conducted at BGI Genomics Co., Ltd (Shenzhen, China). More than 2 Gb of clean data of 150 bp pair-end reads were generated for each sample. De-novo assemblies were constructed using GetOrganelle v.1.7.0 (Jin et al., 2020), and connection and annotation were subsequently conducted using Bandage 0.8.1 (Wick et al., 2015) and Geneious 8.0.2 (Kearse et al., 2012). Collinearity among the plastomes was evaluated through the progressiveMauve algorithm and default parameters in Geneious 8.0.2 (Darling et al., 2010). All the sequences were aligned using MAFFT v.7 (Katoh and Standley, 2013).

2.2. Data matrices and phylogenetic analyses

We implemented phylogenetic analyses using six matrices (Table S5). Firstly, we constructed two matrices consisting of all 94 complete plastid genomes by half gaps deletion (Matrix 1) or all gaps deletion (Matrix 2) with Gblocks (Talavera and Castresana, 2007). Secondly, we constructed two gene matrices with 115 gene sequences (Matrix 3), and 85 coding-gene sequences (CDS) + four rRNA genes (Matrix 4). Thirdly, the CDS sequences of 85 protein-coding genes were translated into amino acid (AA) sequences. Furthermore, three plastid regions (rbcL, rps4-trnS, and trnL-F) of 61 Dryopteridaceae taxa were downloaded from NCBI (Table S2), and added to Matrix 1 as Matrix 6.

All matrices were analyzed using Maximum Likelihood (ML) analysis, Maximum Parsimony (MP), and Bayesian Inference (BI). ML analyses were conducted using IQ-TREE v.1.6.12 (Nguyen et al., 2015) with 10, 000 ultrafast bootstrap replicates. The MP analyses were implemented in PAUP∗ 4.0d100 (Swofford, 2002). All characteristics were weighted equally and gaps were treated as missing data. 1000 replicates were performed with 10 tree-bisection-reconnection (TBR) searches per replicate and a maximum of 100 trees held per TBR search. MP Bootstrap support for nodes was estimated with 1000 heuristic replicates and tree bisection–reconnection branch swapping. BI analyses were conducted with MrBayes 3.2.6 (Ronquist et al., 2012), using ten million generations with one tree sampled every 1000 generations; four runs with four chains were performed in parallel. The first 25% of trees were discarded as burn-in. The Markov Chain Monte Carlo (MCMC) output was examined to check for convergence and to ensure that all the effective sample size (ESS) values were above 200 in TRACER v.1.7 (Rambaut et al., 2018).

2.3. Morphological characters coding and ancestral state reconstruction

We conducted field observation of the living plants of Dryopteridaceae, examined specimens deposited in the herbaria of BM, CHS, IBSC, K, KUN, MO, P, PE, PYU, TI, and TNS, and plant pictures on the websites (e.g., GBIF: https://www.gbif.org/; CVH: https://www.cvh.ac.cn/). Firstly, we made a statistical observation of the morphological features used to identify different genera of Dryopteridaceae (e.g., rhizome shape, fronds morphology, shape of rachis and costae, scales, sori and indusia) in previous studies (Pichi-Sermolli, 1977; Ching, 1978; Tryon and Tryon, 1982; Kramer et al., 1990; Wu and Ching, 1991). In order to further understand the relationship between the morphology-based classifications and molecular phylogeny of Dryopteridaceae, we further checked more morphological features of lamina division, and other appendages (hairs and glands) on costa and lamina. A total of 25 morphological features were preliminarily analyzed and 13 morphological characters (Tables S3 and S4) were finally mapped on the phylogeny tree and analyzed using RASP v.4.2 (Yu et al., 2020).

2.4. Distribution and historical biogeography

The ancestral range reconstruction of Dryopteridaceae was conducted using RASP 20200510 (Yu et al., 2020) based on the phylogenetic tree generated by Matrix 6 using the dispersal extinction-cladogenesis model (DEC; Ree and Smith, 2008). We recognized four biogeographic areas: A, Eurasia; B, Americas; C, Africa and Indian Ocean Islands; D, Australasia and Tropical Asia.

2.5. Estimation of divergence times

We estimated the divergence times of major lineages of Dryopteridaceae based on Matrix 1. One secondary molecular calibration and one fossil record were applied in divergence time analyses. The secondary calibration point was derived from earlier study of divergence times of Dryopteridaceae (128.97 (112.63–145.81) million years ago (Ma)) (Du et al., 2021). The fertile leave fossil of Elaphoglossum miocenicum Lóriga, A.R. Schmidt, R.C. Moran, K. Feldberg, H. Schneid. & Heinrichs (Lóriga et al., 2014) (15.97 Ma) was utilized to calibrate the crown of Elaphoglossum.

The estimation of divergence times was carried out using Bayesian methods with BEAST v.2.6.3 (Bouckaert et al., 2014), using the GTR + G + I nucleotide substitution model, birth-death tree prior, and lognormal uncorrelated relaxed clock model. The stem of Dryopteridaceae was assigned as 128.97 Ma with a uniform prior and 112.63–145.81 Ma as the minimum–maximum age constraints. The fossil age of 15.97 Ma was incorporated as an "offset" in a lognormal distribution, with a "Mean" value of 3.0 relative to the fossil age and a "Standard deviation" of 1.0. One run consisting of two billion generations with sampling every 10, 000 generations was conducted. Convergence was achieved within one billion generations, with Effective Sample Size (ESS) values exceeding 200 for all parameters. The initial 200 million generations were discarded as burn-in, and the subsequent 80, 000 trees were used to construct the maximum clade credibility (MCC) tree employing TreeAnnotator v.2.6.3 (utilizing mean node heights) (Bouckaert et al., 2014).

3. Results 3.1. Characteristics of plastomes in Dryopteridaceae

A total of 53 complete plastome sequences of Dryopteridaceae were newly generated in this study (Table S1). The size of the plastomes ranged from 147, 271 bp [Arachniodes hainanensis (Ching) Ching] to 166, 133bp [Elaphoglossum yoshinagae (Yatabe) Makino]. The GC content of each genus was similar, with the differences of less than 1% (Table S1). The GC content of novel plastomes ranged from 40.0% [Elaphoglossum yunnanense (Baker) C. Chr.] to 43.5% [Arachniodes speciosa (D. Don) Ching] (Table S1).

The plastome collinearity analysis showed that all Dryopteridaceae plastomes were highly conserved in terms of gene content and gene order, and all had a common subregion without gene inversion (Fig. S1). The inverted repeat (IR) boundary of some Ctenitis species [Ctenitis decurrenti-pinnata (Ching) Tardieu & C. Chr., C. rhodolepis (C.B. Clarke) Ching, and C. sinii (Ching) Ohwi] expanded in the LSC direction to include ndhB gene. The IR boundary of six genera (Polybotrya, Cyclodium, Maxonia, Olfersia, Trichoneuron, and Polystichopsis) slightly contracted, and while no contraction was found in Stigmatopteris.

3.2. Molecular phylogeny

Different analytical strategies were employed to minimize systematic errors, such as removing gaps, using the coding sequences (CDS) or AA sequences, and using different tree inference methods (Table S5). The phylogenetic trees constructed using the whole chloroplast genome (deleting half or all gaps) (Matrices 1, 2, 6) had much higher support values than those constructed with only coding genes or AA (Matrices 3–5) (Fig. S2). The clades within Dryopteridaceae recovered from Matrices 1–5 (Fig. S2) were identical to the 24 clades based on Matrix 6 (Fig. 2). These 24 clades were further grouped into seven larger major clades (A–G). Most of the relationships among the major clades and clades were consistent with those reported in the previous studies (Liu et al., 2016), but were fully or strongly supported (Bayesian inference posterior probabilities [BIPP] = 1.00, maximum likelihood ML ultrafast bootstrap support values [UFBS] > 95%, & maximum parsimony bootstrap support [MPBS] > 95%).

Stigmatopteris was strongly supported as sister to the aggregate of the other six clades of the major clade A (BIPP = 1.00, UFBS = 100%, & MPBS = 97%, Fig. 2). Arachniodes bella (C. Chr.) Ching was found fully supported as sister to the polystichoid ferns (including Cyrtomium C. Presl, Phanerophlebia C. Presl, and Polystichum). All analyses resolved Polybotryoideae sensu Liu et al. (2016) into two major clades (B and C) and the Stigmatopteris clade. Cyclodium, Maxonia, Olfersia, and Polybotrya nested within the major clades B, and while Polystichopsis and Trichoneuron nested within the major clade C. Ctenitis was strongly supported as sister to the major clades B and C (BIPP = 1.00, UFBS = 100%, & MPBS = 97%) (Fig. 2). Elaphoglossoideae sensu Liu et al. (2016) were resolved into three major clades (E–G). The major clade E including five genera (Bolbitis, Elaphoglossum, Mickelia, Lomagramma, and Teratophyllum) was fully supported as sister to Pleocnemia (the major clade F). Together, they formed the sister group of the major clade G, which consisted of four genera (Lastreopsis, Megalastrum Holttum, Parapolystichum, and Rumohra Raddi). Two species of Arthrobotrya, A. articulata (Fée) J. Sm. and A. wilkesiana (Brack.) Copel., were nested within Teratophyllum.

3.3. Morphological character evolution

Of the 13 morphological characters (Tables S3 and S4), seven (habit, rhizome shape, frond morphology, rachis-costae architecture, appendages (including hairs, bristle and seta) on stipe base and lamina, and soral arrangement) can be used as diagnostic characters for distinguishing major clades and clades within Dryopteridaceae (Fig. 3, Fig. 4, Fig. 5). Hemi-epiphytic or epiphytic long-creeping rhizome, dimorphic fronds, and acrostichoid sori were exclusively found in the major clades B and E. The hairs from the stipe base to the lamina were synapomorphies unique of the major clade C, which distinguished it from the major clade B. Distinctive ctenitoid hairs occurred only in Ctenitis (the major clade D), and while yellow cylindric glands were observed only in the major clade F.

Different rachis-costae architectures were observed in major clades (Fig. 6 and Table 3). There was V-grooved rachises and costae adaxially (referred to as the dryopteridoid type; Fig. 6B) in the major clades A, B, and C. Nearly rounded or flat adaxial leaf axes (referred to as the pleocnemioid type; Fig. 6D) were found in the major clade D and F. A widened groove with a "U"-shaped bottom (known as the lastreopsid type; Fig. 6C) was observed in the major clade G. Another distinctive feature of rachis-costae architecture, the lower stipe had a typical groove (Fig. 6A1), then transformed to rounded to convex, and eventually developed into adaxial ribs on the upper rachis and costae (Fig. 6A2) (referred to as the bolbitidoid type), found exclusively in the major clade E. Other characters, such as lamina division, arrangement of pinnules above suprabasal pinnae, scales, hairs, and glands on costa and lamina, presence or absence of rachis buds, and indusial shape, showed high homoplasy or irregularity.

3.4. Historical biogeography

The ancestral range reconstruction showed that the most probable ancestral area of Dryopteridaceae was Americas (Figs. 3 and 5). Americas had the largest number of genera (17/24) and species in Dryopteridaceae, followed by Eurasia (12), Africa (9), and Australasia (9). The major clade A had a global distribution and the rest were mainly distributed in tropical to subtropical regions. Arachniodes bella was endemic to Africa (Madagascar). In addition to the major clade B, long-distance dispersal events across continents and oceans occurred throughout the evolutionary history of major clades A–G.

3.5. Estimation of divergence times

The results of the BEAST analysis revealed that all major clades of Dryopteridaceae have originated in the Cretaceous. The initial diversifications of Dryopteridaceae occurred approximately 104.68 (85.15–126.8) Ma, giving rise to two lineages (major clades A–D, E–G). The stem of major clade A was estimated to have emerged around 98.88 (80.7–120.24) Ma during the Cretaceous (Fig. 7, node 1). The divergence between the major clade D and the major clades B–C occurred at 96.66 (78.8–117.57) Ma (Fig. 7, node 2), and then major clades B and C diverged at 73.43 (54.21–94.33) Ma (Fig. 7, node 3). The divergences between major clade G and the major clades E and F, as well as between major clade E and major clade F, occurred at 87.52 (69.4–108.2) Ma (Fig. 7, node 4) and 81.77 (64.54–104.4) Ma (Fig. 7, node 5), respectively.

4. Discussion 4.1. Characteristics of plastomes and major evolutionary lineages in Dryopteridaceae

We reconstructed a solid and robust phylogenetic backbone of Dryopteridaceae using plastomes, effectively resolving the phylogenetic relationships among seven major clades and all 24 clades, and addressing the insufficient support of some generic relationships in previous study (Liu et al., 2016; Fig. 8). The relationships among all 24 clades and seven major clades of Dryopteridaceae were fully or strongly supported (Fig. 2).

The phylogenetic relationships of two uncertain subclades Ctenitis and Stigmatopteris in previous study (Liu et al., 2016) were completely resolved and supported. Ctenitis was resolved as sister to all other genera of Polybotryoideae sensu Liu et al. (2016) excluding Stigmatopteris. A small range of IR boundary expansion was detected in Ctenitis, and while a contraction was found in six genera of Polybotryoideae sensu Liu et al. (2016) (Fig. 8). The major clade A included most genera (except Ctenitis) of Dryopteridoideae sensu Liu et al. (2016), with Stigmatopteris being resolved as their first-diverging lineage. No IR boundary expansion or contraction was observed in the major clade A (Fig. 8). Furthermore, Stigmatopteris shares similar morphological characteristics with species with 1-pinnate-lamina in this major clade. Arachniodes bella was found in a separate clade within major clade A, distinct from other species of Arachniodes, indicating that it represents a distinct lineage.

4.2. Morphological evolution in Dryopteridaceae

Although Dryopteridaceae were previously classified into three subfamilies (Liu et al., 2016), no morphological synapomorphies were found to define each subfamily. However, each of the major clade identified here is morphologically definable (Fig. 8). The rachis-costae architecture remains consistent in fresh plants of different major clades and can be served as an identifying feature of Dryopteridaceae, although distortion occurred in dried specimens, which can lead to potential confusion.

Genera in major clade A are predominantly terrestrial, characterized by the dryopteridoid rachis and costae, monomorphic fronds, and rounded sori (Fig. 8) although a few species of some genera have occasional hairs (including bristles and setae), uniseriate scales, and glands on stipe, rachis, and lamina, anastomosing venation, rachis bud, and exindusiate sori. Stigmatopteris and Arachniodes bella were included in the major clade A based on well-supported relationships and shared morphological characteristics (Fig. 8). Ctenitis (major clade D) has ctenitoid hairs and pleocnemioid rachis-costae type, and was identified as the sister to major clades B and C, necessitating its reclassification outside of Dryopteridoideae.

Both major clades B and C also have dryopteridoid rachis-costae type. However, most genera and species within the major clade B are climbing or hemi-epiphytic, with dimorphic fronds, while species of the major clade C are terrestrial, with monomorphic fronds (Fig. 8). Among the two genera of major clade C, Polystichopsis had been merged in Arachniodes (Kramer et al., 1990; Wu and Ching, 1991) or Dryopteris (Tryon and Tryon, 1982), until recent molecular evidence supported its recognition as an independent genus (Schuettpelz and Pryer, 2007; Prado and Moran, 2015). Morphologically, Polystichopsis has pubescent stipe and lamina, which are distinctly from Arachniodes and Dryopteris, as well as from genera of the major clade B. Prior to the acquisition of molecular sequences, although Trichoneuron was placed in Athyriaceae or Thelypteridaceae, it was considered distinctive among all ferns by Ching (1965b) and Pichi-Sermolli (1977). In fact, this Asia-endemic genus shares many morphological similarities with Polystichopsis, such as terrestrial habitat, monomorphic frond, dryopteridoid rachis-costae type, hairs on stipe and lamina, and rounded sori, but Trichoneuron is distinguished by its sessile or sub-sessile pinnae.

Elaphoglossoideae sensu Liu et al. (2016) were clustered into three major clades (Figs. 2 and S2), which is generally in agreement with previous studies (Labiak et al., 2014; Liu et al., 2016), but with strong support for the relationships among the three major clades. In morphology-based classification, Kramer et al. (1990) placed Elaphoglossoideae within Lomariopsidaceae and the latter two groups (the Lastreopsis and allies, and Pleocnemia) in the tribes Tectarieae and Rumohreae (Rumohra) of Dryopteridaceae. The major clade E encompassed all genera of Bolbitidaceae sensu Tryon and Tryon (1982) and Elaphoglossoideae sensu Pichi-Sermolli (1977) and sensu Ching (1978), as well as Lomagramma of Lomariopsidaceae sensu Pichi-Sermolli (1977) and sensu Ching (1978). All sampled species of the major clade E exhibit the bolbitidoid rachis-costae type—ribbed adaxially on upper rachis and costae. The rachis-costae architecture in the major clade F is the lastreopsid type, and that in the major clade G is the pleocnemioid type (Fig. 6, Fig. 8). In addition to differences in rachis-costae architecture, the habit, rhizome shape, frond morphology, soral arrangement, and venation can also be used to distinguish the major clade E, F, and G (Fig. 8). The species of the major clade E are epiphytic, hemi-epiphytic or climbing, mostly with creeping rhizome, dimorphic fronds, and acrostichoid sori, and while those of major clades F and G are terrestrial plants with erect to short-creeping rhizome, monomorphic fronds, and rounded sori. The pleocnemioid rachis-costae architecture, combined with the yellow cylindric glands, distinguishes Pleocnemia as a distinct clade from all other genera of Dryopteridaceae.

One thing to note is that our morphological statistics and evaluations are based on the vast majority of species within each genus, but there may be extreme variations within genera, as observed in cases such as Dryopteris cochleata (D. Don) C. Chr. with dimorphic fronds, Polybotrya semipinnata Fée with catadromous pinnules, and Elaphoglossum piloselloides (C. Presl) T. Moore with hairs on the stipe, rachis, and lamina.

4.3. Historical spatiotemporal dynamics in Dryopteridaceae

The origin of Dryopteridaceae dates back to the early Cretaceous (Du et al., 2021), when the global climate was shifting towards a new climatic condition characterized by increased rainfall and temperatures (Chaboureau et al., 2014). Ancestral area analysis suggests that this family likely originated in Americas. The early diversification of major clades of Dryopteridaceae was influenced by global climate changes, primarily through dispersal and vicariance events. The stems of all major clades emerged successively in the late Cretaceous (98.88–73.43 Ma) (Fig. 7). Although the precipitation rate decreased slightly between 95 Ma and 70 Ma, it maintained high in the Late Cretaceous (Chaboureau et al., 2014). The emergence of new bioclimatic continents set the stage for plant ecological expansion and may eventually lead to successful ecological radiation and plant diversification (Chaboureau et al., 2014).

In the early Cenozoic period, significant transformations of tropical forests occurred (Azuma et al., 2001; Fritsch et al., 2015; Bansal et al., 2022; Lim et al., 2022) and subtropical evergreen broadleaved forests emerged (Yu et al., 2017; Qin et al., 2023), the ancestors of the major clades then spread to Africa, Australia, Pacific islands, and Asia, forming more genera during the Paleocene and the Eocene. As global temperatures increased in the late Paleocene and early Eocene and forest compositions evolved, the major clades B and E of Dryopteridaceae began to occupy different ecological niches, which may have driven the differentiation of hemi-epiphytic and epiphytic genera to adapt to the new environment. As global temperatures dropped in the late Cenozoic, some genera (e.g., Dryopteris and Polystichum) experienced explosive growth as they adapted to the new temperate ecological niche.

4.4. The systematic relationships of four enigmatic taxa

Due to insufficient samples and sequence data in previous studies, some genera of Dryopteridaceae were found to be not monophyletic (Lu et al., 2019), and the phylogenetic placement of some genera remained to be resolved (Liu et al., 2016). Our results address these questions well and provide solid evidence for resolving the systematic relationships of four enigmatic taxa.

4.4.1. Ctenitis

Ctenitis is a unique genus characterized by ctenitoid hairs on the rachis, pinna rachis and prominent costae on pinnules, and has long been treated as a member of Tectariaceae (or "Aspidiaceae") (Ching, 1978; Kramer et al., 1990; Wu and Ching, 1991). Previous studies based on plastid sequences weakly supported Ctenitis as the sister clade to Dryopteridoideae (Liu et al., 2016), suggesting its tentative placement in Dryopteridoideae (Liu et al., 2016; PPG I, 2016). Our study strongly supported Ctenitis as the sister group to six genera of Polybotryoideae sensu Liu et al. (2016) (the major clades B and C) in the present study. Furthermore, the synteny of the plastomes also distinguishes Ctenitis from other genera within Dryopteridsaceae. The rachis-costae architecture of Ctenitis is the pleocnemioid type, and its erect to ascending rhizome is distinct from that of Polybotryoideae sensu Liu et al. (2016) (Fig. 6, Fig. 8). Therefore, we defined Ctenitis as an independent major clade within Dryopteridaceae.

4.4.2. Stigmatopteris

Based on molecular phylogenetic and morphological evidence, we thoroughly settled the systematic location of Stigmatopteris, and suggested that it was removed from the subfamily Polybotryoideae and placed in the subfamily Dryopteridoideae (Fig. 8). The relationship of Stigmatopteris had remained poorly resolved in previous studies (Labiak et al., 2014; Liu et al., 2016; Moran and Labiak, 2016; Lu et al., 2019), and it was tentatively placed in subfamily Polybotryoideae (Liu et al., 2016; PPG I, 2016). In our study, Stigmatopteris is strongly supported as the sister group to all other clades of the major clade A. Morphologically, Stigmatopteris shares some morphological characteristics with other members of the major clade A in terms of monomorphic fronds and discrete round sori.

4.4.3. Arachniodes bella

Arachniodes bella (Ching, 1962) was originally treated as a member of Dryopteris in the early 20th century (Bonaparte, 1925), or placed in Polystichopsis (Humbert, 1958; Tardieu-Blot, 1958). Morphologically, Arachniodes bella shares similarities with Arachniodes (especially A. superba Fraser-Jenk.) in terms of lamina division, anadromously arranged frond segments, and reniform indusia, and also similar to most species of Dryopteris in terms of erect to ascending rhizomes. Most notably, this species has setiform hairs and glands on the stipe, rachis, lamina and indusium (Fig. 9), which distinguishes it not only from Arachniodes and Dryopteris (Table 4), but also from most genera within Dryopteridoideae (except for Stigmatopteris, which has reduced uniseriate hair-like scales). Based on the comprehensive evidence of morphology, molecular phylogeny, and special distribution area, we suggested that Arachniodes bella should be treated as a new genus within Dryopteridaceae in the hereafter taxonomic treatment.

4.4.4. Arthrobotrya

The classification of Arthrobotrya has always been a controversial topic. It was originally separated from Polybotrya (Smith, 1875) based on the presence of articulate pinnae and pinnules, and included only two species, Arthrobotrya articulata and A. wilkesiana (PPG I, 2016). Holttum (1938) noticed the similarity between Arthrobotrya and Teratophyllum, and merged Arthrobotrya in Teratophyllum. He (Holttum, 1938) further divided Teratophyllum into T. sect. "Euteratophyllum" (=T. sect. Teratophyllum; thin rhizome, biseriate leaves, and pinnate acrophylla) and T. sect. Polyseriatae (thicker rhizome, polyseriate leaves, and bipinnate acrophylla), and included Arthrobotrya in T. sect. Polyseriatae with three species (T. articulatum (Hook.) Mett. ex Kuhn, T. brightiae (F. Muell.) Holttum, and T. wilkesianum (Brack.) Holttum). PPG I (2016) treated Arthrobotrya as a separate genus following Moran et al. (2010a). Our finding indicates that T. sect. Polyseriatae is monophyletic.

The inclusion of Teratophyllum brightiae with intermediate morphological traits in Arthrobotrya will lead to instability of the key morphological characteristics distinguishing Teratophyllum from Arthrobotrya (thin/thicker rhizome, biseriate/polyseriate leaves; pinnate/bipinnate acrophylla). Holttum (1966) also reported that these differences were variable between young and adult plants. Moran et al. (2010a) noted that the type of soral paraphysis differs between Teratophyllum and Arthrobotrya (branched vs. simple). However, this characteristic has not been thoroughly tested in all species. Furthermore, Teratophyllum and Arthrobotrya share similar habits and overlapping distribution areas. Therefore, we support treating Arthrobotrya as a synonym for Teratophyllum.

4.5. Infra-familial classification of Dryopteridaceae

The present results underscore the necessity of redefining all major clades by integrating both molecular phylogeny and morphological data. Many families and genera of ferns with distinctive morphological traits were included within Dryopteridaceae according to current and previous phylogenetic studies (Moran et al., 2010a; Labiak et al., 2014; Moran and Labiak, 2015; Liu et al., 2016). Examples include Peranemataceae (Ching, 1978), Bolbitidaceae (Ching, 1978), Elaphoglossaceae (Pichi-Sermolli, 1977; Ching, 1978), and Lomagramma of Lomariopsidaceae (Pichi-Sermolli, 1977; Ching, 1978), as well as two genera (Ctenitis and Pleocnemia) of Tectariaceae (as "Aspidiaceae" [Pichi-Sermolli, 1977; Ching, 1978]). The three-subfamily classification of Dryopteridaceae was based primarily on molecular phylogenetic relationships of three plastid coding regions, but the relationships between the major clades were not strongly supported (Liu et al., 2016). Each subfamily is difficult to define morphologically. Without morphological evidence, it is very challenging to define each subfamily based solely on phylogenetic relationships. By integrating current phylogenetic results (Fig. 2) with morphological data (Fig. 3), we have two possible taxonomic options: either dividing Dryopteridaceae into seven subfamilies or eliminating all subfamilies. Dryopteridaceae currently encompass about 20% of the fern species, whose member genera were previously assigned to different families (Table 1) displaying distinct, even unique morphological characteristics. Therefore, based on the results of morphological and phylogenetic studies, we propose hereafter in the taxonomic treatment a new classification that divides Dryopteridaceae into seven subfamilies, including four new ones (Fig. 8).

Subfam. 1. Dryopteridoideae (Major clade A).

Subfam. 2. Ctenitidoideae (Major clade D).

Subfam. 3. Elaphoglossoideae (Major clade E).

Subfam. 4. Lastreopsidoideae (Major clade G).

Subfam. 5. Pleocnemioideae (Major clade F).

Subfam. 6. Polybotryoideae (Major clade B).

Subfam. 7. Polystichopsidoideae (Major clade C).

5. Taxonomic treatment

Plants evergreen or deciduous, terrestrial, epilithic, hemi-epiphytic, or epiphytic. Rhizomes erect, ascending, creeping, or climbing, dictyostelic, scaly. Fronds calathiform, caespitose, or remote from one another, with pinnules anadromous at base and anadromously or catadromously arranged distally; stipe, rachises, and pinnules articulate or not, scaly, grooved, round or ribbed adaxially, hairy or not. Lamina monomorphic or dimorphic, 1–5-pinnate, or simple, rarely imparipinnate, scaly, glandular, hairy, or glabrous; thinly papery, papery, or leathery. Venation pinnate and free, or variously anastomosing to form 1 to multiple rows of areoles, with or without included veinlets. Sori acrostichoid or discrete, terminal, subterminal, or dorsal on veins, indusiate or exindusiate. Indusia orbicular or reniform or rarely ovoid, superior, lateral, or rarely inferior, sessile or rarely stalked, entire or toothed. Spores monolete, achlorophyllous, with prominent perispore. Chromosome number x = 41.

Seven subfamilies and 24 genera with more than 2100 species: nearly cosmopolitan, but highest diversity found in temperate regions (Dryopteris, Polystichum) and tropical mountain regions (Elaphoglossum).

5.1. Key to subfamilies of Dryopteridaceae

1a. Rachis, pinna rachis (in 2- to more pinnate leaves), and costae (in simple to 1-pinnate leaves) adaxially grooved……………………………………………………………………2

1b. Rachis, pinna rachis (in 2- to more pinnate leaves), and costae (in simple to 1-pinnate leaves) adaxially not grooved, or only grooved near stipe base…………………………………………………5

2a. Rachis and pinna rachis adaxially grooved, with widened bottom (lastreopsid type)…………………………Lastreopsidoideae

2b. Rachis and pinna rachis adaxially deeply grooved, without widened bottom (dryopteridoid type)……………………………..…3

3a. Root climbers (starting on soil, then later scandent) or hemi-epiphytic, rarely terrestrial; rhizomes short or long creeping, fronds dimorphic or sub-dimorphic, sori acrostichoid, rarely discrete……………………………....…………………Polybotryoideae

3b. Terrestrial; rhizomes erect, ascending or short creeping, fronds monomorphic, sori discrete……………………………………4

4a. Rhizome erect, ascending or short creeping, if creeping, base of the stipe only scaly………………………………Dryopteridoideae

4b. Rhizome short creeping, base of the stipe hairy……………………………...………………Polystichopsidoideae

5a. Sori acrostichoid; fronds dimorphic; Stipe base grooved or not, flat to slightly convex on upper stipe, then turned convex, and finally ribbed adaxially on the upper rachis and costae (bolbitidoid type)…………………………………………………Elaphoglossoideae

5b. Sori discrete; fronds monomorphic; Costae and costules adaxially slightly convex, rounded or nearly flat (pleocnemioid type)…………………………….…………………………………………6

6a. Veins anastomosing, with scales and yellow glands on lamina…………………………...…………………………Pleocnemioideae

6b. Veins free, with scales and multicellular ctenitoid hairs on lamina ….………………………………..………………Ctenitidoideae

5.2. Subfamily 1. Dryopteridoideae B.K. Nayar, Taxon 19: 235. 1970

——Type: Dryopteris Adans.

Plants terrestrial. Rhizomes erect, ascending or creeping, apex densely scaly. Stipes densely scaly on the base and the lower parts, and the upper parts clothed with smaller scales, soft hairs or glands. Fronds caespitose or approximate, monomorphic. Laminae simple to 3 to more pinnatifid, herbaceous, papery or nearly leathery; pinnules anadromous or catadromous, rachis and pinna rachis adaxially grooved. Veins free or anastomosing. Sori orbicular, dorsal or rarely terminal. Indusia present or not, reniform or round, superior or inferior, sessile or with a long stalk.

Genera: Arachniodes Blume, Dryopteris Adans., Cyrtomium C. Presl, Phanerophlebia C. Presl, Polystichum Roth, Pseudarachniodes Z.Y. Zuo & Rouhan, Stigmatopteris C. Chr.

Pseudarachniodes Z.Y. Zuo & Rouhan gen. nov. Type: Pseudarachniodes bella (C. Chr.) Z.Y. Zuo & Rouhan (Baisonym: Dryopteris bella C. Chr.) (Fig. 9).

Plants terrestrial. Rhizomes erect, densely clothed with scales. Scales entire, narrowly lanceolate to oblong, scarious, dark brown to dark castaneous, most often contorted. Stipes up to 30 cm, scaly at the base, covered with sparse to dense hairs, most setiform (straight and white) mixed with some shorter glandular ones. Laminae usually 3- or more pinnate (sometimes 2-pinnate-pinnatifid), to ca. 50 × 45 cm, triangular to lanceate, herbaceous to nearly leathery, most basal pinnae symmetrical, pinnules of suprabasal pinnae anadromous, rachis and costae adaxially grooved, adaxial surfaces of leaves subglabrous, rachises, costae and abaxial surface of leaves and veins with a few linear and dark brown scales in addition to sparse to dense hairs similar to those of stipes (setiform and clear), bright castaneous and spherical glands scattered mostly on abaxial surfaces. Veins free. Indusia orbicular-reniform, glandular, deciduous.

Distribution and diversity: Currently with one species, endemic to Madagascar, P. bella (C. Chr.) Z.Y. Zuo & Rouhan.

Pseudarachniodes bella (C. Chr.) Z.Y. Zuo & Rouhan comb. nov.

Baisonym: Dryopteris bella C. Chr. in Bonap., Notes Pteridol. 16: 164, t.8 (1925).

Polystichopsis bella (C. Chr.) Tardieu, Amer. Fern J. 48(1): 33 (1958).

Arachniodes bella (C. Chr.) Ching, Acta Bot. Sin. 10(3): 261 (1962).

Type: Madagascar, H. Perrier de la Bâthie 7439 (P!).

Additional examined specimens: Madagascar, H. Humbert 18182 (P!) & 18513 (P!); H. Humbert et al. 24.718 (P!), F. Rakotondrainibe 1423 (K!, P!) & 1705 (K!, MO!, P!); G. Rouhan et al. 253 (P!) & 257 (P!).

Key to genera of subfamily Dryopteridoideae

1a. Scales with uniseriate cilia on the margins; laminae with internal punctate glands; veins ending in a conspicuous hydathode; indusia absent………………...…………………………Stigmatopteris

1b. Scales without uniseriate cilia on the margins; laminae with or without internal punctate glands; veins without hydathode; indusia present in most species…….…………………………………2

2a. Indusia (when present) reniform, rarely horseshoe-shaped, globose or subglobose; pinnae and/or pinnules lacking a basal acroscopic lobe; lobes or segments not spinulose-tipped…………………………………………………………………………3

2b. Indusia peltate; pinnae and/or pinnules with a basal acroscopic lobe; lobes or segments spinulose-tipped…………………………………………………………………………6

3a. Pinnules of suprabasal pinnae catadromously arranged…………………………….....…………………………Dryopteris

3b. Pinnules of suprabasal pinnae anadromously arranged………………………………………………………..……………4

4a. Rhizomes creeping…………………………………Arachniodes

4b. Rhizomes erect……………………………..……………………5

5a. Laminar tissue with sparse to dense, setiform hairs (especially abaxially) and some small scales…….……Pseudarachniodes

5b. Laminar tissue glabrous or nearly so, only with small scales………………………..…………Dryopteris (sect. Acrorumohra)

6a. Laminae 1–3-pinnate, apices pinnatifid ………Polystichum

6b. Laminae imparipinnate, with an apical pinna somewhat dissected at base…………………………………………………………7

7a. Veins free or with 1–3 series of marginal anastomoses…………………………………………..……………Phanerophlebia

7b. Veins anastomosing, with 2 to more rows of areoles…………………………………………………….………Cyrtomium

5.3. Subfam. 2. Ctenitidoideae Z.Y. Zuo & Jin Mei Lu, subfam. nov.

——Type: Ctenitis (C. Chr.) C. Chr.

Plants terrestrial. Rhizomes erect to ascending, rarely short-creeping, apex densely scaly. Fronds clustered, monomorphic. Stipes scaly at base or throughout. Laminae 1–4-pinnate, herbaceous or papery, lanceolate to triangular, with small scales and hairs on both surfaces or rarely glabrous; costae of pinnules round, always covered with multicellular ctenitoid hairs (often reddish and jointed, twisted), pinnules catadromous, rachis and pinna rachis adaxially flat to rounded. Veins free. Sori medial or rarely submarginal. Indusia present or not, sometimes very small and hidden by maturing sporangia.

Genus: Ctenitis (C. Chr.) C. Chr.

5.4. Subfam. 3. Elaphoglossoideae (Pic. Serm.) Crabbe, Jermy & Mickel, Fern Gaz. 11: 154, 1975

——Type: Elaphoglossum Schott ex J. Sm.

Plants mostly epiphytic, occasionally hemi-epiphytic or climbing. Rhizomes short to long-creeping, densely scaly at apex. Fronds clustered or widely spaced, dimorphic. Stipes mostly scaly at base. Laminae simple, 1-pinnate, rarely 2-pinnate, rachis and costae glabrate or hairy, pinnules catadromous, lower rachis adaxially with or without grooves, gradually convex upwards, and finally adaxially ribbed on the upper rachis and costae; terminal pinna articulated or not. Veins free or anastomosing. Sori acrostichoid. Exindusiate.

Genera: Bolbitis Schott, Elaphoglossum Schott ex J. Sm., Lomagramma J. Sm., Mickelia R.C. Moran, Labiak & Sundue, Teratophyllum Mett. ex Kuhn.

Key to genera of subfamily Elaphoglossoideae

1a. Laminae simple (rarely divided); veins free, rarely anastomosing or connected by their tips to form a submarginal strand………………………………….…………………Elaphoglossum

1b. Laminae pinnate; veins free or anastomosing………………………………………………….…………………2

2a. Plants climbing, with differentiated bathyphylls (juvenile and lower fronds) and acrophylls (mature upper fronds); rhizomes long-creeping; paraphyses present, simple, or stellate or capitate, long-stalked, the stalk of 1 or 2 rows of cells……………………..…3

2b. Plants epiphytic or terrestrial, without differentiated bathyphylls and acrophylls; rhizome ascending or creeping; paraphyses absent, or rarely present, short-stalked, the stalk of 3 rows of cells……………………………………………………………………...…4

3a. Venation of sterile fronds anastomosing; laminae catadromous (or isodromic) toward apex………..……………Lomagramma

3b. Venation of sterile fronds free; laminae often anadromous toward apex………………………………………...……Teratophyllum

4a. Buds usually present near the apices of pinnae or terminal segments………………………………………………………….Bolbitis

4b. Buds absent or present in the "axil" of the pinna (on the base of the costae)………………………………………………….…Mickelia

5.5. Subfam. 4. Lastreopsidoideae Z.Y. Zuo, subfam. nov.

——Type: Lastreopsis Ching.

Plants terrestrial. Rhizomes erect, decumbent, or creeping, scaly at apex. Stipes scaly at base, stipe and other parts densely clothed in soft hairs. Fronds approximate, monomorphic. Laminae 3 or more pinnate, herbaceous or papery, basal pinnae largest, with hairs on both surface; pinnules anadromous or catadromous, rachis and pinna rachis with widened grooves adaxially. Veins free. Sori terminal on veins. Indusia present or not, sometimes very small and hidden by maturing sporangia.

Genera: Lastreopsis Ching, Megalastrum Holttum, Parapolystichum (Keyserl.) Ching, Rumohra Raddi

Notes: Parapolysticum and Lastreopsis (s.s.) are very similar morphologically, and there are no distinct morphological features that distinguish the two genera (Labiak et al., 2014). We can partially distinguish some species of Parapolystichum by the erect rhizome and the buds on the abaxial rachis, which are absent in Lastreopsis. However, these characteristics are not stable between species within Parapolystichum. The rachis of the lamina, pinna rachises, and costules are pubescent adaxially in Megalastrum (Moran and Prado, 2010). Labiak et al. (2014) summarized the rachis-costa architecture of Megalastrum as the Megalastrum type. We consider it as a special form of the Lastreopsid type—the bottom of the groove on rachis is conspicuous or not, and the groove is shallow.

Key to genera of subfamily Lastreopsidoideae

1a. Lamina nearly leathery, hairless; indusia peltate…Rumohra

1b. Lamina herbaceous to papery, with hairs abaxially; indusia reniform, hypoplastic, or exindusiate…………………………………2

2a. Rachis and pinna rachis slightly grooved with a narrow bottom; pinnae and segments often obtuse, toothless; basal basiscopic lobe of the distal pinnules decurrent on the costae with the distalmost veins arising from the pinna rachis (not the costule)………………………………………………………………Megalastrum

2b. Rachis and pinna rachis grooved with widened bottom; pinnae and segments acuminate, usually dentate; basal basiscopic lobe of the pinnules not decurrent, veins arise from the costule…………………………………………………………....……………3

3a. Rhizomes erect, ascending to creeping; upper portion of rachis usually with buds…………….………………Parapolystichum

3b. Rhizomes creeping; upper portion of rachis never with buds……………………………...……………………………Lastreopsis

5.6. Subfam. 5. Pleocnemioideae Z.Y. Zuo, subfam. nov.

——Type: Pleocnemia C. Presl.

Diagnosis: Plants terrestrial. Rhizomes erect or rarely creeping, apex densely covered with linear scales. Fronds clustered, monomorphic. Stipes only scaly at base. Laminae 2–4-pinnate, herbaceous or papery, basal pinnae largest, with yellow cylindric glands along costules and veins abaxially; pinnules catadromous, rachis and pinna rachis rounded adaxially. Veins along costae anastomosing. Sori dorsal on free veins. Indusia present or not.

Genus: Pleocnemia C. Presl.

5.7. Subfam. 6. Polybotryoideae Hong M. Liu & X.C. Zhang, Plant Syst. Evol. 302(3): 330 (2016)

——Type: Polybotrya Humb. & Bonpl. ex Willd.

Plants climbing or hemi-epiphytic. Rhizomes short- to long-creeping, covered with scales. Fronds clustered or widely spaced, dimorphic or slightly dimorphic. Stipes mostly scaly at base. Laminae 1- to 4-pinnate, rachis and costae loosely to densely hairy (but glabrous in Olfersia), pinnules anadromous or catadromous, rachis, pinna rachis and costa sulcate adaxially. Veins free or rarely anastomosing. Sori acrostichoid or round. Indusia round, or exindusiate.

Genera: Cyclodium C. Presl, Maxonia C. Chr., Olfersia Raddi, Polybotrya Humb. & Bonpl. ex Willd.

Notes: Polybotryoideae are characterized by creeping rhizomes and usually strongly dimorphic sterile and fertile leaves. The perines tend to be finely echinulate. The sori of Olfersia and Polybotrya are exindusiate, whereas those of Cyclodium and Maxonia are indusiate, the indusia being round and peltate. Olfersia is atypical in this subfamily because of its glabrous laminae, conform terminal pinna (imparipinnate laminae), and vein tips connected to form a submarginal strand.

Key to genera of subfamily Polybotryoideae

1a. Sterile and fertile fronds subdimorphic; rhizomes short-creeping………………………………………………………..Cyclodium

1b. Sterile and fertile fronds strongly dimorphic; rhizomes long-creeping…………………………………………………………...………2

2a. Sterile laminae imparipinnate, glabrous; vein tips connected by a submarginal connecting strand……….…………………Olfersia

2b. Sterile laminae 2- to 3-pinnate-pinnatifid; vein tips not connected by a submarginal connecting strand………….…………3

3a. Rhizomes with meristeles surrounded by a dark sclerenchymatous sheath; sori non-indusiate……………………Polybotrya

3b. Rhizomes with meristeles not surrounded by a dark sclerenchymatous sheath; sori and indusial round…………..…Maxonia

5.8. Subfam. 7. Polystichopsidoideae Z.Y. Zuo, subfam. nov.

——Type: Polystichopsis (J. Sm.) C. Chr.

Plants terrestrial. Rhizomes creeping, densely covered with linear scales. Stipes densely clothed in soft hairs, with scales only at base. Fronds approximate, monomorphic. Laminae 3 or more pinnate, herbaceous or papery, basal pinnae largest, with hairs on both surface; pinnules anadromous or catadromous, rachis and pinna rachis sulcate adaxially. Veins free. Sori terminal on veins. Indusia present or not, sometimes very small and hidden by maturing sporangia.

Genera: Polystichopsis (J. Sm.) C. Chr., Trichoneuron Ching.

Notes: Morphologically, Polystichopsis and Trichoneuron are very similar and cannot be distinguished significantly. However, they are typically differentiated by geographical isolation. Polystichopsis is endemic to tropical America, whereas Trichoneuron only occurs in Asia.

Key to genera of subfamily Polystichopsidoideae

1a. Pinnae with distinct stalks, and pinnules anadromous or catadromous; only in the Neotropic……….…………Polystichopsis

1b. Pinnae sessile or sub-sessile, and pinnules catadromous; only in Asia………………………………………...………Trichoneuron

6. Conclusions

Based on extensive field collections, the use of herbarium specimens, and better genome skimming technique, this study provides new insights into the phylogenetic relationships among different lineages of Dryopteridaceae. We reconstructed a robust topology of Dryopteridaceae worldwide and identify some morphological synapomorphies that support the lineages (subfamilies) we have identified. The robust phylogenetic backbone and the reconstructed morphological character evolution here provide an extendable framework for future studies of taxonomy, biogeography, and diversification of Dryopteridaceae.

We present a new classification of Dryopteridaceae recognizing 24 genera in seven subfamilies, of which four subfamilies and one genus are newly established. A recent classification of Selaginellaceae into seven subfamilies and 19 genera (Zhou and Zhang, 2023) is comparable to the classification of Dryopteridaceae presented here. As the fern community prepares to update the PPG classification, pteridologists need to consider how to make the classification of ferns more practical. For example, for taxonomic identification of Dryopteridaceae, Polypodiaceae, and Pteridaceae, which each contain more than 1000 species, how can we first determine the higher taxon (family or subfamily) to which a species belongs and then assign it to a lower taxon (genus), rather than identifying the genus first and then assigning it to the corresponding family. The monophyly with morphological synapomorphy is more suitable to define a "natural" familial or infra-familial group.

Acknowledgements

We would like to thank the curators and staff of the CHS, KUN, MO, P, PE herbaria that offered their facilities and help in examining herbarium specimens, and thank Dr. Ran Wei (Institute of Botany, Chinese Academy of Sciences) for sample collection, Robbin C. Moran (The New York Botanical Garden) for discussions on the morphology of Dryopteridaceae, improving the manuscript, and providing photographs of several neotropical genera. We also thank the Experimental Center of Molecular Biology of the Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences for suggestion of laboratory work, and the iFlora High Performance Computing Center of the Germplasm Bank of Wild Species (iFlora HPC Center of GBOWS, KIB, CAS) for computing. Madagascar collecting permits were granted to GR by Madagascar National Parks and the "Ministère de l'Environnement, de l'Ecologie et des Forêts". The authors are also grateful for field assistance from CNRE-Madagascar and MBG-Madagascar. The MNHN gives access to the collections in the framework of the RECOLNAT National Research Infrastructure. The study was funded by the National Natural Science Foundation of China (Grant No. 31970232) and the National Key Basic Research Program of China (Grant No. 2014CB954100).

Data availability

Data will be made available on request.

CRediT authorship contribution statement

Zheng-Yu Zuo: Writing – original draft, Software, Methodology, Formal analysis, Data curation, Conceptualization. Germinal Rouhan: Writing – review & editing, Resources, Project administration. Shi-Yong Dong: Writing – review & editing, Resources. Hong-Mei Liu: Writing – review & editing, Supervision. Xin-Yu Du: Methodology. Li-Bing Zhang: Writing – review & editing, Resources, Project administration, Investigation. Jin-Mei Lu: Writing – original draft, Writing – review & editing, Visualization, Validation, Supervision, Resources, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare no conflict of interest.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.pld.2024.07.010.