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Research Article
Phylogenetic analyses place the monotypic Dryopolystichum within Lomariopsidaceae
expand article infoCheng-Wei Chen, Michael Sundue§, Li-Yaung Kuo|, Wei-Chih Teng, Yao-Moan Huang
‡ Taiwan Forestry Research Institute, Taipei, Taiwan
§ University of Vermont, Burlington, United States of America
| National Taiwan University, Taipei, Taiwan
¶ Unaffiliated, Nantou, Taiwan
Open Access

Abstract

The monotypic fern genus Dryopolystichum Copel. combines a unique assortment of characters that obscures its relationship to other ferns. Its thin-walled sporangium with a vertical and interrupted annulus, round sorus with peltate indusium, and petiole with several vascular bundles place it in suborder Polypodiineae, but more precise placement has eluded previous authors. Here we investigate its phylogenetic position using three plastid DNA markers, rbcL, rps4-trnS, and trnL-F, and a broad sampling of Polypodiineae. We also provide new data on Dryopolystichum including spore number counts, reproductive mode, spore SEM images, and chromosome counts. Our maximum-likelihood and Bayesian-inference phylogenetic analyses unambiguously place Dryopolystichum within Lomariopsidaceae, a position not previously suggested. Dryopolystichum was resolved as sister to a clade comprising Dracoglossum and Lomariopsis, with Cyclopeltis as sister to these, but clade support is not robust. All examined sporangia of Dryopolystichum produced 32 spores, and the chromosome number of sporophyte somatic cells is ca. 164. Flow cytometric results indicated that the genome size in the spore nuclei is approximately half the size of those from sporophyte leaf tissues, suggesting that Dryopolystichum reproduces sexually. Our findings render Lomariopsidaceae as one of the most morphologically heterogeneous fern families. A recircumscription is provided for both Lomariopsidaceae and Dryopolystichum, and selected characters are briefly discussed considering the newly generated data.

Keywords

Fern, morphology, Papua New Guinea, phylogeny, recircumscription, taxonomy, the Solomon Islands

Introduction

Dryopolystichum Copel., with its single species D. phaeostigma (Ces.) Copel., is distributed along streams in lowland forests in New Guinea, the Bismarck Archipelago, and the Solomon Islands (Copeland 1947; Fig. 1A). Christensen (1937) was the first to point out that D. phaeostigma had been independently described under three different genera or subgenera. All told, generic placements has included Aspidium (≡ Tectaria) (Cesati 1877, Baker 1891), Dryopteris (Christensen 1906, Alderwerelt van Rosenburgh 1908, Copeland 1911, Brause 1920, Alderwerelt van Rosenburgh 1924), and Polystichum (Rosenstock 1911). Copeland (1947) inaugurated the new monotypic genus Dryopolystichum in his Genera Filicum, and argued that it was closest to Ctenitis. Pichi Sermolli (1977) agreed, citing the ctenitoid rachis, free venation, and peltate indusium as critical characters. Holttum included the genus in his “Tectarioid Group” in his list of Malaysian pteridophytes (Holttum 1959), but then omitted it in his 1991 treatment of that group.

Although Copeland did not provide an etymological explanation, the name Dryopolystichum presumably reflects the combination of peltate indusium (which is similar to those of polystichoid ferns) and pinnate-pinnatifid lamina division (which is similar to that of most Dryopteris). Such a combination of characters resulted in taxonomic confusion giving that peltate indusia are never found in Dryopteris, and the laminae of Dryopolystichum do not include prominulous segment apices, the hallmark of polystichoid ferns (Little and Barrington 2003). A peltate indusium is diagnostic of polystichoid ferns, including Phanerophlebia and Polystichum, but also found in a few distantly related genera in Polypodiineae such as Cyclodium, Cyclopeltis, Rumohra, Megalastrum, and Tectaria (Kramer and Green 1990).

Figure 1.

Dryopolystichum phaeostigma (based on SITW10443). A Habitat B Plants C Peltate indusia D Venation E Sulcate rachis-costa architecture F Longitudinal section of the rhizome.

Despite recent advances in fern phylogenetics and classification, the position of Dryopolystichum remains unclear. The thin-walled sporangium with a vertical and interrupted annulus, round sorus, and petiole with several vascular bundles suggest that this genus belongs to suborder Polypodiineae (= eupolypods I) (Sundue and Rothfels 2014, PPG I 2016). However, the remaining prominent features including pinnate-pinnatifid leaf dissection (Fig. 1B), peltate indusium (Fig. 1C), catadromous free veins (Fig. 1D), and sulcate rachis-costa architecture (Fig. 1E), do not clearly place it within any Polypodiinae family (Christensen 1937, Copeland 1947).

One other conspicuous character of Dryopolystichum not emphasized by previous authors is that the distal pinnae are decurrent onto the rachis, and the basal pinnules of its distal pinnae are served by veins that emerge from the rachis, rather than the pinna costa (Fig. 1D). This character is relatively uncommon in the Polypodiineae. It can be found in Dryopteridaceae, mostly in Megalastrum, and less commonly in Stigmatopteris, Ctenitis, and Pleocnemia (Moran et al. 2014, Moran and Labiak 2016). It can also be found in some Tectariaceae such as Pteridrys and Tectaria (Ding et al. 2014). Among these genera, Pleocnemia seems morphologically the most similar to Dryopolystichum because its rachises are adaxially sulcate and narrowly winged laterally. Pleocnemia, however, lacks a peltate indusium (Holttum 1974).

Subsequent to its establishment as a new genus in Genera Filicum (Copeland 1947), and Sermolli’s (1977) contribution, no other substantial argument was made for generic placement of Dryopolystichum. More recent studies maintained Dryopolystichum as a distinct genus, placing it under Dryopteridaceae (Kramer and Green 1990, Smith et al. 2006, Christenhusz et al. 2011). The recently published community-derived classification for extant lycophytes and ferns also places Dryopolystichum in the Dryopteridaceae but without assigning it to subfamily (PPG I 2016).

To resolve the phylogenetic placement of Dryopolystichum, we employ a molecular phylogenetic approach using three chloroplast DNA regions, rbcL, rps4-trnS, and trnL-F. Based on our observations, we further provide new data on Dryopolystichum including spore counts, reproductive mode, spore SEM images, and a chromosome count. Finally, we discuss its diagnostic characters in the light of the inferred phylogeny.

Materials and methods

We examined the morphology of Dryopolystichum phaeostigma using material collected from the Solomon Islands (Braithwaite R.S.S.4557, SING; SITW10443, BSIP, TAIF, TNM) and Papua New Guinea (James & Sundue 1688, BISH, LAE, VT).

Living plants of SITW10443 were transplanted to the Dr. Cecilia Koo Botanic Conservation Center in Taiwan (KBCC). The collection of SITW10443 was made under the “Census and Classification of Plant Resources in the Solomon Islands” project (http://siflora.nmns.edu.tw/). Mitotic chromosomes were counted from these cultivated plants following the protocol of Chen et al. (2014).

Fertile pinnae of SITW10443 were air-dried in an envelope for one day to release the spores. The spores were observed and measured by a tabletop scanning electron microscope (TM-3000 Hitachi, Ibaraki, Japan). The sizes (the length of equatorial axes including the perine ornamentation) of 35 randomly selected spores were measured. Five intact sporangia were observed under a stereo microscope (Leica MZ6, Wetzlar, Germany) to count the number of spores per sporangium.

The genome sizes of spore and leaf nuclei of SITW10443 were examined by flow cytometry in order to infer the reproductive mode (Kuo et al. 2017). The genome size of spore nuclei should be half the genome size of leaf nuclei in the case of sexual and the same size in the case of apomictic reproduction (Kuo et al. 2017). We followed Kuo et al. (2017) for the extraction of leaf nuclei. For extraction of spore nuclei, we used an optimized bead-vortexing treatment with vertex duration of 1 minute and vertex speed of 1,900 rpm, as described by Kuo et al. (2017). An external standard was not necessary since we only need to compare the two phases of the life-cycle to each other.

DNA extraction, amplification and sequencing

Total DNA was extracted using a modified CTAB-Qiagen column protocol (Kuo 2015). Three plastid DNA regions, rbcL, rps4-trnS (rps4 gene + rps4-trnS intergenic spacer), and trnL-F (trnL gene + trnL-trnF intergenic spacer), were amplified and sequenced using the primers “ESRBCL1F” and “1379R” for rbcL (Pryer et al. 2001, Schuettpelz and Pryer 2007), “RPS5F” and “TRNSR” for rps4-trnS (Nadot et al. 1995, Smith and Cranfill 2002), and “FernL 1Ir1” and “f” for trnL-F (Taberlet et al. 1991, Li et al. 2010).

The PCR amplifications were performed in 16 μl reactions containing ca. 10 ng template DNA, 1×Taq DNA Polymerase Master Mix RED solution (Ampliqon, Denmark), and 1 μl each of 10 μM primers. The PCR reactions were carried out in a GeneAmp PCR System 9700 (Applied Biosystems, Carlsbad, California, USA). Thermocycling conditions were the same for PCRs of these three regions and comprised an initial denaturation of 2 minutes at 94°C followed by a core sequence of 35 repetitions of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute followed by a final extension of 10 minutes at 72°C. Resulting PCR products were sequenced using the same PCR primers with BigDyeTM terminator (Applied Biosystems, Carlsbad, California, USA). The newly generated sequences were deposited in GenBank. GenBank accession numbers and voucher information are provided in Appendix.

DNA alignment and phylogenetic analyses

Initial BLAST against the NCBI nucleotide database (Altschul et al. 1990) based on rbcL sequences indicated that Dryopolystichum phaeostigma is closely related to the species of Polypodiineae families, including Lomariopsidaceae, Nephrolepidaceae, Tectariaceae, and Dryopteridaceae. Accordingly, we assembled a data matrix including 250 species representing 36 genera from these families (Appendix). Sampling included all the four genera in which D. phaeostigma has been placed (i.e., Dryopteris, Polystichum, and Tectaria).

Sequences were aligned using Geneious v6.1.8 (Drummond et al. 2011) and then manually checked for errors. The three single-region (rbcL, rps4-trnS, and trnL-F) and dataset combining all three were independently subjected to both maximum likelihood (ML) and Bayesian inference (BI) phylogenetic analyses. Data matrices are available in TreeBASE, study number 20506, at https://treebase.org/. ML tree searches were conducted using RAxML (Stamatakis 2006) employing the GTRGAMMA substitution model through the CIPRES portal (Miller et al. 2010). Five independent searches for the ‘best tree’ and 1,000 bootstrap replicates were performed using a region-partitioned dataset. BI analyses were conducted using MrBayes 3.2.1 (Ronquist and Huelsenbeck, 2003) employing the same substitution model as in ML analysis. Each analysis consisted of two independent runs with four chains for 106 generations, sampling one tree every 1000 generations. Burn-in was set to 10000 based on our preliminary analysis. The convergences of MCMC runs were checked using Tracer v.1.6 (Rambaut et al. 2014).

We addressed the possibility of phylogenetic bias due to long branches following the recommendation of Siddal and Whiting (1999). Since Dracoglossum and Lomariopsis were resolved on long branches in preliminary analyses (not shown), we conducted two additional analyses in which each one of the two long-branched genera, Dracoglossum and Lomariopsis, was excluded to examine whether phylogenetic placement and branch support for Dryopolystichum’s placement changed. Since maximum parsimony (MP) phylogeny is considered to be more susceptible to long-branch attraction (Philippe et al. 2005), we analyzed the concatenated dataset under MP in order to compare those results with our ML phylogeny. The MP analyses were conducted using TNT (Goloboff et al. 2008) following the search strategy detailed in Sundue et al. (2014).

Results

Phylogenetic analyses

All single-region phylogenies resolved Dryopolystichum phaeostigma in Lomariopsidaceae, but with two slightly different topologies. The rbcL and rps4-trnS phylogenies placed D. phaeostigma sister to a clade of Dracoglossum + Lomariopsis with 93% and 72% maximum likelihood bootstrap percentages (BS), respectively (Suppl. materials 2, 3). In comparison, the trnL-F phylogeny placed D. phaeostigma sister to Cyclopeltis (BS = 74%), and Dryopolystichum + Cyclopeltis was sister to Dracoglossum + Lomariopsis (Suppl. material 4). There was no strongly supported conflict between the ML and BI phylogenies (Suppl. materials 14). Both the ML and BI phylogenies based on the combined dataset (Fig. 2, Suppl. material 1) reveal the same topology as those based on the rbcL and rps4-trnS regions. Bootstrap support and posteriori probability (PP) for the above relationships were generally very high except for the branches placing D. phaeostigma, where BS was ≤ 70% and PP were ≤ 0.9 in all the phylogenies.

Figure 2.

Simplified maximum likelihood phylogram of Polypodiineae obtained from the rbcL + rps4-trnS + trnL-F combined dataset. Maximum likelihood bootstrap percentages (BS) are provided at each node. Thickened lines indicate Bayesian inference posterior probability (PP) ≥ 0.9. Original phylogram with support values for all the nodes is available in Suppl. materials 1. Voucher information and GenBank accession numbers are shown in Appendix.

Removing Dracoglossum from the analysis had little effect on the topology within Lomariopsidaceae, and BS supports for the generic placement of Dryopolystichum remained low (≤ 70%, data not shown). In contrast, the removal of Lomariopsis resulted in higher BS values for all clades within Lomariopsidaceae (≥ 99%, data not shown). MP analyses also resulted in a clade comprising all the Lomariopsidaceae genera and Dryopolystichum, but Dryopolystichum was resolved as sister to Cyclopeltis (data not shown).

Karyology, reproductive mode, and spore measurements

All examined sporangia (SITW10443) produced 32 normal spores, and the mean spore length was 64.1 ± 4.5 μm (Fig. 3). The chromosome number of the three sporophyte somatic cells observed was ca. 164 (Fig. 4). Results of flow cytometry revealed that the genome size of spore nuclei is approximately half of those of leaf nuclei (Fig. 5).

Discussion

Phylogenetic placement of Dryopolystichum

The reconstructed maximum likelihood and Bayesian inference phylogenies unambiguously resolved Dryopolystichum within Lomariopsidaceae (Fig. 2), a position not previously suggested (Kramer and Green 1990, Smith et al. 2006, Christenhusz et al. 2011, PPG I 2016). This placement is consistent in all our analyses. Nonetheless, the generic position of Dryopolystichum within Lomariopsidaceae remains poorly resolved. This uncertainty may be partially explained by the incongruence between trnL-F and the other analyzed regions, but our process of removing the long-branched genera showed that low BS was retrieved only when Dryopolystichum and Lomariopsis were both included in the analysis. These results may also be explained by the large amounts of missing data in Lomariopsis; 19 of the 25 species included were represented by trnL-F data alone. We recommend further phylogenetic study using an expanded dataset to resolve the intergeneric relationships within Lomariopsidaceae.

Recircumscription of Lomariopsidaceae

Phylogenetic analyses using DNA sequences have served as the basis for redrawing fern classifications in the 21th century (Smith et al. 2006, Christenhusz et al. 2011, PPG I 2016). With respect to family circumscription, one of the most dramatically changed families is Lomariopsidaceae (Tsutsumi and Kato 2006, Schuettpelz and Pryer 2007, Christenhusz et al. 2013). Just prior to the molecular era, Lomariopsidaceae was treated as one of the largest fern families with six genera and over 500 species (e.g., Kramer and Green 1990) and was strongly supported by the following combination of characters: rhizomes with ventral root insertion, dictyosteles with elongate ventral meristeles, and dimorphic leaves where the fertile leaves had acrostichoid sori (Holttum and Hennipman 1959, Kramer and Green 1990).

Subsequent molecular phylogenetic analyses demonstrated that most genera previously treated in Lomariopsidaceae should be transferred to Dryopteridaceae (Tsutsumi and Kato 2006, Schuettpelz and Pryer 2007). The combination of characters uniting the former Lomariopsidaceae are now interpreted to have evolved multiple times, and to be correlated with dorsiventrality of the rhizome (Moran et al. 2010, McKeown et al. 2012). Meanwhile, Cyclopeltis was transferred from Dryopteridaceae to Lomariopsidaceae as suggested by molecular phylogeny (Schuettpelz and Pryer 2007), although it has none of the characters formerly used to circumscribe Lomariopsidaceae (Holttum and Hennipman 1959, Kramer and Green 1990).

More recently, the neotropical genus Dracoglossum was established (Christenhusz 2007) and later transferred to Lomariopsidaceae from Tectariaceae based on a molecular phylogeny (Christenhusz et al. 2013). This pattern was also unexpected since there are essentially no shared morphological characters by Dracoglossum and Lomariopsis, except for the ribbon-like gametophyte (R. C. Moran pers. com.). Our finding, that Dryopolystichum belongs to Lomariopsidaceae, comes as a further surprise. With these changes, Lomariopsidaceae is a family of five genera (Cyclopeltis, Dracoglossum, Dryopolystichum, Lomariopsis, and Thysanosoria) and ca. 70 species. As far as we can tell, none of the morphological traits commonly used unify these genera (Table 1). In the following paragraphs, we provide a recircumscription of both Lomariopsidaceae and Dryopolystichum, and then discuss selected characters in the light of our phylogenetic placement.

Comparison of morphological characters of the five Lomariopsidaceae genera [based on Holttum and Hennipman (1959), Holttum (1991), Roubik and Moreno (1991), Moran (2000), Christenhusz (2007), Rouhan et al. (2007), and this study].

Genera Cyclopeltis Dracoglossum Dryopolystichum Lomariopsis Thysanosoria
Habit terrestrial terrestrial terrestrial hemiepiphyte hemiepiphyte
Rhizome erect short creeping erect climbing climbing
Frond division* pinnate simple pinnate-pinnatifid pinnate pinnate
Pinnae articulation articulate not articulate articulate articulate
Venation free reticulate, with included veinlet free free free
Rachis-costa architecture prominent prominent grooved grooved or flat grooved
Sporangia form rounded sori form rounded sori form rounded sori acrostichoid form rounded sori
Indusia peltate if present peltate if present peltate absent absent
Perine ornamentation broad folds narrow crests narrow crests various broad folds

Taxonomic treatment

Lomariopsidaceae Alston, Taxon 5(2): 25. 1956.

Type

Lomariopsis Fée, Mém. Foug., 2. Hist. Acrostich.: 10. 1845.

Description

Habit erect, creeping, or climbing; rhizomes dictyostelic, the ventral meristele elongate in transverse section or not; scaly at least when young; scales non-clathrate, basally attached or shallowly peltate, margins entire, toothed, or ciliate; fronds monomorphic or dimorphic; petioles with multiple vascular bundles arranged in a U-shape; laminae simple, pinnate, or pinnate-pinnatifid, provided distally with proliferous buds or not; pinnae articulate to the rachis or not; veins free, ± parallel or pinnate; sori acrostichoid or discrete and then round, with peltate indusia or exindusiate; spores brown, olive or green, chlorophyllous or not, bilateral, monolete, perine loosely attached, variously winged or ornamented.

Five genera and an estimated 70 species. Thysanosoria is included based on its morphological similarity to Lomariopsis (Holttum and Hennipman 1959), but it has not been, to the present, subject to molecular phylogenetic analysis.

Dryopolystichum Copel., Gen. Fil. 125, t. 4. 1947.

Type

Dryopolystichum phaeostigma (Ces.) Copel., Gen. Fil. 125, t. 4. 1947.

Description

Habit terrestrial, on slopes along streams at lowland forests; rhizome short erect, stout and woody, apex densely scaly, blackish sclerenchyma strands visible in sections; scales dark brown, linear-lanceolate, entire, not clathrate; fronds approximate, stipe not articulate, scaly at base, scales similar to those on rhizome; lamina ovate, pinnate-pinnatifid, catadromous, subleathery, nearly glabrous, only very sparse narrow scales on rachis, costa, and costule; rachis and costa grooved adaxially, not connected to each other; veins free, pinnate, veins of basal pinnules on upper pinnae emerge from the rachis rather than costa, all veins terminating in a prominent hydathode, not reaching frond margin; sori round, dorsally on veinlets near hydathode, indusiate; indusia round, persistent, superior, entire, brownish, thick; sporangia long-stalked, annulus with ca. 14 indurated cells, 32 normal spores in each sporangium; spores monolete, 64.1 ± 4.5 μm in lateral view, surface with broadly winged wall; 2n = ca. 164.

Monotypic.

Dryopolystichum phaeostigma (Ces.) Copel., Gen. Fil. 125, t. 4. 1947.

Aspidium phaeostigma Ces., Rend. Ac. Napoli 16: 26, 29. 1877.

Type. Papua New Guinea. Andai, Beccari 12533 (FI [FI013622]).

Dryopteris phaeostigma (Ces.) C.Chr., Index Filic. 284. 1905

Type. Based on Aspidium phaeostigma Ces.

Dryopteris tamatana C.Chr., Index Filic., Suppl. (1906-1912) 40. 1913.

Replaced: Dryopteris kingii Copel., Phillipp. J. Sci., C 6: 73. 1911., not Dryopteris kingii (Bedd.) C.Chr., Index Filic. 273. 1905.

Type. Papua New Guinea. Tamata, C. King 149 (MICH [MICH1287049]).

Polystichum lastreoides Rosenst., Repert. Spec. Nov. Regni Veg. 9: 425. 1911.

Type. Papua New Guinea. C. King 194 (MICH [MICH1190927]).

Dryopteris ledermannii Brause, Bot. Jahrb. Syst. 56: 90. 1920.

Type. Papua New Guinea. Sepik, Ledermann 9619 (B [B_20_005865], L [L0063060], S [S-P-8581]).

Dryopteris cyclosorus Alderw., Nova Guinea 14: 21. 1924.

Type. Indonesia. Irian Jaya, H. J. Lam 1086 (BO [BO1529719, BO1529720], K [K000666126], L [L0051583], U [U0007385]).

Type

Based on Aspidium phaeostigma Ces.

Description

Equal to the genus.

Distribution

New Guinea, the Bismark archipelago, and the Solomon Islands.

Comparison of selected characters of Dryopolystichum

Perine architecture of Dryopolystichum is very similar to that of Dracoglossum plantagineum (Christenhusz 2007, Fig. 3). They are loosely attached, forming thin crests, and having a spiculate microstructure. Perine of Cyclopeltis and Thysanosoria are also similar in being loosely attached and having a spiculate microstructure, but they differ by having broader folds (Holttum and Hennipman 1959, Tryon and Lugardon 1991). The perine characters, however, are not shared by all the taxa of Lomariopsidaceae especially considering the variation of ornamentation existing in Lomariopsis (Rouhan et al. 2007). Moreover, these perine characters also appear in other Polypodiineae lineages particularly in bolbitidoid ferns (Moran et al. 2010) as well as in various Aspleniineae lineages (Sundue and Rothfels 2014, PPG I 2016).

Figure 3.

Spores SEM of Dryopolystichum phaeostigma. A Lateral view of the spore B Detail of surface. Scale bars: A = 50 μm, B = 10 μm.

Blackish sclerenchyma strands are visible in the rhizome sections of Dryopolystichum (Fig. 1F). These are also present in Dracoglossum, Cyclopeltis, and Lomariopsis, but similar characters are known from various groups throughout Polypodiineae (Hennipman 1977, Moran 1986, Hovenkamp 1998). Further studies might reveal variation in these strands to be of systematic value.

The rachis-costae architecture of Dryopolystichum is characterized by an adaxially sulcate rachis with grooves that do not connect to those of the pinna-costae. The rachis is also narrowly winged laterally. Both characters are seen in Thysanosoria and in some species of Lomariopsis (Holttum and Hennipman 1959, Moran 2000). In contrast, Dracoglossum and Cyclopeltis have non-winged and non-sulcate rachises (Holttum 1991, Christenhusz 2007).

The chromosome number in somatic cells of Dryopolystichum phaeostigma was ca. 164 (Fig. 4). The base numbers for Lomariopsidaceae genera (Cyclopeltis, Dracoglossum, and Lomariopsis) are 40 or 41 (Walker 1985, Kato and Nakato 1999, Moran 2000), suggesting that D. phaeostigma is a tetraploid.

Figure 4.

Chromosome number of Dryopolystichum phaeostigma. A Chromosomes at mitosis metaphase, 2n = ca. 164 (SITW10443) B explanatory illustration of A. Scale bars = 10 μm.

Our flow cytometry and spore count results indicate that Dryopolystichum phaeostigma is sexually reproducing and has 32 spores per sporangium (Fig. 5). In Polypodiales, sporogenesis leading to the formation of 64 spores in a sporangium is by far the most common pattern of sexually reproducing species, e.g., Aspleniaceae (Gabancho et al. 2010), Athyriaceae (Kato et al. 1992, Takamiya et al. 1999), Davalliaceae (Chen et al. 2014), Dryopteridaceae (Lu et al. 2006), Polypodiaceae (Wang et al. 2011), Pteridaceae (Huang et al. 2006), and Thelypteridaceae (Ebihara et al. 2014). Cases of sporogenesis resulting in 32 spores per sporangium are known from a few Polypodiales ferns but all belong to the suborders Lindsaeineae and Pteridineae, i.e., Lindsaeaceae (Lin et al. 1990), Cystodiaceae (Gastony 1981), and Ceratopteris (Pteridaceae; Lloyd 1973). Our study provides the first confirmed case of a sexual reproduction with 32 spores per sporangium in the suborder Polypodiineae.

Figure 5.

Relative DNA contents of Dryopolystichum phaeostigma spore and leaf nuclei inferred by flow cytometry.

Conclusion

We have shown, based on molecular phylogenetic evidence, the placement of Dryopolystichum within Lomariopsidaceae. A revised description was provided for both Lomariopsidaceae and Dryopolystichum resulting from a review of literature and our own observations. Future studies using an expanded dataset are necessary to resolve intergeneric relationships in Lomariopsidaceae.

Acknowledgements

We are grateful to Kathleen Pryer’s lab for sharing the material of Dracoglossum. Peter Hovenkamp, Thais Almeida, David Barrington, and two anonymous reviewers provided valuable comments on an earlier draft of this manuscript. The curators and staffs of herbaria BSIP, SING, TAIF, and VT for providing access to their collections. We also thank Robbin Moran and Wita Wardani for checking specimens at NY and BO, respectively. Field work in Solomon Islands was supported by Taiwan International Cooperation and Development Fund (TH410-2012-085), Taiwan Forestry Research Institute (102AS-4.1.1-FI-G1), and Dr. Cecilia Koo Botanic Conservation Center (KBCC) for CWC.

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Appendix

Individuals sampled in this study. For each individual, the species name and GenBank accession numbers (rbcL, rps4-trnS, trnL-F) are provided. A n-dash (–) indicates unavailable information; new sequences are in bold.

Taxon Genbank accession numbers
rbcL rps4-trnS trnL-F
Dryopteridaceae
Arachniodes aristata (G.Forst.) Tindale KJ464418 KJ464592
Arachniodes denticulata (Sw.) Ching KJ464419 KJ464593
Arthrobotrya articulata J.Sm. GU376714 GU376565
Arthrobotrya wilkesiana Copel. GU376719 GU376569
Bolbitis acrostichoides (Afzel.) Ching KJ464420 GU376644 GU376500
Bolbitis aliena (Sw.) Alston GU376646 GU376502
Bolbitis angustipinna (Hayata) H.Ito GU376654 GU376509
Bolbitis appendiculata (Willd.) K.Iwats. GU376647 GU376503
Bolbitis auriculata (Lam.) Alston KJ464421 GU376649 GU376505
Bolbitis bipinnatifida (J.Sm.) K.Iwats. GU376676 GU376530
Bolbitis fluviatilis (Hook.) Ching GU376656 GU376510
Bolbitis gemmifera (Hieron.) C.Chr. GU376657 GU376511
Bolbitis heteroclita (Pr.) Ching GU376659 GU376513
Bolbitis heudelotii (Bory) Alston GU376662 GU376515
Bolbitis humblotii (Baker) Ching KJ464422 GU376663 GU376516
Bolbitis lonchophora (Kunze) C.Chr. GU376664 GU376517
Bolbitis major (Bedd.) Hennipman GU376665 GU376518
Bolbitis portoricensis (Sprengel) Hennipman GU376670 GU376523
Bolbitis salicina (Hook.) Ching GU376671 GU376525
Bolbitis semipinnatifida (Fée) Alston GU376672 GU376526
Bolbitis serratifolia (Mertens) Schott GU376673 GU376527
Bolbitis sinuata (C.Presl) Hennipman GU376675 GU376529
Bolbitis tibetica Ching & S.K.Wu GU376677 GU376531
Ctenitis eatonii (Baker) Ching KF709483 KJ196645
Ctenitis sinii (Ching) Ohwi KJ196643
Ctenitis subglandulosa (Hance) Ching KJ196655
Ctenitis yunnanensis Ching & Chu H.Wang KJ196715
Cyclodium heterodon (Schrad.) T. Moore var. heterodon KJ464425 KJ464596
Cyclodium rheophilum A.R.Sm. KJ464426 KJ464597
Dryopteris apiciflora (Wall. ex Mett.) Kuntze KJ196641
Dryopteris christensenae (Ching) Li Bing Zhang KJ196679
Dryopteris heterolaena C.Chr. KJ196623
Dryopteris integriloba C.Chr. KJ196701
Dryopteris mariformis Rosenst. KJ196686
Dryopteris nidus (Baker) Li Bing Zhang KJ196687
Dryopteris patula (Sw.) Underw. KJ464427 KJ464598
Dryopteris polita Rosenst. KJ196700
Dryopteris squamiseta (Hook.) Kuntze GU376678 KJ196632
Dryopteris wallichiana (Spreng.) Hyl. KJ464428 GU376680 KJ464599
Elaphoglossum amygdalifolium (Mett.) Christ GU376681
Elaphoglossum burchellii (Baker) C.Chr. GU376682 GU376533
Elaphoglossum decoratum (Kunze) T.Moore KJ464429 GU376683 KJ464600
Elaphoglossum guentheri Rosenst. GU376684 GU376535
Elaphoglossum langsdorffii T.Moore GU376685 GU376536
Elaphoglossum lloense (Hook.) T.Moore GU376686 GU376537
Elaphoglossum luridum Christ GU376538
Elaphoglossum squamipes (Hook.) T.Moore GU376539
Lastreopsis amplissima (C.Presl) Tindale KJ464432 KJ464604
Lastreopsis decomposita (R.Br.) Tindale KJ464439
Lastreopsis hispida (Sw.) Tindale KJ464446 KJ464614
Lastreopsis killipii (C.Chr. & Maxon) Tindale KJ464448 KF709505
Lastreopsis marginans (F.Muell.) Tindale KJ464449 GU376691 KJ464616
Lastreopsis poecilophlebia (Hook.) Labiak, Sundue & R.C.Moran KJ464423 GU376692 KJ464594
Lastreopsis tenera (R.Br.) Tindale KJ464467 GU376699 KJ464636
Lastreopsis tripinnata (F.Muell. ex Benth.) Labiak, Sundue & R.C.Moran KJ464491 GU376700
Lastreopsis walleri Tindale KJ464472 GU376701
Lastreopsis wurunuran (Domin) Tindale KJ464474 GU376704
Lomagramma brooksii Copel. GU376705 GU376542
Lomagramma cordipinna Holttum GU376707 GU376543
Lomagramma lomarioides (Blume) J.Sm. GU376550
Lomagramma matthewii (Ching) Holttum KJ464476 KJ464640
Lomagramma perakensis Bedd. GU376552
Lomagramma pteroides J.Sm. GU376555
Lomagramma sinuata C.Chr. GU376556
Lomagramma sumatrana Alderw. GU376558
Maxonia apiifolia (Sw.) C.Chr. KJ464477 GU376709 KJ464641
Megalastrum abundans (Rosenst.) A.R.Sm. & R.C.Moran KJ464478 KJ464642
Megalastrum atrogriseum (C.Chr.) A.R.Sm. & R.C.Moran KJ464479 GU376710 KJ464643
Megalastrum connexum (Kaulf.) A.R.Sm. & R.C.Moran KJ464481 KJ464645
Megalastrum lanatum (Fée) Holttum KJ464483 KJ464647
Megalastrum littorale R.C.Moran, J.Prado & Labiak GU376651 GU376561
Megalastrum macrotheca (Fée) A.R.Sm. & R.C.Moran KJ464484 GU376697 KJ464648
Megalastrum vastum (Kunze) A.R.Sm. & R.C.Moran KJ464487 GU376658 KJ464651
Mickelia bernoullii (Kuhn ex Christ) R.C.Moran, Labiak & Sundue GU376666 GU376506
Mickelia guianensis (Aubl.) R.C.Moran, Labiak & Sundue GU376667 GU376548
Mickelia hemiotis (Maxon) R.C.Moran, Labiak & Sundue GU376512
Mickelia nicotianifolia (Sw.) R.C.Moran, Labiak & Sundue KF667557 GU376519
Mickelia oligarchica (Baker) R.C.Moran, Labiak & Sundue KJ464489 GU376520
Mickelia scandens (Raddi) R.C. Moran, Labiak & Sundue GU376696 GU376547
Olfersia cervina Kunze KJ464493 DQ153079 KJ464652
Parapolystichum acuminatum (Houlston) Labiak, Sundue & R.C.Moran KJ464430 KC977454 KJ464601
Parapolystichum boivinii (Baker) Rouhan KJ464435 KJ464607
Parapolystichum confine (Maxon ex C.Chr.) Labiak, Sundue & R.C.Moran KJ464438
Parapolystichum effusum (Sw.) Ching KJ464441
Parapolystichum effusum (Sw.) Ching subsp. divergens (Willd. ex Schkuhr) Tindale KJ464440
Parapolystichum excultum (Mett.) Labiak, Sundue & R.C.Moran KF709501 GU376541
Parapolystichum glabellum (A.Cunn.) Labiak, Sundue & R.C.Moran KJ464445 KF709503 KJ464613
Parapolystichum microsorum (Endl.) Labiak, Sundue & R.C.Moran KJ464451 GU376712 KJ464617
Parapolystichum perrierianum (C.Chr.) Rouhan KJ464455 KJ464623
Parapolystichum rufescens (Blume) Labiak, Sundue & R.C.Moran KJ464461 KJ464629
Parapolystichum vogelii (Hook.) Rouhan KJ464470
Parapolystichum windsorensis (D.L.Jones & B.Gray) Labiak, Sundue & R.C.Moran KJ464473 KJ464639
Pleocnemia conjugata C.Presl GU376713 KF709510
Pleocnemia cumingiana C.Presl KJ196828 KJ196705
Pleocnemia dahlii (Hieron.) Holttum KJ196829 KJ196706
Pleocnemia hemiteliiformis (Racib.) Holttum KF709482 KF667560 KF709511
Pleocnemia irregularis (C.Presl) Holttum KF709491 KF709513
Pleocnemia leuzeana (Gaudich.) C.Presl KJ196830
Pleocnemia olivacea (Copel.) Holttum KJ464495
Pleocnemia presliana Holttum KJ464496 KF667561
Pleocnemia rufinervis Nakai JF303976 KF667562
Pleocnemia winitii Holttum EF460686 KF709515
Polybotrya alfredii Brade KJ464497 KF667563 KJ464653
Polybotrya andina C.Chr. KJ464498 KP271084 KJ464654
Polybotrya pubens Mart. KJ464499 KP271085
Polystichum tsus-simense (Hook.) J.Sm. var. mayebarae (Tagawa) Sa.Kurata AB575224 DQ150408
Pseudotectaria biformis (Mett.) Holttum KF897951
Pseudotectaria decaryana (C.Chr.) Tardieu KF897952
Rumohra adiantiformis (G.Forst.) Ching KJ464500 KJ464655
Rumohra berteroana (Colla) J.J. Rodr. KJ464503 KJ464657
Stigmatopteris ichthiosma (Sodiro) C.Chr. KJ464504 KJ464658
Stigmatopteris killipiana Lellinger KJ464505 KJ464659
Stigmatopteris lechleri (Mett) C.Chr. KJ464506 KP271087 KJ464660
Stigmatopteris sordida (Maxon) C.Chr. KJ464507 KJ464661
Teratophyllum koordersii Holttum GU376566
Teratophyllum ludens (Fée) Holttum GU376567
Teratophyllum wilkesianum Holttum KJ464508
Nephrolepidaceae
Nephrolepis abrupta (Bory) Mett. HM748137 KF667559
Nephrolepis acutifolia (Desv.) Christ. HM748139
Nephrolepis biserrata (Sw.) Schott AB575227 GU376688
Nephrolepis brownii (Desv.) Hovenkamp & Miyam. KR816691
Nephrolepis cordifolia (L.) C.Presl AB575228
Nephrolepis davalliae Alderw. HM748147
Nephrolepis davallioides Kunze HM748148 GU376690
Nephrolepis exaltata (L.) Schott HM748149
Nephrolepis falcata (Cav.) C.Chr. HM748150
Nephrolepis falciformis J.Sm. AB232404
Nephrolepis lauterbachii (Christ) Christ HM748153
Nephrolepis pectinata (Willd.) Schott HM748155
Nephrolepis pendula (Raddi) J.Sm. HM748156
Nephrolepis radicans (Burm.) Kuhn HM748157
Nephrolepis rivularis (Vahl) Mett. HM748158
Nephrolepis undulata J.Sm. HM748159
Lomariopsidaceae
Cyclopeltis crenata (Fée) C.Chr. DQ054517 EF540718 DQ51448
Cyclopeltis novoguineensis Rosenst. KY397974 KY397978 KY397970
Cyclopeltis semicordata (Sw.) J.Sm. EF463234 KY397977 KY397969
Dracoglossum plantagineum (Jacq.) Christenh. KC914564 KY397979 KY397971
Dracoglossum sinuatum (Fée) Christenh. KU605106
Dryopolystichum phaeostigma (Ces.) Copel. KY397972 KY397976 KY397968
Lomariopsis crassifolia Holttum DQ396559
Lomariopsis guineensis (Underw.) Alston KJ628952 DQ396560
Lomariopsis hederacea Alston DQ396561
Lomariopsis jamaicensis (Underw.) Holttum DQ396562
Lomariopsis japurensis (C.Martius) J.Sm. DQ396563
Lomariopsis kunzeana (Underw.) Holttum DQ396569
Lomariopsis latipinna Stolze DQ396571
Lomariopsis lineata (C.Presl) Holttum DQ396572
Lomariopsis longicaudata (Bonap.) Holttum Q396573
Lomariopsis madagascarica (Bonap.) Alston DQ396575
Lomariopsis mannii (Underw.) Alston DQ396577
Lomariopsis marginata (Schrad.) Kuhn AY818677 DQ396578
Lomariopsis maxonii (Underw.) Holttum DQ396580
Lomariopsis muriculata Holttum DQ396582
Lomariopsis palustris (Hook.) Mett. ex Kuhn HM748162 DQ396585
Lomariopsis pervillei Kuhn DQ396586
Lomariopsis pollicina (Willemet) Mett. ex Kuhn EF463235 DQ396588
Lomariopsis prieuriana Fée DQ396590
Lomariopsis recurvata Fée DQ396592
Lomariopsis rossii Holttum DQ396594
Lomariopsis salicifolia (Kunze) Lellinger DQ396595
Lomariopsis sorbifolia (L.) Fée EF463236
Lomariopsis spectabilis Mett. AB232401 KJ196685
Lomariopsis vestita E.Fourn. DQ396598
Lomariopsis wrightii Mett. DQ396600
Tectariaceae
Arthropteris altescandens J.Sm. KF667636 KF667550 KF667606
Arthropteris articulata (Brack.) C.Chr. KC977367 KC977437 KC977411
Arthropteris beckleri (Hook.) Mett. U05605 KF667607
Arthropteris cameroonensis Alston KF667638
Arthropteris guinanensis H.G.Zhou & Y.Y.Huang KC977364 KC977442 KC977404
Arthropteris monocarpa (Cordem.) C.Chr. HM748132 KF897941
Arthropteris orientalis (Gmel.) Posth. HM748133 KC977435 KC977420
Arthropteris palisotii (Desv.) Alston AB575230 KC977427 KC977406
Arthropteris parallela (Baker) C.Chr. EF463266 KC977453 KC977425
Arthropteris paucivenia (C.Chr.) H.M.Liu, Hovenkamp & H.Schneid. EF463268 KC977426
Arthropteris repens (Brack.) C.Chr. KC977368 KC977438 KC977412
Arthropteris tenella (G.Forst.) J.Sm. ex Hook.f. KC977363 KF011547 KC977424
Hypoderris brauniana (H.Karst.) F.G.Wang & Christenh. KF667647 KF667618
Hypoderris brownii J.Sm. KF667642 KF667611
Hypoderris nicotianifolia (Baker) R.C.Moran, Labiak & J.Prado KF667653 KF667626
Pteridrys australis Ching KJ196892 KJ196678
Pteridrys cnemidaria (Christ) C.Chr. & Ching KF709488 KF709517
Pteridrys lofouensis (Christ) C.Chr. & Ching EF460687 KF667566
Pteridrys microthecia (Fée) C.Chr. & Ching KJ196848 KF709518
Pteridrys syrmatica (Willd.) C.Chr. & Ching KJ196875 KF709519
Tectaria acerifolia R.C.Moran KF887170 KF897954
Tectaria angulata (Willd.) Copel. KJ196876 KJ196656
Tectaria aurita (Sw.) S.Chandra KJ196849 KJ196631
Tectaria barberi (Hook.) Copel. KJ196846 KJ196628
Tectaria borneensis S.Y.Dong KJ196854 KF667555 KJ196642
Tectaria cicutaria (L.) Copel. KF667649 KF667620
Tectaria coadunata (J.Sm.) C.Chr. KJ196851 KJ196661
Tectaria crenata Cav. KF667650 KF667568 KF667621
Tectaria decurrens (C.Presl) Copel. AB575232 DQ514524
Tectaria devexa (Kunze ex Mett.) Copel. AB575233 KP271088 KF897956
Tectaria dilacerata (Kunze) Maxon KF887173 KF897957
Tectaria fauriei Tagawa AB575234 KJ196658
Tectaria fernandensis C.Chr. KF887174 KF897958
Tectaria gigantea (Blume) Copel. KJ196853 KJ196660
Tectaria griffithii (Baker) Ching KF667652 KF667624
Tectaria grossedentata Ching & Chu H.Wang KJ196882 KP271089 KJ196667
Tectaria harlandii (Hook.) C.M.Kuo AB575231 KJ196648
Tectaria harlandii (Hook.) C.M.Kuo KF887178 KF897961
Tectaria heracleifolia (Willd.) Underw. KF887180 KF897963
Tectaria herpetocaulos Ching & Chu H. Wang KJ196884 KJ196669
Tectaria heterocarpa C.V.Morton KF887181 KF897964
Tectaria impressa (Fée) Holttum KJ196841 KF897965
Tectaria kusukusensis (Hayata) Lellinger EF460681 KF897968
Tectaria labrusca (Hook.) Copel. KJ196818 KJ196692
Tectaria luchunensis S.K.Wu KJ196845 KP271090 KJ196627
Tectaria macleanii (Copel.) S.Y.Dong KJ196810 KJ196680
Tectaria melanocaula (Blume) Copel. KJ196832 KJ196709
Tectaria morsei (Baker) P.J.Edwards ex S.Y.Dong KJ196893 KF667570 KF561675
Tectaria nayarii Mazumdar EF463267 KJ196699
Tectaria paradoxa (Fée) Sledge KF887189 KF897971
Tectaria phaeocaulis (Rosenst.) C.Chr. AB232397 KF709499 KF897972
Tectaria pica (L.) C.Chr. KF887191 GU376715 KF897973
Tectaria polymorpha (Wall. ex Hook.) Copel. KJ196888 GU376716 KJ196657
Tectaria prolifera (Hook.) R.M.Tryon & A.F.Tryon EF463273 KF897974
Tectaria psomiocarpa S.Y.Dong KJ196822 KF667572 KJ196698
Tectaria pubens R.C.Moran KF887193 KF667573 KF897975
Tectaria quinquefida (Baker) Ching KJ196885 KJ396622
Tectaria repanda (Willd.) Holttum KJ196831 KJ196707
Tectaria sagenioides (Mett.) Christenh. KF887194 KF667575 KF561672
Tectaria semipinnata (Roxb.) Morton KJ196817 KF667577 KJ196691
Tectaria simonsii (Baker) Ching AB575236 KF897977
Tectaria singaporiana (Wall. ex Hook. & Grev.) Ching KF887196 KF897978
Tectaria subglabra (Holttum) S.Y.Dong KJ196676
Tectaria subsageniacea (Christ) Christenh. KF887197 KF667576 KF561670
Tectaria subtriphylla (Hook. & Arn.) Copel. AB575237 KF897980
Tectaria tricuspis (Bedd.) Copel. KJ196820 KJ196694
Tectaria variolosa (Wall. ex Hook.) C.Chr. EF460690 KF897982
Tectaria vasta (Blume) Copel. KF667655 KF667628
Tectaria vivipara Jermy & T.G.Walker KF887201 KF897983
Tectaria zeilanica (Houtt.) Sledge AB232395 KF709521
Triplophyllum crassifolium Holttum KF887203 KF897985
Triplophyllum fraternum (Mett.) Holttum KF667657 KF667630
Triplophyllum funestum (Kunze) Holttum EF463276 KF667631
Triplophyllum glabrum J.Prado & R.C.Moran KF887207 KF897989
Triplophyllum heudelotii Pic.Serm. KF897990
Triplophyllum jenseniae (C.Chr.) Holttum KF667660 KF667633
Triplophyllum pentagonum (Bonap.) Holttum KF667662 KF667635
Triplophyllum pilosissimum (J.Sm. ex T.Moore) Holttum KU605127
Triplophyllum securidiforme (Hook.) Holttum KU605128
Triplophyllum vogelii (Hook.) Holttum KF667661 KF667634
Oleandraceae
Oleandra articulata (Sw.) C.Presl KF667644 KF709500 KF667613
Oleandra cumingii J.Sm. KJ196816 KJ196690
Oleandra neriiformis Cav. KJ196815 KJ196689
Oleandra pilosa Hook. KF667646 KF667615
Davalliaceae
Davallodes hirsuta (J.Sm.) Copel. AY096196
Davallodes yunnanensis (Christ) M.Kato & Tsutsumi JX103718 KC914565
Polypodiaceae
Campyloneurum minus Fée KF667665
Microgramma lycopodioides (L.) Copel. KF667664
Niphidium longifolium (Cav.) C.V.Morton & Lellinger KF667663 KF709495