Phylogenetic analyses place the monotypic Dryopolystichum within Lomariopsidaceae

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.

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.
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 andPryer 2007), "RPS5F" and "TRNSR" for rps4-trnS (Nadot et al. 1995, Smith andCranfill 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, Den-mark), 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 BigDye TM 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 10 6 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 .

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 1-4). 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.
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).

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 21 th 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 Hennipman 1959, Kramer andGreen 1990).
Subsequent molecular phylogenetic analyses demonstrated that most genera previously treated in Lomariopsidaceae should be transferred to Dryopteridaceae (Tsutsumi andKato 2006, Schuettpelz andPryer 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 Hennipman 1959, Kramer andGreen 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  Holttum and Hennipman (1959), Holttum (1991), Roubik and Moreno (1991), Moran (2000), Christenhusz (2007), Rouhan et al. (2007), and this study]. 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.
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.

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 Hennipman 1959, Tryon andLugardon 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 Rothfels 2014, PPG I 2016). 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 Hennipman 1959, Moran 2000). In contrast, Dracoglossum and Cyclopeltis have non-winged and non-sulcate rachises (Holttum 1991, Christenhusz 2007.

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.  (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.