Surveys of pteridophytes (i.e., ferns and lycophytes) were carried out over three field seasons (January 2008, 2011, and 2013; 37 days total) at the Nectandra Cloud Forest Reserve, Costa Rica. Most specimens were collected along trails through the reserve. Epiphytes were collected from fallen trees or tree branches, or up to 2 m on tree trunks. Permits for collection were obtained from the Costa Rican government (SINAC No. 04941 and Cites 2014-CR 1006/SJ (#S 1045)). The first set of voucher specimens was deposited at UC, with duplicates at CR, GH, and TI. Herbarium codes follow Thiers (2020). Leaf tissue was preserved on silica gel for DNA extraction. Spores of selected taxa were observed with a standard compound light microscope.

DNA was extracted with the DNEasy plant mini kit following the manufacturer’s protocol (Qiagen). One species per taxon was sampled for morphologically distinct taxa, and up to five specimens per taxon for taxa that are more difficult to identify using standard keys and morphological characters. The plastid rbcL gene was amplified using PCR primers and thermocycler settings of Schuettpelz and Pryer (2007). PCR products were purified with Exo-STAR enzyme (GE Healthcare) and sequenced using the Big Dye Terminator v3.1 Cycle Sequencing Kit (ThermoFisher) with two internal primers, ESRBCL654R and ESRBCL628F (Schuettpelz and Pryer 2007) in addition to the amplification primers. The resulting AB1 trace files were imported into Geneious (Kearse et al. 2012), assembled into contigs, and the consensus sequences exported in FASTA format. A multi-sequence alignment was generated using MAFFT (Katoh et al. 2002), and a phylogenetic tree inferred using IQ-TREE with automatic model selection (Nguyen et al. 2015). For a small number of genera that were not supported as monophyletic in the original phylogenetic analysis (Cyathea and Lellingeria), all available rbcL sequences for closely related taxa (at the family or subfamily level, respectively) were downloaded from GenBank, aligned in combination with the newly generated sequences from Nectandra with MAFFT, and a phylogenetic tree inferred using FastTree on default settings (Price, Dehal, and Arkin 2009, 2010).

Molecular analysis was performed under permits R-CM-RN-001-2014-OT-CONAGEBIO and R-CM-RN-002-2017-OT-CONAGEBIO.

For additional methodological details, see Nitta JH, Ebihara A, Smith AR (in press, PLOS ONE)

For full list of references, see README.txt

Files included:
•    costa_rica_richness.csv: Data on species richness of pteridophytes in protected areas in Costa Rica.
•    cyatheaceae_rbcL.fasta: Aligned rbcL sequences of family Cyatheaceae from the Nectandra Cloud Forest Reserve, Costa Rica and all available sequences on GenBank in FASTA format.
•    cyatheaceae_rbcL.tre: Phylogenetic tree of family Cyatheaceae from the Nectandra Cloud Forest Reserve, Costa Rica and all available rbcL sequences on GenBank in Newick format.
•    grammitidoideae_rbcL.fasta: Aligned rbcL sequences of subfamily Grammitidoideae from the Nectandra Cloud Forest Reserve, Costa Rica and all available sequences on GenBank in FASTA format.
•    grammitidoideae_rbcL.tre: Phylogenetic tree of subfamily Grammitidoideae from the Nectandra Cloud Forest Reserve, Costa Rica and all available rbcL sequences on GenBank in Newick format.
•    JNG4254.fasta: DNA sequence in FASTA format of rbcL gene from Amauropelta atrovirens (C. Chr.) Salino & T.E. Almeida (Nitta 2237).
•    nectandra_gb_template.sbt: Plain text file (submit-block object) containing metadata related to GenBank submission.
•    nectandra_DNA_accessions.csv: DNA accession numbers and specimen accession numbers of pteridophytes from the Nectandra Cloud Forest Reserve, Costa Rica.
•    nectandra_rbcL.fasta: Newly generated rbcL sequences of pteridophytes from the Nectandra Cloud Forest Reserve, Costa Rica in FASTA format.
•    nectandra_rbcL.phy: Aligned rbcL sequences of pteridophytes from the Nectandra Cloud Forest Reserve, Costa Rica in PHYLIP format.
•    nectandra_rbcL.treefile: Phylogenetic tree of pteridophytes from the Nectandra Cloud Forest Reserve, Costa Rica in Newick format.
•    nectandra_specimens.csv: Specimen data of pteridophytes from the Nectandra Cloud Forest Reserve, Costa Rica collected by Joel Nitta.
•    ppgi_taxonomy.csv: Taxonomic system of Pteridophyte Phylogeny Group I (2016) for pteridophytes at the genus level and above.
•    seqids.txt: Newly assigned GenBank accession numbers for sequences generated by this project.

For details, see README file.