A taxonomic and molecular survey of the pteridophytes of the Nectandra Cloud Forest Reserve, Costa Rica.

Floristic surveys are crucial to the conservation of biodiversity, but the vast majority of such surveys are limited to listing species names, and few take into account the evolutionary history of species. Here, we combine classical taxonomic and molecular phylogenetic (DNA barcoding) approaches to catalog the biodiversity of pteridophytes (ferns and lycophytes) of the Nectandra Cloud Forest Reserve, Costa Rica. Surveys were carried out over three field seasons (2008, 2011, and 2013), resulting in 176 species representing 69 genera and 22 families of pteridophytes. Our literature survey of protected areas in Costa Rica shows that Nectandra has an exceptionally diverse pteridophyte flora for its size. Plastid rbcL was selected as a DNA barcode marker and obtained for >95% of pteridophyte taxa at this site. Combined molecular and morphological analyses revealed two previously undescribed taxa that appear to be of hybrid origin. The utility of rbcL for species identification was assessed by calculating minimum interspecific distances and found to have a failure rate of 18%. Finally we compared the distribution of minimum interspecific rbcL distances with two other areas that have been the focus of pteridophyte molecular surveys: Japan and Tahiti. The comparison shows that Nectandra is more similar to Japan than Tahiti, which may reflect the biogeographic history of these floras.

Introduction

Despite its small area (51,100 km2), Costa Rica is home to remarkably high biodiversity, and is ranked as one of the world’s top 25 biodiversity hotspots [1]. It is estimated that vascular plant species richness in Costa Rica exceeds 5,000 spp. per 10,000 km2 [2]. Accordingly, primary taxonomy is an important part of biological research in Costa Rica for the purposes of both documenting this biodiversity and informing conservation practices.

In addition to taxonomic diversity, measures of phylogenetic diversity (PD) provide an important perspective on biodiversity and should be taken into consideration for setting conservation priorities [3]. Therefore, surveys documenting both species richness and molecular phylogenetic diversity are needed. Furthermore, the use of a standard molecular marker as a DNA “barcode” is useful for species identification and taxonomic revision [4]. For example, DNA barcoding surveys have revealed hidden biodiversity in the form of cryptic species in butterflies in Costa Rica [5] and have been used to identify cryptic life-cycle stages in ferns [6,7]. DNA barcodes are being increasingly integrated into biological surveys to document biodiversity at unprecedented scale, rate, and resolution [8-10].

Pteridophytes (i.e., ferns and lycophytes) are an important group of plants to study because many new species are still being discovered in the Neotropics [11] and they play important ecological roles in many ecosystems [12-15]. The pteridophytes of Costa Rica comprise c. 1,200 spp., accounting for one-quarter to one-third of the estimated richness of the Neotropics (c. 3,000 spp. to 4,500 spp.) [11]. While the pteridophyte floras of some sites in Costa Rica are well studied, such as La Selva Biological Station [16,17], other areas in the country have received considerably less attention. We present here the results of surveys conducted over three field seasons on the pteridophyte flora of the recently established Nectandra Cloud Forest Reserve near San Ramon, Costa Rica. We also characterize the phylogenetic diversity at this site and compare it with other pteridophyte floras that have recently been the focus of DNA barcoding surveys.

Materials and methods

Study site

The Nectandra Cloud Forest Reserve (hereafter, “Nectandra”; 10°11’ N, 84°31’ W) is located at 1,000 m to 1,200 m a.s.l. on the Atlantic slope of the Cordillera de Tilarán, Alajuela Province (Fig 1A). It encompasses 158 ha of premontane rainforest (life zones follow Holdridge [18]) and is managed by the Nectandra Institute, whose mission is to conserve and restore cloud forest in northern Costa Rica through grassroots outreach [19]. Approximately three-quarters of Nectandra is primary forest with >98% canopy cover; the remaining area comprises naturally regenerating former coffee and Dracaena plantations (Fig 1B and 1C). Two permanent streams and four seasonal drainages pass through the reserve and empty into the Balsa river. Most of the surrounding land is used for cattle pasture or other agriculture. Previous biological surveys of Nectandra indicate that it has a rich herpetofauna [20] and is home to at least 188 spp. of bryophytes [21].

Fig 1

Location of the Nectandra Cloud Forest Reserve (“Nectandra”).

(a) Map of Costa Rica showing location of Nectandra (yellow diamond) and other protected areas that have been surveyed for pteridophytes (black dots). Other protected areas include Alberto Manuel Brenes Biological Reserve, La Selva Biological Station, Monteverde Cloud Forest Reserve, San Luis Biological Reserve, and the upper watershed of the Savegre River in the Los Santos Forest Reserve. Elevation data downloaded from the CGIAR SRTM 90m digital elevation database [22] using the getData function in the R package raster [23]. Map created using ggplot2 [24]. (b) Aerial photo of Nectandra (c. 1992). Nectandra consists of three parcels of land: The original parcel is in white (c. 80% primary forest), with areas added later in red (“Ocotea” parcel, primary forest) and blue (“Persea” parcel, secondary forest). Note extreme deforestation outside of protected areas. Reprinted under a CC BY license with permission from Instituto Geográfico Nacional de Costa Rica, original copyright 1992. (c) Example of interior of primary forest at Nectandra. Photo courtesy of Evelyne Lennette, reprinted under a CC BY license with permission.

Climate at Nectandra is characterized by extremely high frequency of cloud cover throughout the year. Rainfall peaks during the wet season from November to February. Mean annual precipitation is 3,000 mm yr-1 to 3,500 mm yr-1, with c. 80% fog-saturated days.

Field survey

We carried out surveys of pteridophytes over three field seasons (January 2008, 2011, and 2013; 37 days of sampling total). Most specimens were collected along trails through the reserve. Epiphytes were collected from fallen trees or tree branches, or up to c. 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, TI, and the private collection at Nectandra. Herbarium codes follow Thiers [25]. Leaf tissue was preserved on silica gel for DNA extraction. Spores of selected taxa were observed with a standard compound light microscope.

Taxonomy

We consulted relevant floras [26-28] and recent monographs [29-35] for species identification. In some cases, we also consulted taxonomic experts on particular groups (e.g., grammitid ferns). Genus-level and higher taxonomy follows Pteridophyte Phylogeny Group I [36].

DNA sequencing and phylogenetic analysis

DNA was extracted with the DNEasy Plant Mini kit following the manufacturer’s protocol (Qiagen). We generally sampled one species per taxon from Nectandra for morphologically distinct taxa, and up to five specimens per taxon for taxa that are more difficult to identify using standard keys [26,27] (e.g., Megalastrum, Didymoglossum). We selected the plastid rbcL gene as a barcode marker because it has universal primers available for ferns [37] and has performed relatively well for species identification in ferns relative to other candidate barcode loci [6,38,39]. We amplified rbcL using PCR primers and thermocycler settings of [37] and verified amplification success by gel electrophoresis in 1% TAE. We purified PCR products with Exo-STAR (GE Healthcare) and conducted cycle sequencing using the Big Dye Terminator v3.1 Cycle Sequencing Kit (ThermoFisher) with two internal primers, ESRBCL654R and ESRBCL628F [37], in addition to the amplification primers. We imported the resulting AB1 trace files into Geneious [40], assembled contigs, and exported the consensus sequences in FASTA format. We downloaded rbcL sequences from GenBank if available for any remaining taxa that could not be successfully sequenced (S1 Table). We generated an alignment using MAFFT [41]. For phylogenetic analysis by maximum likelihood, all sites were included in a single partition (no partitioning was specified). We evaluated models of DNA sequence evolution with IQ-TREE [42] (“-m TEST”), which is similar to the model selection process implemented in jModelTest [43]. The Bayesian Information Criterion (BIC) was used to select the best model (IQ-TREE default setting), which was then used by IQ-TREE to infer the tree. Node support was assessed with 1,000 ultra-fast bootstrap (UFboot) [42] and 1,000 Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT) replicates [44] in IQ-TREE. For a small number of genera that were not supported as monophyletic in the original phylogenetic analysis, we also downloaded all available rbcL sequences for closely related taxa (at the family or subfamily level) from GenBank, aligned these in combination with the newly generated sequences from Nectandra with MAFFT, and inferred a tree using FastTree on default settings [45,46]. Molecular analysis was performed under permits R-CM-RN-001-2014-OT-CONAGEBIO and R-CM-RN-002-2017-OT-CONAGEBIO.

Statistical analysis

To assess the completeness of sampling, we constructed species incidence rarefaction-extraction curves using the iNEXT package [47]. Number of collection days was used as the sampling unit.

We compared the number of species at Nectandra with other protected sites in Costa Rica by conducting a literature survey.

We assessed the utility of rbcL for species identification by calculating minimum interspecific distances as follows. We first calculated all raw interspecific distances in the rbcL alignment using the “dist.dna” function in the APE package [48], then extracted the minimum interspecific distance for each species using custom scripts. Species sharing identical rbcL sequences with at least one other species (interspecific distance of zero) were considered failures, i.e., not possible to identify with this marker.

To better characterize observed phylogenetic diversity, we compared the phylogenetic diversity of the pteridophytes of Nectandra with two other pteridophyte floras that have been the subject of DNA barcoding using rbcL: Japan [39,49] and the islands of Moorea and Tahiti, French Polynesia [6]. To ensure that phylogenetic distances were comparable across datasets, we generated a combined rbcL alignment for all species from the three floras together using MAFFT. We then subset the alignment to the species in each flora and calculated minimum interspecific distances per flora as described above.

All analyses were carried out using R v 3.6.1 [50].

Results

Taxonomic survey

Our surveys resulted in 320 individuals representing 176 spp., 69 genera, and 22 families of pteridophytes (S2 Table). Two species included multiple varieties (two each). 169 spp. (94.9%) are ferns and 7 spp. (3.9%) are lycophytes. Most taxa are either epiphytic (n = 94; 52.8%) or terrestrial (n = 74; 41.6%) (Table 1). All taxa are native, except for Macrothelypteris torresiana (Gaudich.) Ching (native to Africa and Asia; introduced in the Americas), which was excluded from the comparison of richness across sites and DNA barcode analysis.

Table 1

Growth habits of ferns and lycophytes at the Nectandra Cloud Forest Reserve, Costa Rica.

Count includes taxa at the species and infraspecies (variety or subspecies) levels (n = 178 total).

	n (percent of totala)
Climbing or clambering	5 (2.8%)
Epipetric	19 (10.7%)
Epiphytic	94 (52.8%)
Terrestrial	74 (41.6%)

aPercentages do not sum to 100% because some taxa have multiple growth habits.

The genera with the most species were Elaphoglossum (14 spp.), Diplazium (12 spp.), Hymenophyllum (12 spp.), and Asplenium (10 spp.). Families with the most species were Dryopteridaceae (28 spp.), Polypodiaceae (28 spp.), and Hymenophyllaceae (25 spp.).

Three taxa could not be matched to any known species: Polyphlebium sp1 (Nitta 123 and Nitta 2378), Campyloneurum sp1 (Nitta 2308), and Megalastrum sp1 (Nitta 727).

Our literature survey identified five other protected areas in mainland Costa Rica that have been surveyed for pteridophytes [17,52-55] (Fig 1, Table 2). The site with the highest species richness is Alberto Manuel Brenes Biological Reserve (281 spp.), with Nectandra (175 spp.) the third-highest after La Selva Biological Station (197 spp.; Table 2). Nectandra has by far the most species per hectare (1.11), with San Luis Biological Reserve second-highest (0.16 species per hectare; Table 2).

Table 2

Species richness of pteridophytes at protected areas in Costa Rica.

A single non-native species, Macrothelypteris torresiana, was excluded from calculations for Nectandra.

	Elevation (m)	Life zone typea	Area (ha)	Richness (no. spp.)	Richness per area (no. spp. per ha)	Reference
Alberto Manuel Brenes Biological Reserve	ca. 1,520	Premontane rainforest	7,800	281	0.04	[54]
La Selva Biological Station	35–130	Tropical wet forest	1,533	197	0.13	[17]
Monteverde Cloud Forest Reserve	1,500–1,640	Lower montane wet forest	3,800	147	0.04	[52]
Nectandra Cloud Forest Reserve	1,000–1,200	Premontane rainforest	158	175	1.11	this study
San Luis Biological Reserve	540–855	Transition between lowland tropical wet forest and premontane wet forest	251	39	0.16	[55]
Upper watershed of the Savegre River in the Los Santos Forest Reserve	2,000–3,491	Montane forest to paramo	10,000	123	0.01	[53]

aLife zones follow Holdridge [18].

The collection curve did not reach an asymptote (Fig 2). Extrapolation of the curve indicates that asymptotic species richness may approach 253 spp. (95% confidence interval 222 spp. to 305 spp.).

Fig 2

Interpolation (solid line) and extrapolation (dashed line) of species richness of pteridophytes at Nectandra Cloud Forest Reserve, Costa Rica.

Point at transition from solid to dashed line indicates observed richness. Gray shading indicates 95% confidence interval.

Phylogenetic analysis

We generated 186 new rbcL sequences of pteridophytes from Nectandra, representing 168 taxa (S1 Table). Mean length of the newly generated sequences was 1,292 bp ± SD 96 bp. rbcL could not be sequenced for 10 taxa, and only partial sequences (ca. 3’ or 5’ half of rbcL) could be obtained for three taxa. Difficulty in sequencing of these samples may be due to degraded condition of DNA or mismatched primer sequences. Of the taxa that we were unable to newly sequence, sequences of two taxa from specimens from Nectandra and three taxa from specimens from other areas were available on GenBank (S1 Table). In total, our rbcL sampling included 173 taxa (97.2%) of pteridophytes occurring at Nectandra.

The rbcL alignment was 1,309 bp long including 591 parsimony-informative sites and 191 sequences. The GTR+I+G4 model was selected for ML phylogenetic analysis according to BIC (the same model was also selected by AIC and corrected AIC).

The rbcL phylogeny (S1 Fig) was generally in agreement with recently published plastid phylogenies [37,56-58] at the family level and above, and families sensu PPGI [36] were monophyletic. One exception was Osmundaceae sister to Gleicheniaceae (SH-aLRT <50%; UFboot 92%), which may be due to poor sampling; we could obtain only a 614 bp rbcL fragment from the sole osmundaceous species at our study site. Some internal nodes had weak support (e.g., Cyatheales [SH-aLRT 64.7%; UFboot 92%], Polypodiidae [leptosporangiates; SH-aLRT <50%; UFboot 87%]). It should be noted that IQ-TREE UFboot support values are not analogous to traditional bootstrap values; only nodes receiving SH-aLRT > = 80% and UFboot > = 95% should be considered reliable [59]. As the purpose of this study was not to robustly infer phylogeny across all ferns and lycophytes, we do not discuss such deep relationships further.

A small number of genera were found to be non-monophyletic but lacked strong support. Sphaeropteris brunei (H. Christ) R.M. Tryon is nested within Cyathea as the sister to Cyathea bicrenata Liebm. (SH-aLRT 79.6%; UFboot 83%). Stenogrammitis limula (Christ) Labiak is nested within Lellingeria as the sister to Lellingeria hombersleyi (Maxon) A.R. Sm. (SH-aLRT <50%; UFboot 87%). These were resolved in their expected genera in broadly sampled trees using all available rbcL sequences on GenBank (S2 Fig and S3 Fig, respectively), so their irregular placement in the Nectandra rbcL tree appears to be an artifact of low sampling rather than poor sequence quality or misidentification.

Sampling of multiple specimens per species for taxa that are morphologically difficult to distinguish revealed several non-monophyletic species (S1 Fig). Megalastrum apicale R.C. Moran & J. Prado, M. atrogriseum (C. Chr.) A.R. Sm. & R.C. Moran, and M. longipilosum A. Rojas are closely related (SH-aLRT 92%; UFboot 100%), and the monophyly of each lacks support. Diplazium carnosum Christ is non-monophyletic with respect to D. urticifolium Christ and D. macrophyllum Desv., but this and most other relationships within Diplazium lacked support. Didymoglossum ekmanii (Wess. Boer) Ebihara & Dubuisson is nested within, and very closely related to D. kapplerianum (J.W. Sturm) Ebihara & Dubuisson (SH-aLRT 100%; UFboot 100%).

Barcode analysis

The pteridophyte flora of Nectandra includes 31 taxa (18%) that share identical rbcL sequences with at least one other taxon. This failure rate is higher than that of the pteridophytes of Moorea and Tahiti (4%), but lower than Japan (22%; Fig 3).

Fig 3

Minimum interspecific rbcL distances for selected pteridophyte floras.

(a) Nectandra, (b) Moorea and Tahiti, (c) Japan (all species), and (d) Japan (sexual diploids only). Red bar indicates interspecific distance of zero, i.e., species that cannot be distinguished using rbcL.

Discussion

Here, we present to our knowledge the first combined taxonomic and molecular survey of pteridophytes of a protected area in Costa Rica. We place our results in a regional and global context by comparing this flora with other protected areas in Costa Rica and two other sites that have been the focus of DNA barcoding: Tahiti and Japan.

Taxonomic diversity

Nectandra has the third-highest species richness of protected areas in Costa Rica with data available for ferns and lycophytes, and by far the greatest number of species per hectare (Table 2). While the number of species per hectare is not a fair measure of biodiversity per se as the species-area curve is not linear [60], it is useful to assess the effectiveness of a given protected area. Clearly, Nectandra is highly effective at protecting a large number of pteridophyte species given its area. Furthermore, the collection curve indicates that additional, unsampled species may be present (Fig 2), adding to the value of this conservation area. A recent survey of the bryophytes of Nectandra found a similar number of species (188 spp.) and also suggested unsampled species remained due to the shape of the collection curve [21].

One reason for the high species richness at Nectandra may be its elevation (1,000 m to 1,200 m). Species richness of pteridophytes in the tropics generally reaches a maximum at mid-elevations on mountains, which is thought to be due to a combination of high humidity and moderate temperature [61]. Plot-based surveys of pteridophytes along elevational gradients in Costa Rica spanning c. 100 m to 3,000 m have found maximum richness at 1,000 m to 1,200 m [62,63]. Another possible explanation is the presence of secondary forest and reserve edges, which may contribute additional species that would otherwise not occur in primary forest at this elevation (i.e., the “edge effect” [64]). Nectandra has a high edge to area ratio due to its small size, and edge effects have been demonstrated for pteridophytes in Mexican montane forests [65]. While we did not collect data to specifically test this hypothesis, the number of taxa restricted to edges at Nectandra is probably no greater than c. 10 spp. (J. Nitta, pers. obs.), and only one non-native species (M. torresiana) was observed occupying disturbed areas. Furthermore, the high number of epiphytes (the most common growth form; Table 1) indicates that high humidity may support large numbers of species at Nectandra. Taken together, these observations suggest effect of elevation is likely more important than edge effects.

Unidentified taxa and putative species complexes

Of the unidentified taxa, two are likely hybrids between distinct species. Polyphlebium sp1 (Nitta 123 and Nitta 2378) shares very similar rbcL sequences with Polyphlebium capillaceum (L.) Ebihara & Dubuisson but differs from this species by having expanded laminae (vs. laminae reduced to a few cells on either side of the veins) and growing on rocks in stream beds (vs. growing epiphytically on tree ferns). Polyphlebium sp1 has 32 spores per sporangium, a condition that often indicates asexual reproduction via apogamy [66]. This may allow it to reproduce by spores despite not being able to complete normal meiosis. To our knowledge, a count of 32 spores per sporangium has not been previously reported from Polyphlebium, and more detailed study is needed to confirm the reproductive mode of this taxon. Campyloneurum sp1 (Nitta 2308) matches in rbcL exactly with Campyloneurum angustifolium (Sw.) Fée, but differs in morphology from C. angustifolium at Nectandra (Nitta 782) by having wider fronds (c. 1.5 cm. vs. c. 0.75 cm in C. angustifolium) and more rows of sori between the costa and the margin (3–4 vs. 1–2 in C. angustifolium). Clear, misshapen spores were observed in Campyloneurum sp1, whereas C. angustifolium is a sexual diploid (2n = 72) with oblong spores [67]. The chloroplast is generally maternally inherited in ferns [68-70]. Therefore, it is likely that each of these taxa is a hybrid between the species with which they share rbcL as the mother and another unknown species as the father.

Megalastrum sp1 (Nitta 727) shares common aspects of morphology with M. longipilosum, but the rbcL sequences of M. apicale, M. atrogriseum, and M. longipilosum are extremely similar. The low divergence in rbcL and non-monophyly of some taxa (M. apicale) are consistent with the status of this group as a species complex, comprising several closely related diploid taxa and their hybrids; alternatively, rbcL alone may be too slowly evolving to distinguish recently diverged taxa in this group. Similarly, Diplazium also showed a high degree of morphological diversity but extremely low divergence in rbcL sequences, and at least one species, D. carnosum, appears to be non-monophyletic. Further study, in particular with regards to ploidal level and reproductive mode, is needed to determine the status of Megalastrum and Diplazium at Nectandra as possible species complexes. Also, sequencing of nuclear genes and more variable plastid markers is needed to clarify relationships for the putative hybrids (Polyphlebium sp1 and Campyloneurum sp1) as well as these putative species complexes.

Molecular diversity and DNA barcode suitability

Unlike animals, there is no single DNA barcode available for plants that works to reliably identify species across all taxonomic groups [71]. rbcL has relatively high phylogenetic informativeness in ferns and lycophytes at the species level and is one of the most frequently sequenced plastid genes in molecular systematic studies of pteridophytes. In previous comparisons with other common plant barcode markers in pteridophytes, rbcL performs better than trnH-psbA and comparably with matK [6,38,39]. However, universal primers for matK in pteridophytes are lacking [38]. rbcL therefore is a reasonable choice for a single barcode marker in pteridophytes. However, there have been relatively few studies comparing the performance of rbcL as a barcode marker across different pteridophyte floras.

We find that the pteridophytes of Nectandra have a species identification failure rate intermediate between that of Moorea and Tahiti (French Polynesia) and Japan (Fig 3). The distribution of minimum interspecific distances may reflect the biogeographic history of each region. Costa Rica and Japan are both mainland areas (or formerly connected with the mainland in the case of Japan), whereas the islands of French Polynesia are extremely isolated and have never been connected to a continent. It is likely that more species in French Polynesia are recent immigrants that have mostly evolved elsewhere, compared to more species that have evolved in situ in Costa Rica and Japan. This would result in the observed distribution of high interspecific divergences for species from French Polynesia and low interspecific divergences for species in Costa Rica and Japan.

Furthermore, the high failure rate in Japan is likely due to a high rate of apogamy combined with the taxonomic practice of splitting apogamous and sexual forms into separate species [49]. When only sexual diploids are included, the failure rate in Japan drops to 7% and the distribution of minimum interspecific distances more closely resembles that of Nectandra (Fig 3).

The patterns identified here show that DNA barcoding strategies in different areas may need to take into account the biogeographical history of the study site. Whereas rbcL alone may be largely sufficient in oceanic islands like French Polynesia, a second, more variable barcode marker such as trnLF [72] or matK [38] is probably needed to improve species identification rates of recently diverged taxa in continental areas such as Japan or Costa Rica. The second marker need not have universal primers, since rbcL will indicate genus or family, and primers for the second marker can then be selected appropriately.

Conclusions

The Nectandra Cloud Forest Reserve is clearly an important site for biodiversity of ferns and lycophytes in Costa Rica, harboring a high number of species for its relatively small area. Our surveys have already revealed multiple taxa that appear to be new to science, and more likely remain to be discovered. We demonstrated that rbcL can be used to reliably distinguish species in c. 82% of cases at Nectandra. This will enable future studies to investigate the ecology of neotropical pteridophytes in more depth, particularly with regards to the gametophytic phase. Other studies using rbcL as a DNA barcode to identify fern gametophytes to species have revealed particular clades and morphologies that tend to occur long distances from conspecific sporophytes [6,7], but such patterns have yet to be observed in the Neotropics. We hope our study will lead to more molecular ecological research incorporating field observations of gametophytes and contribute to the systematics of neotropical pteridophytes.

Maximum-likelihood phylogenetic tree of ferns and lycophytes at the Nectandra Cloud Forest Reserve, Costa Rica inferred using IQ-TREE with 1,000 SH-like approximate likelihood ratio test (SH-aLRT) and ultra-fast bootstrap (UFboot) replicates each.

Tree rooted on lycophytes. Numbers at nodes indicate SH-aLRT support (%)/UFboot support (%); values less than 50% shown with “-”; values of 100% shown with “*”; completely blank nodes indicate identical sequences. For phylogram on right side, scale bar shows expected number of changes per site. Numbers after species name are J. H. Nitta specimen collection numbers except for Abrodictyum rigidum (J.-Y. Dubuisson HV 1997–3, Venezuela), Trichomanes polypodiodes (M. Kessler 8808, Bolivia), and Radiovittaria remota (R. Moran 3180a, Costa Rica), which could not be sequenced successfully, so GenBank sequences were used instead (accessions AY095108, AY175795, and U21289, respectively). All J. H. Nitta specimens from Nectandra.

(PDF)

Click here for additional data file.

Maximum-likelihood phylogenetic tree of the family Cyatheaceae including all available rbcL sequences on GenBank and newly sequenced taxa from the Nectandra Cloud Forest Reserve, Costa Rica inferred using FastTree.

Tree rooted on species from Nectandra in family Dicksoniaceae. Numbers at nodes indicate local support values computed with the Shimodaira-Hasegawa test; values less than 50% not shown. For phylogram on right side, scale bar shows expected number of changes per site. Numbers after species names are GenBank accession numbers for sequences downloaded from GenBank or J. H. Nitta specimen collection numbers for sequences newly obtained by this study. Newly obtained ingroup sequences highlighted in yellow.

(PDF)

Click here for additional data file.

Maximum-likelihood phylogenetic tree of grammitid ferns (subfamily Grammitidoideae) including all available rbcL sequences on GenBank and newly sequenced taxa from the Nectandra Cloud Forest Reserve, Costa Rica inferred using FastTree.

Tree rooted on species from Nectandra in subfamily Polypodioideae. Numbers at nodes indicate local support values computed with the Shimodaira-Hasegawa test; values less than 50% not shown. For phylogram on right side, scale bar shows expected number of changes per site. Numbers after species names are GenBank accession numbers for sequences downloaded from GenBank or J. H. Nitta specimen collection numbers for sequences newly obtained by this study. Newly obtained ingroup sequences highlighted in yellow.

(PDF)

Click here for additional data file.

GenBank accession numbers of sequences analyzed in this study.

All specimens from the Nectandra Cloud Forest Reserve, Alajuela Province, Costa Rica, except for Abrodictyum rigidum (Sw.) Ebihara & Dubuisson (J.-Y. Dubuisson HV 1997–3, Venezuela), Radiovittaria remota (Fée) E.H. Crane (R. Moran 3180a, Costa Rica), and Trichomanes polypodiodes L. (M. Kessler 8808, Bolivia). Accessions beginning with ‘MW’ were newly generated by this study.

(CSV)

Click here for additional data file.

A checklist of ferns and lycophytes observed at the Nectandra Cloud Forest Reserve, Costa Rica.

All voucher specimens deposited in UC with duplicates when available at CR, GH, and TI. For additional data on each voucher specimen, see Dryad repository [51].

(CSV)

Click here for additional data file.

16 Jul 2020

PONE-D-20-11593

A taxonomic and molecular survey of the pteridophytes of the Nectandra Cloud Forest Reserve, Costa Rica

PLOS ONE

Dear Dr. Nitta,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please, consider  especially  rev.#1's comments on improving bibliographic references and the taxonomic survey. Further details on Material & Methods are also welcome.

Please submit your revised manuscript by Aug 30 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Paulo Takeo Sano, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

3.We note that [Figure(s) 1] in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

1.    You may seek permission from the original copyright holder of Figure(s) [1] to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

2.    If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

Additional Editor Comments (if provided):

Please consider all comments from both reviewers, especially rev.#1's comments on improving bibliographic references and the taxonomic survey. Rev.#1 also asked for further clarification on some methods.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Most of my suggestions were provided in the annotated file provided. Some major comments are below:

1- I think many relevant citations are missing for the Taxonomic part of the manuscript. Besides the general floras published by Lellinger and Moran et al., I strongly recommend the authors to consult, and cite, the relevant monographs (taxonomic revisions) that are available for many of the taxa, as well as recent taxonomic works on Costa Rican ferns. I think it is important to acknowledge the contribution of these studies to the understanding of biodiversity.

2- Line 102. Please, list the taxa that you considered the most difficult to identify.

3- Line 105. I think it would be nice to explain why rbcL was chosen for this study in the Mat & Meth Section.

4- Please, include something in the Mat & Meth about model calculations. By the way, why you used BIC for the ML analysis? Why not to use AIC?

5- Taxonomic survey: Some improvements could be made to this part. For instance, mention how many are ferns and how many are lycophytes, and what is the amount of epiphytes, terrestrial, epipetric, etc.. Also, are there exotic/invasive taxa that might be contributing to the diversity of Nectandra? Were those included in your analysis?

6- Barcode analysis: This is why rbcL may not be the best to infer species boundaries, and it may not be enough to state that there are "species complexes" based on the phylogenetic results. (Even though I think this might be true in some cases...). Please, consider clarifying this paragraph.

7- About what would explain the richness in Nectandra, what about humidity? Because this region is located at the Atlantic slopes, it has a high humidity along the year, which contributes to the occurrence of many epiphytes. Because it has been noted that epiphytes represent a considerable part of the diversity in some areas, I wonder whether this is not the case also in Nectandra. By the way, this is another reason it would be interesting to mention how many species are epiphytes, terrestrial, epipetric, etc.

8- As for the comparison provided for Tahiti, Japan and Nectandra’s floras, I think another possibility is the isolation of Tahiti, which would preclude recurrent events of dispersal from other areas, keeping the isolation of local populations. In both CR and Japan, there are more possibilities of gene flow between other areas, adding to the complexity of local populations.

Reviewer #2: The authors present a checklist to a reserve that is accomplished through a combination of field work, herbarium study, and DNA barcoding using the chloroplast rbcL marker. The work appears to have been conducted with the utmost care. It is clearly presented and well written. It will act as a gold standard by which similar projects will be compared. I have made some additional comments, but I have a hard time finding errors in this work I congratulate the authors on a job well done.

Signed,

Michael Sundue

I am happy to see that this work adheres to open and reproducible science. The effort and thoroughness of providing the fully reproducible manuscript using Docker and Drake are appreciated. Nonetheless, I was unable to reproduce the manuscript. The address https://github.com/joelnitta/nectandra_ferns

returned a 404 page not found error and attempting docker pull joelnitta/nectandra_ferns

returned a ‘manifest unknown error’. Perhaps I am missing something simple.

195 change “was in generally good agreement” to “generally in agreement”

The evidence in favor of the new taxa is clear and the arguments used to explain them is logical.

While I appreciate the brevity of the paper. Personally, I would like to see more discussion of the potentially novel taxa with reference to the current state of the taxonomy in each group. That said, the authors are perfectly justified in maintaining their brief discussion if they prefer.

The trees presented in the supplement show species of tree ferns that are highlighted in yellow some of which are non-monophyletic. Yet there is no discussion of these nor any other tree fern for that matter. Why were these results excluded from the discussion? The same is true for some polypodiaceae.

Your Mycopteris taxifolia may be M. costaricense. See Sundue 2014 “Mycopteris, a new neotropical genus of grammitid ferns”

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Paulo Labiak

Reviewer #2: Yes: Michael Sundue

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

28 Sep 2020

2020-09-29

Dear Dr. Paulo Takeo Sano,

Thank you very much for handling our MS. We are happy for the chance to respond to the reviewers. Please see our responses below (all line numbers in our responses refer to the revised MS unless otherwise indicated).

There is also a summary of other changes we made to the MS besides those requested by the reviewers following our responses.

Best Regards,

Joel Nitta

Joel H. Nitta, Ph.D.

Project Research Associate

Iwasaki Lab

Department of Biological Sciences

The University of Tokyo

nitta@bs.s.u-tokyo.ac.jp

https://www.joelnitta.comhttp://iwasakilab.bs.s.u-tokyo.ac.jp/

---

Reviewer #1: Most of my suggestions were provided in the annotated file provided. Some major comments are below:

1- I think many relevant citations are missing for the Taxonomic part of the manuscript. Besides the general floras published by Lellinger and Moran et al., I strongly recommend the authors to consult, and cite, the relevant monographs (taxonomic revisions) that are available for many of the taxa, as well as recent taxonomic works on Costa Rican ferns. I think it is important to acknowledge the contribution of these studies to the understanding of biodiversity.

RESPONSE: We have added one more flora and seven recent monographs (line 102).

2- Line 102. Please, list the taxa that you considered the most difficult to identify.

RESPONSE: We have added examples of taxa that were difficult to identify (Megalastrum and Didymoglossum, line 110).

3- Line 105. I think it would be nice to explain why rbcL was chosen for this study in the Mat & Meth Section.

RESPONSE: We have added our reasons for selecting rbcL (lines 111-113).

4- Please, include something in the Mat & Meth about model calculations. By the way, why you used BIC for the ML analysis? Why not to use AIC?

RESPONSE: We have added more details about the procedure used for model selection (lines 123-127). We used BIC because it is the default in IQ-tree. Practically speaking, in our analysis, the same model was selected by AIC, AICc, and BIC (GTR+I+G4), so it makes no difference which one was used. We now mention this in the results (line 216).

5- Taxonomic survey: Some improvements could be made to this part. For instance, mention how many are ferns and how many are lycophytes, and what is the amount of epiphytes, terrestrial, epipetric, etc.. Also, are there exotic/invasive taxa that might be contributing to the diversity of Nectandra? Were those included in your analysis?

RESPONSE: We have added a count of the number of ferns vs. lycophytes (line 173). We have added a description in the text of the number of taxa observed with different growth habits (lines 173-174), as well as a new table summarizing this information (new Table 1). There is only one non-native species (Macrothelypteris torresiana), which we now mention. We also now exclude the non-native species from the comparison of richness across sites and the DNA barcode analysis.

6- Barcode analysis: This is why rbcL may not be the best to infer species boundaries, and it may not be enough to state that there are "species complexes" based on the phylogenetic results. (Even though I think this might be true in some cases...). Please, consider clarifying this paragraph.

RESPONSE: Thanks to the reviewer for pointing out this possibility. While it is not clear which paragraph the reviewer is referring to, we have clarified the text in several areas to reflect that rbcL alone may not be sufficient for species discrimination at Nectandra, as follows:

- We removed "indicative of species complexes" (line 216, original MS) from the Results.

- We now say "consistent with the status of this group as a species complex" (instead of "indicative") in the Discussion (line 309).

- We now state "alternatively, rbcL alone may be too slowly evolving to distinguish recently diverged taxa" in the Discussion (lines 310-311).

- We added a paragraph to the Discussion about the need for a second marker in continental areas like Costa Rica (lines 347-354).

7- About what would explain the richness in Nectandra, what about humidity? Because this region is located at the Atlantic slopes, it has a high humidity along the year, which contributes to the occurrence of many epiphytes. Because it has been noted that epiphytes represent a considerable part of the diversity in some areas, I wonder whether this is not the case also in Nectandra. By the way, this is another reason it would be interesting to mention how many species are epiphytes, terrestrial, epipetric, etc.

RESPONSE: Thanks to the reviewer for pointing this out. We have now added mention of humidity to the Discussion, including the observation of high numbers of epiphytes at Nectandra (lines 283-284). We also added a table summarizing growth habits (new Table 1).

8- As for the comparison provided for Tahiti, Japan and Nectandra’s floras, I think another possibility is the isolation of Tahiti, which would preclude recurrent events of dispersal from other areas, keeping the isolation of local populations. In both CR and Japan, there are more possibilities of gene flow between other areas, adding to the complexity of local populations.

RESPONSE: We already explicitly state "the islands of French Polynesia are extremely isolated" in the sentence directly preceding the commented text. We do not see any need to revise the text further.

---

Following are our responses to comments from Reviewer 1 included as annotations in the PDF. All line numbers for the reviewer's comments refer to the original MS.

Lines 69-70: Why, specifically, the herpetofauna and bryophytes were mentioned here? are these the only surveys carried out in the reserve? If so, please, include this information.

RESPONSE: Yes; these are the only published surveys previously carried out at the reserve. Providing information about the biodiversity of the site for other organisms is useful background information. We now mention that these are previous biological surveys carried out at Nectandra (line 69). However, saying they are the "only" previous studies seems superfluous. We believe the reader would understand that we are citing all relevant literature and haven't left out anything on purpose.

Line 97: Haven't you asked any experts for help with some particular groups? Also, I think it is important to cite the relevant monographs and recent taxonomic works on Costa Rican ferns.

RESPONSE: Yes! Indeed, Paulo Labiak answered a query about Lellingeria hombersleyi while we were preparing the manuscript (in case there is any question, this communication took place prior to submission, was strictly about this specimen only, and did not include any other information about this manuscript). We apologize for forgetting to mention it in the original MS. We have added consulting taxonomic experts to the methods (lines 103-104) and mention any such contributions in the Acknowledgments (lines 371-373). We have also added additional references used for identification (see response to Reviewer 1, Point 1).

Line 102: Please, list the taxa that you considered the most difficult to identify.

RESPONSE: See response to Reviewer 1, Point 2.

Line 103-105 (with regards to specimens from other areas in Costa Rica): Is this listed somewhere? I think it is important to specify which ones are not from Nectandra.

RESPONSE: We have removed these specimens from analysis (see “additional corrections” at the end of this document).

Line 114 (with regards to phylogenetic analysis): No model was estimated?

RESPONSE: More details have been added (see response to Reviewer 1, Point 4).

Line 159: Please, specify how many are ferns and how many are lycophytes.

RESPONSE: This has been added (see response to Reviewer 1, Point 5).

Line 187 (with regards to difficulty in sequencing some samples): What about primer specificity?

RESPONSE: We now mention this possibility (line 209).

Line 193-194 (with regards to model of DNA evolution): This needs to be mentioned in the Mat& Met section. What program was used? Also, I think it would be more appropriated to use AIC for ML analysis, instead of BIC.

RESPONSE: We disagree that the particular model used must be reported in the materials and methods, and not results. The outcome of model selection is a result of an algorithm (as well as used in phylogenetic analysis). Furthermore, more details about the model selection procedure have been added to the methods (see response to Reviewer 1, Point 4).

Line 216 (with regards to putative species complexes): Couldn't it be due to the lack of resolution based on the rbcL sequences instead? rbcL is a very conservative gene, and perhaps not the best one to be used at the species level.

RESPONSE: See response to Reviewer 1, Point 6.

Lines 225-226 (with regards to statement that 18% of species at Nectandra share identical rbcL sequences with at least one other taxon): See, this is why rbcL may not be the best to infer species boundaries, and it may not be enough to state that there are "species complexes" based on the phylogenetic results. (Even though I think this might be true in some cases...)

RESPONSE: See response to Reviewer 1, Point 6.

Lines 253-254 (with regards to additional species in secondary forest and reserve edges): Are there non-native species recorded for these disturbed areas? Could they be contributing to the diversity in Nectandra? I think it is worth including a sentence about it.

RESPONSE: There is only one non-native species (Macrothelypteris torresiana). We now mention this in the discussion of diversity at Nectandra (line 282).

Line 259 (with regards to the effect of elevation on number of species): What about humidity? Because this region is located at the Atlantic slopes, it has a high huidity along the yearm which contributes to the occurence of many epiphytes. Because it has been noted that epiphytes represent a considerable part of the diversity in some areas, I wonder whether this is not the case also in Nectandra?

By the way, I think it would be interesting to mention how many species are epiphytes, terrestrial, epipetric, etc...

RESPONSE: See response to Reviewer 1, Point 7.

Line 271 (with regards to Campyloneurum sp1): Alexander ARojas, a Costa Rican pteridologist, has recently published some new species in this complex. Perhaps your specimen could be one of them?

RESPONSE: We checked the species in Rojas 2017 (Am. J. Plant Sci.) and confirmed that our specimen is not one of these. We also added Rojas 2017 (Ref. 35) to the list of monographs that we consulted for species identification (line 102).

Lines 290-291, 296-297 (with regards to use of rbcL in molecular systematics in ferns and as a barcode marker): These two sentences are kind of contradictory. Please, rephrase.

RESPONSE: We disagree with the reviewer on this point. The first sentence establishes that rbcL is widely used in molecular systematic studies of pteridophytes; the second says that there have been relatively few studies comparing the performance of rbcL as a barcode marker across different pteridophyte floras. We see no contradiction in these statements; systematic studies and barcode studies are two different things.

Lines 304-306: I think that another possibility is the isolation of Tahiti. This would preclude recurrent events of dispersal from other areas, keeping the isolation of local populations. In both CR and Japan, there are more possibilities of gene flow between other areas, adding to the complexity of local populations.

RESPONSE: See response to Reviewer 1, Point 8.

Lines 362-364: Authors?

RESPONSE: We have corrected this reference (line 405), as well as others that were formatted incorrectly in the last version.

---

Reviewer #2: The authors present a checklist to a reserve that is accomplished through a combination of field work, herbarium study, and DNA barcoding using the chloroplast rbcL marker. The work appears to have been conducted with the utmost care. It is clearly presented and well written. It will act as a gold standard by which similar projects will be compared. I have made some additional comments, but I have a hard time finding errors in this work I congratulate the authors on a job well done.

RESPONSE: We thank the reviewer for the encouraging comments. Indeed, we hope this method will be applied in other areas and contribute to our understanding of biodiversity.

I am happy to see that this work adheres to open and reproducible science. The effort and thoroughness of providing the fully reproducible manuscript using Docker and Drake are appreciated. Nonetheless, I was unable to reproduce the manuscript. The address https://github.com/joelnitta/nectandra_ferns returned a 404 page not found error and attempting docker pull joelnitta/nectandra_ferns returned a ‘manifest unknown error’. Perhaps I am missing something simple.

RESPONSE: The github repository is still set to private, so that is why the attempting to open the URL resulted in a 404 error. The github repository will be made public upon acceptance. We knew this would happen, so that is why we made the code available via a dropbox link for reviewers to use in the meantime (line 154, original manuscript). Apparently the reviewer did not notice this. We apologize for any confusion. We corrected the docker image settings: "docker pull joelnitta/nectandra_ferns" should now work properly.

195 change “was in generally good agreement” to “generally in agreement”

RESPONSE: Done (line 217, revised MS).

The evidence in favor of the new taxa is clear and the arguments used to explain them is logical. While I appreciate the brevity of the paper. Personally, I would like to see more discussion of the potentially novel taxa with reference to the current state of the taxonomy in each group. That said, the authors are perfectly justified in maintaining their brief discussion if they prefer.

RESPONSE: Unfortunately, it was beyond the scope of this paper to do a thorough investigation of the potentially novel taxa. We therefore choose to keep our discussion of these to a minimum and suggest directions for further study.

The trees presented in the supplement show species of tree ferns that are highlighted in yellow some of which are non-monophyletic. Yet there is no discussion of these nor any other tree fern for that matter. Why were these results excluded from the discussion? The same is true for some polypodiaceae.

RESPONSE: We believe this is already clear in the original text: The purpose of the trees in Figs S2 and S3 is to check the placement of genera that were recovered as non-monophyletic in the sampling of species only at Nectandra with a broader sampling, as described in the methods (lines 129-133). We state in the Results that these are recovered in their expected genera in the broadly sampled trees (lines 232-236), and explain the yellow highlighting in the caption for the supplemental figures (lines 608-609, 618-619). There is no need to discuss these trees further in the Discussion, since they do not pertain directly to analysis at Nectandra, and have been presented in the Results.

Your Mycopteris taxifolia may be M. costaricense. See Sundue 2014 “Mycopteris, a new neotropical genus of grammitid ferns”

RESPONSE: Thanks to the reviewer for pointing this out. Upon closely re-examining species originally identified as Mycopteris taxifolia, we realized that the reviewer is correct, and that these are indeed Mycopteris costaricensis ("costaricense" in the reviewer's comment is a spelling error). We have updated our data and the manuscript appropriately.

---

We made the following additional corrections to the manuscript (all line numbers refer to the revised manuscript):

Line 4: Correct affiliation (from "University of Tokyo" to "The University of Tokyo")

Line 23: Change the number of previously undescribed taxa that appear to be of hybrid origin from three to two.

Lines 77-79: Add references for source of elevation data and software used to create map.

Line 80: Correct date of photo from 1995 to 1992.

Lines 83-86: Add CC BY license for photos.

Line 97: Correct "TNS" to "TI".

Line 113, others subsequently: In the original submission, we included new sequences of two Costa Rican specimens collected from areas outside of the Nectandra reserve. We realized that this was not covered by our molecular permits (R-CM-RN-001-2014-OT-CONAGEBIO and R-CM-RN-002-2017-OT-CONAGEBIO), which were specifically for specimens from Nectandra. We have removed these sequences from the analyses, removed all references to them from the MS, and will not be submitting them to GenBank. This has no impact on the main findings of the paper. To see other places where edits were made to the MS regarding these specimens, please see “nectandra_ferns_changes_2020-04-22_to_2020-09-29.docx".

Line 134: Add one more permit (R-CM-RN-002-2017-OT-CONAGEBIO).

Line 287: Change heading to from "Unidentified taxa and species complexes" to "Unidentified taxa and putative species complexes"

Lines 297-301: Clarify differences in morphology between Campyloneurum sp1 and Campyloneurum angustifolium by mentioning the number of rows of sori between the costa and margin, and adding measurements for lamina width.

References: Fix several references.

Submitted filename: response_to_reviewers.rtf

Click here for additional data file.

12 Oct 2020

A taxonomic and molecular survey of the pteridophytes of the Nectandra Cloud Forest Reserve, Costa Rica

PONE-D-20-11593R1

Dear Dr. Nitta,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Paulo Takeo Sano, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The text improved a lot after the changes made. Thank you for the informations. I wish you success with this paper.

Reviewers' comments:

10 Nov 2020

PONE-D-20-11593R1

A taxonomic and molecular survey of the pteridophytes of the Nectandra Cloud Forest Reserve, Costa Rica

Dear Dr. Nitta:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Paulo Takeo Sano

Academic Editor

PLOS ONE