American Journal of Botany 101(9): 1466–1475, 2014. THE FIRST FOSSIL OF A BOLBITIDOID FERN BELONGS TO THE EARLY-DIVERGENT LINEAGES OF ELAPHOGLOSSUM (DRYOPTERIDACEAE)1 JOSMAILY LÓRIGA2, ALEXANDER R. SCHMIDT3, ROBBIN C. MORAN4, KATHRIN FELDBERG2, HARALD SCHNEIDER5, AND JOCHEN HEINRICHS2,6 2University of Munich (LMU), Systematic Botany and Mycology, Menzinger Str. 67, 80638 Munich, Germany; 3University of Göttingen, Courant Research Centre Geobiology, Goldschmidtstraße 3, 37077 Göttingen, Germany; 4New York Botanical Garden, Bronx, New York 10458-5126 USA; and 5Botany Department, Natural History Museum, London, UK • Premise of the study: Closing gaps in the fossil record and elucidating phylogenetic relationships of mostly incomplete fossils are major challenges in the reconstruction of the diversification of fern lineages through time. The cosmopolitan family Dryopteridaceae represents one of the most species-rich families of leptosporangiate ferns, yet its fossil record is sparse and poorly understood. Here, we describe a fern inclusion in Miocene Dominican amber and investigate its relationships to extant Dryopteridaceae. • Methods: The morphology of the fossil was compared with descriptions of extant ferns, resulting in it being tentatively assigned to the bolbitidoid fern genus Elaphoglossum. This assignment was confirmed by reconstructing the evolution of the morphological characters preserved in the inclusion on a molecular phylogeny of 158 extant bolbitidoid ferns. To assess the morphology-based assignment of the fossil to Elaphoglossum, we examined DNA-calibrated divergence time estimates against the age of the amber deposits from which it came. • Key results: The fossil belongs to Elaphoglossum and is the first of a bolbitidoid fern. Its assignment to a particular section of Elaphoglossum could not be determined; however, sects. Lepidoglossa, Polytrichia, and Setosa can be discounted because the fossil lacks subulate scales or scales with acicular marginal hairs. Thus, the fossil might belong to either sects. Amygdalifolia, Wrightiana, Elaphoglossum, or Squamipedia or to an extinct lineage. • Conclusions: The discovery of a Miocene Elaphoglossum fossil provides remarkable support to current molecular clock-based estimates of the diversification of these ferns. Key words: ancestral state reconstruction; bolbitidoid fern; Dominican amber; Elaphoglossum; eupolypods I; fossil fern; Miocene; Polypodiales. Molecular clock-based studies have been increasingly employed to explore macroevolution and macroecology of ferns including aspects of their diversification in the past 120 Myr (e.g., Schneider et al., 2004, 2010; Schuettpelz and Pryer, 2009; Sessa et al., 2012; Liu et al., 2014). These studies challenged the fossil record as the main source of information about fern diversification by using molecular-based estimates of diversification times of extant lineages using DNA sequences. Although most of these studies incorporate one or more fossils as time constraints, little attention has been given to the consistency of the obtained hypotheses and the known fossil record. Recent reviews of the fern fossil record document a limited availability of reliably determined fossils especially for derived ferns (Collinson, 2001; Skog, 2001). In fact, some authors consider the 1 Manuscript received 9 June 2014; revision accepted 19 August 2014. The authors thank Jonathan Wingerath for providing access to the Dominican amber collections at the Smithsonian Institution and Jann Thompson and Scott L. Wing (Washington, D.C.) for granting our loan request. Ariel Rodríguez assisted with statistical analyses in R. This is publication number 135 from the Courant Research Centre Geobiology that is funded by the German Excellence Initiative. The participation of R.C.M. in this work was partially funded by a grant from the U.S. National Science Foundation (DEB-1020443). 6 Author for correspondence (e-mail: jheinrichs@lmu.de) doi:10.3732/ajb.1400262 fern fossil record inadequate for comprehensive time calibrations of molecular topologies (Lehtonen et al., 2012). This view, however, has not been backed up by exploring the information from published fossils, which have not yet been used for calibration purposes, or by newly discovered fossils using an integrative approach as suggested in Schneider et al. (2009). The Dryopteridaceae provide an outstanding example to explore the impact of newly discovered fossils on our understanding of fern diversification as outlined in molecular clock-based studies (Schneider et al., 2004; Schuettpelz and Pryer, 2009; Sessa et al., 2012; Liu et al., 2014). With about 1700 species in some 36 genera, the family is one of the most species-rich among derived ferns (Smith et al., 2006; Liu et al., 2007; Moran et al., 2010a, b; Christenhusz et al., 2011; McHenry et al., 2013). Phylogenetic studies reported two core lineages of Dryopteridaceae (Schuettpelz and Pryer, 2007; Lehtonen, 2011; Liu et al., 2014). The first lineage corresponds to the Dryopteridoideae and contains genera such as Arachniodes, Ctenitis, Dryopteris, and Polystichum. The second lineage corresponds to the subfamily Elaphoglossoideae (Christenhusz et al., 2011) and contains genera such as Polybotrya, Megalastrum, and Stigmatopteris. It also includes the well-supported, speciesrich, and almost entirely tropical clade known as the bolbitidoid ferns (Schuettpelz and Pryer, 2007; Moran et al., 2010a; Liu et al., 2014). This clade is characterized morphologically by the synapomorphies of dorsiventral rhizomes with an elongated American Journal of Botany 101(9): 1466–1475, 2014; http://www.amjbot.org/ © 2014 Botanical Society of America 1466 September 2014] LÓRIGA ET AL.—ELAPHOGLOSSUM FOSSIL 1467 Fig. 1. Holotype of Elaphoglossum miocenicum sp. nov. in Miocene Dominican amber (USNM 414283). (A) Upper surface of the leaf. (B) Lower surface of the leaf with sporangia covering the blade. (C, D) Basal petiolar scales. (E) Middle petiolar scale. (F) Syninclusion of fungal conidiophores at the margin of the leaf. (G) Sporangia in oblique-lateral view showing the vertical annulus and the transversal stomium. (H) Sporangium in dorsal view showing fungal conidiophores emerging between the annulus cells. (I) Cross section of the 3-seriate sporangium stalk. (J) Spore with continuous broadly folded perine. Scale bars = 1 mm (A, B), 100 µm (C–E, G, H), and 10 µm (F, I, J). 1468 [Vol. 101 AMERICAN JOURNAL OF BOTANY Fig. 2. Morphological characters observed in the amber fossil of Elaphoglossum miocenicum sp. nov. Black squares represent the character states present in the fossil as used in the ancestral character state reconstruction (see Fig. 4). (in transverse section) ventral meristele, roots borne only from this ventral meristele, lack of hairs on the leaves, sterile–fertile leaf dimorphy, and acrostichoid sporangial arrangement, i.e., the sporangia are distributed over the lower surface of the blade (Moran et al., 2010a). Within the bolbitidoid ferns, the largest genus is Elaphoglossum, a largely epiphytic, pantropical genus. The other bolbitidoid genera are typically either terrestrial (Bolbitis) or climbing from the soil up tree trunks (Arthrobotrya, Lomagramma, Mickelia, and Teratophyllum) (Moran et al., 2010a). So far, few fossils have been attributed to the Dryopteridaceae, and no fossils of bolbitidoid ferns have been documented (Collinson, 2001). Some fossils previously assigned to the family (see discussion of these in Collinson, 2001) are unlikely to belong to the Dryopteridaceae as defined by Smith et al. (2006). This is largely because earlier authors used the wider definition of Dryopteridaceae provided by Kramer (1990), a definition that includes genera now considered to belong to families in eupolypods I and II, such as Athyriacae, Onocleaceae, Tectariaceae, Thelypteridaceae, and Woodsiaceae (Smith et al., 2006; Schuettpelz and Pryer, 2007; Lehtonen, 2011). Late Miocene Dryopteris fossils (Sessa et al., 2012) and Eocene fossils assigned to the extant genus Rumohra (Collinson, 2001) appear to be the most reliable fossils of the Dryopteridaceae. The family placement of these fossils, however, has not been determined by reconstructing the evolution of the fossils’ characters on a phylogenetic tree. This approach is now widely considered crucial to achieve reliable assignments of fossil taxa and to overcome shortcomings of the previously used similarity assignments (Parham et al., 2012). In the present study, we describe an inclusion of a fertile fern in amber from the Dominican Republic. The amber has been dated as early Miocene, 20 to 15 Myr old (IturraldeVinent and MacPhee, 1996), and was exuded by resin-bearing species of Hymenaea in the Fabaceae (Poinar, 1991; Langenheim, 1995). We identify the fossil as Elaphoglossum, a member of the bolbitidoid lineage of the Dryopteridaceae. We use a molecular phylogeny of bolbitidoid ferns to reconstruct the ancestral states of characters preserved in the fossil. Finally, we examine whether the fossil’s age is consistent with estimated divergence times of bolbitidoid ferns based on calibrations from other fossils used previously in other large-scale phylogenetic analyses of ferns. MATERIALS AND METHODS The fossil is from the Dominican Republic and preserved in the amber collection of the U. S. National Museum of Natural History at the Smithsonian September 2014] LÓRIGA ET AL.—ELAPHOGLOSSUM FOSSIL 1469 Fig. 3. Time-calibrated phylogeny of bolbitidoid ferns. Nodes with a posterior probability ≥0.95 are marked by asterisks. The mean age (million years from present) of these nodes is indicated; bars represent the 95% highest posterior density (HPD) credibility intervals. Dashed vertical lines represent the age range estimated for Dominican amber. The geologic timescale follows Gradstein et al. (2012): PA, Paleocene; PI, Pliocene; PE, Pleistocene; H, Holocene; QN, Quaternary. Mean ages and 95% HPD credibility intervals of every node are provided in Appendix S2. 1470 AMERICAN JOURNAL OF BOTANY [Vol. 101 September 2014] LÓRIGA ET AL.—ELAPHOGLOSSUM FOSSIL Institution (coll. no. USNM 414283). The amber inclusion was investigated using a Zeiss Stemi 2000 dissection microscope and a Zeiss AxioScope A1 compound microscope, each equipped with a Canon 60D digital camera. In most instances, incident and transmitted light were used simultaneously. The images of Fig. 1 are digitally stacked photomicrographic composites of up to 40 individual focal planes obtained using the software package HeliconFocus 5.0 (HeliconSoft, http://www.heliconsoft.com) for a better illustration of the threedimensional inclusions. The fossil was compared with published morphological descriptions of extant ferns (e.g., Rouhan et al., 2004, 2008; Moran et al., 2007; Vasco et al., 2009a, 2013; Vasco, 2011; Lóriga et al., 2014) and putatively assigned to Elaphoglossum. This assignment was investigated with two independent approaches. First, divergence times of bolbitidoid ferns were estimated without incorporating the fossil as a time constraint. Second, the evolution of the fossil’s morphological characters was reconstructed on a phylogeny of the bolbitidoid ferns. These approaches also tested the morphology-based assignment of the fossil to certain clades (sections) within Elaphoglossum, and the consistency of molecular clock-based time estimates with the age of the amber as determined by geologists (Iturralde-Vinent and MacPhee, 1996). The taxonomic samping of bolbitidoid ferns was based on those species included in published phylogenies (Rouhan et al., 2004, 2007; Skog et al., 2004; Vasco et al., 2009b, in press; Moran et al., 2010a; Lóriga et al., 2014). The genera included were Arthrobotrya (2 species), Bolbitis (13 species), Lomagramma (8 species), Mickelia (5 species), Elaphoglossum (127 species), and Teratopyllum (2 species). The sampling of Elaphoglossum included all sections recognized by Rouhan et al. (2004) (i.e., sects. Amygdalifolia, Elaphoglossum, Lepidoglossa, Polytrichia, Setosa, Squamipedia), with the addition of sect. Wrightiana recognized by Lóriga et al. (2014). Noncoding intergenic plastid DNA sequences of the rps4-trnS and trnL-trnF regions of all investigated 158 bolbitidoid species were downloaded from GenBank (Appendix 1) and aligned with the program Muscle 3.6 (Edgar, 2004) under default parameters implemented in the program MEGA 5.1 (Tamura et al., 2011). The resulting alignment was manually edited in BioEdit 7.0.5.3 (Hall, 1999), and ambiguous positions were excluded. The final alignment with 712 bp (rps4-trnS, 371 bp; trnL-trnF, 341 bp) is available at TreeBase (http://treebase.org, study 16183). Divergence time estimates were performed with the BEAST v1.8.0 package (Drummond et al., 2006; Drummond and Rambaut, 2007) by assigning nodeage information from Schuettpelz and Pryer (2009) for the split of Bolbitis and the rest of the bolbitidoid ferns at 46.3 Ma, and the split of Elaphoglossum and Mickelia at 32.7 Ma. Because the results of Liu et al. (2014) indicated somewhat older ages for this split than those estimated by Schuettpelz and Pryer (2009), a minimum-age approach was adopted by modeling the age constraint for the root as a truncated normal prior distribution with a mean of 46.3 Ma, a standard deviation of 10, and a truncation from 46.3–1000 Ma (Knoop and Müller, 2009). The age constraint for Elaphoglossum had a truncated normal prior distribution with a mean of 32.7 Ma, a standard deviation of 10 and a truncation from 32.7–1000. The TVM+G model of evolution was chosen using the Bayesian information criterion of the program jModeltest v2.1.4 (Darriba et al., 2012), with PhyML implemented (Guindon & Gascuel, 2003). The analysis setup was done with the program BEAUTi 1.8.0, employing the above constraints, a lognormal relaxed clock and a birth–death model for incomplete sampling (Stadler, 2009). The analysis was run for 200 million generations and a sampling of every 20 000th tree. After a burnin of 25%, a maximum credibility tree was assembled with the program TreeAnnotator v1.8.0. The performance of the analysis was examined with the program TRACER 1.5 (Rambaut and Drummond, 2007). ESS values > 200 were regarded as good support. FigTree (http://tree/bio.ed.ac.uk/software/figtree) was used to depict the maximum credibility tree. The ancestral state of six discrete morphological characters preserved in the fossil (Fig. 2) was reconstructed to identify the relationships of the fossil. Three of these characters related to the ornamentation of the perine and were coded following Moran et al. (2007, 2010c). Information on the characters of most 1471 species is available online at http://www.plantsystematics.org/index.html or in online databases of the herbaria B, NY, and MNHN. The morphological character matrix is provided in Appendix S1 (see Supplemental Data with the online version of this article). Ancestral character state reconstructions (ASR) were carried out using the ace function of the ape package in R (Paradis et al., 2004). The maximum likelihood method for ASR (Pagel, 1994, 1999) was used over the time-calibrated consensus tree obtained from the Bayesian divergence time analysis. We implemented a model with equal rates of transition between states. Intermediate character states were treated as a new state. Missing data and not applicable characters were coded as lacking. RESULTS Of the 712 character sites in the concatenated DNA matrix, 140 were constant and 450 parsimony informative. All six bolbitidoid genera were resolved monophyletic (Figs. 3, 4), with Mickelia in a sister relationship to Elaphoglossum. Two monospecific sections of Elaphoglossum, sects. Amygdalifolia and Wrightiana, were placed in serial sister relationships to the rest of the genus. Section Elaphoglossum was placed sister to a clade with sects. Squamipedia, Setosa, Polytrichia, and Lepidoglossa. Section Squamipedia was recovered as sister to a clade comprising sect. Lepidoglossa and the sister sects. Setosa and Polytrichia. Divergence time estimates (Fig. 3) indicated an Eocene origin of Elaphoglossum, an Oligocene age of its core group (all sections with the exception of the monospecific sect. Amygdalifolia), and the presence of all sectional lineages in the middle Miocene. Node mean ages and 95% highest posterior density (HPD) credibility intervals are provided in Appendix S2 (see online Supplemental Data). Reconstruction of ancestral character states (Fig. 4) suggested that the most recent common ancestor of all bolbitidoid ferns had divided fertile blades (PL = 1.00) (proportional likelihood values [PL] are provided in Appendix S2, node identification numbers in online Appendix S3). All bolbitidoid genera retained this ancestral character state except Elaphoglossum. The divided blades of E. bifurcatum and E. cardenasii were secondarily derived. It is ambiguous whether the most recent common ancestor of Elaphoglossum and Mickelia had either entire or divided fertile blades (PL = 0.53 vs. PL = 0.47). The perine folds of the most recent common ancestor of Mickelia were reconstructed as thin cristate with a probability of PL = 0.85. It is equivocal whether the perine folds of the most recent common ancestor of Elaphoglossum and Mickelia were thin and cristate or broad and rounded (PL = 0.51 vs. PL = 0.31). The ancestors of several early-diverging lineages of Elaphoglossum most likely exhibited the same set of characters observed in the fossil; namely, perine nonappressed and with continuous, broad, rounded folds (characters of all Eupolypod ferns), fertile blades simple (characters of nearly all species of Elaphoglossum), and petiolar scales not rolled at the base and lacking unicellular marginal teeth (Fig. 1). Within Elaphoglosssum, the sections that exhibit these characters include sects. Amygdalifo- ← Fig. 4. Time-calibrated phylogeny of bolbitidoid ferns presented in Fig. 3 showing the ancestral state reconstruction of morphological characters observed in Elaphoglossum miocenicum sp. nov. Morphological characters are displayed in the terminals of the tree in the same order as described in Fig. 2 (squares with two colors indicate intermediate states; gray squares indicate not applicable characters or lack of data). Rectangles at main internal nodes of the tree represent the proportional likelihoods of character presence for characters 1–6 as inferred by the ancestral state reconstructions. Morphological states scored for every species are provided in Appendix S1, and proportional likelihood values of character presence in every node is provided in Appendix S2. 1472 AMERICAN JOURNAL OF BOTANY lia, Lepidoglossa, Squamipedia, and Wrightiana. The most recent common ancestor of sects. Setosa and Polytrichia had basally enrolled scales (PL = 1.00), whereas the scales were flat in the fossil and the rest of bolbitidoids. The most recent common ancestor of Elaphoglossum sect. Lepidoglossa probably had scales with acicular marginal appendages consisting of a single cell (PL = 1.00), whereas in the fossil and the rest of bolbitidoids marginal teeth were formed by the upturned ends of two adjacent cells. DISCUSSION AND TAXONOMIC TREATMENT The fossil has simple and entire leaves and an acrostichoid arrangement of sporangia (Fig. 1), suggesting it is an Elaphoglossum, a bolbitidoid fern genus in the Dryopteridaceae. The most recent common ancestor of Elaphoglossum and Mickelia was reconstructed to have had either entire or divided fertile blades (PL = 0.53 vs. PL = 0.47), but the perine folds of the ancestor of Mickelia were reconstructed as thin and cristate (PL = 0.85). The fossil had broadly rounded perine folds (Fig. 1J), which are frequent in Elaphoglossum (Moran et al., 2007). Elaphoglossum is the only fern genus characterized by the combination of simple and entire leaves, the presence of phyllopodia, an acrostichoid arrangement of sporangia, and free veins. Unfortunately, petiole bases, which would allow determination of the presence or absence of phyllopodia, were not present in the fossil, and venation was not visible on the fertile lamina preserved in the inclusion. Simple and entire leaves with acrostichoid sori occur also in several genera belonging to distinct lineages such as Dipteridaceae, Polypodiaceae, and Tectariaceae. The Dipteridaceae can be discounted because the fossil is not a simple and entire-leaved Cheiropleuria. That genus has 4-seriate sporangial stalks, and slightly oblique, complete annuli, and tetrahedral, trilete spores (Smith et al., 2006). It also lacks foliar scales. In contrast, the fossil has three-seriate sporangium stalks, vertical annuli interrupted at the stalks (i.e., not bypassing the stalk and completely encircling the sporangial capsules), and bean-shaped monolete spores (Fig. 1G–J). In the Polypodiaceae, some species of Leptochilus (including Colysis) have simple blades with an acrostichoid arrangement of sporangia, but unlike the fossil (Fig. 1A–E), these ferns lack scales on the petioles of fertile leaves (R. C. Moran and H. Schneider, personal observations). Moreover, Leptochilus, like most Polypodiaceae, has a thin perine tightly appressed to the exine (Tryon and Lugardon, 1991), not a broadly folded perine as found in the fossil. Finally, it seems unlikely that the fossil belongs to the Tectariaceae. Laminar scales, such as are common on the fossil, are rare or absent in that family, as are also sporangia with an acrostichoid arrangement (R. C. Moran, personal observations). Given the evidence, the fossil most likely belongs to Elaphoglossum. It could not be assigned to a section within Elaphoglossum because important characters were not preserved or visible, such as rhizome habit and presence/absence of hydathodes (Rouhan et al., 2004; Moran et al., 2010a; Lóriga et al., 2014). Assuming consistency of sectional character states through time, however, the fossil can be excluded from three sections. Elaphoglossum sect. Lepidoglossa can be eliminated because the scales of the fossil lack unicellular marginal teeth, which occur in all extant species of this section (Vasco et al., 2009b), and were estimated to be present in the most recent ancestor of the section with a probability of PL = 1.00 (Fig. 4). [Vol. 101 Similarly, sects. Polytrichia or Setosa can be excluded because they have subulate (longitudinally enrolled) scales on the leaves (Mickel and Atehortúa, 1980). Some species in these two sections bear flat scales on parts of the lamina, especially the margins. These flat scales, however, are always accompanied by subulate scales elsewhere on the same leaf, in contrast to the consistently flat scales of the fossil. Given the elimination of these three sections, the fossil belongs either to one of the remaining sections of Elaphoglossum (i.e., sects. Amygdalifolia, Elaphoglossum, Squamipedia, or Wrightiana), or to an extinct lineage not part of any extant section. Previous divergence time analyses (Schuettpelz and Pryer, 2009; Liu et al., 2014) provided evidence for a Paleogene origin of Elaphoglossum. This is consistent with our newly obtained divergence-time analysis (Fig. 3) and the interpretation of the fossil as a member of Elaphoglossum. The morphology of the fossil does not exclude the possibility that it belongs to an extant species of Elaphoglossum. Miocene amber inclusions of bryophytes from the Dominican Republic have frequently been assigned to extant species (Gradstein, 1993; Frahm and Newton, 2005), although uncertainty remains since these inclusions show only a subset of the features visible in living plant material (Heinrichs et al., 2013). To assess whether the fossil belongs to an extant species, we estimated the ages of the sectional crown groups of Elaphoglossum. Our divergence-time analysis allowed slightly older ages than those presented in other studies (Schuettpelz and Pryer, 2009); however, despite this conservative approach, we found the extant species of the sectional crown groups to be younger than the fossil. Hence, the fossil is considered to represent a stem lineage element of one of the above sections, or an extinct member of the early-diverging sects. Amygdalifolia and Wrightiana. These two sections are monospecific and may or may not represent survivors of the early divergence of the genus (Lóriga et al., 2014). Finally, the possibility cannot be ruled out that the fossil belongs to an extinct lineage of Elaphoglossum that is not part of an extant section. Given the above analyses that provide evidence the amber inclusion is an extinct crown group representative of Elaphoglossum, we describe it here as a new species. New species— Elaphoglossum miocenicum Lóriga, A. R. Schmidt, R. C. Moran, K. Feldberg, H. Schneid. & Heinrichs, sp. nov. Holotype— National Museum of Natural History of the Smithsonian Institution, amber inclusion no. USNM 414283. Fragment of fertile leaf with acrostichoid arrangement of sporangia (Fig. 1). Type locality: Dominican Republic, Santiago area. Age and stratigraphic position: Early Miocene, about 15 to 20 Myr ago. Syninclusions: Conidiophores of a fungus and the leafy liverworts Bazzania sp. (Lepidoziaceae) and Cheilolejeunea antiqua (Lejeuneaceae). Diagnosis— Bolbitidoid fern with simple, entire, fertile leaves and sporangia covering the lower surface of the blade; leaf scales flat, with toothed margins, unicellular marginal teeth lacking; perines with broad, continuous folds. Description— The fossil consists of a fragment of a fertile leaf including the petiole and the basal half of the blade. Fertile leaf simple, entire; petiole 0.9 cm long, 1.5 mm broad; scales September 2014] LÓRIGA ET AL.—ELAPHOGLOSSUM FOSSIL scattered on the petiole and blade, lanceolate to irregularly shaped, becoming larger towards the petiole base, up to 3.6 mm long, basifixed, narrowly lanceolate, brown, margin entire to dentate; blade wedge-shaped, base long-decurrent. Leptosporangia densely covering the abaxial surface of the blade (acrostichoid sporangial arrangement), stalks 3-celled; annulus vertical, interrupted at the stalk, stomium transverse; spores monolete, reniform, equatorial diameter 36.0 (25.0–45.0) × 21.7 (20.0– 25.0) μm, perine with continuous, broad folds. Perspectives—Dominican amber is a well-known source of plant microinclusions and especially famous for its numerous liverwort and moss fossils that indicate a conserved generic composition of epiphytic bryophyte communities during the Miocene of the Caribbean (Frahm and Newton, 2005; Heinrichs et al., 2014). In contrast, only a few fern inclusions have been recognized so far (Grimaldi, 1996), of which only one has been treated taxonomically, as Grammitis succinea (Gómez, 1982). The present study documents the second fern genus in Dominican amber and the first fossil of a bolbitidoid fern. 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Bolbitis acrostichoides (Afzel. ex Sw.) Ching; GU376644; GU376500. B. aliena (Sw.) Alston; GU376646; GU376502. B. appendiculata (Willd.) K. Iwats.; GU376648; GU376504. B. auriculata (Sw.) Alston; GU376649; GU376505. B. bipinnatifida (J. Sm.) K. Iwats.; GU376676; GU376530. B. fluviatilis (Hook.) Ching; GU376656; GU376510. B. gemmifera (Hieron.) C. Chr.; GU376657; GU376511. B. heteroclita (C. Presl) Ching; GU376660; GU376514. B. humblotii (Baker) Ching; GU376663; GU376516. B. major (Bedd.) Hennipman; GU376665; GU376518. B. portoricensis (Spreng.) Hennipman; GU376670; GU376523. B. serratifolia (Mert. ex Kaulf.) Schott; GU376673; GU376527. B. tibetica Ching & S.K. Wu; GU376677; GU376531. Elaphoglossum achroalepis (Baker) C. Chr.; AY540225; AY536288. E. acrostichoides (Hook. & Grev.) Schelpe; EF040628; EF040614. E. aff. ciliatum (C. Presl) T. Moore; EU907748; EU907813. E. affine (M. Martens & Galeotti) T. Moore; AY536169; AY534841. E. albescens (Sodiro) Christ; GU376678; GU376532. E. alismaefolium (Feé) T. Moore; KF212425; KF212399. E. amygdalifolium (Mett. ex Kuhn) Christ; AY536173; AY534845. E. angulatum (Blume) T. Moore; AY540230; AY536293. E. asterolepis (Baker) C. Chr.; AY540231; AY536294. E. aubertii (Desv.) T. Moore; EF040622; EF040608. E. auricomum (Kunze) T. Moore; AY536145; AY534817. E. auripilum Christ; EF040626; EF040612. E. avaratraense Rakotondr.; AY540233; AY536296. E. backhouseanum T.Moore; AY540234; AY536297. E. bifurcatum (Jacq.) Mickel; EU907737; AY194070. E. biolleyi Christ; AY540235; AY536298. E. burchellii (Baker) C. Chr.; EU907738; EU90780. E. cardenasii W.H. Wagner; AY536131; AY534802. E. cardiophyllum (Hook.) T. Moore; AY53617; AY534842. E. cf. longifolium (Jacq.) J. Sm.; KF212426; KF212402. E. ciliatum (Hook.) T. Moore ex Diels; EU907745; EU907810. E. cismense Rosenst.; AY540237; AY536300. E. concinnum Mickel; KJ528151; KJ528179. E. conspersum Crhist; AY540238; AY536301. E. coriaceum Bonap.; EF040627; EF040613. E. coursii Tardieu; AY540240; AY536303. E. crinitum (L.) Christ; AY536134; AY534805. E. croatii Mickel; AY540241; AY536304. E. cubense (Mett. ex Kuhn) C. Chr.; KF212429; KF212404. E. cuspidatum (Willd.) T. Moore; EU907750; EU907815. E. davidsei Mickel; AY540242; AY536305. E. decaryanum Tardieu; AY540243; AY536306. E. deckenii (Kuhn) C. Chr.; AY540244; AY536307. E. decoratum (Kunze) T. Moore; GU376681; GU376534. E. dendricola (Baker) Christ; EU907751; EU907816. E. dimorphum (Hook. & Grev.) T. Moore; EU907753; EU907817. E. doanense L.D. Gómez; AY540245; AY536308. E. dussii Underw. & Maxon; EU907755; EU907819. E. edwallii Rosenst.; AY536144; AY534816. E. eggersii (Baker) Christ; KF212431; KF212406. E. erinaceum (Fée) T. Moore; KF212432; KF212407. E. eximium (Mett.) Christ; AY536132; AY534803. E. firmum (Mett. ex Kuhn) Urb.; KF212382; KF212408. E. flaccidum (Fée) T. Moore; AY540246; AY536309. E. forsythiimajoris Christ; EF040620; EF040606. E. fournierianum L.D. Gómez; AY540248; AY536311. E. gayanum Mickel; AY534838; AY536166. E. glabellum J. Sm.; AY536167; AY534839. E. gramineum (Jenman) Urb.; KF212383; KF212409. E. grayumii Mickel; AY540250; AY536313. E. guatemalense (Klotzsch) T. Moore; AY536164; AY534836. E. guentheri Rosenst.; GU376682; GU376535. E. herminieri (Bory & Fée) T. Moore; KF212435; KF212410. E. heterolepis T.Moore; AY540251; AY53631. E. hoffmannii (Mett. ex Kuhn) Christ; AY540252; AY536315. E. hornei C.Chr.; AY540253; AY536316. E. huacsaro (Ruiz) Christ; HG425357; KF212419. E. humbertii C. Chr.; EU907771; EU907834. E. hybridum (Bory) Brack.; EU907772; EU907835. E. ipshookense Mickel; EU907773; EU907836. E. lanatum Lorence; AY540258; AY536321. E. lancifolium (Desv.) C.V. Morton; AY540259; AY536322. E. langsdorffii (Hook. & Grev.) T. Moore; GU376536; GU376683. E. leucolepis (Baker) Krajina ex Tardieu; AY540261; AY536324. E. lindenii (Bory ex Fée) T. Moore; AY536130; AY534801. E. lingua (C. Presl) Brack.; AY540262; AY536325. E. lloense (Hook.) T. Moore; GU376684; GU376537. E. luridum (Fée) Christ; AY540263; AY536326. E. macropodium (Fée) T. Moore; AY54026; AY536327. E. malgassicum C. Chr.; AY540265; AY536328. E. marojejyense Tardieu; AY540266; AY536329. E. marquisearum Bonap.; AY540267; AY536330. E. martinicense (Desv.) T. Moore; KF212386; KF212411. E. maxonii Underw. ex Maxon; KF212438; KF212413. E. metallicum Mickel; AY536160; AY534832. E. micropogon Mickel; AY540268; AY536331. E. mitorrhizum Mickel; AY540269; AY536332. E. moorei (E. Britton) Christ; KJ528150; KJ528208. E. nervosum (Bory) Christ; EU907775; EU907837. E. nidiformis Mickel; EF040629; EF040616. E. nidusoides Rouhan & Rakotondr; EF040618; EF040604. E. nigrescens (Hook.) T. Moore ex Diels; EU907781; EU907843. E. nigrocostatum Mickel; AY536152; AY534824. E. oblanceolatum C. Chr.; AY540271; AY536334. E. ocoense C. Chr.; KF212414; KF212441. E. orbignyanum (Fée) T. Moore; EU907783; EU907845. E. ovalilimbatum Bonap.; AY540272; AY536335. E. ovatum (Hook. & Grev.) T. Moore; AY540273; AY536336. E. paleaceum (Hook. & Grev.) Sledge; EU907784; EU907846. E. palmeri Underw. & Maxon; KF212442; KF212415. E. papillosum (Baker) Christ; AY536129; AY534800. E. peltatum (Sw.) Urb.; KF212444; KF212417. E. petiolatum (Sw.) Urb.; AY540275; AY536338. E. phanerophlebium C. Chr.; AY540276; AY536339. E. piloselloides (C. Presl) T. Moore; KF212445; KF212418. E. pilosius Mickel; AY540277; AY536340. E. poolii Christ; AY540278; AY536341. E. potosianum Christ; EU907786; EU907849. E. prestonii (Baker) J. Sm.; AY534810; AY53481. E. pringlei (Davenp.) C. Chr.; EU907716; EU907850. E. productum Rosenst.; AY540279; AY536342. E. pusillum (Mett. ex Kuhn) C. Chr.; HG428762; KF212420. E. pygmaeum (Mett. ex Kuhn) Christ; AY540281; AY536344. E. richardii (Bory) Christ; EF040621; EF040607. E. rufidulum C. Chr.; AY540285; AY536348. E. russelliae Mickel; AY540286; AY536349. E. rzedowskii Mickel; EU907788; EU907851. E. samoense Brack.; AY540287; AY536350. E. sartorii (Liebm.) Mickel; AY536161; AY534833. E. scolopendriforme Tardieu; AY540288; AY536351. E. setigerum (Sodiro) Diels; AY540289; AY536352. E. sieberi (Hook. & Grev.) T. Moore; AY540290; AY536353. E. siliquoides (Jenman) C. Chr.; AY536127; AY534798. E. smithii (Baker) Christ; AY540291; AY536354. E. spatulatum (Bory) T. Moore; EF040623; EF040609. E. splendens Brack.; AY540296; AY536359. E. squamipes (Hook.) T. Moore; AY536157; AY534829. E. squamipes (Hook.) T. Moore; AY536158; AY534830. E. subsessile (Baker) C. Chr.; AY540298; AY536361. E. succisifolium (Willd.) T. Moore; AY540299; AY536362. E. tectum (Humb. & Bonpl. ex Willd.) T. Moore; AY536142; AY534813. E. tomentosum (Bory ex Willd.) Christ; AY540300; AY53636. E. tripartitum (Hook. & Grev.) Mickel; AY536156; AY534828. E. vestitum (Schltdl. & Cham.) T. Moore; AY536146; AY534818. E. vieillardii (Mett.) T. Moore; AY54030; AY536364. E. wawrae C. Chr.; AY540302; AY536365. E. welwitschii (Baker) C. Chr.; AY540303; AY536366. E. wrightii (Mett. ex D.C. Eaton) T. Moore; KF212447; KF212423. E. yungense de la Sota; EU907796; EU907859. Lomagramma brooksii Copel.; GU376691; GU376542. L. cordipinna Holttum; GU376695; GU376546. L. lomarioides (Blume) J. Sm.; GU376699; GU376550. L. matthewii (Ching) Holttum; GU376700; GU376551. L. perakensis Bedd.; GU376703; GU376554. L. pteroides J. Sm.; GU376704; GU376555. L. sinuata C. Chr.; GU376706; GU376557. L. sumatrana Alderw.; GU376708; GU376559. Mickelia bernoullii (Kuhn ex Christ) R.C. Moran, Labiak & Sundue; GU376651; GU376506. M. guianensis (Aubl.) R.C. Moran, Sundue & Labiak; GU376698; GU376549. M. nicotianifolia (Sw.) R.C. Moran, Labiak & Sundue; GU376669; GU376522. M. oligarchica (Baker) R.C. Moran, Labiak & Sundue; GU376668; GU376521. M. scandens (Raddi) R.C. Moran, Labiak & Sundue; GU376696; GU376547. Teratophyllum koordersii Holttum; GU376715; GU376566. T. ludens (Fée) Holttum; GU376717; GU376568.