Filmy Ferns (Hymenophyllaceae) and Associated Spike-Mosses (Selaginellaceae) from the Mid-Cretaceous Kachin Amber, Myanmar

Simple Summary Three new species of filmy ferns are described in mid-Cretaceous Kachin amber, which represent the first fossil record of Hymenophyllaceae from tropical Asia. These filmy ferns and the syninclusions of spike-mosses greatly expand the diversity of the cryptogams in Kachin amber and provide additional evidence regarding the paleoenvironment. Together with other contemporaneous findings, the present fossils indicate that Hymenophyllaceae have already accumulated some notable diversity in the Cretaceous. Abstract Filmy ferns (Hymenophyllaceae) are the most diverse lineage of the early-diverging leptosporangiate ferns with ca. 430 species widely distributed around the world but with the highest diversity in the humid tropics. However, their fossil record is scarce because of the low preservation potential of the delicate, membranous laminae. So far, no Hymenophyllaceae fossils have been reported from tropical Asia. Here, we describe some fern remains and their syninclusions (spike-mosses) in four pieces of Kachin amber from the mid-Cretaceous of Hukawng Valley, Northern Myanmar, as Hymenophyllites angustus sp. nov., H. kachinensis sp. nov., H. setosus sp. nov. (Hymenophyllaceae) and Selaginella alata sp. nov. (Selaginellaceae), respectively. These fern remains are assigned to Hymenophyllaceae based on the filmy, one-cell thick, decompound pinnatifid laminae and dichotomous venation. They represent the first fossil record of Hymenophyllaceae in tropical Asia. The growth habits of these ferns and associated spike-mosses and their implication for paleoenvironment are discussed. Our study expands the diversity of the cryptogams in mid-Cretaceous Kachin amber. Together with other contemporaneous findings, the present fossils indicate that Hymenophyllaceae have already accumulated some notable diversity in the Cretaceous.

Remarks: The present fern remains display the most distinctive characteristic of the family Hymenophyllaceae, viz., membranaceous, translucent laminas which are only one cell layer thick between the veins. Due to the lack of material of fertile fronds with indusia, it is really difficult to nail down the systematic relationships with the subfamilies of the Hymenophyllaceae. However, some characters allow at least the exclusion of most genera of the subfamily Trichomanoideae. For example, the present fern remains differ from Abrodictyum in having more than three rows of cells between midribs and laminar margins; they differ from Cephalomanes in having tripinnatifid lamina; they have glabrous lamina, while Callistopteris and Vandenboschia have hairs on stipes and rachises; the absence of false veinlets in the present fern remains differentiate them from most species of Crepidomanes and Didymoglossum [41,80]. Additionally, the presence of anadromous venation of pinnules also makes our fern remains more consistent with Hymenophylloideae, because most species of extant Hymenophylloideae have anadromous venation of pinnules, whereas, in Trichomanoideae, both anadromous and catadromous venation is present [60]. Due to the lack of fertile materials, we tentatively place our fern remains in the fossil genus Hymenophyllites, which displays great resemblance to the extant Hymenophyllum but still lacks some characters of it. Hymenophyllites represents a putative member of Hymenophyllaceae reported from the Carboniferous to Cretaceous [54,57,[63][64][65] Description: Five lamina fragments are preserved as sterile pinnae or pinnules in two pieces of amber (Figure 2A,B). The pinnae or pinnules are ca. 1.2-1.3 cm long, once pinnatifid, membranous, one cell layer thick between the veins, glabrous, distinctly segmented into simple or forked ultimate segments which are characterized by one to several lobes ( Figure 2C-F). The lobes are flat, slightly elongate to oblong, 0.7-1.0 mm wide, entiremargined with obtuse, truncate to retuse apices. Each lobe is vascularized by a single veinlet. Veins are dichotomous, forming zigzag or curved costules; false veinlets are absent. Lamina cells are polygonal, isodiametrical to elongated, 109-124 µm long and 40-76 µm wide, with straight to slightly wavy cell walls; stomata are absent ( Figure 2G). A juvenile sorus is born at apex of ultimate lobe in leaf margin ( Figure 2H).
Remarks: The present fern remains are similar to Hymenophyllites angustus sp. nov. in gross morphology, but slightly differs in having larger pinnae and/or pinnules, wider lobes of the ultimate segments and comparatively thinner veins. These characters are often used to distinguish extant species of Hymenophyllum [41] and thus support the present fossils as a new species of Hymenophyllites. The discovery of a juvenile sorus with a likely bivalved indusium born in our leaf margin indicate the present fossils may belong to the extant Hymenophyllum of the subfamily Hymenophylloideae. More fertile materials are still needed to finally assign them into the extant genus. Some sterile and fertile shoots of Selaginella (Figure 2A,B), as well as a fern sporangium ( Figure 2D), are preserved as syninclusions with Hymenophyllites kachinensis sp. nov. The sporangium is ovate, sessile, 181 µm long and 146 µm wide, with an uninterrupted annulus ( Figure 2I  ated segment margin. Some species of Hymenophyllum subg. Hymenophyllum, e.g., H. rolandi-principis Rosenst., have a marginal seam of smaller, rounded cells that may even be darkened/indurated. Thus, the fern remain studied here closely resembles extant lineages of Hymenophyllum in vegetative characters and could be tentatively assigned to the fossil genus Hymenophyllites due to the lack of fertile characters. The subfamily assignment also awaits future confirmation when fertile material is available. The presence of bristlelike hairs, darkened submarginal lines and slightly thicker leaf lamina make the present species very easy to distinguish from the previous two new species Hymenophyllites angustus and Hymenophyllites kachinensis ( Figure 4).    Figure 3A,B). Both adaxial and abaxial surfaces are sparsely covered with bristle-like hairs ( Figure 3C,D). The hairs are single, straight to curved, ca. 68-292 µm long, erect to leaning. The pinna or pinnule is distinctly segmented into 7 ultimate segments. Individual segments are simple, characterized by single sterile lobes, closely spaced, alternate, 0.7-1.3 mm wide; acroscopic and basiscopic parts are shallowly and deeply lobed, respectively; apices are obtuse, rounded to retuse; margin is generally entire but with some hairs located very close to or nearly on leaf margin ( Figure 3C,E), and sometimes folded to form darkened submarginal lines ( Figure 3E). Veins are dichotomous, forming a zigzag costule and seven lateral veins; lateral veins are simple or dichotomous, reaching high up to approach segment apex, ending just below apex; submarginal false veinlets are absent; internal false veinlets are absent. Lamina cells are polygonal; stomata are absent ( Figure 3F).
Remarks: The membranaceous, translucent lamina fragment indicates that the present fern remain probably belongs to the family Hymenophyllaceae. Leaf surface sparsely covered with single setose hairs is characteristic of Hymenophyllum subgenus Sphaerocionium (C. Presl) C. Chr., which is cosmopolitan with ca. 70 species, highly diversified in the Neotropics [42]. In subgenus Sphaerocionium today, the majority of species have stellate hairs but there are also some species with exclusively unbranched single hairs [42]. The darkened submarginal lines in our fossil look like marginal false veinlets of the extant genera Crepidomanes and Didymoglossum in the subfamily Trichomanoideae of Hymenophyllaceae [41,42,80]. But these two extant genera have glabrous leaf surface except D. tahitense (Nadeaud) Ebihara et K. Iwatsuki which has many dark brown hairs along veins [41]. The darkened submarginal lines can also be interpreted as the folds of the differentiated segment margin. Some species of Hymenophyllum subg. Hymenophyllum, e.g., H. rolandi-principis Rosenst., have a marginal seam of smaller, rounded cells that may even be darkened/indurated. Thus, the fern remain studied here closely resembles extant lineages of Hymenophyllum in vegetative characters and could be tentatively assigned to the fossil genus Hymenophyllites due to the lack of fertile characters. The subfamily assignment also awaits future confirmation when fertile material is available. The presence of bristle-like hairs, darkened submarginal lines and slightly thicker leaf lamina make the present species very easy to distinguish from the previous two new species Hymenophyllites angustus and Hymenophyllites kachinensis (Figure 4).    Age: Late Albian-early Cenomanian, mid-Cretaceous.
Repository: Collection Department of Nanjing Institute of Geology and Paleontology, Chinese Academy of Sciences, Nanjing, China.
Diagnosis: Shoot anisotomously branched. Vegetative leaves dimorphic, arranged in four ranks, two ranks of smaller ascending median leaves and two other ranks of larger spreading lateral leaves. Median leaves non-carinate with aristate apex and denticulate to ciliolate margin. Lateral leaves non-carinate, lanceolate, with acute to acuminate apex and entire to serrulate to ciliolate margin. Strobili born at apex of the fertile shoots, compact, tetragonal. Sporophylls arranged in four ranks, monomorphic, with long acuminate apex, dentate margin, alary serrulate keel.
Description: The two fertile shoots are 1.0 cm and 0.5 cm long and bear four and three strobili respectively ( Figure 5A-C). Stems are anisotomously branched, without articulations. Rhizophores are not observed. Leaves are dimorphic, arranged in four ranks, two ranks of median leaves on dorsal or upper side of stem and branch and two other ranks of lateral leaves on lateral or lower side ( Figure 5D-G). Ligules are not observed. Axillary leaves are symmetrical, lanceolate, 1.6 mm long and 0.6 mm wide (measurement of one leaf), with non-auriculate bases and ciliolate margin (with 52-81 µm long cilia) ( Figure 5D,E). Median leaves are smaller, ascending, non-carinate, ovate to obovate, 0.6-0.8 mm long and 0.3-0.4 mm wide (measurement of four leaves), with aristate apex and denticulate to ciliolate margin (with 21-64 µm long teeth or cilia) ( Figure 5F,G). Lateral leaves are larger and spreading, non-carinate, lanceolate, 1.2-2.0 mm long and 0.6-0.8 mm wide (measurement of eight leaves), with acute to acuminate apex and entire serrulate to ciliolate margin (with 6-88-µm-long teeth or cilia that occur mainly at the acroscopic base) (Figure 5D-G). Strobili are solitary, born at apex of main stem or branch, compact, tetragonal, 1.7-4.6 mm long and 1.4-1.8 mm wide ( Figure 5H,I). Sporophylls are arranged in four ranks, monomorphic, ovate-lanceolate, 0.8-1.1 mm long and 0.1-0.2 mm high, with long acuminate apex, dentate margin (with 21-47 µm long teeth) and alary serrulate keel (with 14-32 µm long teeth) ( Figure 5J). Sporophyll-pteryx is not observed. Sporangia are single per sporophyll, mature, and deeply two-valved nearly to the base. Macrospores are not observed.
Remarks: Selaginella is the sole extant genus of Selaginellaceae and includes approximately 750 herbaceous species, widely distributed around the world but with its highest diversity in the tropics [81][82][83][84]. Selaginella is an ancient lineage with fossils dating back to the Carboniferous or even Devonian [53,85]. Schmidt et al. [23,25] discovered the presence of hyperdiverse Selaginella in the mid-Cretaceous Kachin amber, Myanmar, including ten species of Selaginella subgenus Stachygynandrum with anisophyllous (bilateral) strobili and eleven species with isophyllous strobili. All these fossil species are erected based on the combination of a series of key characters [23,25]. Furthermore, Li et al. [24] described a new species of Selaginella subgenus Stachygynandrum with anisophyllous strobili from Kachin amber. Compared with all these Selaginella fossils from Kachin amber, our fossils are clearly different from the eleven species with anisophyllous strobili by having isophyllous strobili, and distinct from the two species S. isophylla A.R. Schmidt et L. Regalado and S. wunderlichiana A.R. Schmidt et L. Regalado with monomorphic and decussately arranged vegetative leaves in having dimorphic vegetative leaves arranged in four rows [25]. Our fossils resemble the rest nine species in the aspects of dimorphic vegetative leaves and isophyllous strobili, but none of the nine species has alary keel on the abaxial side of the midrib of sporophyll [25]. Alary keel is also not documented in the extant species of Selaginella [81,84].
The laminae are at least twice pinnatifid in Hymenophyllites kachinensis, but they are only once pinnate-pinnatifid in the Eocene species Hymenophyllum axsmithii [67]. Hymenophyllites setosus sp. nov. is distinctive for its leaf surface covered with single setose hairs.  [60] ? means unknown character, and -stands for inapplicable character.
Although the subfamily Trichomanoideae are a high diversified lineage with eight genera and an estimated 184 species [39], unequivocal fossils of this subfamily are extremely rare, only with Eogonocormus as a possible member. The thalloid fan-like form of the frond, marginal sori borne on fanlike pinnule lobes, and in situ trilete papillate spores indicate that Eogonocormus is close to the extant genus Gonocormus Bosch [58]. At present, the genus Gonocormus has been merged into Crepidomanes within the subfamily Trichomanoideae, but some members of it were transferred into Hymenophyllum, such as Hymenophyllum nitidulum (Bosch) Ebihara et K. Iwatsuki [41]. These new taxonomic treatments document how misleading morphology alone can be in this group of plants.

Ecological and Putatively Evolutionary Implications
It is suggested based on ancestral state reconstructions of habit that filmy ferns (Hymenophyllaceae) were ancestrally terrestrial, with epiphytism having evolved several times independently during the Cretaceous [89]. The development of more humid climates in the increasingly closed canopy forests caused by the rapid rise of angiosperms in the Cretaceous may have provided optimal conditions for epiphytic filmy ferns to diversify [89]. It is likely that Hymenophyllites angustus sp. nov., Hymenophyllites kachinensis sp. nov. and Hymenophyllites setosus sp. nov. were epiphytic ferns, because the extant species of Hymenophyllum are almost uniformly epiphytic [89]. Hymenophyllites kachinensis was always found together with the spike-mosses (Selaginella), which are understory herbaceous plants, normally growing on open soil or exposed rocks but also occasionally epiphytic in moist forests [84]. These filmy ferns and associated spike-mosses probably lived on tree trunks and therefore could easily be incased in resin.
Due to the thin filmy laminas, the extant Hymenophyllaceae predominantly live in moist mossy forest [45,90], and the evolution of the filmy ferns shows a tendency towards progressive reduction in size, which may be considered as adaptive to the extremely moist mossy zone [45]. Additionally, the Kachin Selaginella fossils also possess several characters suggestive of humid conditions, such as the relatively thin leaves often with fungal colonization [25]. Therefore, the discovery of the filmy ferns and associated spike-mosses from Kachin amber provides additional evidence for the presence of a tropical humid forest located close to the seashore [70,76,91] [26][27][28][29][30][31][32]36].
Divergence time estimates based on molecular data suggest that the stem group of Hymenophyllaceae may have diverged from other leptosporangiate ferns as early as the Carboniferous to Triassic [3,37,38,89,92,93], with the divergence between Trichomanoideae and Hymenophylloideae occurring during the Middle Jurassic [89]. Based on the analysis of rbcL sequence data, the family Hymenophyllaceae probably arose and first diverged in the Paleotropics, possibly in Asia [90] and subsequently dispersed from there [45]. However, prior to this study, Hymenophyllaceae fossils have never been reported from modern tropical Asia. The rarity of fossils found in tropical regions today is because of the high rate of chemical weathering under present per humid tropical conditions that dissolves any calcium or carbon-based fossils in the rocks. Amber is chemically rather inert and thus the best chance for modern tropical fossils. Hymenophyllites angustus sp. nov., Hymenophyllites kachinensis sp. nov. and Hymenophyllites setosus sp. nov. represent the first fossil record of Hymenophyllaceae from tropical Asia. Although much of the extant diversity of Hymenophyllaceae appears to have accumulated later in the angiospermdominated forests of the Cenozoic [89,94], the present fossils, along with other Cretaceous fossil records [57,58,60], indicate that Hymenophyllaceae have already accumulated some notable diversity in the Cretaceous. The study of Dubuisson et al. [93] supported the Early Cretaceous as a critical time for early diversification in Hymenophyllum.

Conclusions
Here, we describe fern remains from the mid-Cretaceous Kachin amber, Myanmar as three new species of Hymenophyllites (Hymenophyllaceae), namely H. angustus sp. nov., H. kachinensis sp. nov. and H. setosus sp. nov., and also describe associated Selaginella alata sp. nov. (Selaginellaceae) embedded together with Hymenophyllites kachinensis. The presence of these filmy ferns and associated spike-mosses are suggestive of high humidity in the source forests of this amber. These fern remains of Hymenophyllaceae represent the first fossil record of the family in tropical Asia. Our results greatly expand the known diversity of the fern flora in Kachin amber, Myanmar. Together with other contemporaneous findings, the present fossils indicate that Hymenophyllaceae have already accumulated some notable diversity in the Cretaceous.  Data Availability Statement: All data dealing with this study are reported in the paper.