First Fossil Record of Trichomanes sensu lato (Hymenophyllaceae) from the Mid-Cretaceous Kachin Amber, Myanmar

Hymenophyllaceae (filmy ferns), with ca. 430 species, are the most species-rich family of early diverging leptosporangiate ferns but have a poor fossil record dating back to the Late Triassic period. Traditionally, Hymenophyllaceae comprise two species-rich genera or clades: Hymenophyllum (hymenophylloids) and Trichomanes sensu lato (s.l.) (trichomanoids). Unequivocal fossils of Hymenophyllum have been reported from the Early Cretaceous of central Mongolia and the early Eocene of Okanogan Highlands, Washington, USA. However, despite being a highly diversified lineage with an estimated 184 extant species, Trichomanes s.l. lack a definitive fossil record, which severely affects the reliability of the molecular dating of this group. Here, we report the first unequivocal fossil record of Trichomanes s.l. as T. angustum comb. nov. on the basis of fertile material with tubular involucres and long exserted receptacles from the mid-Cretaceous Kachin amber, Myanmar. This species was previously tentatively assigned to Hymenophyllites due to a lack of fertile evidence. Inferred to be an epiphytic fern, T. angustum further enriches the species diversity of the epiphytic palaeocommunities in the mid-Cretaceous Kachin amber, which are mainly composed of Porellalean leafy liverworts and Dicranalean and Hypnodendralean mosses. Fossil records indicate that Hymenophyllaceae probably originated in the tropical Pangea at the latest in the Triassic when all continents were coalesced into a single landmass and had already accumulated some notable diversity in low-middle latitude areas of Laurasia by the mid-Cretaceous period.

Although it is clear that filmy ferns are ancient lineages according to their nearbasal phylogenetic position in early-diverging leptosporangiate ferns [1,[5][6][7][8]20], reliable fossils of this family are scarce due to low fossilization potential of the delicate, membranous laminae [9,11,[21][22][23][24], hampering detailed reconstructions of the origins of their extant diversity.The earliest convincing fossils of the Hymenophyllaceae were reported as Hopetedia praetermissa Axsmith et al. from the Late Triassic (Carnian) Pekin Formation of North Carolina, USA [21].Hopetedia Axsmith et al. display a character state mosaic of the two extant genera, namely Trichomanes-like funnel-shaped indusia and Hymenophyllum-like included receptacles [21].Eogonocormus Deng from the Lower Cretaceous of northeastern China is a small thalloid plant with creeping rhizomes and marginal sori with in situ spores, borne on fanlike pinnule lobes, which indicate convincingly that this genus belongs to the Hymenophyllaceae [25].Reliable filmy fern fossils also include Hymenophyllites H.R.Goeppert from the mid-Cretaceous of Kazakhstan and Myanmar [26,27] and Acrostichopteris Fontaine from the Early Cretaceous of Spain, China and USA [28][29][30][31].However, it is uncertain with respect to the affinities between the abovementioned fossil genera and the extant genera of Hymenophyllaceae.The earliest fossils of Hymenophyllum have been described as H. iwatsukii Herrera et al. based on abundant and exceptionally well-preserved lignified material from the Early Cretaceous (Aptian-Albian) of Mongolia [11].Hymenophyllum axsmithii Pigg et al. was described from the early Eocene Okanogan Highlands, Washington, USA [24].However, despite being a highly diversified lineage with an estimated 184 extant species [1], Trichomanes s.l.lack a definitive fossil record to calibrate its internal nodes, which severely affects the reliability and the dating of this group of ferns.
Recently, Li et al. [27] described some sterile lamina fragments from the mid-Cretaceous Kachin amber, Myanmar, as three new fossil species of Hymenophyllites, including H. angustus Y. Li  In the present paper, we transfer H. angustus to Trichomanes s.l.based on newly found fertile material from Kachin amber, Myanmar.Trichomanes angustum comb.nov.displays typical trichomanoid characters, namely tubular involucres and long exserted receptacles, and thus represents the first unequivocal fossil trichomanoid filmy fern known so far.We also discuss its palaeoecological implications and infer the palaeogeographic history of Hymenophyllaceae based on fossil records.

Materials and Methods
Kachin amber originates from several amber mines about 20 km southwest of the village of Tanai in the Hukuang Valley of Kachin State, northern Myanmar [32,33].Kachin amber deposits are currently the most important source of Cretaceous amber-preserved paleobiota [32,34] and yield a large number of plant and animal inclusions [35,36].The age of Kachin amber is regarded as the late Albian-early Cenomanian, based on the evidence of the ammonite Puzosia Matsumoto and palynomorphs [37,38].The U-Pb dating of zircons suggests the earliest Cenomanian age (98.79 ± 0.62 Ma) for the amber-bearing horizon of Kachin amber [34].
The fossils studied here include two pieces of Kachin amber from Myanmar and are housed at the Collection Department of Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, Nanjing, China, under accession numbers PB200744 and PB201715.Concerning the recent conflicts in Myanmar [39], we declare that we followed the recommendations by Haug et al. [40].All Kachin amber pieces mentioned in this study were acquired in compliance with the laws of Myanmar and China, including Myanmar's are more than 3 rows between the midrib and margins, polygonal, isodiametrical to slightly elongated with thin and straight cell walls, 25-88 µm long and 14-66 µm wide outside the venation area; however, they are also elongated fusiform to rectangular inside the venation area (Figures 1F and 2D).Differentiated marginal elongated cells are absent.Stomata are absent.Sori are paratactic, borne at the apex of the first arising lobes of the pinnule or its segments, oriented upward, nearly straight to the plane of the lamina (Figures 2A,B and 3A-E).Involucres are tubular, slightly curved, immersed in lobes or segments, winged throughout, 1.0-2.2mm long and 0.3-0.6 mm wide, ca.3-5 times as long as wide (Figures 3 and 4).Involucre mouths are usually not dilated (Figures 3C,E-G and  4).Receptacles are filiform, long exserted, and up to 3.1 mm long (Figures 3B,C,F,G and  4).

Remarks:
We made this description based on eight sterile lamina fragments in PB200744 and two fertile lamina fragments in PB201715.Although Ebihara et al. [3] subdivided the Trichomanes s.l.into eight genera mainly based on the results of molecular phylogeny [3,13], it was virtually impossible to find macro-morphological characters that consistently discriminated these genera for all species [16].It is especially difficult to put this classification into practice for the present fossils because they apparently lack their rhizome part: a very important character for generic identification.So, we adopted the concept of Trichomanes s.l.[17][18][19], which is still used today [16,41].Remarks: Although Ebihara et al. [3] subdivided the Trichomanes s.l.into eight genera mainly based on the results of molecular phylogeny [3,13], it was virtually impossible to find macro-morphological characters that consistently discriminated these genera for all species [16].It is especially difficult to put this classification into practice for the present fossils because they apparently lack their rhizome part: a very important character for generic identification.So, we adopted the concept of Trichomanes s.l.[17][18][19], which is still used today [16,41].It is strange that some pollen grains adhere on the lamina surface in PB201715 (Figure 5A,B), where filmy fern sporangia and spores are completely absent, although there are rich sori and involucres.These pollen grains are monoporate, with a round to rounded triangle, 31-65 µm in diameter (Figure 5C-F).Monoporate pollen is indeed present in some Mesozoic gymnosperm taxa, such as Admolia Batten and Perinopollenites Couper [42].The former is an unknown gymnosperm reported from the Early Cretaceous of the UK and China, while the latter belongs to taxodioid Cupressaceae ('Taxodiaceae') and is widely distributed around the world during the Jurassic to the Cretaceous period [42].However, the lack of ornamentation details hampers a definite identification.It is strange that some pollen grains adhere on the lamina surface in PB201715 (Figure 5A,B), where filmy fern sporangia and spores are completely absent, although there are rich sori and involucres.These pollen grains are monoporate, with a round to rounded triangle, 31-65 µm in diameter (Figure 5C-F).Monoporate pollen is indeed present in some Mesozoic gymnosperm taxa, such as Admolia Batten and Perinopollenites Couper [42].The former is an unknown gymnosperm reported from the Early Cretaceous of the UK and China, while the latter belongs to taxodioid Cupressaceae ('Taxodiaceae') and is widely distributed around the world during the Jurassic to the Cretaceous period [42].However, the lack of ornamentation details hampers a definite identification.Although highly challenging, we made a great effort to narrow down this identification according to the classification of Ebihara et al. [3], who subdivided Trichomanes s.l.into eight genera, namely Abrodictyum C.Presl, Callistopteris Copel., Cephalomanes C.Presl, Crepidomanes C.Presl, Didymoglossum Desv., Polyphlebium Copel., Trichomanes L. sensu stricto (s.str.), and Vandenboschia Copel., each with one to four subgenera.Firstly, our fern remains could be distinguished from the Palaeotropical genera Callistopteris and Cephalomanes as well as the predominantly Neotropical genus Trichomanes s.str.by having tubular involucres, while the latter three genera had campanulate involucres [3,43].In addition, our remains were divided at least tripinnate with one cell layer thick pinnae as well as anadromous venation, while Cephalomanes only has a once-pinnate lamina with asymmetric pinnae and Trichomanes s.str.also displayed other different features in its five subgenera.For example, once-pinnate to bi-pinnate-pinatifid lamina was found in subgenera Afrotrichomanes and Lacostea, more than one cell layer thick lamina was identified in subgenera Davalliopsis and Feea, and usually, catadromous venation is found in subgenus Trichomanes [3].Secondly, our fern remains differ from Abrodictyum in having more than three rows of laminar cells between the midrib and margins and having straight internal cell walls, while the lamina of most Abrodictyum species is reduced to less than three rows of cells between the midrib and the margins and internal cell walls are wavy or pitted [3,14,44].Finally, our fern remains could be confined to a hemiepiphytic/epiphytic clade within the trichomanoids, comprising Polyphlebium, Didymoglossum, Crepidomanes, and Vandenboschia [10]; however, the absence of false veinlets clearly differentiated our fern remains from Didymoglossum that always have false veinlets [3,14].Therefore, we could only narrow down the identification of our fossils to three candidate genera, namely Polyphlebium, Crepidomanes, and Vandenboschia.Unfortunately, we have not found any other traits in our fossils that allow the clear-cut identification of these three genera by lamina characters alone (Figure 6).To identify the genus, we needed to add more evidence, such as rhizome and rachis characters.
Crepidomanes include two subgenera, Crepidomanes and Nesopteris, and ca.30 species, distributed from Palaeotropics to northern temperate regions [3].In addition, one species in the Neotropics (C.pyxidiferum (L.) Dubuisson et Ebihara) was found [50].The subgenus Crepidomanes includes three sections and differs from our fern remains as follows.In section Crepidomanes, false veinlets are often present, and involucre mouths are usually bilabiate; section Gonocormus has campanulate involucre with dilate mouths; section Crepidium has double rows of elongate marginal cells [3].It is to be noted that some members of section Crepidomanes lack false veinlets, e.g., C. schmidtianum (Zenker ex Taschner) K.Iwats.(Figure 6C) and C. vitiense (Baker) Bostock.The subgenus Nesopteris has rather large and more than 15 cm long fronds [3], but the relatively long involucres in our fossils reminded us of this subgenus (Figure 6D).
Vandenboschia is a pantropical genus with two subgenera, Vandenboschia and Lacosteopsis, and ca.15 species that are often hemiepiphytic and occasionally terrestrial [3,10,46].The subgenus Vandenboschia owns bipinnate or more finely divided lamina (Figure 6B), while the subgenus Lacosteopsis has once pinnate lamina [3].Thus, the identification of the subgenus Lacosteopsis could be excluded.In addition, this genus always has minute clavate hairs on its rachises [3].If our two large fossils (Figures 1A,B and 2A,B) stem from the lamina portion rather than from single large lateral pinnae, the identification of subgenus Vandenboschia could also be excluded owing to its minute clavate hairs on rachises.[3,10,46].The subgenus Vandenboschia owns bipinnate or more finely divided lamina (Figure 6B), while the subgenus Lacosteopsis has once pinnate lamina [3].Thus, the identification of the subgenus Lacosteopsis could be excluded.In addition, this genus always has minute clavate hairs on its rachises [3].If our two large fossils (Figures 1A,B and 2A,B) stem from the lamina portion rather than from single large lateral pinnae, the identification of subgenus Vandenboschia could also be excluded owing to its minute clavate hairs on rachises.
There are still some intriguing aspects of T. angustum that remain unknown, including the host phorophytes.However, it is likely that T. angustum grew on resin-producing gymnosperm trees so that it was easily captured by the resin, and the monoporate pollen adhering on the fern lamina surface (Figure 5) represents pollen rain from the phorophyte trees or nearby trees.The mid-Cretaceous Kachin amber forest was dominated by various gymnosperm trees, including members of Araucariaceae and Cupressaceae [32,37].Nuclear magnetic resonance spectroscopic studies and anatomical analyses of fossil wood fibers have indicated that araucarioid trees of the Araucariaceae, especially Agathis Salisb., were determined as the source of Kachin amber [71].

Paleobiogeographic History of Hymenophyllaceae
Copeland [72] suggested an Antarctic origin for the Hymenophyllaceae according to the fact that most filmy ferns grow in the Southern Hemisphere, with numerous monotypic groups in austral regions.Iwatsuki [9,73] believed that filmy ferns evolved in the tropics and subsequently dispersed from there.From the analysis of rbcL sequence data, Dubuisson et al. [74] inferred a basal position for Asian groups in Trichomanes, while the most basal taxa within Hymenophyllum are also Paleotropical and/or austral [75].Thus, Dubuisson et al. [74] speculated that Hymenophyllaceae probably arose and first diverged in the Paleotropics, possibly in Asia.Divergence time estimates indicate that Hymenophyllaceae might have evolved during the Carboniferous to the Triassic period [2,5,6,10,20,43] and split into hymenophylloids and trichomanoids in the Middle Jurassic period [10].Numerous Paleozoic and Mesozoic Hymenophyllaceae-like fronds were assigned to the extinct genera Hymenophyllites, Trichomanides, and Trichomanites [76][77][78][79] or to the extant genus Hymenophyllum [80], but the affinity of these fossils to Hymenophyllaceae is quite uncertain owing to the lack of definite evidence of a membranaceous habit or marginal indusiate sori [21,81].
The earliest reliable Hymenophyllaceae fossils are reported from the Late Triassic Pekin Formation of North Carolina, USA [21], followed by an apparent gap in the Jurassic period (Table 1; Figure 7).Hymenophyllaceae diversified in the Early to middle Cretaceous period of Laurasia with several fossil genera and species reported from Spain, Kazakhstan, Mongolia, China, and the USA [11,[25][26][27][28][29][30][31] (Table 1; Figure 7).Some younger fossils of Hymenophyllaceae have been recently reported to be from the early Eocene of Okanogan Highlands, Washington, USA [24] (Table 1; Figure 7).It is strange that all unequivocal fossils of Hymenophyllaceae were reported from the Northern Hemisphere.Despite being a worldwide fern lineage predominantly distributed in tropical mountains and south-temperate areas, Hymenophyllaceae lack a definitive fossil record in the Southern Hemisphere (Figure 7).Although some putative fossils of Hymenophyllaceae were reported as Trichomanides laxum Tenison-Woods and T. spinifolium Tenison-Woods from the Jurassic of Queensland, Australia [76] and Hymenophyllum priscum Menéndez from the Late Cretaceous of Chile [80], the identification to Hymenophyllaceae has been thought to be wrong or less convincing [21].Although the bulk of the extant diversity of Hymenophyllaceae appears to have accumulated later in angiosperm-dominated forests during the Cenozoic [10], the reliable fossil record of Hymenophyllaceae is scarce in the Cenozoic (Table 1; Figure 7).
All in all, present fossil evidence indicates a tropical Pangea origin for Hymenophyllaceae at the latest in the Triassic when all continents coalesced into a single landmass Pangea under a global hothouse climate [82] (Figure 7), and some notable diversity was already developed in low and middle latitude areas of Laurasia in the mid-Cretaceous when the earth experienced a hothouse climate again [82] (Figure 7).

Conclusions
Here, we report the first definitive fossil species of Trichomanes s.l. as T. angustum comb.nov., which is combined from the previously described Hymenophyllites angustus, based on newly found fertile material from the mid-Cretaceous Kachin amber, Myanmar.

Conclusions
Here, we report the first definitive fossil species of Trichomanes s.l. as T. angustum comb.nov., which is combined from the previously described Hymenophyllites angustus, based on newly found fertile material from the mid-Cretaceous Kachin amber, Myanmar.Morpho-logical comparisons suggest that T. angustum can be confined to a hemiepiphytic/epiphytic clade within the trichomanoid.Inferred to be an epiphytic fern, T. angustum further enriches the species diversity of the epiphytic palaeocommunities in mid-Cretaceous Kachin amber, mainly comprising Porellalean leafy liverworts and Dicranalean and Hypnodendralean mosses.Fossil records indicate that Hymenophyllaceae probably originated in the tropical Pangea at the latest, in the Triassic and accumulated some notable diversity in low and middle-latitude areas of Laurasia by the mid-Cretaceous.

Life 2023 , 16 Figure 3 .
Figure 3. Fertile pinnae and sori of Trichomanes angustum comb.nov.from mid-Cretaceous Kachin amber.PB201715b.(A,B) A fertile pinna in abaxial and adaxial views.(C) Enlargement of pinna in adaxial view showing apical portions of sori.(D) Enlargement of pinna in abaxial view showing basal portions of sori.(E) Several sori in lateral view.(F,G) Apical portions of two sori showing nondilated involucre mouths and filiform, long exserted receptacles.

Figure 3 .
Figure 3. Fertile pinnae and sori of Trichomanes angustum comb.nov.from mid-Cretaceous Kachin amber.PB201715b.(A,B) A fertile pinna in abaxial and adaxial views.(C) Enlargement of pinna in adaxial view showing apical portions of sori.(D) Enlargement of pinna in abaxial view showing basal portions of sori.(E) Several sori in lateral view.(F,G) Apical portions of two sori showing non-dilated involucre mouths and filiform, long exserted receptacles.

Author Contributions:
Conceptualization, Y.L. and Y.-M.C.; methodology, Y.L. and Y.-M.C.; software, Y.L. and Y.-M.C.; validation, Y.L.; formal analysis, Y.L.; investigation, Y.L. and A.E.; resources, Y.L., N.N. and Z.-Z.T.; data curation, Y.L.; writing-original draft preparation, Y.L.; writing-review and editing, Y.L., A.E., N.N., Z.-Z.T. and Y.-M.C.; visualization, Y.L. and A.E.; supervision, Y.L. and Y.-M.C.; project administration, Y.-M.C.; funding acquisition, Y.-M.C. and N.N.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the National Natural Science Foundation of China, grant number 42002023, the institutional research project of the BIN RAS (Saint Petersburg, Russia), grant number 122011900029-7 and the State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology, CAS, grant number 20192101.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement: All data are reported in this paper.