A New Species of Krameropteris (Dennstaedtiaceae) from Mid-Cretaceous Myanmar Amber ^†

Abstract

Krameropteris is an extinct fern genus found in mid-Cretaceous Myanmar amber, assigned to the Dennstaedtiaceae, with only one previously described species, i.e., K. resinatus. This study describes a new species, K. calophyllum, also preserved in Myanmar amber. The new species is characterized by branched venation with free veinlets terminating in thickened tips, multicellular hairs along the pinnule margins, and submarginal or medial exindusiate sori at the ends of veinlets. Its sporangia are polypod-type, producing trilete spores. This new species represents the second record of an extinct fern genus of the early-diverging Dennstaedtiaceae lineage found in Myanmar amber. Krameropteris calophyllum differs from K. resinatus in its pinnule epidermal characteristics and spore morphology, with the latter exhibiting a conspicuously ornamented perine, covered with sparse tubercles and ridges. The new species provides insights into potential herbivory interactions and the relationship between mid-Cretaceous polypod ferns and the smallest mite ever recorded. These findings suggest that Dennstaedtiaceae exhibited species-level diversity by the mid-Cretaceous, indicating an earlier origin of the family, potentially as early as the Early Jurassic, consistent with recent DNA-based time divergence estimates.

1. Introduction

Ferns constitute the second largest group of vascular plants, after angiosperms, and are nearly globally distributed. The order Polypodiales accounts for more than 80% of all extant fern species diversity, reflecting its diversity in ferns, which parallels the dominance of angiosperms among vascular plants [1,2,3,4]. DNA sequence-based divergence time estimates indicate that most lineages recognized today within the Polypodiales originated in the Late Cretaceous or earlier, coinciding with the rise of many angiosperm crown groups [1,3,4,5,6,7,8,9]. However, fossil evidence of Polypodiales from the Cretaceous, which could substantiate or challenge these DNA-based estimates, remains extremely limited. Therefore, the fossil records serve as an essential independent source of evidence, providing additional calibration constraints for age estimations. Recent studies of Myanmar amber fossils, dating from the late Albian to earliest Cenomanian (~100 Ma), have yielded remarkable specimens representing five of the six suborders of Polypodiales: Polypodiineae (including Dryopteridaceae [10,11,12]), Aspleniineae (including Thelypteridaceae [13]), Dennstaedtiineae (including Dennstaedtiaceae [5,14,15]), Lindsaeineae (including Cystodiaceae [16,17] and Lindsaeaceae [18,19]), and Pteridineae (including Pteridaceae [20]).

Among these suborders of Polypodiales, only Dennstaedtiineae exhibits the highest genus-level diversity, with three described fossil genera—Krameropteris [5], Cladarastega [14], and Microlepia [15]—along with one additional genus under description (Prodennstaedtia, unpublished), each containing only one species. Krameropteris, with the species K. resinatus, was the first fossil, regarded as the best match, to be assigned to Dennstaedtiaceae, reported by Schneider et al. [5] based on a single fertile leaf fragment preserved in mid-Cretaceous amber from Myanmar. In this study, we describe a new species of Krameropteris from the same amber deposits, further demonstrating species-level diversity within Dennstaedtiaceae. Additionally, we present evidence of potential herbivory and discuss the likely interactions between mid-Cretaceous polypod ferns and arthropods, based on further information obtained from the new fossil specimen.

2. Materials and Methods

The pinnule encased in amber analyzed in this study was collected in 2016, prior to the onset of issues related to amber from Myanmar, and therefore complies with all ethical standards for the research of such fossils [21,22]. The amber sample was obtained from the Hukawng Valley in Tanai Township, Myitkyina District, Kachin State, Myanmar (Figure 1A,B). Regarding the age of the Kachin amber, Grimaldi et al. [23] estimated its age to be approximately Cenomanian to Turonian based on the stratigraphic distributions of Cretaceous insect families. Cruickshank and Ko [24] identified a Middle or Upper Albian ammonite, Mortoniceras. Shi et al. [25] conducted U-Pb zircon dating on the sedimentary matrix of the amber-bearing beds, further assigning an earliest Cenomanian age of 98.79 ± 0.62 Ma to the Myanmar amber. More recently, Yu et al. [26] constrained the age of the Myanmar amber to the Upper Albian to Lower Cenomanian based on biostratigraphy and radioisotope data (Figure 1C). The amber specimen is housed at the Fossil Repository of Nanjing Institute of Geology and Paleontology, Chinese Academy of Sciences, Nanjing, China, under the accession number PB203297. Regarding ethical concerns related to Myanmar amber [21,22], we declare that this amber specimen was acquired in full compliance with both Myanmar’s import/export regulations on jewelry and China’s fossil laws, following the recommendations set forth by Haug et al. [27].

The amber sample was prepared using a water-fed saw, followed by grinding and polishing with a lap to expose the inclusions. The inclusions were examined under a ZEISS Axio Zoom V16 and M2 microscopes (Carl Zeiss AG, Oberkochen, Germany), equipped with high-resolution digital cameras. Both incident and transmitted lights were used simultaneously for imaging. All photomicrographs were digitally stacked composites of ca. 15–120 individual focal planes, processed using ZEN 2.3 pro software to better illustrate the three-dimensional structure of the inclusions. Terminology for describing the fern fossils follows the classic books [28,29,30,31] and Schneider et al. [5]. All images were arranged and labeled on plates using Adobe Photoshop CS2023.

3. Results

Systematic Paleontology

For the present classification, we follow that by Pteridophyte Phylogeny Group, PPG I [2].

Class: Polypodiopsida Cronquist, Takhtajan & Zimmermann 1966

Order: Polypodiales Link 1833

Suborder: Dennstaedtiineae Schwarstburd & Hovenkamp 2016

Family: Dennstaedtiaceae Lotsy 1909

Genus:Krameropteris Schneid, Schmidt & Heinrichs 2016

Species: Krameropteris calophyllum C. X. Li, sp. nov. (Figure 2A, Figure 3, Figure 4, Figure 5 and Figure 6A)

Holotype: Specimen No. PB203297, a pinnule inclusion in Myanmar amber

Type locality: Amber mines near Tanai, Ledo Road, 105 km northwest of Myitkyina, Kachin State, Myanmar (26°20′N, 96°36′E). This site is within the Hukawng Basin, which is composed of folded sedimentary (volcanic) rocks of the Cretaceous and Cenozoic Ages.

Type horizon: Myanmar amber, lowest Upper Cretaceous, lowest Cenomanian, absolute age 98.79 ± 0.62 million years ago, established by U-Pb dating of zircons from the rind of the unprocessed amber.

Etymology: The specific epithet calophyllum is proposed to represent the beautiful and well-preserved pinnae fragment. In Old Greek, kallos means “beauty”, while phyllum denotes “leaf”.

Repository: The holotype is deposited in the Nanjing Institute of Geology and Paleontology, Chinese Academy of Sciences (abbreviation of NIGPAS), Nanjing, China.

Diagnosis: Fern pinnule pinnate with free branched veins, not reaching the segment margin, tips swollen. Pinnules hypostomatic, short multicellular hairs along pinnule margins, orbicular simultaneous maturity exindusiate sori at ends of veins, polypod sporangia, and trilete tetrahedral–globose spores.

Description: The amber inclusion consists of a single fertile pinnule fragment, measuring 6.4 mm long and 5.2 mm wide. The pinnule is pinnately lobed. The lobes are elliptical with acute apices and slightly thickened margins. The lobes near the apex of the pinnule gradually transition into slightly pinnatifid or crenate forms (Figure 2A). The venation of the pinnule is pinnate; lateral veins branch 1–3 times with free veinlets. The veinlets terminate at thickened ends located ca. 0.2–0.4 mm from the margins. The sori are abaxial and exindusiate and occur singly, with simultaneous maturity (Figure 3C,D). They are orbicular, with a maximum diameter of 1.2 mm, and typically one to two sori per pinnule lobe. The more apical pinnule lobes tend to bear a single sorus, while the larger, more basal lobes may contain up to two sori. The sori are submarginal, terminating at the tips of the veinlets, and are located approximately 0.3–1.1 mm from the margins (Figure 2A). Each sorus contains a somewhat raised receptacle and lacks paraphyses (Figure 3C,D). Each sorus contains up to 42 mature polypod sporangia, which are nearly globose, measuring 157.3 µm in length and 142.6 µm in width (average of 13 sporangia). The sporangia have stalks with three rows (Figure 3A), and the annulus is vertical and broken at the stalk, with ~21 darkened and thickened annulus cells (Figure 3A,B). Some sporangia contain trilete spores, which are tetrahedral–globose in shape, with a conspicuously ornamented perine covered with sparse tubercles and ridges (Figure 4A,B). Dispersed trilete spores are also present on the abaxial surface of the pinnule (Figure 4C), with an average diameter of 26–31 μm (measured from seven spores) both in sporangia and on the pinnule abaxial surface. The epidermal cells of the pinnule have undulating or waved anticlinal walls (Figure 5A). The stomata are hypostomatic and anomocytic, without subsidiary cells, and the guard cells measure 25–40 µm in length (Figure 3A and Figure 5B). Short, multicellular hairs, ranging from 18–28 µm in length, are also present along the pinnule margins (Figure 5C).

Remarks: The fossil represents one terminal fragment of a fertile pinna, containing six sori with developing sporangia and spores. Syninclusions include the remains of being attacked by invertebrates, as well as mites on the pinnule abaxial surface and between the sporangia (Figure 2). Extant species of Monachosorum (Dennstaedtiaceae) share similarities with the Krameropteris fossils in terms of trilete spores, exindusiate sori, and the simultaneous maturity of polypod sporangia. Except for M. maximowizcii [29], which differs, the remaining Monachosorum species have pinnules resembling those of the fossil. Given the uncertainties regarding the evolution of these characteristics, the fossil may not represent a member of Monachosorum stem lineage, as suggested by Schneider et al. [5], but could instead be more appropriately placed within the extinct genus Krameripteris.

4. Discussion

4.1. Comparisons to Fossils from Myanmar Amber

Currently, nine genera of Polypodiales have been described from Myanmar amber: Cretacifilix [10,11], Krameropteris [5], Cystodium [16,17], Holttumopteris [13], Prosperiflix [12], Cladarastega [14], Microlepia [15], Proodontosoria [19], and Heinrichsia [20]. Of these, only Krameropteris [5] and Prosperiflix [12] share the characteristic of lacking indusiate structures. However, Prosperiflix [12] is distinguished by its bilateral, monolete spores with a tuberculate surface. Therefore, only the Krameropteris exhibits the combination of exindusiate sori and a simultaneous sporangial maturity, with polypod sporangia producing trilete spores (Figure 2A, Figure 3, Figure 4, Figure 5 and Figure 6).

The fossil genus Krameropteris is characterized by pinnate, branched lateral veins with free veinlets, multicellular hairs, exindusiate sori at ends of veins, and polypod sporangia producing trilete spores. The new species, K. calophyllum, shares all of these features, justifying its placement within the genus Krameropteris. However, K. calophyllum can be easily distinguished from the previously described species, K. resinatus, by several different characteristics: it has larger sori with up to 42 sporangia, spores with a conspicuously ornamented perine covered with sparse tubercles and ridges, short multicellular hairs along pinnule margins (as opposed to the longer hairs along the veins in K. resinatus), and veins that end in thickened tips, not reaching the segment margins (Figure 2A, Figure 3C,D,Figure 4A,B, Figure 5 and Figure 6).

4.2. Early Diversification of Dennstaedtiaceae and Their Potential Interactions with Arthropods

Fossil evidence is essential for confirming the presence of early-diverging lineages of polypod ferns, such as Dennstaedtiaceae. The timing of divergence between Dennstaedtiaceae and its sister group remains uncertain, with different molecular dating analyses yielding varying results. Two recent studies illustrate this discrepancy: Schwartzburd et al. [32] proposed this divergence occurred in the Cretaceous, around 135.78 Ma, while Lu et al. [33] estimated a Jurassic divergence at approximately 162.43 Ma. The discovery of two Krameropteris species in the mid-Cretaceous not only supports the genus establishment of Dennstaedtiaceae by this time but also contributes to the growing body of fascinating and remarkable fossilized flora from Myanmar amber. This finding further suggests that Dennstaedtiaceae began to diversify at the species level prior to the mid-Cretaceous, leading to at least two distinct species. This new fossil evidence is consistent with the later molecular dating estimates.

The extant genus Monachosorum (Dennstaedtiaceae) is characterized by exindusiate sori at the ends of veins and the simultaneous maturity of polypod sporangia producing trilete spores. These features are also found in the extinct genus Krameropteris. The current distribution of Monachosorum spans Japan, China, Indochina, the Indian subcontinent, and parts of the Malay Archipelago [29] (Figure 1C). Therefore, the newly discovered fossil provides further evidence that this fern lineage has persisted in Southeast Asia since the Early Cretaceous, as indicated by Schneider et al. [5], who first reported the Krameropteris fossil.

So far, very little is known about insect herbivores of fossil ferns. That makes the partial remains of an immature blattoid adjacent to the pinna of another extinct genus of Dennstaedtiaceae, Cladarastega, particularly interesting [14]. While the relationship between this immature blattoid and Cladarastega remains unclear, Poinar [14] speculates that certain extant fern herbivores, such as sawflies (Tenthredinidae: Hymenoptera), gall gnats (Ceccidiomyiidae: Diptera), and aphids (Aphididae: Hemiptera) [34]—whose fossil records date back to at least the Early Cretaceous [35]—may have fed on ferns preserved in Myanmar amber. However, most research on arthropod–plant interactions has focused on predation, typically inferred from feeding traces on fossilized plant foliage. Such studies often examine herbivory evidence in the form of feeding traces found on plant fossils [36,37,38,39,40]. Our new fossil not only provides evidence of potential herbivory, including remains of arthropod attacks and frequent marginal feeding by herbivorous insects (Figure 2A), but also reveals evidence of mite presence on the leaf surface and between sporangia (Figure 2A,B). We hypothesize that the mites inhabited the fronds of Krameropteris calophyllum. Despite evidence of frond consumption, the small size of the mites—averaging 70 μm in length and 42 μm in width (based on idiosoma measurements from two specimens)—makes them the smallest mite fossils ever recorded [41,42,43]. These dimensions, comparable to spore sizes (average diameter of 26–31 μm), suggest that the mites likely used the fronds as a habitat rather than a food source. This observation warrants further investigation into the ecological relationship between ferns and mites.

5. Concluding Remarks

The new Krameropteris specimen indicates that species-level diversity within Dennstaedtiaceae was already present in the mid-Cretaceous, supporting the hypothesis that this family originated before the mid-Cretaceous, probably as early as the Early Jurassic. This is consistent with the recent DNA-based divergence time estimates [33]. The new fossil also suggests potential herbivory and interactions between the early diverging lineages of polypod ferns and arthropods. In particular, the mites inhabiting the fronds of the new species K. calophyllum probably used its fronds as a habitat rather than a food source. The discovery and detailed description of fossil specimens are crucial for understanding the evolutionary relationships between extant groups and their ancestors, as well as for dating key evolutionary events inferred from studies of modern ferns.