Significant features of the mines and the possible culprit
The Cladophlebis mining structures from the Momonoki Formation are leaf mines by holometabolous insects and can be distinguished from other feeding methods (e.g., surface-feeding) and also from features of taphonomic origin because they more or less satisfy the following criteria for shapes of insect leaf-mines51: an oviposition site at one end of the mining structure; an enlarged oviposition area as a blotch or curvilinear trace; a sign of the evacuated leaf tissue; width of evacuated plant tissue and frass trail increases from one end to the other; the presence of a frass trail, either particulate or fluidized; response tissue along with the marginal tissue; a distinctive terminus, such as an expanded region (chamber). The three mines on the same pinna (Fig. 2A–B) are probably produced by the same insect taxon as they share features of the frass trails. Each mine is composed of a continuous, single frass trail of which the size of pellets subsequently expands at the end. The abrupt change in the faecal shapes can be interpreted as larval development while mining.
Mine morphology and host-plant range often provide us with keys to identify leaf-mining insects because leaf-mining insects tend to be associated with a relatively narrow range of plants and show stereotyped, taxon-characteristic behavioural patterns in oviposition and feeding2. However, the convergence among different insect orders/families and variation among closely related species make it difficult to differentiate miners based on their mine shapes51.
The taxonomic affinity of the mines at Momonoki is herein examined in the light of mine shape, the extant groups of fern-pinnule miners, and the chronological origins of possible culprits. The overall mine shape is not comparable to those of known mining structures by extant fern-miners52. Extant fern-mining insects are found in four orders: Diptera, Coleoptera, Lepidoptera, and Hymenoptera53. In particular, the evidence currently available is not consistent with an affinity to Diptera or Hymenoptera. Flies are relatively diverse as miners of fern pinnules (and stems), represented by Chirosia (Anthomyiidae), Agromyzidae, and Cecidomyiidae53. For example, Chirosia is a genus of which all extant taxa are fern-miners as larvae; they consume either fronds or stems of a range of fern taxa52,54–59. Dipteran leaf mines tend to contain fluidized frass, which is often deposited as two discontinuous rows of pellets19; these are not seen in the Cladophlebis mine. No evidence for the presence of dipteran leaf-mining taxa is available for the Late Triassic, although nematocerans and some of the earliest groups of brachycerans are markedly diverse44,45. The mine producer is less likely to be affiliated with Hymenoptera, although this order was already diverse by the end of the Triassic60, with the oldest fossils dating back to the Middle Triassic61. The extant fern-feeders of Hymenoptera do not include pinnule-miners, and only Blasticotomidae and Tenthredinidae, as petiole-borers and internal fern-feeders, respectively, are known53.
Altogether, the shape of the Cladophlebis mine is not clearly comparable with typical mines of Diptera and Hymenoptera; instead, it can better be assigned to those of Coleoptera or Lepidoptera in terms of the time of appearance and the mine shapes. Beetles became widespread worldwide in the Middle to Late Triassic62–64. The earliest beetle group, Protocoleoptera, are found from the Momonoki Formation34,43, although they are thought to be saproxylic (i.e., borers of decaying wood), based on some circumstantial evidence65. Linear mines containing granular faecal pellets from the Triassic are often assigned to beetles, assuming some lineages of Polyphaga are the candidates66.
Lepidopteran mines exhibit considerable variation in mine shape, tissue consumption, and contents (e.g., faecal pellets). Notably, leaf mines of Ectoedemia (Nepticulidae) typically start as fine, strongly meandering galleries that subsequently become broad blotches67. In addition, typical nepticulids generally avoid leaf veins and leave granular pellets, with abrupt changes in the accumulation pattern in some species. Importantly, these mine features are seen in the Cladophlebis mine from the Momonoki Formation. Regarding the evolutionary history of nepticulids, based on a recent fossil-calibrated molecular phylogeny68, the appearance of leaf-mining moth clades, including Nepticuloidea, dates back to the latest Triassic; for calibrating this phylogeny, wing-scale fossils of Coelolepida from the Triassic–Jurassic boundary of Germany were used69. However, the Cladophlebis mine is not straightforwardly comparable to those of extant nepticulids because no extant nepticulid species feeds on ferns.
The stoichiometric footprint of the studied plant–insect interaction
The elemental analyses indicate quantitative variability in some elements (Si, P, S) that may partly be responsible for physiological processes in nutritional cycles (Fig.3); plant tissues are deposited and then partly removed by an insect and then the insect metabolizes and excretes the undigested substances. The content of frass is thus the product of absorption, metabolism, and excretion. Compared to the leaf vein, the fossilized frass (coprolites) are shown to be highly phosphatic.
Another notable point is the varying intensity of Si among sample points. For the leaf lamina, biogenic and lithologic Si may be conjugated, and thus caution is needed in interpreting our result that the highest intensity of Si was found in the leaf lamina. However, the incremental difference of Si between the frass and leaf vein may be the result of biogenic silica contained in the frass. Biomineralization of silica, especially in the form of phytoliths (SiO2, nH2O), is found in many clades of pteridophytes70,71, and these phytoliths can enhance plant resistance to herbivore feeding72,73. This relatively higher content of Si in the frass coprolites may therefore reflect undigested defensive compounds.
The elemental analyses shed new light on how this plant–herbivore interaction which occurred 220 million years ago, is mediated by plant chemicals. Furthermore, this study illustrates that ecological stoichiometry, a method that traces the flow of energy and elements in ecosystems, can be applied to interactions between plants and endophytic herbivores in the fossil record. Future investigations of plant chemical landscapes (nutrition and defence) across various temporal and spatial settings would provide new insights into the macroevolutionary patterns of combat between plants and herbivores.
Origin and early history of leaf-mining
Credible leaf mines are absent or very rare before the Late Triassic27. From the Palaeozoic, at least two types of trace fossils have been previously assigned as possible leaf mines, although this is currently not supported. One type concerns U- or V-shaped structures on pinnules of medullosans from the Upper Carboniferous, or Lower Permian74,75, which were later attributed to fungal or bacterial infection27,76,77. The other type represents a series of small and extensive serpentine structures74,78,79, the ichnotaxon name of which is Asteronomus (?) meandriformis; currently, these are acknowledged as structures of taphonomic origin27. A notable example from the Early Permian is a possible mine on a megaphyll of Glossopteris cf. indica from the Rio Bonito Formation, Morro do Papaléo Mine, Brazil; this structure contained the possible frass of the miner and ended with a terminal expansion, which was possibly a larval/pupal chamber (Adami-Rodrigues et al., 2004). Another example is a U-shaped contour on the foliage of Vjaznikopteris rigida from the Volga River Basin (the P–Tr boundary), European Russia (Krassilov and Karasev, 2008). However, the identity of these traces as leaf mines is disputable because the above-mentioned criteria for insect mines are not met.
From the Middle–Late Triassic, several distinct shapes of mining structures are known (Fig.4), and some of them are described as distinct damage types (DT)82. Several sites of the Molteno Formation (Carnian) are a rich source of herbivory and oviposition trace fossils66,83. Two types of leaf mines, one of each from Heidiphyllum elongatum foliage (DT41, DT71) and one from Sphenobaiera schenckii (DT139), have been recorded from this locality66(Fig. 4A); additionally, this locality bears an undescribed, well-preserved mine on Cladophlebis fern pinnules (Labandeira, C. C., pers. comm.), although its shape differs considerably from the one from the Momonoki Formation. Another record from the Gondowanan flora is a serpentine mine on Heidiphyllum foliage, Triassohyponomus dinmorensis, from the Blackstone Formation (Carnian) of the Ipswich Coal Measures Group, Queensland, Australia84,85 (Fig. 4B). Some trace fossils from several other localities of the Middle–Late Triassic have also been assigned to leaf mines; for example, a small, semilinear, frass-laden mining structure (DT40) on foliage of the pteridosperm Scytophyllum bergeri, from Monte Agnello, N. Italy (Late Ladinian)86 (Fig. 4C). Some mining structures are also known from the foliage of Nilssonia sturii from the Lunz Formation (Carnian), in the eastern part of the Northern Calcareous Alps, Austria87 (Fig. 4D); also, two types of mines on some gymnosperm (?Glossophyllum) foliage are reported from Dzhayloucho (Ladinian–Carnian), near Madygen, Kyrghyzstan88,89 (Fig. 4E).
The Cladophlebis mine (Fig. 4F) described here represents a novel damage type that serves as the oldest credible fossil mine from the Southern Floristic Region of East Asia, the palaeobotanical assemblage of which is geographically and taxonomically distant from any of the above-mentioned floras (Fig. 4G). Our finding, therefore, reinforces the view that leaf-mining had become a pervasive feeding method for plant-feeding insects by the Late Triassic. By this time, they had already colonized a wide range of plant groups: conifers, pteridosperms, cycadophytes, ginkgophytes, and ferns.