Oldest leaf mine trace fossil from East Asia provides insight into ancient nutritional flow in a plant–herbivore interaction

The Late Triassic saw a flourish of plant–arthropod interactions. By the Late Triassic, insects had developed all distinct strategies of herbivory, notably including some of the earliest occurrences of leaf-mining. Herein we describe exceptionally well-preserved leaf-mine trace fossils on a Cladophlebis Brongniart fern pinnule from the Momonoki Formation, Mine Group, Japan (Middle Carnian), representing the oldest unequivocal leaf-mines from East Asia. The mines all display a distinctive frass trail—a continuous meandering line, which later becomes a broad band containing spheroidal particles—demonstrating larval development. Although the shapes of the frass trails are generally comparable to those of Lepidoptera or Coleoptera, they cannot be unequivocally assigned to a specific extant leaf-mining taxon. Furthermore, elemental analyses by X-ray fluorescence (XRF) reveals that the frass trail comprises phosphate coprolites. The quantitative variations in P, S, and Si between coprolites and leaf veins may reflect physiological processes (e.g., consumption, absorption, and excretion) mediated by plant chemicals. Our findings reinforce the idea that leaf-mining had become a pervasive feeding strategy of herbivorous insects by the Late Triassic.

. The star denotes the fossil locality, Okubata.
Palaeobiological setting. Plant remains from this locality are primarily represented by impressions of foliage, stem fragments with leaves, and, occasionally, fructifications. The plant assemblage of the Momonoki Formation is composed of ferns, sphenophytes, cycadophytes, ginkgophytes, and conifers 40 . There are three characteristics of the Momonoki Formation that are distinct from some other coeval fossil plant assemblages 41 : Sphenopsida (e.g., Neocalamites Halle and Equisetites Sternberg), ferns (e.g., Camptopteris Presl, Clathropteris Brongniart, Dictyophyllum Lindley et Hutton, Cladophlebis), and conifers (e.g., Podozamites Braun, Cycadocarpidium Nathorst) are well represented; cycadophytes and ginkgophytes 42 are common; Danaeopsis Schimper and Symopteris Xu (Marattiaceae) ferns are lacking. Liverworts (Pallaviciniites Schuster) are also present 43 . The floral composition of the Momonoki Formation is part of the Southern Floristic Region of East Asia 41,44 ; the floristic region extends over a broad geographic range, from as far south as Indonesia and north to a small area of northeastern China, and is characterized by the occurrence of Dictyophyllum and Clathropteris 41 .
A diverse fossil insect fauna has been found at the Momonoki Formation 36,45 . These fossils are borne from specific stratigraphic layers of dark, fine-grained siltstones that are devoid of coarse detritus 31 . Most of the insect fossils are isolated wings, but nearly complete bodies are also found 36 . The recorded insects encompass at least 12 orders: Odonata, Ephemeroptera, Blattodea, Plecoptera, Paraplecoptera, Reculida, Neuroptera, Hymenoptera, Coleoptera, Hemiptera, Mecoptera, Diptera 32 . In particular, there are rich yields of wings of Coleoptera (Taldycupedidae) and Blattodea (Mancusoblattidae, Mesoblattinidae) amongst the insect fauna of this deposit 32,34 . Notably, the Momonoki Formation yields true flies 36 , albeit undescribed, which have only been recorded from a few deposits from the Middle-Late Triassic [46][47][48][49][50][51] . Although more than 6000 specimens of fossil insects are recorded from this formation 30,36 , the taxonomic diversity is largely unexplored, and numerous terrestrial and aquatic insect specimens remain undescribed. At present, 18 species in ten families belonging to seven orders are described [32][33][34][35]45 . The insect fauna of the Mine Group is thought to have an affinity with four remote localities 32,33 : the Mount Crosby Formation in Australia, the Madygen Formation in Kyrgyzstan, the Djam Djun Formation in Vietnam, and the Yan-Chen Formation in China.

Results
Description of trace fossil. Three leaf mines with similar structures are found on five consecutive pinnules of the basiscopic pinna (white arrowheads in Fig. 2A,B). The plant tissues, including the epidermis and veins, stand out sharply against the profoundly weathered matrix of the slab. The mines broadly cover the lamina, although the margins of mined and remaining areas are not clearly distinguished as breached epidermal tissue, indicating that the miners left some tissue layers unconsumed. The mines originate either from the proximal or distal end of the lobes. Starting points (i.e., oviposition site) are obscure or unseen (orange arrowheads in Fig. 2C,D), lying on a secondary leaf vein. Frass trails appear as narrow sinusoidal threads with a width of 0.5-1.3 mm (1.1 mm on average), accompanying many close-set hairpin curves with occasional loops. The frass trails subsequently expand into broad bands of width 0.5-0.8 mm, with the dense accumulation of faecal material; individual frass pellets are hardly discernible. The frass trails are single, continuous lines but accompanied with a few gaps; altogether, each frass trail covers nearly the entire distal part of a pinnula. Although the trajectories of the frass trails freely extend across the secondary veins, they do not traverse the first veins (i.e., midribs) except those near the leaf apex; such behavioural characteristics thus give the entire mining structure on a pinnule a U-shape. These mines connect to the neighbouring pinnule through the widened base of the pinnule (Fig. 2D-E). The fact that the leaf veins within the mined area are not distorted dictates that the vascular bundles are left unconsumed. The darkened tissue at the end of the mine indicates a possible pupal chamber (pc, Fig. 2D).

Material.
A single part-and-counterpart specimen (MMHF11-00001a, MMHF11-00001b; Fig. 2A  Remarks. The focal Cladophlebis frond is targeted by external foliage feeders in addition to miners. Excisions bordered by evident reaction rims (the area defined by two yellow asterisks in Fig. 2A) indicate signs of external feeding by a mandibulate folivore.

X-ray fluorescence (XRF) analyses.
Elements were quantified at four sample points: (A) frass trail, (B) leaf vein, (C) leaf lamina, (D) rock matrix (Fig. 3). The XRF spectrum detected peaks of ten elements (Al, Si, P, S, K, Ca, Ti, Fe, Sr, Zr); the other peaks were of Rh gas used for the analyses. The values for four elements (Si, P, S, Ca) were particularly notable because these elements could be at least partly biogenic and involved in physiological processes. The leaf lamina was composed of just a thin film of plant tissue, and thus the elemental composition might include that of rock beneath the lamina. The results for the leaf lamina generally showed a similar trend with that of rock; however, P and S, presumably of biogenic origin, were detected, confirming that leaf tissue was preserved on the rock. We report the results below as the total amount of X-ray per sec (cps). www.nature.com/scientificreports/ Marked variations were found in Si; it was highest in the leaf lamina (376.457 ± 5.394 cps), slightly higher than in rock (366.144 ± 5.323 cps), and lowest in the leaf vein (114.689 ± 3.029 cps). The highest P was recorded in the leaf vein (11.680 ± 1.149 cps), which was followed by P in frass (7.923 ± 0.992 cps), whereas peaks for P were not detected in the lamina or rock. S showed a similar trend as P, but with peaks in frass (29.034 ± 1.614 cps) and the vein (11.680 ± 1.149 cps) being higher than those of P. Ca was consistently detected from all measured sample points and showed only minor differences among them; it was highest in the leaf vein (19.380 ± 1.381 cps), followed by frass (17.519 ± 1.286 cps), rock (16.851 ± 1.251 cps), and leaf lamina (13.895 ± 1.158 cps).

Discussion
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-mines 53 : 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). In this case, features of the frass trails are shared by the three mines on the same pinna ( Fig. 2A,B), which are most likely to be produced by the same insect taxon who has a stereotyped strategies for consumption and excretion. Each mine is composed of a continuous, single frass trail, the width of which subsequently expands at the end. The abrupt change in the shapes of faecal tracks can be interpreted as larval development while mining. In the mined pinnules, boundaries between mined and unmined areas are obscure because the frass trails frequently cut across the secondary leaf veins without distorting them. This can be interpreted in two ways: the larvae might mine only epidermal cells and they did not consume mesophylls, as in the case of Phyllocnistis Zeller (Lepidoptera: Gracillariidae) 54,55 ; otherwise, the larvae could mine mesophyll avoiding vascular tissues (e.g., some agromyzid flies 56 ). The latter strategy is possible because the mesophyll of ferns is anatomically well-differentiated into palisade and spongy tissues 57 .
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, taxoncharacteristic behavioural patterns in oviposition and feeding 2 . However, in general, the convergence among different insect orders/families and variation among closely related species make it difficult to differentiate miners based on their mine shapes 53 .
The taxonomic affinity of the mines from the Momonoki Formation is herein examined in the light of mine shape, systematic and evolutionary backgrounds of leaf-mining clades, 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-miners 58 . Extant fern-mining insects are found in four orders: Diptera, Coleoptera, Lepidoptera, and Hymenoptera 59 .
Leaf-mining flies are diverse, encompassing three infraorders, Culicomorpha (e.g., Chironomidae), Bibinomorpha (e.g., Sciaridae), and Muscomorpha (Empidoidea-Muscoidea). Among them, Agromyzidae contain an overwhelming number of leaf-mining taxa. 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 diverse 46,47 . Typical leaf mines of Agromyzidae, to which all fossil dipteran leaf mines have been assigned 60 , make linear-blotch mines 61 , and mine through mesophylls leaving major veins 62 ; they tend to contain fluidized frass, which is often deposited as two discontinuous rows of pellets 19 . However, the forms of leaf mines by Agromyzidae greatly vary among taxa 63 . Flies are relatively diverse as miners of fern pinnules (and stems), represented by Chirosia Rondani (Anthomyiidae) 64 , Agromyzidae (e.g., Chromatomyia Hardy, Phytoliriomyza Hendel), and Cecidomyiidae 59 . Among them, Chirosia is a predominant component that use fronds or stems of a range of fern taxa 58,[65][66][67][68][69][70] . In mines of some species of Phytoliriomyza, the frass trail displays a shift from a narrow meandering line to a wider band of the faecal pellets, which, to some extent, resemble those of the mines from the Momonoki Formation.
Leaf-mining taxa of Hymenoptera are mainly composed of sawflies (Tenthredinidae). This order was already diverse by the end of the Triassic 71 , with the oldest fossils dating back to the Middle Triassic 72 . Leaf-mining sawflies produce large conspicuous blotch mines 53,73 and the larvae deposit cylindrical frass pellets which are scattered irregularly about the mine cavities 58,74 . The extant fern-feeders of Hymenoptera do not include pinnuleminers, and only Blasticotomidae and Tenthredinidae, as petiole-borers and internal fern-feeders, respectively, are known 59 .
The coleopteran leaf miners are known from Buprestoidea, Chysomeloidea, Curculionoidea, and a few other clades 60 ; fern-pinnule mining taxa are reported from these superfamilies 59 . Beetles became widespread worldwide in the Middle to Late Triassic [75][76][77] . The earliest beetle group, Protocoleoptera, are found from the Momonoki Formation 34,45 , although they are thought to be saproxylic (i.e., borers of decaying wood), based on some circumstantial evidence 78 . Linear mines containing granular faecal pellets from the Triassic are often assigned to beetles, e.g., Polyphaga 79 . Notably, buprestid mines have some distinctive features that are comparable to those of the described mines. The buprestid mines are typically blotchy; they often are full-depth mines, avoiding epidermal and vascular tissues; the frass trails are generally long and stringy when freshly deposited, which later can be fragmented and become granular 58,[80][81][82][83] .
Lepidopteran leaf-miners mainly consist of microlepidopteran groups, including Nepticuloidea, Gracillaroidea, and part of Yponomeutoidea. Based on a recent fossil-calibrated molecular phylogeny 84  www.nature.com/scientificreports/ Germany were used 85 . The earliest presumed nepticulid leaf mine fossils are known from the Dakota Formation of the Early Cretaceous (102 Ma) 18,86,87 . The 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 blotches 87 . In addition, typical nepticulids generally 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. The Gracillariid moths produce serpentine or blotch mines and the mode of leaf-mining is conserved at the subfamily level 88 . Many gracillariid taxa (Acrocercopinae, some Gracillariinae, and Ornixolinae, and Lithocollectinae) make a narrow linear mine during the sap-feeding phase of early instars which later become a simple blotch mine. With regard to the fossil and molecular evidence, it is therefore unlikely that a member Nepticulidae caused the Late Triassic mine, although the Nepticuloidea or another early leaf-mining moth group cannot be ruled out. Altogether, the shape of the Cladophlebis mines does not conform to typical mines of Diptera (Agromyzidae) and Hymenoptera; instead, it shares more features with those of Coleoptera or Lepidoptera in terms of the mine shapes. The assignment of a fossil leaf mine to a particular taxonomic group of insects is subject to uncertainty and limitations 19 . Available biological accounts of leaf-mining insects are limited, and leaf mines are often not photographed or illustrated. Particularly, in this case, a series of diagnostic features-oviposition habit (e.g., oviposition scar, deposition of eggs), pupal chamber, mining tissue types (e.g., epidermis, parenchyma), relative position of the frass trail in the mine-were not readily recognizable. Furthermore, the mine recorded herein predates the estimated divergence time of major clades of leaf-mining insects in the modern, as examined above. Overall, the potential leaf-miner may be Coleoptera or Lepidoptera, in terms of the general resemblance of the mine shape, the time of appearance, and the presence of records at the order level, although the possibility of Hymenoptera and Diptera is not completely excluded, due to the lack of conclusive diagnostic features. Future taxonomic studies on the insect fossils from the same deposit would provide support for ascertaining the suspect leaf-miner.

The stoichiometric footprint of the studied plant-insect interaction. The elemental analyses indi-
cate 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, which is consistent with the fact that the coprolites are largely composed of calcium phosphate.
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 (SiO 2 , nH 2 O), is found in many clades of pteridophytes 89,90 , and these phytoliths can enhance plant resistance to herbivore feeding 91,92 . This relatively higher content of Si in the frass coprolites may therefore reflect undigested defensive compounds.
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 which occurred 220 million years ago. Similarly, the synchrotron X-ray Fluorescence (SRS-XRF) analyses for exceptionally preserved leaf fossils of Acer pseudoplatanus L. from the Green River Formation (Eocene, ca. 50 Ma) 93 have revealed a high concentration of some metal elements (e.g., Cu, S, Zn) in the plants and the faecal materials; however, phosphorus in the leaves was below the detection limits. 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 Triassic 27 . 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 Permian 94,95 , which were later attributed to fungal or bacterial infection 27,96,97 . The other types represent a series of small and extensive serpentine structures 94,98,99 , the ichnotaxon names of which are Asteronomus meandriformis Müller and A. divergens Müller; currently, these are acknowledged as structures of taphonomic origin 27 . 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 100 . Another example is a U-shaped contour on the foliage of Vjaznikopteris rigida Naugolnykh from the Volga River Basin (the P-Tr boundary), European Russia 101 . 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) 102 . Several sites of the Molteno Formation (Carnian) are a rich source of herbivory and oviposition trace fossils 79,103 . Two types of leaf mines, one of each from Heidiphyllum elongatum (Morris) foliage (DT41, DT71) and one from Sphenobaiera schenckii (Feistmantel) Florin (DT139), have been recorded from this locality 79 (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 Gondwanan flora is a serpentine mine on Heidiphyllum Retallack foliage, Triassohyponomus dinmorensis Rozefelds et Sobbe, from the Blackstone Formation (Carnian) of the Ipswich Coal Measures Group, Queensland, Australia 104,105 (Fig. 4B) 106 (Fig. 4C). Some mining structures are also known from the foliage of Nilssonia sturii Krasser from the Lunz Formation (Carnian), in the eastern part of the Northern Calcareous Alps, Austria 107 (Fig. 4D); also, two types of mines on some gymnosperm (?Glossophyllum) foliage are reported from Dzhayloucho (Ladinian-Carnian), near Madygen, Kyrghyzstan 108,109 (Fig. 4E).
The Cladophlebis mines (Fig. 4F) described here represent 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.  44

Methods
Plant-arthropod interactions were censused for ca. 200 full storage boxes (ca. 536 × 336 cm) of fossil specimens. Thus, the examined surface area of fossils came to no less than 3600 m 2 ; the specimens were collected from several localities of the Momonoki Formation. Although many plant-insect interactions were found and Cladophlebis fern fronds were a dominant component of the fossil floral assemblage, the leaf mines described in this paper were found on only one specimen. Three leaf mines were found on a shale, as one part and counterpart specimen. The fossil type specimen was identified by H.Y. The shale was collected from National Route 435 by H.T. with obtaining permission (Fig. 1). The fossil type specimen is deposited and publicly available in the Mine City Museum of History and Folklore (MMHF), Yamaguchi prefecture, Japan; the catalogue numbers are MMHF11-00001a and MMHF11-00001b. The geological map (Fig. 1) was made by H.Y. based on relevant studies 38,39 , and later slightly modified by Y.I., using Adobe Illustrator® 2021. The field study on fossil plants comply with relevant institutional, national, and international guidelines and legislation. Photographs of materials were taken by N.O. with a Nikon D850 using three types of Macro-NIKKOR lens (120 mm F 46.3, 55 mm f 2.8, and 65 mm f 4.5). Photos were later edited by Y.I. with Adobe Photoshop® 2021 to increase the contrast and to erase the background. Areas and lengths were measured by Y.I. with Fiji (Fiji Is Just Image J) 110 . For preparing Fig. 4A-F, illustrations were made by Y.I. by tracing photos from the relevant literature with water-based drawing pens, scanned with a CanoScan LiDE 400 (Canon) at a resolution of 300 dpi, and edited with Adobe Photoshop® 2021.
To examine the stoichiometric footprint of this ancient plant-insect interaction, elements of the mine trace were analysed by H.Y. via energy-dispersive X-ray fluorescence (XRF) using a EA6000VX High Sensitivity XRF Analyzer (Hitachi High-Tech Science Corporation). An XRF spectrometer measures elements between Na and U in order of increasing atomic number. Elemental compositions were quantitatively measured from three points of the specimen where organic compounds derived from fossil leaves remained (one point was set on a frass trail of a leaf mine where insect's coprolites were visible; each one sample point was set on leaf vein and lamina of the same pinnule), and, as a control point, from the host sedimentary rock. The measurement duration was set at 120 s using a 0.2 × 0.2 mm collimator under an excitation voltage of 50 kV and a tube current of 1000 μA. Output data were visualized by Y.I. with ggplot2 111 and formatted with Adobe Illustrator® 2021.

Data availability
The fossil type specimen is housed in the Mine City Museum of History and Folklore (MMHF), Yamaguchi prefecture, Japan: catalogue numbers MMHF11-00001a and MMHF11-00001b. In addition, the original XRF analysis data are provided in Supplementary Information S1-S4.