﻿Diversity and larval leaf-mining habits of Japanese jewel beetles of the tribe Tracheini (Coleoptera, Buprestidae)

﻿Abstract From the Japanese Archipelago, 12 Habroloma and 20 Trachys species (Buprestidae: Tracheini) have been recorded. Two new Habroloma species were found, which are associated with Elaeocarpaceae and Loranthaceae, also new host plant families/orders for Tracheini. The two new species are described as Habrolomaelaeocarpusisp. nov. and Habrolomataxillusisp. nov., and the latter is the first Tracheini species shown to be associated with epiphytes. Leaf mines of 31 Tracheini species are also reported in this work, including new records of leaf mines for 16 Tracheini species. The larvae of all these recorded species are full-depth linear-blotch mesophyll miners of mature leaves and pupate within their mines. The mining habits of Habroloma species associated with Symplocos (Symplocaceae) are unique: the young larvae bore into midribs and petioles and cause leaf fall, and the larvae then mine the fallen leaves.


Introduction
The coleopteran family Buprestidae is a species-rich clade whose larvae are xylophagous wood-borers, while the leaf-mining habit has evolved (Hering 1951). The tribe Tracheini of the subfamily Agrilinae is one of these leaf-mining clades and has great diversity, especially in Asia, Europe, and Africa. The two genera of Tracheini, Trachys and Habroloma, comprise more than 650 and 300 species worldwide, respectively (Bellamy 2008). Continental Asia is home to diverse trachyine species (Obenberger 1918(Obenberger , 1929 and the number of trachyine species has been underestimated. In recent years, 14 Trachys and 33 Habroloma species have been newly described from China (Peng 2020(Peng , 2021a(Peng , b, c, d, 2022a. By contrast, the Japanese Archipelago harbors 20 Trachys and 12 Habroloma species (Buprestidae: Tracheini) (Ohmomo and Fukutomi 2013), and no new taxa have been added since the monograph by Kurosawa (1959).
Leaf-mining habits in Buprestidae are believed to have evolved from wood-boring habits (Frost 1924), and the switch from wood-boring to leaf-mining has occurred several times in Buprestidae (Evans et al. 2015). The leaf-mining habits of Japanese trachyine species are characterized by full-depth blotch mining of the leaves of woody plants such as Malus, Rosa, Prunus, Ulmus, Zelkova, Aphananthe, Broussonetia, Quercus, Castanopsis, Platycarya, Salix, and Deutzia, or subwoody climbing plants such as Pueraria, Amphicarpaea, and Desmodium (Yano 1952), while in Europe many leaf-mining trachyine species are associated with herbaceous plants such as Fragaria, Potentilla, Scabiosa, Stachys, Malva (Frost 1924), and Geranium (Schaefer 1950).
To shed light on the diversity and host plant associations of trachyine species in Japan, we have conducted extensive rearing of leaf-mining larvae on diverse plants and a substantial collection of mined leaves. From the accumulated materials, we identified two undescribed trachyine species. The two new species are associated with two new plant orders and families for Tracheini: Oxalidales (Elaeocarpaceae) and Santalales (Loranthaceae). Furthermore, we detected leaf mines for 31 trachyine species, including new leaf mine records for 18 trachyine species. In this paper, we describe the two new species, as well as the leaf mines of 31 trachyine species, and discuss the diversity and evolution of plant utilization patterns of trachyine species in the Japanese Archipelago.

Materials and methods
We have conducted extensive sampling of buprestid leaf mines from the Japanese Archipelago since the 1980s. By rearing the leaf-mining larvae, we obtained 400 adult buprestid beetles. All of the specimens were collected by MK unless otherwise noted. The leaves containing leaf mines were dried, and the dried herbarium specimens have been deposited in the Kyoto University Museum (KUM).
The morphology of adult specimens was examined under a microscope (VHS-7000; Keyence). Specimens were photographed by synthesizing virtual images from a sequence of corresponding depth images. To observe male genitalia, the specimens were macerated in hot water and dissected under a microscope. The abdomen was removed from the body and then cleaned in 5% KOH solution for ~ 12 h at room temperature. After washing in distilled water, the terminalia extracted from the abdomen were mounted on slides with glycerol.

Systematics of Japanese Habroloma Thomson, 1864
Among adult beetles that emerged from collected leaf mines, we identified two new Habroloma species. We describe the two species using the following key to Japanese species. In this key, Habroloma hikosanensis is missing because it is within the morphological variation of Habroloma yuasai.
Diagnosis. A small wedge-shaped species (length 3.1-3.3 mm) having pronotum with posterior margin trisinuate. Elytra rather flattened, ornamentation consisting of white pubescence; on posterior half with three transverse bands, anterior one obliquely zigzag, two posterior ones transversely straight. Male genitalia with slender tegmen with paramere setiferous on anterior margin and slender pennis with rounded apex. Larvae mine leaves of Elaeocarpus japonicus.
Head, seen from above, transverse, broadly and sharply excavated between the eyes, with the inferior rim of the eyes strongly and rather suddenly produced; frons with the median impression distinct; fovea just above each antennal cavity obsolete and indistinct; surface rather smooth, sparsely scattering laterally with traces of variolate and ocellate punctures, and sparsely clothed with whitish recumbent hairs; clypeal suture transverse, somewhat arcuate exteriorly; clypeus transverse, ~ 2.6× as wide as long, with the anterior margin somewhat arcuately emarginate; antennal cavities surrounded posteriorly with elevated carina; antennae short and compact, with the third segment ~ 1.5× as long as the fourth, with apical five segments serrated.
Pronotum transverse, widest just before the base, distinctly wider than elytra, and ~ 3.2× as wide as long; sides slightly but distinctly expanded just before the base, then crescent-shaped and strongly attenuated to the anterior angles, which are acute and strongly produced in dorsal aspect; anterior margin deeply, broadly and arcuately emarginate; posterior margin trisinuate, produced and subtruncate, narrowly and slightly emarginate just before scutellum; posterior angles acute and produced posteriorly; disk dilated laterally, broadly and obsoletely depressed at the anterior half of the lateral dilation on each side, but without fovea, and obsoletely impressed along the basal lobe causing the middle of the disk to be somewhat convex; surface lustrous, punctured with traces of large, obsolete, shallow, somewhat ocellate punctures, and sparsely clothed with whitish hairs. Scutellum smooth and triangular.
Elytra rather deplanate, widest at the base, ~ 1.3× as long as wide and ~ 4.3× as long as pronotum; sides feebly sinuate and narrowed or subparallel to the anterior 2/5, and then arcuately attenuated to the apex, but the attenuation somewhat angulate near the apex; sutural margin not elevated entirely; humeri slightly prominent; basal depressions along the base transverse; lateral carinae subparallel to the lateral margin; disk constricted behind humeri, narrowly and obsoletely impressed along the inferior side of each lateral carina; surface rather uniformly but coarsely punctate with shallow, ill-defined, irregularly sized punctures, with the punctuation being somewhat rugous at the sides; ornamentation consisting of white, yellowish-grey, and blackish hairs, with the whitish hairs being predominant. Ornamentation consisting of white pubescence arranged on each elytron as follows: at base with two irregular spots, at mid length near suture with one irregular spot, toward side with one narrow, wavy, and irregular strip, on posterior half with three transverse bands, anterior one obliquely zigzag, two posterior ones transversely straight.
Body beneath scattered with very fine inconspicuous cinereous hairs. Prosternal process inverted trapezoidal, narrow toward the base, ~ 1.3× broader than long, with the apex almost truncate. Metasternum slightly convex coarsely punctate with variolate and obsolete punctures at the middle. Abdomen beneath rather uniformly punctate with shallow, obsolete variolate punctures. Legs normal; posterior coxae depressed entirely, with the latero-posterior angles acute and produced latero-posteriorly.
Head, seen from above, transverse, broadly excavated between the eyes, with the inferior rim of the eyes strongly produced; frons with the median impression distinct; fovea just above each antennal cavity obsolete and indistinct; surface rather smooth, sparsely scattered laterally with traces of variolate and ocellate punctures, and sparsely clothed with recumbent yellowish-grey hairs; clypeal suture transverse, somewhat arcuate exteriorly; clypeus transverse, ~ 2.6× as wide as long, with the anterior margin somewhat arcuately emarginate; antennal cavities surrounded posteriorly with elevated carina; antennae short and compact, with the third segment ~ 1.5× as long as the fourth, and five apical serrated segments.
Pronotum transverse, widest just before the base, as wide as elytra, and ~ 2.4× as wide as long; sides slightly but distinctly expanded just before the base, then crescentshaped and strongly attenuated to the anterior angles, which are acute and strongly produced in dorsal aspect; anterior margin deeply, broadly, and arcuately emarginate; posterior margin trisinuate and subtruncate, narrowly and slightly emarginate just before scutellum; posterior angles acute and produced posteriorly; disk dilated laterally, broadly and obsoletely depressed at the anterior half of the lateral dilation on each side, but without fovea, and obsoletely impressed along the basal lobe causing the middle of the disk to be somewhat convex; surface lustrous, punctured the traces of large, obsolete, shallow, somewhat ocellate structures, and sparsely clothed with yellowish gray hairs. Scutellum smooth and triangular.
Elytra slightly convex along base, widest at the base, ~ 1.4× as long as wide and ~ 3.3× as long as pronotum; sides feebly sinuate and narrowed or subparallel to the anterior 2/5, and then arcuately attenuated to the apex but with the attenuation somewhat an- gulate near the apex; humeri slightly prominent; basal depressions along the base transverse; lateral carinae subparallel to the lateral margin; disk constricted behind humeri, narrowly and obsoletely impressed along the inferior side of each lateral carina; surface rather uniformly but coarsely punctate with shallow, ill-defined, irregularly sized punctures, but the punctuation somewhat rugous at the sides. Ornamentation consisting of yellowish grey pubescence arranged on each elytron as follows: at base with two irregular spots, on posterior 2/3 with three transverse bands, first and second ones obliquely zigzag, apical one slightly transversely waved; with transverse irregular spot apically.
Body beneath scattered with very fine inconspicuous cinereous hairs. Prosternal process rounded, ~ 1.27× broader than long. Metasternum slightly convex and coarsely punctate, with variolate and obsolete punctures at the middle. Abdomen beneath rather uniformly punctate with shallow, obsolete variolate punctures. Legs normal; posterior coxae depressed entirely, with the latero-posterior angles acute and produced latero-posteriorly.
Male genitalia: not studied. Female. Like the male, but more robust. Ornamentation of elytra is similar but pubescence more whitish in female. Body length: 2.5-2.7 mm, width: 1.5-1.7 mm.
Distribution. Japan (Amami-Oshima Island, known only from the type locality).

Leaf mines of Japanese Tracheini species
Leaf mines of Tracheini species have the following characteristics. The mined leaves are mature leaves that have completed expansion and hardening. An egg is laid on the upper side of a leaf and covered by a circular brown glossy coating, which is secreted by an adult female. Pupation takes place within the mine. Hereafter, we describe leaf mines of the 14 Habroloma and 20 Trachys species in Japan.  (Yano 1952), Rubus palmatus, Rubus buergeri (Kurosawa 1959). Leaf mine. Brown full-depth blotch mine on mature leaf. Egg is laid at a distance from leaf margin and the mine expands in the leaf blade. Frass is thread-like.

H. atronitidum (Gebhardt, 1929)
Leaf mine. Brown full-depth blotch mine on mature leaf. Egg is laid near leaf margin, and the mine expands along the leaf margin. Frass is thread-like.

H. lewisii (E. Saunders, 1873)
Leaf mine. Brown, full-depth sometimes bluish, linear-blotch mine on mature leaf. Egg is laid along leaf margin, and the mine expands along leaf margin. Frass is thread-like, coiling or undulating for an extended length.

H. griseonigrum (E. Saunders, 1873)
Leaf mine. Brown full-depth blotch mine on mature leaf. Egg is laid near leaf margin of leaf base, and the mine expands upwards along leaf margin. Frass is thread-like.
Leaf mine. Brown full-depth linear-blotch mine on mature leaf. Egg is laid near leaf margin, and the mine expands along leaf margin. Frass is thread-like, excreted from the mine through cracks of upper epidermis.

H. nixilla insulicola Y. Kurosawa, 1959
Fig. 5H, I Host plant. Lythraceae: Lagerstroemia subcostata (Kurosawa 1959). Leaf mine. Brown full-depth linear-blotch mine on mature leaf. Egg is laid along leaf margin of leaf base, and the mine expands along leaf margin. Frass is thread-like, coiling in the mine.
Leaf mine. Unknown.  (Kurosawa 1976), while the identification seems to be incorrect; S. caudata (new record). Leaf mine. Pale brown full-depth linear-blotch mine on mature leaf. Egg is laid near midrib of leaf base, and the hatched larva enters midrib and bores into petiole, causing the leaf to fall off from the branch by being abscised at the petiole base. After the leaf-fall, the mine departs from the midrib and slowly expands upwards along leaf margin or along midrib. After advancing halfway, the mine abruptly expands to become a blotch mine. Frass is thread-like, going in a zigzag in the early linear mine, and becomes thick cord-like without undulating as the mine expands. The fallen leaf is kept green for ca. two weeks, during which the larva completes its development.

H. liukiuense
Leaf mine. Pale brown full-depth linear-blotch mine on mature leaf. Egg is laid near midrib of leaf base, and the hatched larva enters midrib and bores into petiole, causing the leaf to fall off from the branch by being abscised at the petiole base. After the leaf-fall, the mine departs from the midrib and slowly expands upwards along leaf margin or along midrib. After advancing halfway, the mine abruptly expands to become a blotch mine. Frass is thread-like, going in a zigzag in the early linear mine, and becomes granular. The fallen leaf is kept green for ca. two weeks, during which the larva completes its development.
Trachys species checklist Fig. 7A Host plant. Fabaceae: Pueraria montana var. lobata (Yano 1952).  Leaf mine. Gray full-depth blotch mine on mature leaflet. Egg is laid in an inner area of leaf blade, and mine expands toward leaf margin. Frass is granular and distributed all over the mine.

Trachys tokyoensis Obenberger, 1940
Leaf mine. Brown full-depth blotch mine on mature leaflet. Egg is laid along anterior margin of a leaflet, and the mine expands toward leaf base. Frass is granular and distributed all over the mine.

Trachys toringoi Y. Kurosawa, 1951
Leaf mine. Brown blotch mine on mature leaf. Egg is laid in an inner basal area of leaf blade, and the mine expands toward leaf top. Frass is granular and distributed all over the mine.

Trachys inconspicuus E. Saunders, 1873
Leaf mine. White full-depth blotch mine on mature leaf. Egg is laid along leaf margin, and the mine expands along leaf margin. Frass is granular and distributed all over the mine.

Trachys pecirkai Obenberger, 1925
Leaf mine. Brown full-depth blotch mine on mature leaf. Egg is laid near midrib, and the mine expands along lateral vein and then along leaf margin. Frass is granular and distributed all over the mine.

Trachys cupricolor E. Saunders, 1873
Leaf mine. Brown full-depth blotch mine on mature leaf. Egg is laid along leaf margin, and the mine expands along leaf margin. Frass is granular and distributed all over the mine.

Trachys griseofasciatus E. Saunders, 1873
Leaf mine. Dark brown full-depth blotch mine on mature leaf. Egg is laid along anterior leaf margin, and the mine expands along leaf margin. Frass is thread-like and distributed all over the mine.

Trachys robustus E. Saunders, 1873
Leaf mine. Gray full-depth linear-blotch mine on mature leaf. Egg is laid along midrib near leaf tip, and the mine expands downwards along leaf margin. Frass is granular and distributed all over the mine.

Trachys aurifluus Solsky, 1875
Leaf mine. Brown full-depth linear-blotch mine on mature leaf. Egg is laid near leaf margin, and the mine expands along leaf margin. Frass is granular and distributed all over the mine.
Material examined. Iyari, Inao, Omachi, Nagano Pref., 1-VII-2013 (vacant mine of Tilia maximowicziana) (Fig. 10G). Leaf mine. Brown full-depth blotch mine of whole layers of leaf blade. Egg is laid along leaf margin near leaf base, and the mine expands upwards along leaf margin. Frass is granular and loosely connected.

Trachys tsusimae Obenberger, 1922
Leaf mine. Brown full-depth blotch mine on mature leaf. Egg is laid along leaf margin, and the mine expands along leaf margin. Frass is granular and distributed all over the mine.
The host plant genera of the two new trachyine species are Elaeocarpus and Taxillus, belonging to Elaeocarpaceae (Oxalidales) and Loranthaceae (Santalales), respectively, and representing the first records in Tracheini for both families and orders, while both plant families have been recorded as hosts for Agrilus (Jendek & Poláková, 2014). The record on Taxillus is also the first record of buprestids associated with epiphytic, plantparasitic plants.
Our records of leaf mines suggest that those of trachyine species are generally full-depth blotch mines on mature leaves of woody or subwoody plants, except for one species (Trachys pseudoscrobiculatus) associated with Viola. These leaf mines contrast with upper-layer mines on young leaves formed by agromyzids, epidermal/mesophyll mines on young leaves formed by gracillariids, thin full-depth linear mines formed by Lyonetiidae, and full-depth linear-blotch mines on young leaves formed by Eriocraniidae.