Systematics of Phyllocnistis leaf-mining moths (Lepidoptera, Gracillariidae) feeding on dogwood (Cornus spp.) in Northeast Asia, with the description of three new species

Abstract During an ongoing DNA-barcoding campaign of the leaf-mining moths that feed on woody plants in Northeast Asia, four lineages of the genus Phyllocnistis (Gracillariidae, Phyllocnistinae) were discovered on dogwood (Cornus spp): P. cornella Ermolaev, 1987 on C. controversa Hemsl. (Japan: Hokkaido) and three new species – one feeding on C. controversa, C. florida L. and C. macrophylla Wall. in Japan (Honshu, Shikoku, Kyushu), a second species on C. macrophylla in China (Yunnan) and a third on Siberian dogwood Cornus alba L. in Russia (Siberia). All these species showed differences in morphology, in the barcode region of the cytochrome c oxidase I gene and in two nuclear genes (histone H3 and 28S ribosomal RNA). No correlation was found between the deep mitochondrial splits observed and the Wolbachia infection pattern. Based on both morphological and molecular evidence, the three recently discovered lineages are described here as new species: P. indistincta Kobayashi & Triberti, sp. n. (Japan), P. saepta Kirichenko, Ohshima & Huang, sp. n. (China) and P. verae Kirichenko, Triberti & Lopez-Vaamonde, sp. n. (Russia). In addition, the authors re-describe the adult morphology of P. cornella, provide the first record of this species from Japan and highlight the diagnostic characters that allow these Cornus-feeding Phyllocnistis species to be distinguished.


Introduction
Leaf-mining micromoths of the family Gracillariidae have been the focus of recent taxonomic studies in particular in the Palaearctic region (Langmaid and Corley 2007, Triberti 2007, Laštůvka et al. 2013, Doorenweerd et al. 2014, Kirichenko et al. 2015, Huemer et al. 2016. Amongst the 100 plus genera of gracillariids known worldwide, the genus Phyllocnistis Zeller, 1848 belongs to the subfamily Phyllocnistinae Herrich-Schäffer, 1857 (Kawahara et al. 2017) and still remains poorly studied (Davis and Miller 1984, Brito et al. 2012. Phyllocnistis adults are small, with wingspan generally less than 5 mm and difficult to identify to species level since they show little morphological differentiation in genitalia (De Prins and Kawahara 2009). However, some studies have shown that the morphology of the pupal head and abdomen can provide valuable diagnostic characters , Kobayashi et al. 2011a.
To date, 109 species have been recognised within the genus Phyllocnistis (De Prins and De Prins 2017, Brito et al. 2017a, 2017b, Fochezato et al. 2017. Amongst them, 22 new species have been discovered and described in the last decade: 16 species in South America (i.e. Brazil, Costa Rica, Colombia, French Guiana) , Brito et al. 2012, 2017a, 2017b, Fochezato et al. 2017, three in the USA , two in Japan (Kobayashi et al. 2011b, Kobayashi and and one in Europe (Langmaid and Corley 2007).
Phyllocnistis species are found in six biogeographical realms: 36 species are known from the Oriental realm, 28 species from the Neotropics, 18 species from the Palaearctic, 17 species from Australasia, 16 species from the Nearctic and four species from the Afrotropics; five species occur in more than one realm, partly through introductions . Host plants are known for 74 species , Brito et al. 2017a, 2017b, Fochezato et al. 2017. Larvae develop on plants from 21 orders and 34 families. Despite the broad host plant range, Phyllocnistis species show a high level of host plant specificity, often feeding on a single host plant genus or species.
Until recently, only one species, P. cornella Ermolaev, 1987, has been recorded feeding on dogwood Cornus (Cornales: Cornaceae). It was described from the Russian Far East, from Kunashir, the southernmost island of the Kuril Islands, on C. controversa (Ermolaev 1987). V.P. Ermolaev based his description on the morphology of the male genitalia and forewing pattern, providing a short description of the species.
The Eastern part of Eurasia, with its vast forests and little explored mountain ranges, still holds many new species of Lepidoptera. During an ongoing DNA-barcoding campaign of Gracillariidae leaf-mining moths from Northeast Asia, four divergent Phyllocnistis lineages feeding on Cornus spp. were detected: two from Japan, one from China and one from Russia. DNA barcoding, nuclear data and morphology of adults confirmed the presence of three new species that are described here: P. indistincta Kobayashi & Triberti sp. n. (Japan: Honshu, Shikoku, Kyushu), P. saepta Kirichenko, Ohshima & Huang sp. n. (China: Yunnan) and P. verae Kirichenko, Triberti & Lopez-Vaamonde, sp. n. (Russia: Siberia). In addition, the authors also re-describe the adult morphology of the closely related East Asian P. cornella, highlighting diagnostic characters that help to distinguish Cornus-feeding Phyllocnistis and provide the first records of P. cornella from Japan (Hokkaido). Female genitalia and pupal morphology of P. cornella are described here for the first time.

Materials and methods
In total, 212 specimens have been studied (Suppl. material 1). One hundred and seventy nine of them were represented by Cornus-feeding Phyllocnistis and the remaining 33 specimens belonged to closely related Phyllocnistis species feeding on other host plants: P. labyrinthella (Bjerkander, 1790), P. citrella Stainton, 1856, P. extrematrix Martynova, 1955, P. gracilistylella Kobayashi, Jinbo & Hirowatari, 2011, P. unipunctella (Stephens, 1834 and Phyllocnistis spp. All these species are distributed in Eurasia and are known in the eastern part. The specimens of the East Asian species P. citrella originated from Europe and the USA (Suppl. material 1).

Cornus-feeding Phyllocnostis were collected in the following countries:
Japan. Leaf mines and individuals of P. cornella were sampled on Hokkaido (2 specimens) and P. indistincta on Honshu (137 specimens), Shikoku (1 specimen) and Kyushu (19 specimens) on different Cornus species (Suppl. material 1). Leaves with mining larvae and cocoon folds were sampled from March to November 2008-2017. Larvae and pupae found in mines on leaves were reared in the laboratory in plastic cups containing wet cotton, at constant conditions (20 °C, 55 % RH, LD 16:8 h photoperiod). Three larvae and six pupae were preserved in 99 % ethanol and 95 adults were pinned. In addition, specimens collected by Dr H. Kuroko in Osaka Prefecture University (OPU) and collections of N. Hirano (Matsumoto, Nagano Prefecture) were examined.
China. Leaf mines of P. saepta with one living larva and two pupae were sampled on C. macrophylla in one location in Yunnan Province (Weixi) in July 2016 (Suppl. material 1). One larva and one pupa were preserved in 99 % ethanol and one adult emerged from the mine and was pinned for further studies (Suppl. material 1).
Russia. Leaf mines with individuals of P. verae were collected on Siberian dogwood C. alba in Siberia (Krasnoyarsk, near village Borovoe, along the river Yenisei) in a forested area, in June-July 2015. Overall, 11 larvae were preserved in 96 % ethanol and six adults were pinned (Suppl. material 1). To obtain adults, leaves with mines were kept in glass jars at constant conditions (22 °C, 55 % RH, LD 18:6 h photoperiod), following the protocol in Ohshima (2005). In addition, 20 leaves with mines, some of them with larvae or pupae inside mines, were preserved in herbarium collections as described in Kirichenko (2014). In June-July 2015-2017, an extensive survey of Cornus spp. was undertaken throughout the Asian part of Russia: in Siberia -Tyumen, Sugrut, Tomsk, Omsk, Novosibirsk, Kemerovo, Barnaul, Irkutsk, Ulan-Ude and Chita, in the Russian Far East -Blagoveshensk, Vladivostok, Gornotayejnoe and on the Island Sakhalin -Yuzhno-Sakhalinsk. Dogwood trees (especially C. alba and C. controversa), growing in botanical gardens and arboreta, in city plantations and in forests, were checked for the presence of Phyllocnistis mines.
Photographs of leaf mines were taken in the field and in the laboratory using an OLYMPUS μ1060 digital camera (in Japan) and a digital camera Sony Nex3 (in Russia). Additionally, leaf mines of P. indistincta were scanned using an EPSON GT7400. Pupae of P. indistincta were dried under room temperature and sputter-coated with a 60:40 mixture of gold-palladium for examination with a scanning electron microscope (SEM) HITACHI SU1510 (Hitachi Ltd., Tokyo, Japan), with a lanthanum hexaboride (LaB 6 ) source, at an accelerating voltage of 15 kV. Mounted moths of Cornus-feeding species were photographed using an OLYMPUS E-500 digital camera (P. indistincta). Other species were photographed using Zeiss Axiocam MRc 5 digital camera mounted on a Zeiss V.20 stereo microscope. Phyllocnistis cornella, P. saepta and P. verae specimens were photographed up to 30 times and then focus stacking was applied using Helicon Focus (http://www.heliconsoft.com/) or Zeiss Axiovision software.
Twenty five genitalia slides of P. cornella (two slides), P. indistincta (16), P. saepta (one) and P. verae (six) were prepared and analysed (Suppl. material 1). For genitalic preparation, abdomens were heated in a 10 % potassium hydroxide solution, stained with acetocarmine and then slide-mounted in Canada balsam. For P. cornella specimens, the abdomens were removed from the specimens and used in non-destructive DNA extraction (Doorenweerd et al. 2016). Female genitalia were stained with chlorazol black and slide mounted in euparal. Genitalia were photographed with a Leica DFC 450 digital camera through a Leitz Diaplan GMBH microscope. On the same microscope, a Leitz drawing tube was attached for drawings of genitalia. Environmental scanning electron microscope (ESEM) digital images were taken with a Hitachi TM1000. Drawing of genitalia was made by PT and SK. P. cornella specimens were studied with a motorised Zeiss AxioImager with Zeiss Axiocam MRc 5 camera. All images were processed in Adobe Photoshop CS5 Extended.
(Macherey-Nagel, Germany) and sequenced by the Sanger method with Abi Prism Big Dye Terminator 3.1 cycle sequencing kit (25 cycles of 10 s at 96 °C, 5 s at 50 °C, 4 min at 60 °C). Sequencing was carried out using a 3500 ABI genetic analyser. The COI amplification and sequence reactions for the RMNH specimens were carried out at Naturalis Biodiversity Centre (Leiden, The Netherlands). All the remaining reactions were carried out at INRA (Orléans, France). All sequences were aligned using Codon-Code Aligner 3.7.1. (CodonCode Corporation) or Geneious R6.
DNA sequences, along with the voucher data, images and trace files, were deposited in the Barcode of Life Data Systems (BOLD) (Ratnasingham and Hebert 2007) and the sequences were deposited in GenBank. All data are available in BOLD through the public dataset: dx.doi.org/10.5883/DS-PHYLCORN.
Barcode Index Numbers (BINs) (Ratnasingham and Hebert 2013) were obtained from BOLD. Intra-and interspecific genetic distances were estimated using the Kimura 2-parameter. RaxML v8 (Stamatakis 2014) was used to estimate the best-scoring tree and perform a multiparametric bootstrap test with extended majority-rule stopping criterium, following the PhylOStack v1.7 protocol (Doorenweerd 2017). As outgroups, one specimen of Phyllonorycter connexella (Zeller, 1846) from Salix sp. was used for the COI tree and one specimen of Phyllocnistis labyrinthella from Populus balsamifera L. for both the H3 and 28S analyses. Both specimens were collected in Siberia (Russia: the Republic of Khakassia, Abakan) (Suppl. material 1). Outgroups were sequenced following the protocol described above.
The sequences of Cornus-feeding species formed distinct clusters, with a maximum intraspecific divergence varying from 0 to 1.2 % versus a nearest-neighbour distance, varying from 6.1 to 13.6 %, when comparing Cornus-feeding species in pairs ( Table  2). The minimum interspecific distance was smaller in the pairs P. saepta -P. verae (6.1 %) and P. cornella -P. indistincta (7.3 %) and doubled in comparisons across these pairs (Table 2). Diagnostic mutations separating Cornus-feeding species ranged from 32 to 68 (Fig. 1). No evidence for mitochondrial introgression was found.
The closest neighbour to the Cornus-feeding Phyllocnistis group turned out to be an undescribed species (i.e. Phyllocnistis sp. 2) collected from Vitaceae in Japan, with a minimal interspecific distance of 8.9 % between P. verae and sp. 2. The cluster of Salicaceae-feeding Phyllocnistis was another close neighbour to the cluster of Cornaceae-feeding Phyllocnistis, with the minimal interspecific divergence (13.1 %) observed between P. verae and P. labyrinthella (Table 2).
Besides three new species on Cornus, DNA barcoding data revealed four putative new species in the east: three species in Japan feeding on Parthenocissus (Vitaceae), Ligustrum (Oleaceae) and Derris (Fabaceae), respectively and one in the Russian Far East feeding on Salix (Salicaceae) (Fig. 1, Suppl. material 1).

Nuclear genes
Sequences of the nuclear genes histone H3 and 28S were obtained for previously barcoded 14 specimens of Cornus-feeding Phyllocnistis spp. (Fig. 2). H3 explicitly delimited four clusters, corresponding to the four lineages defined by COI: P. cornella, P. indistincta, P. saepta and P. verae. The gene 28S supported the clusters of P. saepta and P. verae, but it did not show any divergence between P. cornella and P. indistincta (Fig. 2).
In both H3 and 28S, the number of diagnostic mutations was the least in the pairs: P. cornella -P. indistincta, (two mutations in H3 and no mutation in 28S) and P. saepta -P. verae (four mutations in H3 and two in 28S). The highest number of mutations was detected in the pair P. cornella -P. saepta, i.e. 15 and 19 mutations in 28S and H3, respectively.

Wolbachia test
Out of the 14 specimens of the four Cornus-feeding Phyllocnistis species screened for the genes wsp and fbpA, only one specimen of P. indistincta (MICRU069-16) from Japan (Honshu, Nara, Tateriko, ex Cornus kousa) showed the presence of an infection (Suppl. material 2).

Species descriptions
The four species described below, all feeding on Cornus, are morphologically very similar. The forewing pattern is characterised by a longitudinal fascia (lf ), more or less defined, running along the costal margin proximally and then bent inwardly distally. Three costal and four apical ciliary strigulae are present, the first costal forming a transverse fascia (tf ) crossing the wing. The male genitalia have slender valvae, rounded apically and a membranous phallus, finely pleated in the distal half, without cornuti and with a long phallobase/ductus ejaculatorius. The female genitalia have a cup-shaped antrum, a thin ductus and a large bursa with two flattened signa usually bearing a short median projection. The set of these characters allows this group of species to be differentiated quite easily from other congeners in the Palaearctic (Martynova 1955, Liu and Zeng 1989, Kobayashi et al. 2011b but their poor interspecific differences make identification of each species rather difficult. Recent studies (Kawahara et al. 2009, Davis and have shown that important identifying characters are sometimes present in pupae. However, only pupae of P. indistincta were available, so that it was not possible to use these characters. Identification keys of adults are given below, based on forewing pattern.  11,12,13,14,16,18 Etymology. This Latin adjective, declined in the feminine gender, means "indistinct". It is related to the longitudinal fascia in the forewing pattern that is basally indistinct in this species. Diagnosis. Forewing lustrous-white with a longitudinal white-yellow fascia and with an indistinct inner margin, three costal and four apical ciliary strigulae; male genitalia with phallus about as long as phallobase; female corpus bursae with two signa, similar in shape, the distal signa larger than the central signa.

Key to adults
Unlike other Cornus-feeding Phyllocnistis, the forewing pattern of P. indistincta has a well-defined outer margin of lf and an indistinct inner margin. Male genitalia differ from P. saepta by the phallus (about as long as phallobase or slightly shorter in P. indistincta); female genitalia are only distinguishable from P. verae by the size and the shape of the two signa.   Description of adult. (Fig. 9A-E). Wing span 5.0 mm in holotype, 4.0-6.5 mm in paratype; forewing length 2.4 mm in holotype, 2.0-3.0 mm in paratypes.
Head. Frons and vertex smooth, lustrous white. Antennae and labial palpi white yellowish. Thorax. Tegulae, thorax and legs white. Forewing lustrous white, subapical area orange with a small apical black spot; a yellow-orange lf along costa from base to middle, margined with dark brown on both sides, then bent inwards distally, inner margin often indistinct in basal 2/3 not touching transverse fascia. Cilia white with a tf from costal 2/3 to dorsal 1/2, sometimes interrupted in the middle and three dark brown costal strigulae before apex; a black apical spot, giving origin to four divergent dark brown apical strigulae, one extending to upper part of costal cilia, the second and third to apex, the fourth to upper part of terminal cilia; terminal cilia white with a fuscous fringe line near termen. Hindwing lustrous white. This species and other members of the genus Phyllocnistis share an R1 vein arising from the apical half of the discoidal cell in the forewing ( Fig. 13; Kawahara et al. 2017: Fig. 4C).
Abdomen. Mostly white yellowish dorsally, white ventrally. In the male, coremata present on segment 8, consisting of a pair of elongate, dilated extensions bearing a terminal cluster of long slender scales. In the female, dorsally on segment 8, a pair of tufts of scales longer than those covering the segment.
Female genitalia (Fig. 16). Anterior apophyses slightly longer than posterior apophyses; ostium bursae opening in membrane between sternum 7 and 8; antrum membranous, funnel-shaped, narrowing slightly up to the size of the ductus bursae; ductus bursae completely membranous, slender, as long as antrum and terminating in the caudal area of corpus bursae; two signa are here present, usually similar in shape, the caudal bigger than central one, each with a single, short, median projection, but sometimes this projection, usually spine-shaped, is furcate or very reduced; on the wall around signa, minute scattered sclerites, thinner in the remainder of corpus bursae; ductus seminalis elongate, slightly larger than ductus bursae and arising from anterior end of corpus bursae; ductus spermathecae (not figured) with efferent canal short, forming 3 coils of equal diameter before vesicle.
Pupa. (Fig. 18). Maximum length 2.8 mm, diameter 0.8 mm. Vertex with a triangular frontal process (cocoon-cutter), minutely serrated in profile and with elongated and thin apex curved towards dorsum (Fig. 18A-F); a single pair of short setae at base of frons (clypeus) (Fig. 18A); antenna extending to abdominal segment A6. A pair of relatively long setae latero-dorsally on meso-and metathorax; forewing extending to A6-7. A pair of minute dorsal setae from A1 to A8 with a second pair of large thornlike setae, more distal, from A3 to A8, delimiting an elongated area of minute spines, projected posteriorly, from A3 to A8 (Fig. 18G-I); each abdominal segment with a pair of long, lateral, sensory setae, probably covered by the wings in A1; a pair of divergent lobate processes from caudal apex of A10 (Fig. 18J-L).
Biology. (Figs 3-6). The larvae mine leaves of Cornus species, forming a long serpentine mine parallel to leaf veins (Figs 3, 5D-F and L); about 10 cm in length, 2.0-5.0 mm in width, with a brownish frass line 1.0 mm in width. There were one to two mines per leaf (Figs 3, 5D and J). The mines were usually observed on the lower side of leaf on C. controversa (Figs 3A-D, 4B-D), more often on upper side of leaf on C. florida and C. kousa (Fig. 5D-F, J-L). Late instar larvae are about 4.0-6.0 mm long and pale yellow in colouration (Fig. 4H). The final instar spins a white cocoon at the leaf margin, the leaf margin slightly curled upwards by contraction of the cocoon silk. The pupal cocoon fold is 5.0 mm in length, 1.0-1.1 mm in width (Fig. 4I). Dr H. Kuroko collected adult moths of this species in Mt. Daisen, Tottori Prefecture, from flower bracts with mines containing larvae. In the present study, two bracts were observed of C. florida with similar serpentine mines (pale pink to ocherous, about 15 cm in length, 0.5-2.0 mm in width, brownish frass line: 0.1-0.2 mm in width) (Figs 3K, 6C-E). A pupal cocoon fold (whitish to ocherous, 5.7 mm in length, 1.0 mm in width) was formed along the bract margin (Fig. 6F). A mine on the upper side of leaf was observed in the same tree (Fig. 6B).
In the Nasu Imperial Villa of Tochigi Prefecture, Arita et al. (2009) collected this species as Phyllocnistis sp. 1, "Mizuki-Kohamoguri" and noted some aspects of forewing pattern and leaf mine on Cornus controversa and C. kousa.
Phenology. In Japan in 2008-2016, larvae were observed from June to October. The overwintering form of this species is unknown.
Remarks. In the type series of P. indistincta, some specimens have a forewing pattern which differs in the following points: 1) a well-defined inner margin of lf (Figs 11C, F, 12C, E-G, K), 2) a fuscous to dark orange dorsal spot at dorsum 1/4 (Figs 11C, F, 12F), 3) tf is indistinct or interrupted in the middle (Figs 11C, 12B, C, G and I), i.e. more similar to that of the other Cornus-feeding Phyllocnistis. The forewing pattern differences were recorded in the type locality of this species, Menashi, Nara Prefecture (Figs 11D, E, 12A-C), in Ohshirakawa, Nagano Prefecture (Figs 11C, F, 12E) and in Nasu Imperial Villa, Tochigi Prefecture ( Fig. 12F; see also Arita et al. 2009: Pl. 3, Fig. 17). Judging from the genital characters, the differences in forewing pattern are treated as individual variation. The Indian and Japanese species, P. toparcha has a similar forewing pattern, with individual variation as P. indistincta, but P. toparcha is separated from P. indistincta by well-defined inner margin of strigulae and rather large lf. Accoding to Kuroko (1982), the autumn generation of P. toparcha has bolder strigulae and scattering fuscous to dark scales from base to dorsum 1/2 than the summer generation, but SK collected adult moths of P. toparcha having a dark dorsal spot or blotch and a darker patch in the lower half of the apical portion in summer (Kobayashi, unpublished data). Diagnosis. Forewing lustrous-white with a complete lf, three costal and four apical ciliary strigulae, tf interrupted; male genitalia with phallus shorter than phallobase; female corpus bursae with two signa, similar in size and shape.

Phyllocnistis verae
Forewing pattern of P. verae is distinguished from P. saepta and P. cornella by the interrupted tf. In male genitalia, length of the phallus and the phallobase is similar to P. indistincta, but with a higher number of ventral setae (42-50). In the female genitalia, the two signa are very similar in shape and size, while they are different in the other Cornus-feeding species.
Type material. Description of adult. (Figs 10A and B). Wing span 6.0-6.1 mm (6.1 mm in holotype). Head, thorax, legs and hindwing do not differ from the other Cornus-feeding species. Forewing lustrous white, subapical area orange with a small dark spot; lf welldefined on both sides; cilia white with tf always interrupted in the middle, three dark brown costal and four apical strigulae.
Female genitalia (Fig. 17A-C). The whole structure is similar to the other species. Bursa copulatrix with two flattened signa bearing usually a short median projection, both very similar in shape and size.
Pupa. Not studied. Biology. (Fig. 7). The mine is similar to that of other Phyllocnistis species: a very long serpentine subepidermal tunnel, slightly widening to the end, not intersecting itself (Fig. 7B, C, I). Black grains of frass form a rather wide central line (Fig. 7F). The mine is on the lower side of the leaf, often following secondary veins, crossing them closer to the leaf margin where veins are thinner (Fig. 7B-G). Young and late instar larvae are yellow (Fig. 7D-H). The tunnel ends upon the leaf margin or 10-15 mm away from it, where the mine slightly widens (Fig. 7I, J). Here, silk is deposited across the tunnel that causes contraction of this part of the mine, where pupation takes place (Fig. 7K-L).  Phenology. In 2015, by the 5th of July, when insect mines were found in nature, most larvae were at their final stage and some already had pupated. It suggests that larval development of the first generation may have started in late May. Thus, adults of the first generation can be on the wing in mid July. There are no records of the second generation. The overwintering stage remains unknown. Ecology and host plants. (Fig. 7A). In Central Siberia, the moth inhabits the forested areas. In Krasnoyarsk, mines were found on bushes growing along the river (Fig. 7A) Etymology. The name saepta is the past participle of the Latin verb saepio, that means "to block" and refers to the strigulae-shaped blotch present on the dorsum of the forewing. Diagnosis. Forewing lustrous white, lf well-defined, touching tf without interrupting it, a dorsal dark brown blotch, strigula-shaped, is present in the first third, while the inner margin of lf, apically, shows a hint of a dark line along the cell; male genitalia with a small number of ventral setae (14) and phallobase about twice the length of phallus.  P. saepta is distinguished from other Cornus-feeding species by the presence of a strigula-shaped blotch on the dorsum of the forewing. It also differs from P. cornella by a long lf, touching tf, from P. verae by tf not interrupted and from P. indistincta by well-defined lf. Description of adult. (Fig. 9F). Wing span 5.0 mm. Head. Like P. indistincta. Thorax. Tegulae and thorax white, legs not present. Forewing lustrous white, subapical area orange with a small apical black spot; lf well-defined, touching tf without interrupting it. A dorsal dark brown blotch, strigula-shaped, is present in the first third, while the inner margin of lf, apically, shows a hint of a dark line along the cell (Fig. 9G). Cilia white, tf not interrupted in the middle, three dark brown costal and four apical strigulae. Hindwing lustrous white.
Female genitalia. Unknown. Pupa. Not studied. Biology. (Fig. 8). The mine is similar to that of other Phyllocnistis species, on lower or upper side of the host leaf, often following secondary veins, crossing them closer to the leaf margin (Fig. 8B, C, E). Pupation takes place in the mine near leaf margin (Fig. 8D, F).
Phenology. In 2016, in China (Yunnan) late instar larvae were found at the end of July.
Ecology and host plants. (Fig. 8A). In China, the moth inhabits a forested area. Host plant is C. macrophylla.
Distribution. Only one location is known so far in China -Yunnan Province, Weixi.

Phyllocnistis cornella Ermolaev, 1987
Figs 10C-D, 15F, 17D-E Phyllocnistis cornella Ermolaev, 1987: 39-40;Seksyaeva, 1997: 429. Diagnosis. Forewing lustrous white, lf from base to 1/2, then bent inwards, not touching tf, inner margin indistinct basally; in female genitalia, bursa copulatrix with two signa usually similar in shape or smaller, with a more strongly curved median projection, the caudal signum up to twice as large as the central one.   P. cornella is very similar to P. saepta. It mainly differs in the forewing pattern, with lf not reaching tf. Hokkaido specimens show a similar forewing pattern. Unlike P. verae, tf is not interrupted. The male genitalia, drawn by Ermolaev, do not show any particular differential characters. Females are indistinguishable from P. indistincta, but differ from P. verae by size and shape of signa.
Abdomen. As in P. indistincta. Male genitalia (Fig. 15F). From the Ermolaev's figure, it is not possible to see any differential character. The authors did not have any male specimen from Hokkaido.
Female genitalia (Fig. 17D, E). Very similar to P. indistincta. Bursa copulatrix with two signa usually similar in shape, or the smaller with a more strongly curved median projection, the caudal signum up to twice as long as the central one (Fig. 14D, E); both signa have a spine-shaped median projection, about as long as the signum. On the wall around signa, minute scattered sclerites, thinner on the remainder of corpus bursae; ductus seminalis elongate, slightly longer than ductus bursae and arising from anterior end of corpus bursae.
Pupa. Not studied. No description was provided in Ermolaev (1987) and the authors were unable to preserve the pupa from the rearings of the specimens collected in Hokkaido.
Biology. Original description of mine: "Mine is green whitish without frass, more often on the lower side of the leaves of Cornus controversa" (Ermolaev 1987). In the present study, the larvae form a serpentine epidermal mine; frass line is pale brown to black. Two mines were found on the upper side of the leaf on C. controversa. The final instar larva spun a white cocoon at the leaf margin, the leaf margin slightly curled upwards by contraction of the cocoon silk.
Phenology. No data were provided in the original description of Ermolaev (1987). In Japan (Hokkaido), the pupa was found in late October; there are likely two generations annually. The overwintering form of this species is unknown.
Ecology and host plants. The host plant is Cornus controversa Hemsl. (Ermolaev 1987). In Japan, Hokkaido, the species has also been found on this plant.
Remarks. In his paper, Ermolaev (1987) indicated that the holotype was stored in the collection of the Zoological Institute of the Russian Academy of Sciences (ZIN RAS), Saint Petersburg, Russia. The curator at the time, who also collaborated with Ermolaev, Dr V.I. Kuznetzov, has unfortunately passed away. The current curator, Dr S.V. Baryshnikova (Seksyaeva) informed the authors that the holotype of P. cornella is not in the collection of ZIN RAS and has never been deposited there. As Ermolaev worked in Vladivostok, the authors tried to locate the specimens in the collections at the Biological and Soil Institute of the Far Eastern Branch of the Russian Academy of Sciences (present: Federal Scientific Center of the East Asia Terrestrial Biodiversity FEB RAS), but Dr M.G. Ponomarenko kindly informed the authors that there are no any specimens of P. cornella in the Institute's collection. Ermolaev was mostly active in the 1970s and 1980s and left science almost 30 years ago, together with all his collections (including P. cornella) and nobody knows if he is still living or where he might be.
In Figure 1 in the original description, Ermolaev (1987) draws three apical ciliary strigulae, but, in the text, he writes that there are four, which the authors find more likely to be corrrect. It is believed that the two female specimens that were sampled in Japan, Hokkaido correspond to P. cornella. Hokkaido is geographically close to Kunashir and has a similar flora and fauna and the specimens closely fit the original description of the species (Ermolaev 1987). As only two females were sampled and the type locality has not been explored, the neotype selection has been postponed until more material has been collected on Hokkaido and Kunashir Islands.

Discussion
Overall, 17 species of Phyllocnistis are presently known in the Asian part of Russia, China and Japan (Table 1). Amongst them, so far only one Phyllocnistis species, P. cornella, has been known to develop on plants of the family Cornaceae (Table 1). Here the authors confirm the existence of three other Cornaceae-feeding Phyllocnistis: P. indistincta (Japan), P. saepta (China) and P. verae (Russia), which were discovered during a DNA barcoding campaign for leaf-mining insects of Northeast Asia. The average interspecific divergence for the DNA barcode fragment found within the complex of Phyllocnistis spp. feeding on Cornus reaches 13.8%, which is comparable to other members of the Phyllocnistinae ( Table 2). The divergent split in COI was supported by the two sequenced nuclear genes H3 and 28S (except that, for P. indistincta and P. saepta, no difference was found in 28S). In addition to the high genetic divergence, morphological differences were found in wing pattern, male and female genitalia, allowing the authors to describe the three new species developing on Cornus in Northeast Asia and distinguish them from the previously known P. cornella.
Endosymbiotic bacteria like Wolbachia may manipulate host reproduction and significantly affect mitochondrial divergence within insect species (Kodandaramaiah et al. 2013, Ritter et al. 2013. In this case, deep mitochondrial splits observed in Cornus-feeding Phyllocnistis species were not associated with Wolbachia infections, since amongst the 14 specimens of the four Phyllocnistis species, only one specimen (i.e. P. indistincta, MICRU069-16; Honshu, Japan) showed evidence of Wolbachia infection (Suppl. material 2). Such mitochondrial divergences could be the result of geographic Phyllocnistis species develop on different Cornus species: P. indistincta on C. controversa, C. kousa, C. macrophylla (native to Japan) and on C. florida (introduced in Japan from North America), P. saepta on C. macrophylla (native to China) and P. verae on C. alba (native to Siberia). The related species P. cornella was described from the Russian Far East from Cornus controversa. The above-mentioned Cornus species correspond to four subgenera: Kraniopsis (C. alba and C. macrophylla), Mesomora (C. controversa), Syncarpea (C. kousa) and Cynoxylon (C. florida) (Xiang et al. 2006). The related nature of host plants may partly explain the phylogenetic proximity of some Cornus-feeding Phyllocnistis species. Amongst four Phyllocnistis species, the species from China and Russia are the closest neighbours on the COI tree (minimum intraspecific distance 6.1%) and they both feed on plants from the same subgenus, Kraniopsis. Such a relationship is, however, not evident for the other two Cornusfeeding Phyllocnistis species from Japan.
Dogwoods are widely used for ornamental purposes in gardens and landscaping and their distribution is ubiquitous in temperate Eurasia. The North American C. florida is planted in Japan as an ornamental tree. Cornus alba, that is native in Russia, Mongolia, Northeast China, Manchuria and North Korea, has been introduced in Europe (Drake 2009). During the course of the study, the authors did not observe dense populations of Phyllocnistis on Cornus in the surveyed locations and, therefore, it is unclear whether or not they may impact their hosts in urban ecosystems. Abundant populations of another leaf mining moth, Antispila sinensis Liu & Wang, 2017 (Heliozelidae), recently described from Cornus walteri Wangerin in China, noticeably damaged the foliage of shade trees in Chinese city parks (Liu and Wang 2017).
[−] no data because a single specimen was sequenced.
In addition to the three new species of Phyllocnistis discovered on Cornus in Northeast Asia, the analysis of DNA barcodes allowed the detection of a further four putative new species of Phyllocnistis in this region, though on plant families other than Cornaceae (Table 1, Suppl. material 1). One species (mentioned in the text as Phyllocnistis sp. 1) was found in the Russian Far East, Primorskiy Kray on Salix (Salicaceae) (N. Kirichenko leg.), while the other three were sampled as single individuals in Japan: Phyllocnistis sp. 2 on Parthenocissus (Vitaceae) (E.J. van Nieukerken leg.), Phyllocnistis sp. 3 on Derris (Fabaceae) and Phyllocnistis sp. 4 on Ligustrum (Oleaceae) (A. Kawakita leg.). The putative new species on Ligustrum represents the first record of Phyllocnistis feeding on Oleaceae (Table 1). Additional specimens will be needed for the formal description of these species.