DNA Barcoding of the Palaearctic Elfin Butterflies (Lepidoptera, Lycaenidae) with a Description of Four New Species from Vietnam

Simple Summary We studied the Palaearctic elfin butterflies, a group of hairstreaks (family Lycaenidae, subfamily Theclinae, tribe Eumaeini) comprising about 50 species belonging to three genera, Ahlbergia Bryk, 1947, Cissatsuma Johnson, 1992, and Novosatsuma Johnson, 1992, known mostly from China. Members of this group are still underexplored, and the taxonomy of the Palaearctic elfin butterflies is complicated both at the species and the generic level. We performed an analysis based on a 658 bp region of the COI mitochondrial gene, covering the previously considered genera Ahlbergia, Cissatsuma, Novosatsuma and Callophrys Billberg, 1820, in order to properly discriminate the Palaearctic elfin butterfly species and reveal their phylogenetic positions. The phylogenetic analysis revealed Holarctic Callophrys sensu lato as a strongly supported monophyletic group, but none of the usually treated Palaearctic elfin butterfly genera was recovered as a monophyletic unit. Additionally, we revealed and described four new species of Palaearctic elfin butterflies from Northeast Vietnam. Abstract Phylogenetic analysis is provided for the first time for 12 species of Palaearctic elfin butterflies, members of the previously recognized genera Ahlbergia Bryk, 1947, Cissatsuma Johnson, 1992, and Novosatsuma Johnson, 1992, based on the barcoding region of the mitochondrial cytochrome C oxidase subunit I gene (COI). Comparison of the COI barcodes revealed very low levels of genetic divergence between the species of the Palaearctic elfin butterflies and Callophrys Billberg, 1820 sensu stricto. COI-based phylogeny revealed that Palaearctic Callophrys and the Palaearctic elfin butterflies, except Cissatsuma, are polyphyletic. Four new sympatric species, namely, Callophrys (Ahlbergia) hmong sp. n., C. (A.) tay sp. n., Callophrys (Cissatsuma) devyatkini sp. n. and C. (A.) dao sp. n. are described from Ha Giang Province, North Vietnam, based on wing colouration, the morphologies of the male and female genitalia, and differences in COI sequences. Discovery of the new species expands the distribution range of the group towards the southeast, beyond the Palaearctic region.


Introduction
The term "Palaearctic elfin butterflies" is traditionally used for three genera of the Eumaeini hairstreaks, Ahlbergia Bryk, 1947, Cissatsuma Johnson, 1992, andNovosatsuma Johnson, 1992, inhabiting mountains of East Asia, with most species in China, mainly species and reveal their phylogenetic positions, we used an integrative taxonomic approach combining a COI barcoding-based molecular phylogenetic analysis and an analysis of morphological characters.
In this paper, we (1) provide for the first time a hypothesis of the phylogeny of the Palaearctic elfin butterflies based on a 658 bp region of the COI mitochondrial gene, covering the previously distinguished genera Ahlbergia, Cissatsuma, Novosatsuma and Callophrys; (2) describe the revealed new species on the basis of COI barcoding data and analysis of morphology; and (3) discuss the taxonomy of the Palaearctic elfin butterflies.

Nomenclature Used in This Study
In the present work, we follow the concept of a diverse Holarctic genus Callophrys [17][18][19] and for taxonomic purposes tentatively treat Callophrys sensu stricto, Ahlbergia, Cissatsuma and Novosatsuma as subgenera of the genus Callophrys, leaving all the combinations previously used within this group [13] at subgeneric level to avoid taxonomic confusions and to make a framework for future studies.

Sampling, DNA Extraction, PCR and Sequencing
Our ingroup dataset included 12 species of all previously recognised genera of the Palaearctic elfin butterflies, namely, Ahlbergia, Novosatsuma and Cissatsuma. Eleven of them (37 specimens) were sequenced during this study (Table 1), and three COI sequences (OL457026, MW785858 and MW785859) of Callophrys (Ahlbergia) frivaldszkyi were obtained from GenBank. Table 1. Specimens of the genus Callophrys studied in the current research and their COI GenBank accession numbers. Specimens with genitalia dissected are marked with asterisks. HT, holotype; PT, paratype; n/a, not available. GenBank accession numbers of the specimens obtained in the current study are in bold. Additionally, the COI sequences of the genus Callophrys (sensu lato) available from GenBank were included in the phylogenetic analysis: two Palaearctic Callophrys species, namely, Callophrys (Callophrys) rubi and C. (Rhymnaria) baidula Zhdanko, 2000 was used as the outgroup. Thus, the final dataset for the phylogenetic analysis included 53 specimens.

Code
One leg of each specimen was taken for DNA extraction using a QIAamp DNA Investigator Kit (Qiagen, Venlo, The Netherlands), following the manufacturer's protocol. The mitochondrial DNA barcode (a 658 bp fragment of the COI gene) was amplified using LCO1490/HCO2198 [32] and LepF/LepR primer pairs [33]. In case standard lepidopteran barcode primers failed to yield a sufficient product, we amplified full-length barcode fragments using the primer pair combinations LepF/MH-MR1 + MH-MF1/LepR and LCO1490/MH-MR1 + MH-MF1/HCO2198 [34]. For specimens collected a long time ago, self-designed primer pairs, amplifying six short overlapping fragments, were used to obtain targeted fragments. The primers used in this study are listed in Table 2.

Sequence Processing and Phylogenetic Analysis
The sequences were aligned with the MUSCLE algorithm implemented in GENEIOUS v.7.1.9 [35]. Phylogenetic analyses were performed using the Bayesian inference (BI) approach. The Bayesian estimation of posterior probability was performed in MrBayes v.3.2.5 [36], applying the GTR + G + I evolutionary model, as suggested by PartitionFinder v.2.1.1 [37]. Markov chains were sampled at intervals of 500 generations. Two runs of ten million generations with four chains (one cold and three heated) were performed. The first 25% of sampled trees were discarded as burn-in. We regarded tree nodes with BI posterior probabilities (PPs) > 0.95 to be sufficiently resolved a priori.

Morphology and Distribution
Morphological characters of 44 specimens of the Palaearctic elfin butterflies representing 12 species previously delimitated on the basis of morphology (including four new ones and one of uncertain status, C. (A.) sp.) of the previously recognised genera Ahlbergia, Cissatsuma and Novosatsuma are studied within this work.
The characters used for the species delimitation of the Palaearctic elfin butterflies were selected following Johnson [1], Huang and Zhou [11] and Huang and Zhu [13]. The nomenclature for the genitalia and wing patterns was adapted after Johnson [1] and Krupitsky [15]. The nervuration nomenclature follows the Comstock-Needham system adapted for butterflies [39]. For the examination of the male genitalia, the abdomens of the studied specimens were removed and macerated in 10% KOH. After cleaning in water and dehydration in 96% EtOH, a genital capsule with valvae and separated aedeagus were placed in a drop of glycerol, covered with a cover glass and photographed. In the case of the genital capsule, photos were taken in ventral and lateral views, and in lateral view in the case of the aedeagus.
The images of the studied specimens were taken with a Canon EOS 5D mark II digital camera (Canon Inc., Tokyo, Japan) equipped with a Sigma 150 mm f2.8 lens (Sigma Corporation, Kawasaki, Japan), using an originally developed light system and a Canon Speedlight 430 EX flash (Canon Inc., Tokyo, Japan) with a diffuser. The images of the genitalia were taken with a Canon EOS 6D digital camera (Canon Inc., Tokyo, Japan) equipped with a Canon MP-E 65 mm f/2.8 lens (Canon Inc., Tokyo, Japan), using two Micromed Dual Goose illuminators (Micromed, St. Petersburg, Russia). Obtained images were edited using Adobe Photoshop CC 2014.2.2 software.
The distribution map was generated using SimpleMappr (http://www.simplemappr. net, accessed on 31 August 2022) on the basis of the literature [12][13][14]23,40] and label data for the studied specimens and was edited using Adobe Photoshop CC 2014.

Phylogenetic Relationships
The phylogenetic analysis revealed Holarctic Callophrys sensu lato as a strongly supported monophyletic group ( Figure 1). The Palaearctic clade comprising elfin butterflies and Callophrys sensu stricto was also recovered with high support, with C. (Cisincisalia) johnsoni as a sister species. Within the Palaearctic clade, all recovered lineages received PPs ≥ 0.96. The genera Callophrys, Ahlbergia and Novosatsuma were recognised as polyphyletic groups.  Two deep-branching lineages within the Palaearctic clade were recovered on the phylogenetic tree. The first lineage comprises Callophrys avis and four Ahlbergia species, including the type species of the genus, A. ferrea (Butler, 1866). Three new species tentatively classified as members of the subgenus Ahlbergia based on the analysis of morphology belong to this clade. One of them (described below as C.  (Table 3).    [14]), and rather long narrow male genitalia with distal part of the valva with sides parallel and then gradually tapering to apex (vs. very short and broad male genitalia with distal part of valva gradually tapering to apex, cf. Figure 8 in [14]).

Taxonomy
Additionally, C. (A.) hmong sp. n. is somewhat similar to C. (A.) haradai Igarashi, 1973, comb. nov., known from the holotype "female" (male, in fact, as evidenced by the androconial spot and the shape of the abdomen depicted in the photo included with the original description) from Nepal [40] and a couple of photos in nature [41]. Based on limited diagnostic information from the mentioned images of C. (A.) haradai, it can be concluded that the new species differs from the latter in the less developed blue field of the male forewing limited by the postdiscal area (vs. blue field reaching the submarginal area in C. (A.) haradai, cf. Figure 1 in [40]) and the less contrasted beige disc (vs. darkened disc in C. (A.) haradai, cf. Figure 2 in [40] Despite the COI barcode shared with C. (A.) tay sp. n., such characters as the rounded hindwing nearly entirely covered with blue scales and inwardly directed rounded anal Thorax: dark brown with bluish-grey hairs dorsally, densely covered with brown and rusty-brown hairs ventrally; femur dark brown, tibia and tarsus whitish with dark-brown stripes.
Abdomen: dark brown with whitish hairs dorsally, whitish ventrally, tip brown. Forewing: triangular, with rounded apex and termen. Forewing length 14.0 mm in holotype, 11.0-15.0 mm in paratypes. Dorsal side black, cell and discal area in spaces M3-2A densely covered with celestial-blue scales; veins dark brown; base of costal area with suffusion of rusty-brown scales; outer margin same as background; fringe wavy, convex at veins, dark brown with admixture of beige scales; basal area of wing and inner margin covered with rather long whitish hairs, tornus with long dark-brown scales; androconial spot leaf-like, dark brown to beige, length about 2.0 mm. Ventral side brown, densely covered with whitish scales, base and costa with rusty-brown scales; postmedial line wavy, dark brown with white scales proximally and rusty-brown scales distally; spaces R3-CuA1 with crescent line consisting of dark-brown elements with groups of whitish scales distally; submarginal area with brown scales; outer margin brown; fringe as on dorsal side.
Hindwing: costa straight, with basal lobe, apex rounded, termen wavy; anal lobe well-developed, rounded. Dorsal side black with broad celestial-blue field in spaces Rs-CuA2 extending to submarginal area in spaces M3-CuA2, with V-shaped black incisions separating it from margin; veins black; spaces 2A-3A greyish blue; outer margin black, bordered by blue scales, more intensively in spaces M3-CuA2, and with whitish scales in half of space CuA2 and 2A near anal lobe; basal half of wing and inner margin covered with long sparse whitish hairs; fringe dark brown proximally, whitish distally, with long dark-brown scales at veins; anal lobe with long rusty-brown scales. Ventral side with brown base, postbasal marks dark brown with whitish scales proximally, developed in space Sc + R1 and cell, connected with marginal band of disc in space CuA2; marginal band of disc consisting of dark-brown broken lines, bold in spaces Sc + R1, Rs, CuA2-3A, with projection in space M3; space between postbasal marks and marginal band of disc covered with greyish scales; crescent line black, developed in spaces M3-2A, traced in other spaces; limbal area with mixture of whitish and dark-brown scales; wing between crescent line and limbal zone whitish in Sc + R1-M3, and with beige scales and bold rusty-brown lunules in spaces CuA1-2A.
Male genitalia ( Figure 3A). Falx stout; lobes of uncus rather long, with well-developed inner processes; genital capsula elongated; vinculum broadened, with well-developed inner lobes; valva covered with hairs of different lengths, robust, with very broad ovoid basal part reaching vinculum and abruptly turning into distal part as long as 1.5 lengths of basal part; proximal half of distal part of valva with nearly parallel sides; distal half of valva in outer edge tapering to rounded apex; in bilobed configuration of valvae, their distal parts nearly gradually tapering after medial flexure; valvae strongly jointed, with small groove between apices; saccus triangular with rounded apex, 1/4 as long as genitalia; aedeagus rather slender, arcuate, about 1.8 times as long as genital capsule, with serrated slightly deflected cornuti.
Female ( Figure 2B). Generally similar to male but blue field broader, ventral side lighter and less contrasted. Forewing length 16.0 mm.
Female genitalia ( Figure 4A). Lamella postvaginalis very long and broad, semicircular; anterior edge with small depression; lamella antevaginalis small, membranous; junctures of lamellae well-developed; ductus bursae with slightly broadened antrum, two times as long as lamella postvaginalis, slightly contracting towards bursa; corpus bursae with two very small two-spined signi.
Etymology. The new species is named after the Hmong people, the largest ethnic group of Ha Giang Province. The type locality of the new species is situated near Sa Phin commune, which was the site of the residence of the Hmong kings.

Description
Male ( Figure 2C,E). Head: antenna black, white-ringed at bases of segments, antennal club black; eye surrounded with white stripe, dark brown with very short sparse hairs; head dark brown, with white scales and hairs; palpus: palpomere 2 black with whitish hairs and scales on outside, white inside, palpomere 3 black.
Thorax: dark brown with blueish-grey hairs dorsally, densely covered with grey hairs ventrally; femur dark brown, tibia and tarsus black with white stripes dorsally, whitish ventrally.
Abdomen: dark brown with grey scales and hairs dorsally, whitish ventrally. Forewing: triangular, with pointed apex and rounded termen. Forewing length 12.0 mm in holotype, 12.0-14.0 mm in paratypes. Dorsal side dark brown, cell and discal area in spaces M3-2A densely covered with dark-blue scales; veins dark brown; outer margin coloured as background; fringe wavy, convex at veins, dark brown with admixture of whitish scales; basal area of wing and inner margin covered with rather long whitish hairs, tornus with long dark-brown scales; androconial spot narrow, lanceolate, brown to dark brown, length about 2.0-2.5 mm. Ventral side brown to greyish brown, densely covered with grey scales, base and costa dark brown; postmedial line wavy, dark brown with grey scales distally; crescent line more or less developed in spaces R3-CuA1, consisting of dark-brown elements; submarginal area with grey scales; outer margin dark brown; fringe as on dorsal side.
Hindwing: costa nearly straight, apex rounded, termen wavy, anal lobe well-developed, rounded. Dorsal side dark brown with broad dark-blue field in spaces Rs-CuA2 extending to postdiscal or submarginal area in spaces M3-CuA2; veins black; spaces 2A-3A; outer margin black, bordered by blue scales, more intensively in spaces M3-CuA2, and with grey scales in half of space CuA2 and 2A near anal lobe; basal half of wing and inner margin covered with long, sparse, whitish hairs; fringe dark brown proximally, whitish distally, with long black scales at veins; anal lobe with long black scales. Ventral side with dark-brown base and grey spots near postbasal marks, postbasal marks and marginal band of disc bold, black, discal band strongly darkened, especially in spaces CuA2-3A; postdiscal band grey, varying from light grey to grey-brown; crescent line black with grey scales, usually well-developed in spaces M3-2A, traced in other spaces; limbal area with mixture of whitish and dark-brown scales; wing between crescent line and limbal zone as postdiscal band in Sc + R1-M3, and with darker scales and bold brown lunules in spaces CuA1-2A, anal lobe with large brown spot; fringe as on dorsal side.
Male genitalia ( Figure 3B). Falx rather narrow, pointed; lobes of uncus rather short; genital capsule rather compact; vinculum broadened, with well-developed inner lobes reaching distal part of valva; valva covered with hairs of different length, narrow, with slightly broadened basal part barely reaching vinculum and distal part 1.5 times as long as basal part; in bilobed configuration of valvae, their distal parts nearly gradually tapering after small medial flexure, apices of valvae disjoined; saccus triangular with rounded apex, as long as 1/5 of genitalia length; aedeagus rather slender, slightly curved mesially, about 1.5 times as long as length of genital capsule, with small serrated slightly deflected cornuti.
Female ( Figure 2D). Generally similar to male but blue field broader and ventral colouration lighter, ground colour light grey, discal band of disc and limbal area grey. Thorax: brown with whitish hairs dorsally, densely covered with rusty-brown and whitish hairs ventrally; legs brown with light-brown scales.
Abdomen: brown with whitish hairs dorsally, whitish ventrally, tip rusty brown. Forewing: triangular, with pointed apex and rounded convexity at cells M3-CuA1. Forewing length 15.0 mm. Dorsal side dark brown; basal, postbasal and discal area except costal zone covered with blue scales; outer margin same as background; fringe dark brown, with admixture of rusty-brown scales, wavy, with projections at veins; basal area of wing and inner margin covered with rather long whitish hairs. Ventral side motley; cell and spaces R1-CuA1 rusty brown before postmedial line, brown in other spaces; postmedial line wavy, rusty brown with whitish scales distally; marginal area with traces of dark-brown crescent line surrounded with whitish scales; wing covered with whitish and light-brown scales, more densely near costa and after postmedial line; fringe as on dorsal side.
Hindwing: costa straight, apex and termen rounded. Dorsal side dark brown with large steel-blue field and diffused blue scales mostly limited by postdiscal area and penetrated by dark-brown veins with large blue field mostly limited by postdiscal area and penetrated by dark-brown veins, spaces 2A-3A light brown with admixture of greyish-blue scales, outer margin dark brown bordered by blue scales, most intensively in spaces CuA1-2A; basal half of wing and inner margin covered with long sparse whitish hairs; apical and anal lobes well-developed, prominent, rusty brown; fringe wavy, dark-brown and rusty proximally, whitish distally, dark at convexities near veins.
Ventral side: base rusty brown; postbasal marks well-developed, wavy, dark brown, with beige scales proximally; marginal band of disc strongly uneven, rusty brown, consisting of blurred spot in spaces Sc + R1 and Rs, two parallel straight brown lines in spaces M1-M2, projection in space M3 and bold wavy line in spaces CuA1-2A; space between postbasal and marginal band of disc brown with beige scales; postdiscal area motley, with beige, whitish, brown and rusty-brown scales; crescent line uneven, consisting of groups of brown and rusty-brown scales; submarginal (limbal) area dark brown with whitish scales, separated from crescent line by beige and rusty-brown scales; anal lobe with rusty-brown spot; outer margin dark brown; fringe as on dorsal side; most of wing covered with long whitish and greyish hairs.
Female genitalia ( Figure 4C). Lamella postvaginalis long and broad, trapezoid with rounded corners and two lateral convolutions; anterior edge with large semicircular de-pression; lamella antevaginalis small, membranous; junctures of lamellae well-developed; ductus bursae rather stout and long, three times as long as lamella postvaginalis, with slightly broadened antrum and base; corpus bursae with two small unispined signi.
Male ( Figure 2F). Forewing length 12.0-14.0 mm. Generally similar to female, but blue field narrower, especially on forewing, cell blue-grey with admixture of blue scales, discal area in spaces CuA2-2A with blue scales; veins dark brown; costal area with suffusion of beige scales, more intensive near base. Androconial spot very thin, lanceolate, brown, length about 2.0 mm. Hindwing with large steel-blue field and diffused blue scales mostly limited by postdiscal area and penetrated by dark-brown veins. In one specimen, spaces M1-M2 with one straight brown line.
Male genitalia ( Figure 3C). Falx stout; lobes of uncus broad, rather short; genital capsula rather large; valva covered with hairs of different lengths, robust, with ovoid basal part abruptly turning into distal part 1.5 times as long as basal part; in bilobed configuration of valvae, their distal parts nearly gradually tapering after medial flexure, apices of valvae broadly disjointed; saccus triangular with thin oblong apex, 1/4 times as long as genitalia length; aedeagus rather stout, arcuate, about two times as long as genital capsule, with serrated cornuti; lower cornutus strongly deflected.
Etymology Description Female ( Figure 2H). Head: antenna broken off; eye surrounded with white stripe, dark brown with very short sparse hairs; head dark brown, with white scales and hairs; palpus: palpomere 2 black with white hairs and scales, palpomere 3 black.
Thorax: brown with blueish-grey hairs dorsally, densely covered with grey hairs ventrally; leg rusty brown with whitish scales.
Abdomen: brown with grey scales and hairs dorsally, whitish ventrally. Forewing: triangular, with rounded termen and apex; forewing length 14.0 mm. Dorsal side dark brown, cell and discal area in spaces M3-2A densely covered with light-blue scales; veins dark brown; outer margin same as background; fringe slightly wavy, convex at veins, dark brown with admixture of whitish scales; basal area of wing and inner margin covered with rather long whitish hairs, tornus with long dark-brown scales. Ventral side rusty brown, darker at base, with whitish scales along inner margin; cell marking brown; postmedial line wavy, rusty brown with scarce whitish scales distally; crescent line absent; submarginal area with dark-brown scales and whitish scales in CuA2-2A; outer margin rusty brown; fringe as on dorsal side.
Hindwing: costal area with small basal lobe, nearly straight, apex rounded, termen wavy, anal lobe well-developed, rounded. Dorsal side grey-brown with very broad lightblue field covering nearly entire wing surface except postdiscal area in cells Sc + R1 and submarginal area in cells Rs-M1 covered with grey-brown scales, cells 2A-3A covered with blueish-grey scales; veins grey-brown; outer margin black, bordered by blue scales, more intensively in spaces M3-CuA2, and with grey-blue scales in half of space CuA2 and 2A near anal lobe; basal half of wing and inner margin covered with long sparse whitish hairs; fringe dark-brown proximally, whitish distally, with long black scales at veins; anal lobe with long black scales. Ventral side with dark rusty-brown base, rusty brown near postbasal marks, postbasal marks blurred, brown, discal band darkened, with whitish scales in space Sc + R1, with rusty-brown, brown and dark-brown scales in other spaces; marginal band of disc strongly uneven, consisting of blurred dark-brown spots in spaces Sc + R1-M2, strong projection in space M3 and bold wavy dark-brown line in spaces CuA1-2A; postdiscal band rusty brown with whitish scales in spaces 2A-3A; crescent line blurred, dark brown, well-developed in spaces M3-2A, traced in other spaces; limbal area with dark-brown scales and admixture of whitish scales; wing between crescent line and limbal area rusty brown; anal lobe with rusty-brown spot; outer margin black; fringe as on dorsal side; basal half of wing and inner margin covered with short light hairs.
Female genitalia ( Figure 4D). Lamella postvaginalis broad, trapezoid with rounded corners; lamella antevaginalis small, membranous; junctures of lamellae small; ductus bursae with slightly broadened antrum, about two times as long as lamella postvaginalis, straight, gradually broadened to base; corpus bursae with two very small two-spined signi.
Etymology. The new species is named after the Dao people, the third-largest ethnic group of Ha Giang Province.
Distribution. The new species is known only from the type locality in Dong Van Karst Plateau in Northeastern Vietnam, Ha Giang Province, about 1500 km south from the somewhat morphologically similar C. (A.) liyufeii described from Baoji City, Shaanxi Province, China.

Discussion
Northeastern Vietnam (including Ha Giang Province, where the type locality of all four species described in this paper is situated) east of the Red River, a major biogeographical boundary, is known as a biodiversity hotspot. It hosts a number of endemic species of butterflies, including Lycaenidae, or species endemic to a region common for the mountains of Northeastern Vietnam and Guangxi Province in China [28,42]. The discovery of four new species of the Palaearctic elfin butterflies in Vietnam is important in terms of biogeography: it expands the range of the group towards the southeast, beyond the Palaearctic region, and clarifies some aspects of its distribution ( Figure 5).
According to the morphological characters mentioned in the diagnosis and our phylogenetic analysis, C. (A.) hmong sp. n. is closely related to the Chinese species C. (A.) clarolinea and C. (A.) bijieensis. It is geographically isolated from both species by the rather low eastern part of the Yunnan-Guizhou Plateau, which is characterised by a mostly subtropical climate unsuitable for such temperate insects as elfin butterflies. C. (A.) hmong sp. n. or a closely related new species also can be found in the mountains of Laos and Northern Thailand, e.g., the elfin butterfly specimen from Chiang Mai mentioned by Ek-Amnuay [24] as "Callophrys sp." shares some diagnostic characters of the species in question and probably belongs to an undescribed species. Unlike the latter case, the results of our study do not reject the concept of Cissatsuma sensu Johnson [1], but this clade appears polyphyletic, as the morphologically different C. (A.) frivaldszkyi is united with the Cissatsuma species in our analysis. The type species of Cissatsuma, C. (Cissatsuma) albilinea (Riley, 1939), comb. nov., is rare in collections and was inaccessible for the phylogenetic analysis, but we analysed the recently described morphologically close species C. (C.) berezowskii.
In the case of C. (C.) devyatkini sp. n., its placement in a clade together with C. (C.) berezowskii is supported by the morphologies of the male and female genitalia. The genitalia of both species, as well as those of C. (C.) zhoujingshuae, which are somewhat similar to those of C. (C.) devyatkini sp. n., fit the diagnosis of Cissatsuma by Johnson [1]: the valvae in ventral view with straight or concaved lateral margins in most parts, except at the apex, with the ductus bursae robust and relatively wider and longer than in Ahlbergia. As in the case of C. Although the COI-based phylogenetic analysis does not resolve relations of all involved groups, it reveals that the genus Callophrys sensu stricto is also polyphyletic. The polyphyly of the Palaearctic Callophrys sensu stricto was demonstrated earlier by ten Hagen and Miller [19]. In their neighbour-joining tree, C.  Callophrys (Ahlbergia) tay sp. n. shares some details of the colouration with C. (N.) monstrabila, the type species of the genus Novosatsuma sensu Johnson [1], known with certainty from the sole holotype from the Naga Hills on the border of India and Myanmar. Such an affinity is peculiar in terms of biogeography, as the Naga Hills and the type locality of the latter new species in Ha Giang Province are separated by a distance of over 1000 km. In its turn, the darkened basal disc, the light spot in cell 2A and the shape of the valva in the male genitalia make C.  [11], the difference in the male genitalia between Ahlbergia and Novosatsuma is not clearly marked, and the differences in the female genitalia are overvalued. A robust ductus bursae with a welldefined fluted antrum is the most important diagnostic character of the genus Novosatsuma sensu Johnson [1], but this character was not based on the type species of the genus (as it was known only from the male holotype). The female genitalia of C. (A.) tay sp. n. do not possess any diagnostic characters at the generic or subgeneric level and do not fit in with Johnson's concept of Novosatsuma, so the status of this taxon remains questionable and requires further study with more species involved in the phylogenetic analysis.
Unlike the latter case, the results of our study do not reject the concept of Cissatsuma sensu Johnson [1], but this clade appears polyphyletic, as the morphologically different C. (A.) frivaldszkyi is united with the Cissatsuma species in our analysis. The type species of Cissatsuma, C. (Cissatsuma) albilinea (Riley, 1939), comb. nov., is rare in collections and was inaccessible for the phylogenetic analysis, but we analysed the recently described morphologically close species C. (C.) berezowskii.
In the case of C. (C.) devyatkini sp. n., its placement in a clade together with C. (C.) berezowskii is supported by the morphologies of the male and female genitalia. The genitalia of both species, as well as those of C. (C.) zhoujingshuae, which are somewhat similar to those of C. (C.) devyatkini sp. n., fit the diagnosis of Cissatsuma by Johnson [1]: the valvae in ventral view with straight or concaved lateral margins in most parts, except at the apex, with the ductus bursae robust and relatively wider and longer than in Ahlbergia. As in the case of C. (A.) hmong sp. n., the new Cissatsuma species is isolated from C. (C.) zhoujingshuae by the eastern part of the Yunnan-Guizhou Plateau.
According to the morphological characters, C. (A.) dao sp. n. belongs to the C. (A.) leechi species group. Based on the pattern of the dorsal side of the hindwing, it is related to C. (A.) liyufeii from Shaanxi Province, China, from which it is geographically isolated by the rather low eastern part of the Yunnan-Guizhou Plateau.
Although the COI-based phylogenetic analysis does not resolve relations of all involved groups, it reveals that the genus Callophrys sensu stricto is also polyphyletic. The polyphyly of the Palaearctic Callophrys sensu stricto was demonstrated earlier by ten Hagen and Miller [19]. In their neighbour-joining tree, C. (Callophrys) avis was a sister species to C. (Cissatsuma) tuba, and C. (A.) frivaldszkyi was included in the C. (Callophrys) rubi clade. Surprisingly, in our reconstruction, C. (C.) avis is united with the type species of Ahlbergia, C. (A.) ferrea. This result can be explained by the connected range of the ancestor of the group in the past. The same example is known in the tribe Theclini of the subfamily Theclinae: the monotypic Mediterranean genus Laesopis Rambur, 1858 is morphologically similar to the East Asian genus Artopoetes Chapman, 1909 [43]. Despite the generally similar colouration and wing pattern, the polyphyly of the genus Callophrys sensu stricto is also supported by the morphologies of the genitalia of C. (C.) avis, C. (C.) mystaphia and C. (C.) mystaphioides, which are characterised by the complicated valva structure in the male genitalia and the very simple lamella postvaginalis appearing similar to those of the Palaearctic elfin butterflies and some Nearctic Callophrys [44,45]. As the recent genome-wide phylogenetic analysis of the Nearctic Callophrys has shown, a similar wing pattern can be implemented in unrelated clades of this group independently [18], such as the green colouration of the Palaearctic Callophrys sensu lato and species of the recently established Nearctic subgenus Greenie Grishin, 2021.
Values of the interspecific uncorrected pairwise genetic divergences (p-distances) of the studied Callophrys (sensu lato) species ranging from 0.15% to 2.72% are less than the species threshold of about 3% empirically found for Lepidoptera in general [46]. In the subfamily Theclinae, shared or very close COI mitochondrial barcodes in morphologically different species were also found in the Palaearctic Callophrys sensu lato [19], as well as in the Tomares hairstreaks [47]. This pattern can be explained by incomplete lineage sorting or hybridisation leading to mitochondrial introgression, which occurs occasionally in Lycaenidae [48][49][50], including the genus Callophrys sensu lato [22]. The mitochondrial introgression from C. (A.) tay sp. n. to C. (A.) dao sp. n. belonging to the morphologically distinct C. (A.) leechi species group is an explanation of the shared COI mitochondrial barcode.
Another possible explanation of the shared or very close mitochondrial haplotypes within the studied group is an influence of the rickettsial bacterium Wolbachia Hertig, 1936, an endosymbiont associated with mitochondria and thus maternally inherited. Wolbachia can cause selective sweeps in mitochondrial haplotypes owing to genetic hitchhiking, leading to mitochondrial introgression and reduced mitochondrial diversity [51].

Conclusions
Our results demonstrate a need for a complex analysis of genetic markers and morphology for the delimitation and taxonomic rearrangement of the Palaearctic elfin butterflies and Callophrys. The final taxonomic solution of the subgeneric classification should be based on a multilocus molecular phylogenetic analysis covering all the species groups of the Palaearctic Callophrys sensu lato and provide a robust phylogenetic scheme for this genus.