Integrative taxonomic study reveals a new species of Maculolachnus (Hemiptera: Aphididae) from South Korea

Abstract

The genus Maculolachnus Gaumont, 1920 (Hemiptera: Aphididae: Lachninae) exhibits substantial hidden diversity, yet its life history remains poorly understood. Here, we present an integrative taxonomic study of Maculolachnus in South Korea, revealing a previously unrecognized species closely related to M. submacula and providing the first biological insights into the group. We combined detailed morphological examinations, mitochondrial COI sequence analyses, and field observations of colony structure and phenology to clarify species boundaries and elucidate life history traits. Populations formerly identified as M. submacula exhibited distinct morphological characters and significant COI divergence. Phylogenetic reconstructions (BI, ML, NJ), species delimitation methods (ABGD, ASAP, bPTP), and haplotype network analyses consistently supported four distinct species: M. submacula, M. sijpkensi, M. paiki, and a new species described herein as Maculolachnus koreanus sp. nov. The COI distance between M. koreanus sp. nov. and M. submacula exceeded 2.4%, surpassing the typical aphid barcode gap. We redescribe the rare alate viviparous female of M. paiki, which is endemic to South Korea. Observations on M. koreanus sp. nov. revealed ant attendance and seasonal reproductive strategies. Scanning electron microscopy (SEM) provided the first detailed characterization of sensorial structures. This study highlights the value of integrative taxonomy in revealing hidden diversity within Lachninae.

International Commission on Zoological Nomenclature (ICZN). International Code of ZoologicalNomenclature 4th edn, i–xxix, 1–306 (The International Trust for Zoological Nomenclature,1999). urn:lsid:zoobank.org:pub:F4BF9295-FF83-434C-9A29-74FDB7A64A9E

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-40274-3.

Introduction

Hidden diversity and fragmentary biological knowledge have long hindered a comprehensive understanding of aphid systematics and evolution, resulting in persistent taxonomic ambiguity across several lineages¹. The genus Maculolachnus Gaumont, 1920 (Hemiptera: Aphididae: Lachninae) exemplifies this issue, representing a taxonomically obscure lineage of large, bark-inhabiting aphids that feed exclusively on woody hosts within the family Rosaceae^2,3. Despite their distinctive external morphology and restricted host range, Maculolachnus species remain among the least investigated groups of Lachninae⁴. Their life histories, reproductive modes, and seasonal phenologies are almost entirely undocumented, and most available records are limited to apterous viviparous females collected from Rosaceae plants³. Consequently, both the species diversity and biological attributes of Maculolachnus are likely to be substantially underestimated across its Eurasian range.

According to the literature, four nominal species are recognized within the genus: M. blackmani Kanturski & Chakrabarti, 2022, M. submacula (Walker, 1848), M. sijpkensi Hille Ris Lambers, 1962, and M. paiki Seo, 1994⁵. Among these, M. submacula exhibits the broadest geographic range, being reported throughout Europe, Pakistan, India, and Asia, including the Korean Peninsula^3,6–10. However, recent field collections and integrative analyses of Korean populations reveal consistent morphological distinctions and substantial mitochondrial divergence, suggesting previously unrecognized diversity within what has historically been treated as M. submacula.

The biology of Maculolachnus species remains equally enigmatic³. The life cycle of M. paiki, known solely from the alate viviparous female, has never been elucidated, and its specific host associations remain uncertain⁸. Furthermore, despite its original description from India and Pakistan, M. blackmani has not been rediscovered, and its phylogenetic position remains unresolved owing to the absence of molecular data². These persistent knowledge gaps underscore the need for a modern, integrative reassessment incorporating morphological, molecular, and ecological perspectives.

In this study, we present a comprehensive revision of Maculolachnus in South Korea through an integrative taxonomic framework. Our aims are to clarify species boundaries, reassess the identity of populations historically attributed to M. submacula, and document the rediscovered alate viviparous female of M. paiki, a species endemic to Korea. We further describe Maculolachnus koreanus sp. nov., a distinct lineage supported by both morphological and molecular evidence. This work provides the first detailed insights into the hidden diversity and life history of Maculolachnus and highlights the importance of integrative taxonomy for uncovering overlooked biodiversity within Lachninae (Table 1).

Table 1.

Morphometric and morphological differences between apterous and alate viviparous females of Maculolachnus submacula and Maculolachnus koreanus sp. nov.

Character	Apterous viviparous females		Alate viviparous females
	Maculolachnus submacula	Maculolachnus koreanus sp. nov	Maculolachnus submacula	Maculolachnus koreanus sp. nov
Color in life	Head and thorax dark brown	Head and thorax light brown	–	–
Abdomen membranous	Smooth cuticle	polygonal reticulation	Smooth cuticle	Polygonal reticulation
GP	More or less bilobed	An indentation on the proximal end	–	–
URS	Straight	Triangular	Straight	Triangular
PT/BASE	0.29–0.33	0.35–0.37	0.35	0.22–0.30
URS/HT II	0.58–0.65	0.56–0.57	0.59	0.53–0.57
ANT IV/ANT III	0.33–0.38	0.44–0.45	0.40	0.47–0.48
Forewing	–	–	Base of CuA1, CuA2, and Rs colorless, dark spot under the pterostigma, extending to 1/3 of the wing	Base of CuA1, CuA2, and Rs brown, dark spot under the pterostigma, extending to the middle of the wing

Results

Taxonomic account

Lachninae Herrich-Schaeffer, 1854.

Lachnini Herrich-Schaeffer, 1854.

Maculolachnus Gaumont, 1920.

Maculolachnus koreanus sp. nov.

Maculolachnus submacula Paik, 1972⁶; Lee et al., 1994⁷; Seo, 1994⁸.

Apterous viviparous female (description, holotype)

(Figs. 1b, 2b, and 3c, h, j; Table 1 2).

Fig. 1.

Maculolachnus koreanus sp. nov. in life: (a) fundatrix, (b) apterous viviparous female, (c) alate viviparous female, (d) oviparae, (e) male (nymph), (f) egg, (g) colony of fundatrix on the bark of young branches of Rosa hybrida, (h) colony of alate viviparous females on the bark of young branches of Rosa multiflora, (i) colonies of sexual morph on the bark of young branches of Rosa sp., (j-l) host plants (Rosa spp.), (m–n) ant tunnels and ant access near the ground.

Fig. 2.

Maculolachnus koreanus sp. nov.: (a) fundatrix (MKFX-1), (b) apterous viviparous female (MKAP-1), (c) alate viviparous female (MKAL-4), (d) oviparous viviparous female (MKOV-5), (e) male (nymph) (MKMA-1).

Fig. 3.

Comparison of the main morphological differences between Maculolachnus submacula and M. koreanus sp. nov.: (a) ANT of apterous viviparous female of M. submacula, (b) ANT of alate viviparous female of M. submacula, (c) ANT of apterous viviparous female of M. koreanus sp. nov. (MKAP-1), (d) ANT of alate viviparous female of M. koreanus sp. nov. (MKAL-6), (e) forewing of alate viviparous female of M. submacula, (f) forewing of alate viviparous female of M. koreanus sp. nov. (MKAL-10), (g) GP of apterous viviparous female of M. submacula, (h) GP of apterous viviparous female of M. koreanus sp. nov. (MKAP-1), (i) URS of apterous viviparous female of M. submacula, (j) URS of apterous viviparous female of M. koreanus sp. nov. (MKAP-1).

Table 2.

Biometric measurements of Maculolachnus species (min.–max.) mean length (mm).

Species	Maculolachnus koreanus sp. nov				M. paiki	M. blackmani	M. sijpkensi	M. submacula
Character	fx. (n = 5)	apt. (n = 1)	al. (n = 10)	ovi. (n = 5)	al. (n = 4)	desc. by Kanturski & Chakrabarti, 2022	apt. (n = 3)	apt. (n = 6)	al. (n = 1)
BL	(2.70–3.20) 2.96	2.90	(2.38–3.18) 2.95	(3.20–3.57) 3.36	(2.56–2.86) 2.67	2.87–3.55	(3.20–3.57) 3.36	(3.07–3.78) 3.35	3.32
ANT III	(0.46–0.55) 0.51	(0.52–0.53) 0.52	(0.45–0.55) 0.51	(0.43–0.53) 0.48	(0.52–0.57) 0.55	0.45–0.60	(0.60–0.66) 0.63	(0.52–0.64) 0.60	0.52
ANT IV	(0.18–0.23) 0.21	(0.23–0.24) 0.23	(0.18–0.24) 0.22	(0.17–0.22) 0.19	(0.18–0.21) 0.19	0.21–0.26	(0.27–0.31) 0.29	(0.20–0.24) 0.22	0.21
ANT V	(0.21–0.27) 0.23	(0.24–0.24) 0.24	(0.20–0.25) 0.23	(0.20–0.24) 0.22	(0.22–0.25) 0.23	0.23–0.29	(0.31–0.34) 0.32	(0.25–0.31) 0.28	0.26
BASE	(0.17–0.20) 0.19	(0.17–0.18) 0.17	(0.15–0.21) 0.19	(0.16–0.19) 0.18	(0.18–0.21) 0.19	0.18–0.23	(0.24–0.27) 0.26	(0.21–0.24) 0.22	0.22
PT	(0.04–0.05) 0.05	(0.06–0.06) 0.06	(0.04–0.06) 0.05	(0.04–0.05) 0.04	(0.08–0.09) 0.09	0.06–0.08	(0.06–0.06) 0.06	(0.07–0.07) 0.07	0.08
URS	(0.17–0.18) 0.18	0.19	(0.16–0.17) 0.17	(0.16–0.19) 0.18	(0.17–0.18) 0.18	0.21–0.22	(0.21–0.23) 0.22	(0.20–0.22) 0.21	0.20
FEMORA III	(1.02–1.22) 1.10	(1.19–1.24) 1.21	(1.00–1.22) 1.15	(0.96–1.18) 1.07	(0.98–1.04) 1.01	1.05–1.37	(1.32–1.41) 1.35	(1.17–1.54) 1.37	1.27
TIBIAE III	(1.74–1.98) 1.88	(2.01–2.03) 2.02	(1.75–2.12) 1.98	(1.60–1.97) 1.77	(1.62–1.80) 1.73	1.80–2.20	(2.34–2.48) 2.40	(1.97–2.46) 2.24	2.25
HT Ib	(0.04–0.05) 0.04	(0.05–0.05) 0.05	(0.04–0.05) 0.05	(0.04–0.04) 0.04	(0.04–0.05) 0.04	0.04–0.05	(0.05–0.06) 0.05	(0.05–0.07) 0.06	0.05
HT Id	(0.04–0.04) 0.04	(0.04–0.05) 0.04	(0.04–0.05) 0.04	(0.03–0.04) 0.04	(0.03–0.04) 0.03	0.03–0.04	(0.07–0.08) 0.07	(0.07–0.08) 0.07	0.07
HT Iv	(0.11–0.12) 0.11	(0.12–0.12) 0.12	(0.11–0.12) 0.11	(0.09–0.12) 0.11	(0.09–0.10) 0.09	0.10–0.12	(0.14–0.15) 0.14	(0.14–0.16) 0.14	0.14
HT Ii	(0.07–0.08) 0.08	(0.07–0.08) 0.07	(0.07–0.07) 0.07	(0.07–0.08) 0.07	(0.06–0.06) 0.06	0.07–0.08	(0.08–0.09) 0.08	(0.07–0.10) 0.08	0.07
HT II	(0.30–0.34) 0.32	(0.33–0.34) 0.34	(0.29–0.34) 0.31	(0.29–0.32) 0.30	(0.28–0.29) 0.29	0.31–0.34	(0.32–0.34) 0.32	(0.33–0.39) 0.37	0.34
SIPH sclerite	(0.25–0.32) 0.29	(0.30–0.36) 0.33	(0.20–0.35) 0.29	(0.23–0.31) 0.27	(0.23–0.29) 0.25	0.30–0.35	(0.35–0.49) 0.40	(0.32–0.40) 0.37	0.30
GP W	(0.45–0.55) 0.50	0.51	(0.38–0.49) 0.47	(0.57–0.64) 0.60	(0.38–0.44) 0.40	–	(0.77–0.84) 0.80	(0.57–0.69) 0.62	0.50
Forewing	-–	–	(2.82–3.69) 3.28	–	(3.28–3.43) 3.35	–	–	–	3.8

(fx, fundatrix; apt, apterous viviparous female; al, alate viviparous female; ovi, oviparous female).

Color. In life: head and thorax light brown, abdomen dark brown (Fig. 1b). Eyes black. ANT I–VI dark brown. Femora light brown with 2/3 distal end dark brown. Tibiae dark brown. Tarsi dark brown. SIPH dark brown. Pigmentation in mounted specimen: head, thorax, and abdomen brown. ANT I–VI brown. Femora light brown with 2/3 distal end dark brown. Tibiae brown. Tarsi brown. SIPH, cauda, and GP brown (Fig. 2b). Morphometric characters: body oval (Fig. 2b). HW 0.45–0.46 × ANT. Head with numerous medium-length, fine setae with pointed apices, 0.10–0.12 mm long. ANT 0.52 × BL. ANT III with 2–6 secondary rhinaria, shorter than ANT IV + V + VI. ANT IV shorter than ANT V + ANT VI with 3–4 secondary rhinaria. ANT V a little longer than ANT VI with one rounded primary rhinarium without secondary rhinaria (Fig. 3c). PT 0.35–0.37 × BASE, with one rounded primary rhinarium and 8–9 accessory rhinaria. Other antennal ratios: VI/III 0.45–0.47, V/III 0.45–0.46, IV/III 0.44–0.45. ANT bearing numerous, mostly medium-length, thick, rigid setae with pointed apices. LS ANT III 2.39–2.51 × BD III. ANT VI with 26–27 basal, 3 apical and 2–3 subapical setae. Rostrum reaching ABD TERG III. URS 0.36 × ANT III, 0.78–0.80 × ANT VI and 0.56–0.57 × HT II with 8 fine accessory setae (Fig. 3j). FEMORA III with medium-length, thick, rigid setae with pointed apices, 0.08–0.09 mm long. TIBIAE III with medium-length, thick, rigid setae with pointed apices, 0.08–0.09 mm long. HT I with 14 ventral setae, HT Ib 1.00–1.07 × HT Id, 0.58–0.61 × HT Ii, 0.37–0.39 × HT Iv. HT II 0.64–0.65 × ANT III and 1.38–1.43 × ANT VI. Abdomen membranous with polygonal reticulation and with small mostly irregular and narrow-oval scleroites. Setae arising from scleroites are medium-length, thick, and rigid, with pointed apices. Setae 0.10–0.11 mm long on ABD TERG I–V and 0.10–0.11 mm long on ABD TERG VI–VIII. SIPH low, lying on setose sclerites with irregular edges. ABD TERG VIII in form of narrow stripe on the whole segment width broken in the spinal area, with 19 setae. SIPH cone 7.97–8.67 × SIPH pore. Genital plate with an indentation on the proximal end, with numerous setae (Fig. 3h).

Fundatrix (description)

(Figs. 1a, 2a, 4; Table 2).

Fig. 4.

Morphological characters of Maculolachnus koreanus sp. nov., fundatrix: (a) abdomen sclerotization (MKFX-1), (b) URS (MKFX-1), (c) GP (MKFX-2), (d) SIPH (MKFX-1), (e) ANT (MKFX-1).

Color. In life: head, thorax, and abdomen brown (Fig. 1a). Eyes black. ANT I–VI dark brown. Femora light brown with 2/3 distal end dark brown. Tibiae dark brown. Tarsi dark brown. SIPH dark brown. Pigmentation in mounted specimens: head, thorax, and abdomen brown. ANT I–VI brown. Femora light brown with 2/3 distal end dark brown. Tibiae brown. Tarsi brown SIPH, cauda, and GP brown (Fig. 2a). Morphometric characters: body elongated oval (Fig. 2a). HW 0.44–0.48 × ANT. Head with numerous medium-length, fine setae with pointed apices, 0.10–0.12 mm long. ANT 0.46–0.51 × BL. ANT III with 2–10 secondary rhinaria, shorter than ANT IV + V + VI. ANT IV with 1–5 secondary rhinaria. ANT V with one rounded primary rhinarium with one small rounded secondary rhinaria (Fig. 4e). PT 0.20–0.29 × BASE, with one rounded primary rhinarium and 5–8 accessory rhinaria. Other antennal ratios: VI/III 0.39–0.50, V/III 0.41–0.48, IV/III 0.34–0.44. ANT bearing numerous, mostly medium-length, thick, rigid setae with pointed apices. LS ANT III 2.33–2.85 × BD III. ANT VI with 22–32 basal, 3–5 apical and 2–3 subapical setae. Rostrum reaches ABD TERG II. URS 0.32–0.38 × ANT III, 0.72–0.81 × ANT VI and 0.53–0.59 × HT II with 8–9 fine accessory setae (Fig. 4b). FEMORA III with medium -length, thick, rigid setae with pointed apices, 0.05–0.12 mm long. TIBIAE III with medium-length, thick, rigid setae with pointed apices, 0.06–0.11 mm long. HT I with 15–18 ventral setae, HT Ib 1.02–1.28 × HT Id, 0.45–0.55 × HT Ii, 0.35–0.42 × HT Iv. HT II 0.57–0.65 × ANT III and 1.29–1.46 × ANT VI. Abdomen membranous with polygonal reticulation and with small, mostly irregular and narrow-oval scleroites (Fig. 4a). Setae arising from scleroites are medium-length, thick, and rigid, with pointed apices. Setae 0.09–0.13 mm long on ABD TERG I–V and 0.10–0.12 mm long on ABD TERG VI–VIII. SIPH low, lying on setose sclerites with irregular edges. ABD VIII in form of narrow stripe on the whole segment width broken in the spinal area, with 19–26 setae. Genital plate more or less bilobed with numerous setae (Fig. 4c). SIPH cone 5.53–6.93 × SIPH pore (Fig. 4d).

Alate viviparous female (description)

(Figs. 1c, 2c, 3b, d, f, h, and j; Table  12).

Color. In life: head and thorax dark brown, abdomen brown (Fig. 1c). Eyes black. ANT I–VI dark brown. Femora light brown with 4/5 distal end dark brown. Tibiae dark brown. Tarsi dark brown. SIPH dark brown. Pigmentation in mounted specimens: head, thorax, and abdomen brown. ANT I–VI brown. Femora light brown with 4/5 distal end dark brown. Tibiae brown. Tarsi brown SIPH, cauda, and GP brown. Forewings with a dark spot under the pterostigma (Fig. 2c). Morphometric characters: body elongated oval (Fig. 2c). HW 0.41–0.47 × ANT. Head with numerous medium-length, fine setae with pointed apices, 0.09–0.11 mm long. ANT 0.49–0.53 × BL. ANT III with 9–14 secondary rhinaria, shorter than ANT IV + V + VI. ANT IV with 2–4 secondary rhinaria. ANT V with one rounded primary rhinarium (Fig. 3d). PT 0.22–0.30 × BASE, ANT VI with one rounded primary rhinarium and 3–6 accessory rhinaria. Other antennal ratios: VI/III 0.47–0.48, V/III 0.43–0.48, IV/III 0.35–0.42. ANT bearing numerous, mostly medium-length, thick, rigid setae with pointed apices. LS ANT III 2.82–3.35 × BD III. ANT VI with 24–30 basal, 3–5 apical and 1–4 subapical setae. Rostrum reaches ABD TERG III–IV. URS 0.31–0.35 × ANT III, 0.65–0.74 × ANT VI and 0.53–0.57 × HT II with 8–13 fine accessory setae. FEMORA III with medium-length, thick, rigid setae with pointed apices, 0.07–0.11 mm long. TIBIAE III have medium-length, thick, rigid setae with pointed apices, 0.08–0.12 mm long. HT I with 24–30 ventral setae, HT Ib 1.08–1.31 × HT Id, 0.58–0.66 × HT Ii, 0.37–0.43 × HT Iv. HT II 0.57–0.60 × ANT III and 1.19–1.25 × ANT VI. Abdomen membranous with polygonal reticulation and with small mostly irregular and narrow-oval scleroites. Setae arising from scleroites are medium in length, thick, and rigid, with pointed apices. Setae 0.08–0.13 mm long on ABD TERG I–V and 0.08–0.13 mm long on ABD TERG VI–VIII. SIPH low, lying on setose sclerites with irregular edges. ABD TERG VIII in form of narrow stripe on the whole segment width broken in the spinal area, with 17–27 setae. SIPH cone 5.52–7.08 × SIPH pore. Genital plate with an indentation on the proximal end, with numerous setae. Forewings with media twice branched, base of CuA1, CuA2, and Rs brown; dark spot under the pterostigma, extending to the middle of the wing (Fig. 3f).

Oviparous viviparous female (description)

(Figs. 1d, 2d, 5; Table 2).

Fig. 5.

Morphological characters of Maculolachnus koreanus sp. nov., oviparae (MKOV-5): (a) abdomen sclerotization, (b) URS, (c) SIPH, (d) GP, (e) TIBIAE III and different types of pseudosensoria: rounded (red arrow), 8-shaped (blue arrow), (f) ANT.

Color. In life: head, thorax, and abdomen dark brown (Fig. 1d). Eyes black. ANT I–VI dark brown. Femora light brown with 4/5 distal end dark brown. Tibiae dark brown. Tarsi dark brown. SIPH dark brown. Pigmentation in mounted specimens: head, thorax, and abdomen brown. ANT I–VI brown. Femora light brown with 2/3 distal end dark brown. Tibiae brown. Tarsi brown SIPH, cauda, and GP brown (Fig. 2d). Morphometric characters: body elongated oval (Fig. 2d). HW 0.45–0.53 × ANT. Head with numerous medium-length, fine setae with pointed apices, 0.10–0.12 mm long. ANT 0.48–0.53 × BL. ANT III with 2–5 secondary rhinaria, shorter than ANT IV + V + VI. ANT IV with 2–5 secondary rhinaria. ANT V with one rounded primary rhinarium with one small rounded secondary rhinaria (Fig. 5f). PT 0.19–0.29 × BASE, with one rounded primary rhinarium and 4–8 accessory rhinaria. Other antennal ratios: VI/III 0.43–0.57, V/III 0.43–0.53, IV/III 0.36–0.44. ANT bearing numerous, mostly medium-length, thick, rigid setae with pointed apices. LS ANT III 2.19–2.76 × BD III. ANT VI with 24–27 basal, 3–5 apical and 2–3 subapical setae. Rostrum reaches ABD TERG III–IV. URS 0.34–0.40 × ANT III, 0.71–0.86 × ANT VI and 0.55–0.61 × HT II with 8–12 fine accessory setae (Fig. 5b). FEMORA III with medium-length, thick, rigid setae with pointed apices, 0.05–0.09 mm long. TIBIAE III have medium-length, thick, rigid setae with pointed apices, 0.05–0.09 mm long, and very small 43–66 pseudosensoria which are single, rounded and 8-shaped (Fig. 5e). HT I with 14–15 ventral setae, HT Ib 1.02–1.27 HT Id, 0.54–0.63 × HT Ii, 0.36–0.43 × HT Iv. HT II 0.60–0.68 × ANT III and 1.19–1.45 × ANT VI. Abdomen membranous with polygonal reticulation and with small mostly irregular and narrow-oval scleroites (Fig. 5a). Setae arising from scleroites are medium in length, thick, and rigid, with pointed apices. Setae 0.08–0.12 mm long on ABD TERG I–V and 0.09–0.13 mm long on ABD TERG VI–VIII. SIPH low, lying on setose sclerites with irregular edges. ABD TERG VIII in form of narrow stripe on the whole segment width broken in the spinal area, with 103–116 setae. SIPH cone 4.89–7.58 × SIPH pore (Fig. 5c). Genital plate is in the form of two separate sclerites with numerous setae (Fig. 5d).

Etymology: The specific epithet koreanus is a Latinized form of “Korea,” indicating the country where the species was discovered. The suffix -anus, a masculine singular ending commonly used in zoological Latin, means “pertaining to” or “belonging to”.

Maculolachnus paiki Seo, 1994

Maculolachnus paiki Seo, 1994⁸.

Alate viviparous female (redescription)

(Fig. 6; Table 2).

Fig. 6.

Morphological characters of Maculolachnus paiki, alate viviparous female: (a) body (MPAL-1), (b) URS (MPAL-2), (c) SIPH (MPAL-1), (d) GP (MPAL-1), (e) ABD VI–VIII (MPAL-1), (f) forewing (MPAL-1), (g) ANT (MPAL-2).

Pigmentation in mounted specimens: head, thorax, and abdomen brown. ANT I–VI brown. Femora light brown with 4/5 distal end dark brown. Tibiae brown. Tarsi brown SIPH, cauda, and GP brown. Forewings with a dark spot under the pterostigma (Fig. 5c). Morphometric characters: body elongated oval (Fig. 6a). HW 0.33–0.42 × ANT. Head with numerous medium-length, fine setae with pointed apices, 0.10–0.12 mm long. ANT 0.49–0.57 × BL. ANT III with 3–6 secondary rhinaria, shorter than ANT IV + V + VI. ANT IV with 1–2 secondary rhinaria. ANT V with one rounded primary rhinarium (Fig. 6g). PT 0.44–0.46 × BASE, ANT VI with one rounded primary rhinarium and 2–6 accessory rhinaria. Other antennal ratios: VI/III 0.47–0.53, V/III 0.39–0.44, IV/III 0.32–0.38. ANT bearing numerous, mostly medium-length, fine setae with pointed apices. LS ANT III 2.36–3.55 × BD III. ANT VI with 21–26 basal, 1–3 apical and 3–4 subapical setae. Rostrum reaches ABD TERG II. URS 0.31–0.34 × ANT III, 0.60–0.68 × ANT VI and 0.61–0.64 × HT II with 13–14 fine accessory setae (Fig. 6b). FEMORA III with medium-length, fine setae with pointed apices, 0.09–0.11 mm long. TIBIAE III medium-length to long, thick, fine setae with pointed apices, 0.07–0.10 mm long. HT I with 21–26 ventral setae, HT Ib 1.03–1.50 × HT Id, 0.61–0.80 × HT Ii, 0.38–0.53 × HT Iv. HT II 0.50–0.56 × ANT III and 0.94–1.11 × ANT VI. Abdomen membranous smooth cuticle and with small mostly irregular and narrow-oval scleroites. Setae arising from scleroites are medium in length and fine, with pointed apices. Setae 0.09–0.11 mm long on ABD TERG I–V and 0.10–0.11 mm long on ABD TERG VI–VIII. SIPH low, lying on setose sclerites with irregular edges. SIPH cone 5.08–6.47 × SIPH pore (Fig. 6c). Genital plate with numerous setae (Fig. 6d). ABD TERG VIII in form of narrow stripe on the whole segment width broken in the spinal area, with 11–15 setae (Fig. 6e). Forewings with media twice branched; common node of M1 and M2 under the beginning of the radial sector, dark spot under the pterostigma, extending to 1/3 of the wing (Fig. 6f).

Distribution (Fig. S1).

Maculolachnus koreanus sp. nov.: Andong, Chuncheon, Pyeongchang, Jeongseon (this study), Danyang, Jeju, Muju^6–8.

Maculolachnus paiki: Pyeongchang (this study), Muju^6–8.

Key to apterous viviparous females of the genus Maculolachnus (modified by², apterous viviparous females of M. paiki undescribed).

1. 1. Dorsal abdomen with scleroites at setal bases.                                                                                                  2

• Dorsal abdomen without scleroites at setal bases                                        M. sijpkensi Hille Ris Lambers, 1962.

• 2.Body setae with expanded and slightly flabellate or blunt apices M. blackmani Kanturski & Chakrabarti, 2022

• Body setae with fine, pointed apices.                                                                                                                        3

• 3.ANT IV/ANT III 0.33–0.38, abdomen membranous with smooth cuticle, GP with more or less bilobed M. submacula (Walker, 1848)

• ANT IV/ANT III 0.44–0.45, abdomen membranous with polygonal reticulation, GP with an indentation on the proximal end                                                                                                                           M. koreanus sp. nov.

Key to alate viviparous females of the genus Maculolachnus (alate viviparous females of M. blackmani and M. sijpkensi undescribed).

1. ANT III with more than 7 secondary rhinaria, PT less than 0.40 × BASE                                                           2

• ANT III with 3–6 secondary rhinaria, PT 0.44–0.46 × BASE                                 Maculolachnus paiki Seo, 1994.

• 2.PT 0.22–0.30 × BASE, URS 0.53–0.57 × HT II, ANT IV 0.47–0.48 × ANT III                         M. koreanus sp. nov.

• PT 0.35 × BASE, URS 0.59 × HT II, ANT IV 0.40 × ANT IIIM.                                             submacula (Walker, 1848).

Molecular analyses

Phylogenetic analyses and species delimitation

The Bayesian inference (BI), maximum likelihood (ML), and neighbor-joining (NJ) analyses based on the COI gene revealed that multiple aphid samples were divided into four clades and were identified as the following species (Fig. 7a): Maculolachnus koreanus sp. nov., M. paiki, M. sijpkensi, and M. submacula. The species delimitation methods of ABGD, ASAP, and bPTP each yielded four molecular operational taxonomic units (MOTUs). The pairwise distance gap approach (ABGD) with default settings (X = 0.5) suggested four species with a barcode gap distance of 3.0%. The first ASAP-score (2.5) was chosen, which provides the best-fit scenario at the threshold distance of 1.79% (JC69) with four hypothetical species. The bPTP analysis showed four hypothetical species (Fig. 7a). The distinct separation of clades observed in the study demonstrates that the COI barcode region is an effective tool for identifying closely related species.

Fig. 7.

Molecular data analyses of Maculolachnus species based on 28 COI sequences: (a) Phylogenetic tree inferred using Bayesian inference (BI), representing the primary topology used in this study. Each node is annotated with supporting values from Bayesian inference (BI, posterior probabilities), maximum likelihood (ML: SH-aLRT and UFB bootstrap supports), and neighbor-joining (NJ, bootstrap values), together with the results of species delimitation analyses (ABGD, ASAP, and bPTP), (b) Distribution of genetic divergences based on the Kimura 2-parameter (K2P) model for COI sequences across different taxonomic levels. A total of 137 within-species and 198 between-species pairwise comparisons were calculated, (c) Haplotype network of Maculolachnus species based on COI sequences.

Genetic divergence

Genetic divergences (GD) within all species included in this study are presented in Table 3. The intraspecific and interspecific genetic divergence for four species (Maculolachnus koreanus sp. nov., M. paiki, M. sijpkensi, and M. submacula) were analyzed using COI sequences. The intraspecific genetic divergence, analyzed with 137 comparison pairs, averaged 0.1%, and the maximum intraspecific divergence did not exceed 1.5%. The highest value of intraspecific GD (1.5%) was found in M. sijpkensi. The interspecies genetic divergence was analyzed with 198 comparison pairs. The minimum interspecific genetic divergence among all four species was calculated to be 2.4% between Maculolachnus koreanus sp. nov. and M. submacula, while the maximum interspecific divergence value was 7.6% (averaging 4.3%) between M. sijpkensi and M. paiki. The distribution of their genetic divergence is shown in Table 3. All species analyzed in this study exhibited a distinct barcode gap between the maximum intraspecific GD (1.5%) and the minimum interspecific GD (2.4%) in the COI gene (Fig. 7b). Molecular analyses of the COI gene from Maculolachnus species collected across various countries provide strong support for the identification of the Maculolachnus species found in South Korea as a new species.

Table 3.

Genetic divergences within all species included in this study.

Taxonomic level (no. of comparison pairs)	K2P Pairwise distances
	Maximum	Minimum	Mean
(1) Different taxonomic levels
Within species (137)	1.5	0.0	0.1
Between species (198)	7.6	2.4	4.3

Species	Comparison pairs (CP)	Intraspecific genetic divergence
		Maximum	Minimum	Mean
(2) Intraspecific genetic divergence
Maculolachnus sijpkensi	10	1.5	0.0	0.1
Maculolachnus submacula	6	0.0	0.0	0.0
Maculolachnus paiki	1	0.2	0.2	0.2
Maculolachnus sp. nov	120	0.0	0.0	0.0

Species	M. sijpkensi	M. submacula	M. paiki
(3) Interspecific genetic divergence
M. submacula	CP = 20, .4 (4.2–4.8)
M. paiki	CP = 10, 7.2 (7.0–7.6)	CP = 8, 5.6 (5.5–5.6)
M. sp. nov	CP = 80, 5.1 (4.6–5.3)	CP = 64, 2.4 (2.4–2.4)	CP = 16, 4.9 (4.9–4.9)

Haplotype analysis

The TCS network analysis identified nine haplotypes from the 28 COI sequences analyzed (Fig. 7c). The detailed haplotype distribution across all individuals is provided in Supplementary Table 1. Specimens of Maculolachnus koreanus sp. nov. collected from four regions in South Korea formed a single haplotype (Hap_3), which was distinct from M. submacula (Hap_8) sampled from Europe. Notably, M. submacula (Hap_8) and M. koreanus sp. nov. (Hap_3) did not share any haplotypes, forming separate clusters in the network. M. paiki was composed of two haplotypes (Hap_1 and Hap_2), with two individuals detected among the 28 samples analyzed. In contrast, M. sijpkensi included four haplotypes (Hap_4–7), derived from specimens collected in North America and Europe.

Biology

Maculolachnus koreanus sp. nov. was observed on various Rosa species planted by humans in both mountainous regions and urban areas (Fig. 1j–l). The life cycle of M. koreanus sp. nov. was investigated through direct observations. Fundatrices were observed as adults on June 6 (Figs. 1a, 2a), producing both apterous and alate nymphs. Colonies were primarily established on stems close to the ground and branches (Figs. 1m, n). Workers of Lasius sp. (Formicidae) were frequently observed interacting with the colonies (Fig. 1g), and some aphid colonies were found within ant-constructed shelters (Fig. 1n). Adult apterous and alate viviparous females emerged by June 7 (Figs. 1b, c, 2b, c), with a noticeable increase in the number of alate viviparous females by mid-June (Fig. 1h). The sexual generation is presumed to begin in late October (Figs. 1d, e, i, 2d, e), when oviparous females begin laying eggs on branches and on the spines of branches (Fig. 1f). By late November, oviparous females were no longer detected.

Damage

Colony size peaked in mid-June but declined by early July. In mid-October, colonies increased in size again as the aphids prepared for overwintering. A notable symptom of infestation was the accumulation of black sooty mold on branches, leaves, and flower buds, resulting from the large quantities of honeydew excreted by the aphids.

Notes on SEM morphology and sensilla of alate viviparous female of Maculolachnus koreanus sp. nov.

General characters

Alate viviparous females of M. koreanus sp. nov. are characterized by a densely covered body and appendages with long, fine (hair-like) trichoid sensilla (setae) which, in general view, have very thin, pointed apices (Fig. 8). Compound eyes are rounded, well-developed, protuberant and consist of numerous facets (single ommatidia) whose arrangement is regular. In the middle of the hind part of the compound eyes, a well-developed triommatidium on an ocular tubercle is visible (Fig. 8a–c). On the head of the alate, well-developed, rounded ocelli can be noted two on the dorsal side, above the compound eyes, and one on the frons (Fig. 8a). Alate viviparous females of M. koreanus sp. nov. are characterized by membranous wings with typical venation and many scale-like elements (Fig. 8d, e), and legs with two-segmented tarsi (Fig. 8f). Siphunculi are low but lie on clearly visible oval and hairy sclerites, and have a well-developed flange (Fig. 8g). The abdomen which bears many long, fine, pointed setae, is characterized by short cauda, which with the remaining perianal structures (abdominal tergite VIII with a single row of long setae and a triangular anal plate) has adaptations for ant attendance (Fig. 8h), as described in^10,11 which is clearly visible from the lateral view (Fig. 8h). The shortened cauda and long setae are also visible at the end of the dorsal abdomen (Fig. 8i).

Fig. 8.

Scanning electron micrographs (SEM) of alate viviparous female of Maculolachnus koreanus general morphological characters: (a) lateral side of the head showing antennae (turquoise), compound eyes (yellow), triommatidia (red) and mouthparts including mandibular lamina (green), postclypeus (blue), maxillary lamina (violet), (b) frontal view of the head showing antennae (turquoise), compound eyes (yellow), triommatidia (red), ocelli (purple), and mouthparts including mandibular lamina (green), postclypeus (blue), anteclypeus (orange), maxillary lamina (violet), labrum (yellow) and labium (topaz), (c) structure of the compound eye and ocular tubercle with triommatidium, (d) forewing, (e) hind wing, (f) hind tarsus, (g) siphunculi, (h) lateral view of the abdominal apex showing spiracles (pink), and perianal structures including ABD TERG VIII (blue), cauda (yellow), anal plate (dark green), rudimentary gonapophyses (violet) and genital plate (orange), (i) perianal structures of the abdominal apex from the dorsal view.

Antennal sensilla

The antennal segments of the alate viviparous female of M. koreanus sp. nov. are characterized by the presence of numerous sensilla (including the secondary rhinaria) (Fig. 9a). All segments are covered by long, fine and, in the general view pointed type I trichoid sensilla (setae), and the pedicel additionally bears two other kinds of sensilla. The dorso-lateral side of the pedicel bears a single campaniform sensillum, while on the ventro-lateral side, three rhinariola may be visible (Fig. 9b). Antennal segments III and IV, besides the trichoid sensilla, bear small to large, multiporous placoid sensilla (secondary rhinaria) (Figs. 9c, d), and the fifth antennomere, besides the trichoid sensilla, bears only one large, multiporous sensillum on the distal end (Fig. 9e). On the third antennomere it can be noted that the vast majority of trichoid sensilla cover the dorsal side of the segment, while the small multiparous placoid sensilla are distributed in one row on the ventral side (Figs. 9a, c). The last antennal segment bears the largest number of sensory structures and besides type I trichoid sensilla, type II trichoid sensilla, large and small multiporous placoid sensilla and sunken coeloconic sensilla have been found (Fig. 9f). The rhinariola are of different sizes, in the form of three single and separate openings with a clearly-visible sensillum peg which protrudes above the opening (Fig. 9g). The lower rhinariolum is the largest one, about 10–11 μm in diameter, with the most robust collar, which becomes flat in the inner area of the opening. The sensillum peg, which has the features of a sunken coeloconic sensillum, is tubular, slightly tapering at the apical end, and has about 8–10 projections (Fig. 9h). The middle rhinariolum is similar to the lower one, has almost the same size (9–10 μm), but is characterized by a much flatter collar and lacks a flat area around the sensillum peg (Fig. 9i). The rhinariolum which lies near the pedicel edge is the smallest and flat (only about 5–6 μm). The area of the sensillum opening is rather small, with six, probably cuticular projections and the sensillum has only 5–6 robust projections (Fig. 9j). The third rhinariolum near the pedicel edge may also be visible as a single opening without a protruding sensillum peg (Figs. S2b). The small multiparous placoid sensilla on ANT III and IV (secondary rhinaria) are quite large in relation to the segment width and extremely protuberant with a clearly visible porous surface on their dorsal side (Figs. 9k, l). The large multiparous placoid sensilla (primary rhinarium on ANT V and major rhinarium on ANT VI) have the same large diameter as the small multiparous placoid sensilla but are less protuberant and their porous membrane has several small but clearly visible depressions (Fig. 9m). The pores of the primary rhinaria are mostly rounded or oval and are quite dense, 22–25 per 1 μm² (Fig. 9n). On the other hand, the small multiparous placoid sensilla on ANT VI are quite different from those that form the secondary rhinaria—they are much narrower, club-shaped or bulbous, with a very well-developed sclerotic collar (Fig. 9o). Between the upper and lower small multiparous placoid sensilla on the ANT VI, four sunken coeloconic sensilla can be found. Arrangement of the sunken coeloconic sensilla varies among individuals, but a general scheme is that they are separated into two groups and are lying near the small multiporous placoid sensilla. In some individuals each group was formed by two separate sensilla (Fig. S2c) or three sensilla were lying very close to each other, with a single one separated (Fig. 9p). There are two sunken coeloconic sensilla of type I with 6–8 short projections and type II with 10–12 projections, from which 5–7 are evidently long and about 4–5 much shorter (Figs. 9q, r). As mentioned above, all antennomeres are covered by numerous long, fine, and, in the general view pointed type I trichoid sensilla (Fig. S2a-c). The apical end of the terminal process bears, moreover, short, rigid type II trichoid sensilla (Fig. S2f). The type I trichoid sensilla are set at an angle of about 45° towards the segments’ distal ends. They arise from well-developed, protuberant, flexible sockets, and their surface is ribbed from the very base along the entire length (Fig. S2d, e). Interestingly, at higher magnification, the apical ends of type I trichoid sensilla of M. koreanus turned out to have a smooth surface which is additionally spirally twisted and the very end of the apices is rounded (Fig. S2g). The surface of the type II trochoid sensilla on the terminal process is very similar to the sensilla of the type I sensilla, and their sockets are much more rounded (Figs. S2h, i). Similar to the type I trochoid sensilla, the apical end of the type II sensilla is smooth and has a rounded apex (Fig. S2j).

Fig. 9.

SEM of antennal sensilla of alate viviparous female of M. koreanus I: (a) antennal flagellum with visible type I trichoid sensilla (blue), small multiparous placoid sensilla—secondary rhinaria (red), large multiparous placoid sensilla (yellow) and small multiparous placoid sensilla (green)—primary rhinaria, (b) pedicel with visible type I trichoid sensilla, campaniform sensillum (green) and three rhinariola (pink), (c) fragment of ANT III with type I trichoid sensilla (blue) and small multiparous placoid sensilla (red), (d) ANT IV with type I trichoid sensilla (blue) and small multiparous placoid sensilla—secondary rhinaria (red), (e) ANT V with type I trichoid sensilla (blue) and large multiparous placoid sensillum (yellow)—primary rhinarium, (f) ANT VI with type I trichoid sensilla (blue), type II trichoid sensilla (violet), large multiparous placoid sensillum (yellow)—major rhinarium, small multiparous placoid sensilla (green) and four sunken coeloconic sensilla of two types (pink)—accessory rhinaria, (g) arrangement of the rhinariola on the pedicel, (h, i) ultrastructure of type I rhinariola with short projections, (j) ultrastructure of type II rhinariolum, (k, l) lateral and dorsal view of small multiparous placoid sensilla, (m) structure of large multiparous placoid sensillum on ANT V, (n) ultrastructure of porous membrane of the large multiparous placoid sensillum, (o) ultrastructure of the small multiparous placoid sensillum on ANT VI, (p) sunken coeloconic sensilla on ANT VI, (q) ultrastructure of the type I coeloconic sensillum, (r) ultrastructure of the type II sunken coeloconic sensillum.

Mouthparts sensilla

The labium of M. koreanus sp. nov. is mostly covered by type I trichoid sensilla and characterized by tapering and slightly pointed ultimate rostral segments (especially the segment V) (Fig. S3a). Sensilla cover the whole length and each side of the segment IV, but they are most numerous on the dorsal and ventral side (Fig. S3b, c). The border between the rostral segment IV and V bears three pairs of long, fine and pointed trichoid sensilla (primary setae), one pair on the ventral, one pair at the lateral side and one pair on the dorsal side (Fig. S3e, f). Trichoid sensilla arise from protruding, oval, flexible sockets (Fig. S3g) on segments II–III and from irregular ones on the segment IV (Fig. S3n). The apex of the last rostral segment bears seven pairs of type III basiconic sensilla (Figs. S3h-j). On the basal part of the rostral segment IV one pair of type II basiconic sensilla is well-visible (Fig. S3k). The type II basiconic sensilla are approximately 17–18 μm long and arise from protruding, almost hemispherical sockets (Fig. S3l). The surface of the type II basiconic sensilla is smooth at the very basal part, then ribbed on the remainder of their length, which ends with rounded apex (Fig. S3m). Type III basiconic sensilla on the last rostral segment are nearly the same length (4–5 μm long), except for the first pair, which is much longer (9–10 μm). The surface of the sensilla is smooth, molting pores are clearly visible on their basal parts (Figs. S3o–q) and they end with very regular and rounded apices (Fig. S3r).

Wings characters and sensilla

Alate viviparous females of M. koreanus sp. nov. are characterized by membranous wings on which numerous scale-like elements are visible. The inner basal part of forewings, in the area of wing articulation, is covered by approximately 9–11 campaniform sensilla (Fig. S4a), about 5–6 μm in diameter, which, in contrast to the antennal ones, are characteriszed by an outer collar with well-developed gap (Fig. S4b). Campaniform sensilla, together with trichoid sensilla, are also located on the lower distal border of the pterostigma (Fig. S4d). Trichoid sensilla on the pterostigma border, arising from small, rounded, flexible sockets, are smooth and pointed (Fig. S4d) whereas the campaniform sensilla share the same characteristics (with a gap in the collar) as those on the basal part of the wings (Fig. S4e). Most of the wing membrane surface is covered by numerous and regularly arranged scale-like elements, which are widely crescent-shaped (Figs. S4f, g), and rarer, almost flat or triangular, in the lower area of the wing, especially in the region of the folding apparatus (Figs. S4h, i). The basal part of hindwings also bears 12–14 campaniform sensilla of the same morphological characteristics—i.e., with an open collar (Figs. S4j–l). The claval apparatus of the wings is formed by strengthened and rolled-up membrane of the forewings and four hamuli on hindwings (Figs. S4i, m).

Legs and body surface sensilla

The legs, like other parts of the body of M. koreanus sp. nov. are characterized by the presence of numerous and densely distributed long, fine trichoid sensilla, which in a general are characterized by very fine and pointed apices (Fig. 10a, c, i). In addition to trichoid sensilla, many campaniform sensilla are also found on the inner side of trochantera (two) (Fig. 10b) and on the proximal bases of femora (12–14). Moreover, those on the femora form two separate groups—one on the inner side (about 8–9) and on the ventral side (4–5) (Fig. 10c). The campaniform sensilla are rounded; on the trochanter, they are of the same size, but the two groups on the femora are characterised by smaller sensilla on the inner side and larger ones on the ventral side. The collars of the sensilla are almost closed, with only a small indentation on the cap side (Figs. 10b, d, e). Occasionally, strongly protuberant caps of campaniform sensilla have been observed on the femora (Fig. 10f). The trichoid sensilla on femora are characterized by extremely fine apical ends, which often curved downward (Fig. 10g). They arise from flat, rounded flexible sockets, and their surface are ribbed from the basal part (Fig. 10h). Trichoid sensilla on the tibiae share similar characteristics, with two main differences: they all arise at an angle of 45° towards the distal end of the tibiae, and their sockets are more protuberant (Fig. 10i, j). Their surfaces are ribbed from the basal part along the entire length (Fig. 10k), and similar to the trichoid sensilla on the antennae, their apical ends are spirally twisted with flat, very apical tip (Fig. 10l). Campaniform and trichoid sensilla can also be found on the tarsal segments (Fig. 10m, n). The trichoid sensilla on the first segment of tarsi are arranged in three groups: one pair of dorsal, two pairs of lateral and numerous ventral setae. The ventral setae of the first tarsal segment bear one sense-peg, which is well-distinguishable from the other ventral setae. It is much shorter and arises from more protuberant socket (Fig. 10o, p). On the distal ventral end of the second tarsal segment, well-visible parempodia (empodial setae) can be found (Fig. 10q, r). The cuticle surface varies depending on the body part. On the head, the cuticle is generally smooth with numerous long and fine sensilla (Fig. 10s) which in most cases arise almost perpendicularly from elevated, trapezoidal, flexible sockets (Fig. 10t). The trichoid sensilla on the head and thorax are characterized, like other type I trichoid sensilla of M. koreanus, by a ribbed surface and strongly spirally twisted apical parts with rounded apices (Fig. 10u, v). In contrast, the cuticle of the abdomen is much more wrinkled and, like the head, is covered with long and fine trichoid sensilla, which arise from rounded, oval, and smooth sclerites (Fig. 10w, x). Near the end of the abdomen, the cuticle is characterized by numerous minute denticles arranged in longitudinal rows (Fig. 10y, z).

Fig. 10.

SEM of legs and cuticle sensilla of alate viviparous female of M. koreanus: (a) inner side of hind trochanter with campaniform sensilla (green), (b) ultrastructure of the trochanter campaniform sensilla, (c) hind femur showing numerous campaniforms sensilla (green) on the inner and ventral side, (d-f) ultrastructure of the femoral campaniform sensilla of different characters of the cap, (g) type I trichoid sensilla on femur, (h) ultrastructure of the socket and basal part of the sensillum, (i) type I trichoid sensilla on tibia, (j) ultrastructure of the socket of the sensillum, (k) ultrastructure of the surface of sensillum, (l) ultrastructure of the apical end of the sensillum, (m) lateral view of HT I with dorsal (violet) and lateral setae (turquoise), and one peg-like seta (blue) besides the numerous ventral setae, (n) dorsal view of HT I and proximal part of HT II showing campaniform sensilla (green), dorsal setae (violet), lateral setae (turquoise) and ventral setae (blue), (o) ventral side of HT I with one peg-like seta (blue), (p) difference between the sockets of the peg-like seta and normal ventral setae, (q) end of HT II with claws and empodial setae (yellow), (r) ultrastructure of the empodial setae, (s) head trichoid sensilla, (t) ultrastructure of the socket and basal part of the head trichoid sensilla, (u) ribbed surface of the head trichoid sensilla, (v) ultrastructure of the apical end of the head trichoid sensilla, (w) cuticle and trichoid sensilla of the abdomen, (x) ultrastructure of the scleroite on the abdomen, (y) surface of the dorsal cuticle of the end of abdomen, (z) ultrastructure of the minute denticles.

Discussion

Historical and taxonomic background

The genus Maculolachnus has long been taxonomically problematic owing to pronounced morphological uniformity and a paucity of biological data. In South Korea, Maculolachnus submacula (Walker, 1848) was previously recorded based solely on superficial similarity to European material^6–8. Our integrative reassessment, combining detailed morphology with mitochondrial COI sequences, demonstrates that the Korean specimens differ markedly from M. submacula and represent a distinct, previously undescribed species. Accordingly, the earlier Korean record of M. submacula is most parsimoniously interpreted as a misidentification.

The synonymy historically associated with M. submacula includes several nominal taxa described during the late nineteenth and early twentieth centuries, such as Lachnus incertus (Schouteden, 1906), L. rosae (Cholodkovsky, 1898), L. rosarum (van der Goot, 1912), L. subterraneus (Del Guercio, 1900), and Pterochlorus ogasawarae (Matsumura, 1917)¹². Examination of their original descriptions and subsequent taxonomic treatments indicates that many were established with limited diagnostic information or lack material, which complicates objective comparison with newly collected specimens. In contrast, the Korean population is consistently distinguished by a unique combination of diagnostic morphological characters and mitochondrial COI divergence, providing reproducible evidence that it represents a distinct evolutionary lineage.

Within this historical context, P. ogasawarae, described from an alate viviparous female in East Asia, has remained particularly poorly defined. To clarify its taxonomic relevance, we critically re-examined Matsumura’s original description¹² and extracted all available diagnostic information for direct comparison with the Korean material. Despite efforts to locate or verify primary type material through inquiries to relevant institutional and historical collections, no authentic specimens attributable to this species could be traced. Our assessment therefore relies on the published description and subsequent literature. The taxonomic placement of P. ogasawarae has varied among authors. Some treated it as a synonym of Lachnus (= Pterochlorus) tropicalis^13,14, whereas others transferred it to the synonymy of Maculolachnus submacula^15–17. These differing interpretations likely reflect the absence of type material and the limited diagnostic detail available in the original description. The hypothesis that P. ogasawarae could be conspecific with the Korean Maculolachnus taxa (M. koreanus sp. nov. or M. paiki) is not supported by either biological or morphological evidence. According to the original description¹², P. ogasawarae was collected on Quercus sp. (tentative) in Fagaceae, whereas all species of Maculolachnus recorded worldwide are associated with Rosa species and related hosts within Rosaceae.

Morphometric characters further exclude conspecificity. In P. ogasawarae, body length and antennal length are both 1.6 mm, yielding an antenna-to-body ratio of approximately 1.0. By contrast, alate viviparous females of M. koreanus sp. nov. have a body length of 2.38 to 3.18 mm (mean 2.95 mm) with antennae 1.28 to 1.54 mm (mean 1.39 mm), whereas M. paiki measures 2.56 to 2.86 mm in body length (mean 2.67 mm) with antennae 1.39 to 1.48 mm (mean 1.45 mm). The alate viviparous female of M. submacula itself reaches approximately 3.32 mm in body length, more than twice that of P. ogasawarae. Thus, both Korean species and M. submacula are substantially larger than P. ogasawarae and exhibit markedly different body-to-antenna proportions. Moreover, the alate viviparous female of Lachnus tropicalis sensu Ghosh (1982)¹⁴ is even larger, with a body length of 3.84 to 4.90 mm (mean 4.53 mm) and antennae 1.95 to 2.36 mm (mean 2.17 mm), further underscoring the discordance among these taxa. These size differences are non-overlapping and therefore inconsistent with intraspecific variation. The distribution of secondary rhinaria provides decisive diagnostic evidence. P. ogasawarae is characterized by approximately seven secondary rhinaria on antennal segment III, four on segment IV, and five on segment V. In contrast, M. koreanus sp. nov. bears 9 to 14 secondary rhinaria on segment III, 2 to 4 on segment IV, and none on segment V, while M. paiki possesses 3 to 6 on segment III, 1 to 2 on segment IV, and none on segment V. The consistent presence of numerous secondary rhinaria on antennal segment V in P. ogasawarae, versus their complete absence in both Maculolachnus species, represents a stable and taxonomically significant difference. Taken together, the distinct host-plant associations, non-overlapping size ranges, divergent body-to-antenna ratios, and fundamentally different patterns of secondary rhinaria unequivocally demonstrate that P. ogasawarae is not conspecific with either M. koreanus sp. nov. or M. paiki. The markedly larger dimensions of L. tropicalis further reinforce this conclusion and highlight the incongruence between P. ogasawarae and both competing synonymy concepts.

In the present study, our assessment necessarily relies on the original description of P. ogasawarae. Despite this limitation, P. ogasawarae can be clearly distinguished from the Korean Maculolachnus species and is most reasonably interpreted as an independent taxon. However, its precise taxonomic and phylogenetic placement remains unresolved within the scope of the present study. Although several characters suggest a closer affinity with Calaphidinae rather than Lachninae, this interpretation remains provisional, and clarification of its taxonomic identity and systematic position will require further investigation based on additional specimens and evidence.

Morphological, molecular, and biogeographical differentiation

Detailed comparative analyses between the Korean specimens and European Maculolachnus submacula revealed consistent and well-defined diagnostic differences that unequivocally support the recognition of M. koreanus sp. nov. as a distinct species (Table 1). The most prominent distinctions involve the relative proportions of the antennal segments and associated structures. In apterous viviparous females, M. submacula exhibits a lower ANT IV/ANT III ratio 0.33–0.38 (Fig. 3a) and PT 0.28–0.32 × BASE, whereas M. koreanus sp. nov. shows a markedly higher ANT IV/ANT III ratio 0.44–0.45 (Fig. 3c) and PT 0.35–0.37 × BASE. In alate viviparous females, M. submacula PT 0.35 × BASE (Fig. 3b), while M. koreanus sp. nov. PT 0.22–0.30 × BASE (Fig. 3d). In addition, the forewing spot of M. submacula is distinctly smaller (Fig. 3e) than that observed in M. koreanus sp. nov. (Fig. 3f). Further differences are evident in the genital plate (GP) morphology of apterous viviparous females. In M. submacula, the GP is more or less bilobed (Fig. 3g), whereas M. koreanus sp. nov. possesses a single plate with a shallow indentation along the proximal margin (Fig. 3h). The ultimate rostral segment (URS) also differs between the two species, measuring 0.67–0.73 × ANT VI in M. submacula (Fig. 3i) and 0.78–0.80 × ANT VI in M. koreanus sp. nov. (Fig. 3j). Apterous viviparous female, M. submacula exhibits an ANT IV/ANT III ratio of 0.33–0.38 (Fig. 3a), PT 0.28–0.32 × BASE, whereas M. koreanus sp. nov. shows a higher ratio of 0.44–0.45 (Fig. 3c), PT 0.35–0.37 × BASE. Alate viviparous female, M. submacula PT 0.35 × BASE (Fig. 3b), whereas M. koreanus sp. nov. PT 0.22–0.30 × BASE (Fig. 3d). In M. submacula, the forewing spot is smaller (Fig. 3e) compared to that of M. koreanus sp. nov. (Fig. 3f). Apterous viviparous female, the structure of the genital plate (GP) further distinguishes the two taxa: M. submacula bears a more or less bilobed GP (Fig. 3g), while M. koreanus sp. nov. has a single plate exhibiting a shallow indentation along the proximal margin (Fig. 3h). Apterous viviparous female, M. submacula URS 0.67–0.73 × ANT VI (Fig. 3i), the abdomen is completely membranous and smooth, whereas M. koreanus sp. nov. URS 0.78–0.80 × ANT VI (Fig. 3j), possesses a membranous abdomen with a distinct polygonally reticulated cuticular pattern. These diagnostic features were consistently observed across all examined morphs, underscoring their stability and taxonomic significance.

Molecular evidence provides additional support for the morphological differentiation. The mitochondrial COI sequences of M. koreanus sp. nov. and M. submacula exhibit a genetic divergence exceeding 2.4%, surpassing the interspecific threshold generally applied in aphid taxonomy. Phylogenetic analyses employing Bayesian inference, maximum likelihood, and neighbor-joining algorithms consistently recovered M. koreanus sp. nov. as a monophyletic lineage distinct from M. submacula, M. sijpkensi, and M. paiki. Likewise, species delimitation approaches (ABGD, ASAP, and bPTP) independently supported the recognition of four discrete molecular operational taxonomic units (MOTUs) corresponding to these species. Taken together, the integrative morphological and molecular evidence provides robust confirmation that the Korean population previously attributed to M. submacula represents an independent evolutionary lineage, herein formally described as a new species. Species of Maculolachnus are known to be oligophagous, mainly associated with woody plants of the family Rosaceae, particularly Rosa species. The host plant of M. paiki remains unknown, as no specimens have been observed directly on host plants. Further field investigations focusing on potential rosaceous hosts, especially Rosa spp., will be necessary to determine its host associations and to clarify possible host-related differentiation within the genus.

Morphological insights and taxonomic implications of SEM analyses

The inclusion of scanning electron microscopy provides essential morphological detail that substantially enhances the taxonomic value of the present study. In aphid taxonomy, particularly within morphologically uniform lineages such as Lachninae, SEM offers unparalleled resolution for detecting subtle diagnostic characters that are often overlooked under light microscopy. Although several aphid taxa, such as species of Eulachnus, display diagnostic chaetotaxy that is readily observable using light microscopy¹⁸, many lineages within Lachninae share external characters that are remarkably uniform. In these groups, subtle differences in cuticular structures and sensilla architecture can only be detected through high-resolution SEM imaging. Traditional slide-mounted preparations, which have long been used in aphid taxonomy, often distort the natural configuration of these structures. Consequently, SEM offers an irreplaceable means of documenting unaltered morphology, even when based on a single representative species within a genus. Such morphological datasets provide an important foundation for future comparative studies and for understanding character evolution across the group. Previous works have demonstrated the significance of SEM for resolving taxonomic questions in diverse aphid genera. Detailed SEM analyses have clarified diagnostic traits in Pseudessigella¹⁹, previously poorly known species of Uroleucon²⁰, Miyalachnus²¹, Macromyzus²², and in several studies on the morphology of Cinara^23,24. Furthermore, SEM examinations of a single species have contributed essential data later incorporated into broader systematic revisions, including those of Myzaphis²⁵, Macrosiphoniella in North America²⁶, Sinolachnus²⁷, and Nippolachnus²⁸. This integrated approach to species characterization, established in earlier works by Kanturski¹⁸ and subsequently by^19–29, has also been adopted across other aphid taxonomic studies³⁰ and in unrelated insect groups including Diptera³¹, Hymenoptera^30,32, and other Hemiptera^33,34. Collectively, these studies highlight the value of SEM as a source of high-resolution morphological evidence that strengthens species descriptions and provides a robust platform for future phylogenetic and evolutionary investigations.

Materials and methods

Collection and morphological identification

A total of 26 individuals of Maculolachnus koreanus sp. nov. and Maculolachnus paiki were collected in South Korea. The aphid samples were preserved in 90% ethanol, and slide-mounted specimens were prepared in Canada balsam following the method described by³⁵. Measurements and digital images were obtained using a Leica DMC 5400 (Leica Z16 APO) and Leica DM 4000B camera system (Active Measure version 3.0.3; Mitani Co. Ltd., Japan) cameras system. Abbreviations used for descriptions are as follows: ANT: antennae; ANT I, ANT II, ANT III, ANT IV, ANT V, BASE, and PT: antennal segments I, II, III, IV, V, base of VI, and processus terminalis of antennomere VI respectively; BD III: basal articular diameter of ANT III; LS ANT III: length of the longest setae of ANT III; BL: body length; MaxW: greatest body width; HW: greatest head width across compound eyes; GP: genital plate; HT I: first segment of hind tarsi; HT Ib: basal length of HT I; HT Id: dorsal length of HT I; HT Iv: ventral length of HT I; HT Ii: intersegmental length of HT I; HT II: second segment of hind tarsi; URS: ultimate rostral segment (segment IV + V); FEMORA III: hind femora; TIBIAE III: hind tibiae. ABD TERG I-VIII; abdominal tergite I-VIII. Particular lengths of HT I and their ratios follow^2,36,37.

Material examined

Maculolachnus blackmani, Kanturski & Chakrabarti, 2022.

Holotype: Apterous viviparous female, INDIA, Himachal Pradesh, Kufri (2,510 m) (Shimla), 10.XI.1968, on Rosa sp. (“wild rose” on the slide) (Rosaceae), K. Narayanan & David leg. [NHMUK014843455] (NHM). The material examined is deposited in the Natural History Museum in London, UK (NHM).

Maculolachnus koreanus sp. nov.

Holotype: apterous viviparous female. 1, South Korea, Gangwon-do, Chuncheon-si, Dong-myeon, Sanggyeol-ri, (GPS 37.8618, 127.8498) on Rosa multiflora, 7.vi.2024, leg. M. Lee, [240607-LMH-15], (MKAP-1), (SNU).

Paratypes: fundatrices. 5, South Korea, Gyeongsangbuk-do, Andong-si, Imdong-myeon, Galjeon-ri, (GPS 36.5662, 128.9600), on Rosa hybrida (Rosaceae), 6.vi.2024, leg. M. Lee, [240606-LMH-1], (MKFX-1, 2, 3, 4, 5), (SNU).

alate viviparous females. 2, South Korea, Gyeongsangbuk-do, Andong-si, Imdong-myeon, Galjeon-ri, (GPS 36.5662, 128.9600), on R. hybrida (Rosaceae), 6.vi.2024, leg. M. Lee, [240606-LMH-1], (MKAL-1, 2), (SNU).

3, South Korea, Gangwon-do, Chuncheon-si, Dong-myeon, Sanggyeol-ri (GPS 37.8618, 127.8498), on R. multiflora (Rosaceae), 7.vi.2024, leg. M. Lee, [240607-LMH-15], (MKAL-3, 4, 5), (SNU).

2, South Korea, Gangwon-do, Jeongseon-gun, Gohan-eup (GPS 37.183060, 128.892782), on R. multiflora (Rosaceae), 20.x.2024, leg. M. Lee, [241020-LMH-1], (MKAL-6, 7), (SNU). 2, South Korea, Gyeongsangbuk-do, Andong-si, Imdong-myeon, Galjeon-ri, (GPS 36.5662, 128.9600), on R. hybrida (Rosaceae), 9.xi.2024, leg. M. Lee, [241109-LMH-1], (MKAL-8, 9), (SNU). 1, South Korea, Gangwon-do, Pyeongchang-gun, Daegwanryeong-myeon, Hoenggye-ri, (GPS 37.6831, 128.7299), yellow pan trap. 26.ix.2019, leg. M. Lee, [190926-LMH-1], (MKAL-10), (SNU). 1, South Korea, Chungcheongbuk-do, Danyang-gun, yellow pan trap. 21–30.ix.1969, leg. W. Paik, (MKAL-11), (SNU). 1, South Korea, Jeollabuk-do, Muju-gun, yellow pan trap. 21–31.x.1969, leg. W. Paik, (MKAL-12), (SNU).

oviparous viviparous female. 3, South Korea, Gangwon-do, Jeongseon-gun, Gohan-eup, (GPS 37.183060, 128.892782), on R. multiflora (Rosaceae), 20.x.2024, leg. M. Lee, [241020-LMH-1], (MKOV-1, 2, 3) (SNU). 2, South Korea, Gyeongsangbuk-do, Andong-si, Imdong-myeon, Galjeon-ri, (GPS 36.5662, 128.9600), on R. hybrida (Rosaceae), 9.xi.2024, leg. M. Lee, [241109-LMH-1], (MKOV-4, 5), (SNU).

male (nymph). 3, South Korea, Gangwon-do, Jeongseon-gun, Gohan-eup, (GPS 37.183060, 128.892782), on R. multiflora (Rosaceae), 20.x.2024, leg. M. Lee, [241020-LMH-1], (MKMA-1, 2, 3), (SNU).

The holotype and paratypes of Maculolachnus koreanus sp. nov. are deposited in the College for Agriculture and Life Sciences, Seoul National University Seoul, Korea (SNU). One paratype (alate viviparous female, slide specimen) is deposited in the National Institute of Biological Resources (NIBR). One paratype (alate viviparous female, slide specimen) is deposited at the Zoological Collection of the University of Silesia in Katowice, Poland (DZUS).

Maculolachnus paiki Seo, 1994.

alate viviparous female. 2, South Korea, Gangwon-do, Pyeongchang-gun, Daegwanryeong-myeon, Hoenggye-ri (GPS 37.6831, 128.7299), yellow pan trap. 15.vii.2019, leg. M. Lee, [190715-LMH-1], (MPAL-1, 2), (SNU). alate viviparous female. 1, South Korea, Jeollabuk-do, Muju-gun, yellow pan trap. 21–30.vii.1970, leg. W. Paik, (MPAL-3), (SNU). alate viviparous female. 1, South Korea, Jeollabuk-do, Muju-gun, yellow pan trap. 1–10.vii.1970, leg. W. Paik, (MPAL-4), (SNU).

Maculolachnus sijpkensi Hille Ris Lambers, 1962.

Paratypes. apterous viviparous female. 3, Canada, Ivenhoe Lake, Rosa acicularis (Rosaceae), 6–7.ix.1959, leg. J. Sijpkens, [NHMUK014843464] (NHM). The material examined is deposited in the Natural History Museum in London, UK (NHM).

Maculolachnus submacula (Walker, 1848).

apterous viviparous female. 1, Poland, Branice, Rosa sp. (cultivated) (Rosaceae), 25.v.2020, leg. M. Kanturski. (MSAP-1), (voucher number: Ma 1), (SNU). The material examined is deposited in the College for Agriculture and Life Sciences, Seoul National University Seoul, Korea (SNU). apterous viviparous female. 2, Cambridge, Rosa sp. (Rosaceae), 17.ix.1944, [NHMUK014843582] (NHM). apterous viviparous female. 2, Bennekon., Rosa sp., 3.x.1971, leg. V. Eastop, [VFE 12,957] (NHM). apterous viviparous female. 4, Herts, Tring., 20.iv.1954. leg. G.H.E Hopkins, [15/54] (NHM). apterous viviparous females. 3, Entfield, Rosa sp. (Rosaceae), 12.vii.1987, leg. J.H. Martin, [5103] (NHM), alate viviparous female. 1, Banska Stiarnica, Rosa sp. (Rosaceae), 9.vi.1952, leg. V. Pašek, [BM 1983–340] (NHM). The material examined is deposited in the Natural History Museum in London, UK (NHM).

Molecular analyses

Molecular protocol

Genomic DNA was extracted non-destructively from individual samples collected from each colony using the DNeasy Blood & Tissue Kit (Qiagen, Düsseldorf, Germany) according to a modified manufacturer’s protocol, ensuring that voucher specimens were preserved for morphological examination. A 658 bp fragment of the cytochrome oxidase subunit I (COI) gene was amplified using the following primer pair: LepF 5′- ATTCAACCAATCATAAAGATATTGG-3′ and LepR 5′ TAAACTTCTGGATGTCCAAAAAATCA-3′³⁸. Polymerase chain reaction (PCR) reactions were performed using AccuPower PCR Premix (Bioneer, Daejeon, Republic of Korea) in a total volume of 20 µl. The thermal cycling profile consisted of an initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 45.2 °C for 30 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 5 min. Amplified products were verified on a 1.5% agarose gels, purified, and subsequently sequenced Sanger sequencing by Bionics Inc. (Seoul, Republic of Korea).

Sequence analysis and genetic divergence

In total, 27 COI sequences of Maculolachnus spp. were analyzed, including 20 sequences generated in this study and 7 sequences retrieved from GenBank. Adelges lariciatus (Patch, 1909) (GenBank accession number: JF883971) was used as an outgroup. The sequences were deposited in GenBank (accession numbers PV844038 to PV844056) and are detailed in Table S1. Raw sequences were assembled and edited using SeqMan pro ver. 7.1.0. (DNASTAR, Inc., Madison, Wisconsin, USA). Sequence alignment was performed using MEGA 7³⁹. Intra- and interspecific pairwise genetic distances between the groups were calculated using the pairwise distance method, which is based on the Kimura-2-parameter (K2P) model using MEGA 7³⁹.

Phylogenetic analysis and species delimitation

Phylogenetic analyses were conducted using Bayesian inference (BI), maximum likelihood (ML), and neighbor-joining analysis (NJ). The ML analysis was carried out using IQ-TREE ver. 2⁴⁰ with 1,000 replicates of ultrafast bootstrap approximation (UFB) with the best partition scheme and the best-fit substitution models found by PARTITION-FINDER2⁴¹. The BI analysis was conducted using MrBayes v.3.2.6⁴², the analysis was performed using 10 million Markov chain Monte Carlo (MCMC) generations, and trees were sampled every 1,000 generations. A burn-in of 25% of the sampled trees was applied to ensure adequate mixing of the MCMC chain, using the best partition scheme and the best-fit substitution models identified by PARTITION-FINDER2⁴². Results of both analyses were visualized using FIGTREE v.1.4.4.⁴³. The NJ analysis was conducted using MEGA 7, which is based on the Kimura-2-parameter (K2P) model³⁹. We followed three DNA-based species delimitation methods as outline by⁴⁴: Automatic Barcode Gap Discovery (ABGD), Assemble Species by Automatic Partitioning (ASAP), and Bayesian implementation of the Poisson Tree Processes model (bPTP). ABGD was performed using the ABDG web server (https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.htm, accessed on 15 Jul. 2024) and ASAP was performed using the ASAP web server (https://bioinfo.mnhn.fr/abi/public/asap/, accessed on 15 Jul. 2024) with alignment of sequences as an input file. bPTP was performed with the PTP web server (https://species.h-its.org, accessed on 15 Jul. 2024) using the IQ-TREE result file from the previous analysis as an input.

Population genetic analyses

The identification of variable and parsimony-informative sites and the number of haplotypes (h) were estimated using DnaSP v6.12.03⁴⁵. Based on the haplotype list generated from DnaSP v6.12.03, the number of private haplotypes unique to each population was determined (Supplementary Table 1). Haplotype data were generated in DnaSP v6.12.03 to identify distinct haplotypes. A haplotype network was constructed to estimate gene genealogies using the statistical parsimony approach at the population level using PopART⁴⁶.

Scanning electron microscopy

The methodology applied in this study followed the scanning electron microscopy (SEM) protocols previously described by^23,24 for species of the subfamily Lachninae. Maculolachnus representatives (alate viviparous females) used in this study were directly placed in 80% ethanol during the field studies. After storage, the samples were been dehydrated in ethanol series (80, 90 and 96%) and then transferred to 99,9% ethanol (two times for 10 min each). Dehydrated samples were transferred and kept in chloroform for 24 h in room temperature. Samples after dehydration and purification underwent the drying process using the Leica EM CPD 300 auto critical point dryer (Leica Microsystems, Vienna, Austria). After drying, the samples were mounted on aluminium stubs with double-sided adhesive carbon tape and sputter-coated in a Quorum 150 T ES Plus sputter coater (Quorum Technologies Ltd, Laughton, East Sussex, UK) with a 30 nm gold layer. Analyses were made using the Hitachi SU8010 field emission scanning electron microscope FESEM (Hitachi High–Technologies Corporation, Tokyo, Japan) at 10 kV accelerating voltage with a secondary electron detector in the SEM laboratory of the Institute of Biology, Biotechnology, and Environmental Protection, University of Silesia in Katowice (Katowice, Poland).

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

ML, MK, and SL designed the study. ML collected all the materials, molecular analyses, and wrote the manuscript. MK conducted the scanning electron microscopy (SEM) analysis and wrote the manuscript. SL supervised the whole process. All three authors revised the manuscript and confirmed the final version.

Data availability

The datasets generated and/or analysed during the present study are available in the GenBank repository under accession numbers PV844038–PV844056. All sequence data are publicly accessible at https://www.ncbi.nlm.nih.gov/genbank/.

Declarations

Competing interests

The authors declare no competing interests.