Research Article
Print
Research Article
Description of two new species of Dicranomyia (Erostrata) crane fly (Diptera, Limoniidae) from Korea, with remarks on DNA barcoding and updated taxonomic key
expand article infoJisoo Kim, Yeon Jae Bae
‡ Korea University, Seoul, Republic of Korea
Open Access

Abstract

Two new crane fly species, Dicranomyia (Erostrata) jejuensis sp. nov. and D. (E.) koreana sp. nov., from Korea are described on the basis of morphology and mitochondrial COI sequences. DNA barcode sequences for other four D. (Erostrata) species from Korea are also provided for the first time. The identification key for all known D. (Erostrata) species is presented.

Keywords

Dicranomyia (Erostrata) jejuensis, Dicranomyia (Erostrata) koreana, DNA barcode, Limoniinae, taxonomy

Introduction

Genus Dicranomyia Stephens, 1829, is the largest genus of the Limoniidae and, as such, contains 1,136 species and 24 subgenera, including the subgenus D. (Erostrata) Savchenko, 1976. Twelve species of this subgenus have been reported from the Palearctic, Nearctic, and Oriental regions (Oosterbroek 2023). Adult crane flies are typically found in moist deciduous forests, shrubs along small streams, and abandoned farmlands near streams (Alexander 1931a, b; Podenas et al. 2019, 2020). Meanwhile, larval D. (E.) globithorax has been reported in fungus on decaying logs (Rogers 1930). Data on DNA barcoding for this subgenus is unknown so far.

Alexander (1934) first reported D. (E.) tabashii (as Limonia (Limonia) tabashii) from Suigen (= Suwon) in Korea, and Podenas et al. (2019, 2020) added three and one additional species, respectively, to the recognized fauna of Korea. In Japan, Kato et al. (2018) revised six Dicranomyia species of the subgenus Erostrata including three new species and suggested that Limonia congesta Alexander, 1976 and Limonia striopleura Edwards, 1919 be classified as members of subgenus Erostrata based on non-genitalic characters.

Here, two new D. (Erostrata) crane fly species are described from Korea, providing identification for all Korean members of the subgenus. A DNA barcode (COI) dataset for six Dicranomyia species of the subgenus Erostrata from Korea is also presented for the first time.

Materials and methods

Crane fly sampling and examination

Crane fly adults were collected using insect nets or Malaise traps and preserved in 80% ethanol (Table 1). Wings and legs of selected adults were slide mounted using Euparal. Meanwhile, the genitalia and ovipositors of male and female specimens, respectively, were cleared overnight using 10% KOH and then preserved in micro-vials with glycerol. Specimens were examined using a microscope Olympus SZ51 with a digital camera Canon EOS 6D (Tokyo, Japan) and Olympus BX53 with camera Nikon Z7 (Tokyo, Japan).

Table 1.

Collection data of the Korean Dicranomyia (Erostrata) species used in the barcode analyses of this study.

Species* GenBank accession number Specimen code Locality Date Collector(s) Coordinates
D. (E.) globithorax OM102980 CF21-0149 Gangwon-do, Wonju-si 3 Sep. 2021 J. Kim, D. Lee 37°29'50.88"N, 130°53'23.78"E
D. (E.) jejuensis sp. nov. OM102981 CF21-0150H Jeju-do, Seoguipo-si 14 Jun.–4 Aug. 2021 Y. J. Bae 33°19'49.07"N, 126°37'28.08"E
OM102982 CF21-0150P Jeju-do, Seoguipo-si 4 Aug.–8 Sep. 2021 Y. J. Bae 33°19'49.07"N, 126°37'28.08"E
OM102983 CF21-0151 Jeju-do, Seoguipo-si 4 Aug.–8 Sep. 2021 Y. J. Bae 33°19'49.07"N, 126°37'28.08"E
D. (E.) koreana sp. nov. OM102979 CF21-0148 Jeju-do, Seoguipo-si 13 Jun.–4 Aug. 2021 Y. J. Bae 33°20'57.10"N, 126°29'43.29"E
OP081140 CF21-0152 Gyeongsangnam-do, Sancheong-gun 28 Jul. 2021 J. Kim, C. Lim, D. Lee, W. Lee 35°18'37.83"N, 127°45'05.47"E
D. (E.) submelas OM102978 CF21-0133 Jeju-do, Seoguipo-si 4 Aug.–8 Sep. 2021 Y. J. Bae 33°19'49.07"N, 126°37'28.08"E
D. (E.) tabashii OM102975 CF21-0115 Gyeongsangnam-do, Sancheong-gun 28 Jul. 2021 J. Kim, C. Lim, D. Lee, W. Lee 35°18'37.83"N, 127°45'05.47"E
OM102976 CF21-0115f Gyeongsangnam-do, Sancheong-gun 28 Jul. 2021 J. Kim, C. Lim, D. Lee, W. Lee 35°18'37.83"N, 127°45'05.47"E
D. (E.) yazuensis OM102977 CF21-0132 Gangwon-do, Pyeongchang-gun 28 Jul.–15 Sep. 2020 Y. J. Bae 37°47'05.67"N, 128°34'16.97"E
D. (D.) kandybinae OP093621 CF21-0099 Gangwon-do, Wonju-si 23 Jul.–3 Sep. 2021 Y. J. Bae 37°17'26.50"N, 128°04'54.77"E

The terminologies used to describe the morphology generally follow Cumming and Wood (2017), and de Jong (2017) for wing venation. The species distribution is given according to Oosterbroek (2023).

Specimen depositories are as follows: KUEMKorea University Entomological Museum, Seoul, Republic of Korea; NIBRNational Institute of Biological Resources, Incheon, Republic of Korea.

DNA extraction and sequence generation

Total genomic DNA was extracted from the leg muscle of using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. COI sequences were amplified and sequenced following Suh et al. (2019), except for the use of primers LCO1490 (5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3'; Folmer et al. 1994) and C1-N-2191 (5'-CCC GGT AAA ATT AAA ATA TAA ACT TC-3'; Simon et al. 1994), which targeted a 676-bp region of COI. All sequences were submitted to GenBank (accession numbers: OM102975OM102983; OP081140; OP093621).

DNA barcode sequence analysis

DNA barcode analysis was performed using 11 COI sequences (Table 1), which were generated from six Korean D. (Erostrata) species (10 sequences), and the outgroup species D. (Dicranomyia) kandybinae Savchenko, 1987 (1 sequence). Phylogenetic analyses were conducted using the neighbor-joining (NJ) method and Kimura-2-parameter model (Kimura 1980), with 1,000 bootstrap replicates, in MEGA X (Kumar et al. 2018). Sequence divergence was estimated via pairwise comparison of the uncorrected genetic distances (p-distances) in MEGA X, using the complete deletion option.

Checklist of the world Dicranomyia (Erostrata) crane flies (Oosterbroek, 2023)

Dicranomyia (Erostrata) canis (Alexander, 1931b)

Dicranomyia (Erostrata) cnephosa (Alexander, 1959)

Dicranomyia (Erostrata) congesta (Alexander, 1967)

Dicranomyia (Erostrata) cynotis (Alexander, 1931a)

Dicranomyia (Erostrata) globithorax Osten Sacken, 1869

Dicranomyia (Erostrata) globulithorax Alexander, 1924

Dicranomyia (Erostrata) jejuensis sp. nov.

Dicranomyia (Erostrata) koreana sp. nov.

Dicranomyia (Erostrata) melas (Alexander, 1934)

Dicranomyia (Erostrata) reniformis Kato, Tachi & Gelhaus, 2018

Dicranomyia (Erostrata) striopleura (Edwards, 1919)

Dicranomyia (Erostrata) submelas Kato, Tachi & Gelhaus, 2018

Dicranomyia (Erostrata) tabashii (Alexander, 1934)

Dicranomyia (Erostrata) yazuensis Kato, Tachi & Gelhaus, 2018

Key to the species of Dicranomyia (Erostrata), updated from Kato et al. (2018) and Podenas et al. (2020)

1 Tarsi with white bands Dicranomyia (Erostrata) congesta (India)
Tarsi without bands 2
2 Pleuron with broad, blackish brown lateral stripe Dicranomyia (Erostrata) striopleura (Indonesia, Malaysia)
Pleuron without lateral stripe 3
3 Scutellum obscure yellow 4
Scutellum yellowish brown to blackish brown 6
4 Rostrum black. Wing strongly blackened Dicranomyia (Erostrata) cnephosa (Nepal)
Rostrum pale. Wing tinged with pale brown 5
5 Palpus 2-segmented. Male seventh sternite with strongly darkened internal sac Dicranomyia (Erostrata) tabashii (Japan, Korea, Russia)
Palpus 3-segmented. Male seventh sternite with slightly darkened internal sac with rounded entrance Dicranomyia (Erostrata) koreana sp.nov. (Korea)
6 Gonostylus with black spines on mesal face 7
Gonostylus without black spines 12
7 Gonostylus narrowed to a point, triangular 8
Gonostylus not as above 9
8 Mesal face of gonostylus densely covered with black setae Dicranomyia (Erostrata) cynotis (Philippines)
Mesal face of gonostylus with black setae restricted to distal 1/2 Dicranomyia (Erostrata) canis (Philippines)
9 Paramere distally with rounded tip 10
Paramere distally with pointed tip 11
10 Gonostylus elongate with truncated apex Dicranomyia (Erostrata) globithorax (Canada, Japan, Korea, USA)
Gonostylus elongate with angled apex, length of angled apex ca 1/5 of gonostylus Dicranomyia (Erostrata) globulithorax (Japan, Korea, Russia)
11 Gonocoxite with apically rounded ventromesal lobe. Gonostylus shallowly concaved at inner apical edge, the area 1/5 as long as gonostylus Dicranomyia (Erostrata) melas (Taiwan)
Gonocoxite with apically truncated ventromesal lobe. Gonostylus deeply emarginated at inner apical edge, the area 1/3 as long as gonostylus Dicranomyia (Erostrata) submelas (Japan, Korea)
12 Gonostylus stout and reniform, tip of ventral surface covered with dense, fine setae Dicranomyia (Erostrata) reniformis (Japan)
Gonostylus not reniform, tip of ventral surface without dense, fine setae 13
13 Palpus 2-segmented. Gonostylus tapered in distal 1/3, without apical spine Dicranomyia (Erostrata) yazuensis (Japan, Korea)
Palpus 3-segmented. Gonostylus tapered in distal 1/2, with black apical spine Dicranomyia (Erostrata) jejuensis sp. nov. (Korea)

Taxonomic accounts

Family Limoniidae Speiser, 1909

Subfamily Limoniinae Speiser, 1909

Genus Dicranomyia Stephens, 1829

Erostrata Savchenko, 1976

Dicranomyia (Erostrata) Savchenko in Savchenko and Krivolutskaya 1976: 131–132; Kato et al. 2018: 182; Podenas et al. 2019: 72–73.

Type species

Dicranomyia globithorax Osten Sacken, 1869 (original designation).

Diagnosis

Rostrum is very short or reduced. Number of palpomeres ranges from one to three. Wings have no patterns, even in stigmal region. Third and fourth tarsomere are slightly swollen. Internal sac or notch is located on the male seventh sternite. Gonocoxite has ventromesal lobe. Gonostylus is one paired, with one or two setae arising from small tubercle on outer surface.

Dicranomyia (Erostrata) jejuensissp. nov.

Figs 1, 2

Type material

Holotype : Korea • ♂; Jeju-do, Seogwipo-si, Namwon-eup, Sillye-ri, Iseungi-oreum Volcanic Cone; 33°20.24'N, 126°37.25'E; alt. 450 m; 4 Aug.–8 Sep. 2021; Y. J. Bae leg.; Malaise trap; GenBank: OM102981; CF21-0150H; NIBR.

Paratypes : Korea • 1 ♀; same data as holotype, 14 Jul.–4 Aug. 2021; GenBank: OM102983; CF21-0151; KUEM • 1 ♂; same data as holotype; GenBank: OM102982; CF21-0150P; KUEM.

Diagnosis

Palpus is 3-segmented. Male seventh sternite has shallow V-shaped notch. Outer face of gonostylus has single seta arising from tubercle. Distal lobe of paramere has a hooked tip with a subapical process.

Description

Male (holotype). Body length 4.3 mm, wing length 4.6 mm, antenna length 0.9 mm. General body coloration pale yellow to yellowish brown (Fig. 1A).

Figure 1. 

Dicranomyia (Erostrata) jejuensis sp. nov. A habitus, male (paratype) B head, female (paratype) C wing, male (paratype) D male seventh sternite, ventral view E male terminalia, dorsal view F aedeagal complex, dorsal view G paramere, lateral view H female terminalia, lateral view I tip of hypovalva, lateral view. Abbreviations: ad – aedeagus; cc – cercus; fl – flagellum; gc – gonocoxite; gf – genital fork; gs – gonostylus; hv – hypovalva; pd – pedicel; pl – palpus; pm – paramere; sc – scape; tb – tubercle; t9 – ninth tergite; vlg – ventromesal lobe of gonocoxite. Scale bars: 2 mm (A, C); 0.5 mm (B, D, E, H); 0.1 mm (F, G, I).

Head (Fig. 1B). Dark brown dorsally, pale ventrally. Antennae 14-segmented; scape pale yellow; pedicel yellowish brown; flagellum brown. Rostrum pale, rudimentary. Palpus 3-segmented; basal 2/3 of first palpomere pale; remainder of palpus brown.

Thorax. Prescutum and presutural scutum yellow. Postsutural scutum, scutellum and mediotergite yellowish brown. Pleuron uniformly dull yellow, without lateral stripes. Wing (Fig. 1C) tinged with pale brown; veins brown; Sc ending before middle of Rs; sc-r at tip of Sc; Rs arched at base; R1 and R2 nearly transverse, at the same level; R3 and R4+5 parallel to each other; discal medial cell closed; m-cu before fork of M; CuP ending beyond tip of Sc. Halter pale brown. Legs with coxae and trochanters pale; base of femora pale, remainder of femora brown; tibiae and tarsi brown. Femur II 3.3 mm; femur III 3.8 mm; tibia II 3.5 mm; tibia III 4.0 mm; tarsus II 3.2 mm; tarsus III 2.8 mm. Claw without additional tooth.

Abdomen. Tergites yellowish brown, sternites 1–4 yellow, remaining yellowish brown. Seventh sternite with shallow V-shaped notch with darkened margin (Fig. 1D).

Male terminalia (Fig. 1E–G). Yellow. Ninth tergite with posterior margin rounded (Fig. 1E), medially with distinct emargination, distal part covered with setae. Gonocoxite elongated, approximately 3× as long as width at base, with elongated, setose ventromesal lobe. Gonostylus yellowish brown at base, turning dark distally; distal 1/2 of gonostylus gradually tapered toward apex, with short black spine at tip; dorsal margin near the base with small tubercle bearing pale, stout seta. Paramere (Fig. 1F, G) with basal 1/2 pale and apical 1/2 yellowish, distal lobe darkened apically with hooked tip and subapical projection. Aedeagus (Fig. 1F) as long as gonocoxite, bifid, curved outwards at tip.

Female. Body length 4.5 mm, wing length 4.8 mm, antenna length 0.9 mm (N = 1). General body coloration brighter than male. Femur I 2.8 mm; II 3.2 mm; III 3.4 mm; tibia I 3.2 mm; I: 3.1 mm; III 3.4 mm; tarsus I 3.0 mm; II 2.6 mm; III 2.4 mm.

Female terminalia (Fig. 1H–I). Yellow. Cercus curved upwardly (Fig. 1H), wider at base, narrowing towards acute tip. Genital fork long, ca 1.5× as long as width, extending to base of cercus. Hypovalva wedge-shaped, reaching to ca 2/3 of cercus, with distinct black spot at base. Dorsal and ventral margin of hypovalva serrated near tip (Fig. 1I).

Etymology

Specific name “jejuensis” refers to the type locality, Jejudo Island, Korea.

Distribution

The species is currently only known from Jejudo Island, Korea.

Habitats

Adults of this species are found in deciduous forests with moss-covered rocks along intermittent, rocky mountain streams (Fig. 2) and co-occur with D. (E.) submelas.

Figure 2. 

Habitat of Dicranomyia (Erostrata) jejuensis sp. nov.

Period of activity

Adults were collected from June through early September.

Remarks

Dicranomyia (E.) jejuensis sp. nov. is morphologically similar to D. (E.) yazuensis based on the male genital structures, but it can be distinguished by the following characters: pleuron entirely dull yellow (vs dark dorsally); palpus 3-segmented (vs 2-segmented); distal 1/2 of gonostylus tapered to tip (vs distal 2/3 strongly narrowed toward tip); posterior margin of male seventh sternite with shallow V-shaped notch (vs long triangular notch); distal part of paramere with hooked tip (vs straight tip).

Dicranomyia (Erostrata) koreanasp. nov.

Figs 3, 4

Type material

Holotype : Korea • ♂; Jeju-do, Seogwipo-si, Hawon-dong, Mt. Hallasan; 33°20.95'N, 126°29.72'E; alt. 1220 m; 13 Jun.–4 Aug. 2021; Y. J. Bae leg.; Malaise trap; GenBank: OM102979; CF21-0148; NIBR.

Paratypes : Korea • 1 ♂; Gyeonggi-do, Gapyeong-si, Buk-myeon, Jeokmok-ri, Garim-gyo (Br.); 37°58.60'N, 127°26.55'E; alt. 300 m; 25 Jul.–1 Aug. 2015; Y. J. Bae leg.; Malaise trap; published as D. (E.) tabashii by Podenas et al. (2019); KUEM • 2 ♂♂, 1 ♀; same data as for preceding; 2–8 Aug. 2015; published as D. (E.) tabashii by Podenas et al. (2019); KUEM • 1 ♂; same data as for preceding; 23–29 Jul. 2016; published as D. (E.) tabashii by Podenas et al. (2019); KUEM • 1 ♂; Gangwon-do, Inje-gun, Girin-myeon, Bangdong-ri, Mt. Bangtaesan; 37°54.50'N, 128°24.41'E; alt. 690 m; 30 Jul.–16 Sep. 2019; Y. J. Bae leg.; Malaise trap; KUEM • 1 ♂; Gyeonsangnam-do, Sancheong-gun, Sicheon-myeon, Jungsan-ri, Jungsan-ri Campsite, Mount Jirisan; 35°18.63'N, 127°45.09'E; alt. 700 m; 28 Jul. 2021; J. Kim, C. Lim, D. Lee, W. Lee leg.; sweeping; GenBank: OP081140; CF21-0152; KUEM.

Diagnosis

Palpus is 3-segmented. Center of male seventh sternite has a deep conical internal sac that has a wide, round entrance. Outer face of gonostylus has two setae arising from a small tubercle. Paramere is elongated and narrow, distally with a darkened tip.

Description

Male (holotype). Body length 3.5 mm, wing length 4.5 mm, antenna length 0.7 mm. General body coloration yellow (Fig. 3A).

Figure 3. 

Dicranomyia (Erostrata) koreana sp. nov. A habitus, male (holotype) B head, male (paratype) C wing, male (paratype) D male seventh sternite, ventral view E male terminalia, dorsal view F gonostylus, ventral view G aedeagal complex, dorsal view H paramere, lateral view I female terminalia, lateral view J tip of hypovalva, lateral view. Abbreviation: tb – tubercle. Scale bars: 2 mm (A, C); 0.5 mm (B, D, E, I); 0.1 mm (F, G, H, J).

Head (Fig. 3B). Dark brown dorsally, yellow ventrally. Vertex with a distinct black spot between compound eyes. Antennae 14-segmented; scape pale brown; pedicel yellowish brown; flagellum brown. Rostrum pale, reduced. Palpus 3-segmented, yellowish brown; basal 1/2 of first palpomere pale.

Thorax. Prescutum, scutum and scutellum yellow. Mediotergite yellowish brown. Pleuron entirely pale yellow, without lateral stripes. Wing (Fig. 3C) tinged with pale brown; veins brown; tip of Sc reaching ca 1/3 of Rs; sc-r at tip of Sc; R1 indistinct; R2 ending distinctly beyond tip of R1; discal cell closed; m-cu slightly beyond fork of M. Halter pale brown. Legs with coxae and trochanters pale yellow; femora and tibiae brownish yellow; tarsal segments light brown. Femur I 2.4 mm; femur II 2.8 mm; femur III 3.1 mm; tibia I 2.8 mm; tibia II 2.4 mm; tibia III 2.8 mm; tarsus I 2.9 mm; tarsus II 2.5 mm; tarsus III 2.4 mm. Claw without additional tooth.

Abdomen. Tergites yellow except pale eighth tergite; sternites paler. Seventh sternite (Fig. 3D) with central deep, conical, slightly darkened sac, and rounded entrance.

Male terminalia (Fig. 3E–H). Yellow. Ninth tergite rounded (Fig. 3E), wider basally, narrower apically; posterior margin with two short, setose lateral lobes separated by shallow U-shaped incision. Gonocoxite elongated, ventromesal lobe margin rounded and covered with setae, reaching beyond tip of aedeagus. Gonostylus (Fig. 3E, F) widened at base and narrowed at apex, with short, black dorsal spine at tip; dorsal margin near the base with small protuberance bearing two pale setae. Paramere (Fig. 3G, H) with basal part bilobed, distal lobe elongated and narrow, slightly darkened at tip. Aedeagus (Fig. 3G) apically bent downwards, bifid at tip.

Female. Body length 3.8 mm, wing length 4.7 mm, antenna length 0.7 mm (N = 1). General body coloration lighter than male.

Female terminalia (Fig. 3I–J). Yellow. Cercus curved dorsally (Fig. 3I), gradually tapered to pointed tip. Genital fork broad, as long as width, not extending to base of cercus. Hypovalva elongated, blade-shaped reaching slightly before tip of cercus, with distinct dark spot at basal area. Distal end bearing dorsal and ventral serration (Fig. 3J).

Etymology

Specific name “koreana” refers to the country of its discovery, Korea.

Distribution

The species is widely distributed in Korea, including Jejudo Island.

Habitats

This species is found along intermittent mountain streams in moist mixed forests with grassy vegetation (Fig. 4A) and in wet deciduous forest along the rocky margins of small mountain streams (Fig. 4B). Adults share their habitats with D. (E.) globulithorax on Mount Bangtaesan and with D. (E.) tabashii on Mount Jirisan.

Figure 4. 

Habitats of Dicranomyia (Erostrata) koreana sp. nov. A Jejudo Island B Mt. Jirisan.

Period of activity

Adults are mainly active from July through August.

Remarks

In terms of the shape of the male terminalia, D. (E.) koreana sp. nov. is similar to D. (E.) tabashii, but it can be distinguished by the following characters: palpus 3-segmented (vs 2-segmented); male seventh sternite with weakly darkened, conical internal sac with round entrance (vs strongly darkened, U-shaped internal sac); paramere with darkened tip (vs without). This species is also similar to another species, D. (E.) jejuensis sp. nov. based on the male genital structures, but it can be distinguished by the following characters: male seventh sternite with a deep, conical internal sac (vs shallow, V-shaped notch); gonostylus with two setae from tubercle (vs a single seta); paramere without hook at tip (vs with hook).

The male genitalia of D. (E.) koreana sp. nov. from Mount Bangtaesan differs from other materials of the species based on the shape of the seventh sternite internal sack (shallow conical without rounded mouth) and paramere distal lobe (pointed tip). However, additional specimens are needed to determine whether this difference is due to intra- or interspecific variation.

DNA barcode analysis

The 676-bp COI sequences contained 190 variable sites, of which 156 were parsimony-informative. The interspecific divergences (p-distances) within subgenus D. (Erostrata) ranged from 11.54% to 16.42%, with a mean distance of 13.17% across the entire dataset (Table 2), whereas the intraspecific genetic distances ranged from 0% to 0.59%: from 0% to 0.15% in D. (E.) jejuensis sp. nov., 0.59% in D. (E.) koreana sp. nov., and 0.15% in D. (E.) tabashii. The maximum intraspecific genetic distance (0.59%) was much smaller than the minimum interspecific one (11.54%). The NJ tree (Fig. 5) indicated that the monophyly of each of the new species was highly supported, as was that of subgenus D. (Erostrata) (Fig. 5).

Table 2.

Estimates of genetic divergence (%) between sequences. The number of base differences per site from between sequences are shown. Standard errors (%) are shown above the diagonal and were obtained by a bootstrap procedure (1,000 replicates). All positions containing gaps and missing data were eliminated (complete delete option).

Species Accession number 1 2 3 4 5 6 7 8 9 10 11
1 D. (E.) globithorax OM102980 1.36 1.36 1.36 1.36 1.37 1.28 1.42 1.41 1.37 1.34
2 D. (E.) jejuensis sp. nov. OM102981 13.61 0 0.15 1.37 1.35 1.36 1.34 1.33 1.33 1.45
3 OM102982 13.61 0 0.15 1.37 1.35 1.36 1.34 1.33 1.33 1.45
4 OM102983 13.76 0.15 0.15 1.36 1.34 1.36 1.34 1.33 1.34 1.45
5 D. (E.) koreana sp. nov. OM102979 15.24 14.94 14.94 14.79 0.30 1.38 1.21 1.20 1.31 1.45
6 OP081140 15.38 14.64 14.64 14.50 0.59 1.40 1.22 1.21 1.31 1.45
7 D. (E.) submelas OM102978 11.54 14.64 14.64 14.79 15.09 15.68 1.43 1.43 1.37 1.36
8 D. (E.) tabashii OM102975 16.12 14.94 14.94 14.79 11.69 11.83 16.42 0.15 1.29 1.44
9 OM102976 15.98 14.79 14.79 14.64 11.54 11.69 16.27 0.15 1.30 1.44
10 D. (E.) yazuensis OM102977 15.24 12.43 12.43 12.57 13.46 13.46 13.76 12.87 13.02 1.30
11 D. (D.) kandybinae OP093621 14.50 16.27 16.27 16.42 16.12 16.12 14.50 15.68 15.53 12.57
Figure 5. 

Neighbor-joining (NJ) Kimura-2-parameter tree based on the analysis of the COI of six Korean Dicranomyia (Erostrata) species and D. (Dicranomyia) kandybinae as outgroup. Numbers at the nodes indicate NJ bootstrap support values.

Discussion

This is the first study to use DNA barcoding for the delimitation of the D. (Erostrata) species. The present study identified two new species using both morphological and molecular data. According to the NJ tree (Fig. 5), the subgenus includes two major clades, which can be distinguished based on the presence or absence of numerous black, strong spines on the mesal face of gonostylus. Indeed, D. (E.) jejuensis sp. nov., D. (E.) koreana sp. nov., D. (E.) tabashii, and D. (E.) yazuensis can be distinguished from other members of their subgenus based on the shape and mesal face (without lots of black, strong spines) of their gonostyli. Two hypotheses may be considered: i) this clade can be classified into morphological species groups, or ii) it can be elevated to a new subgenus. Additional materials are needed to more accurately reconstruct phylogenetic relationships within genus Dicranomyia.

Based on our morphological examinations of the materials, we also found that some specimens identified as D. (E.) tabashii by Podenas et al. (2019) are actually specimens of D. (E.) koreana sp. nov. Based on our observation, unknown cryptic species of crane flies could also be detected and identified using molecular data.

Acknowledgements

We are deeply grateful to Dr Sigitas Podenas (Nature Research Centre and Vilnius University, Vilnius, Lithuania), who provided photographs of the D. (E.) tabashii type specimens, and to Dr Pjotr Oosterbroek (University of Amsterdam, Amsterdam, the Netherlands), who provided valuable information through his website, Catalogue of the Crane flies of the World (https://ccw.naturalis.nl/index.php). We are very grateful to members of the Laboratory of Biodiversity and Ecology, and the Korean Entomology Institute (Korea University, Seoul, Korea) for their assistance during field trips. We are also grateful to Dr Pavel Starkevich and Dr Daichi Kato for offering valuable comments and improving the manuscript. This work was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR202102204 and NIBR202203201).

References

  • Alexander CP (1924) New or little-known crane flies from northern Japan (Tipulidae, Diptera). Philippine Journal of Science 24: 531–611.
  • Alexander CP (1931a) New or little-known Tipulidae from the Philippines (Diptera). IX. Philippine Journal of Science 45: 415–448.
  • Alexander CP (1931b) New or little-known Tipulidae from the Philippines (Diptera). X. Philippine Journal of Science 46: 9–38.
  • Alexander CP (1959) Undescribed species of crane-flies from the Himalaya mountains (Tipulidae, Diptera). IV. Journal of the New York Entomological Society 67: 223–235.
  • Alexander CP (1967) New or little-known Tipulidae from eastern Asia (Diptera). LX. Philippine Journal of Science 95: 227–266.
  • Cumming JM, Wood DM (2017) Adult morphology and terminology. In: Kirk-Spriggs AH, Sinclair BJ (Eds) Manual of Afrotropical Diptera. Vol. 1. Introductory chapter and keys to Diptera families. Suricata 4. South African National Biodiversity Institute, Pretoria, 89–133.
  • de Jong H (2017) Limoniidae and Tipulidae (Crane Flies). In: Kirk-Spriggs AH, Sinclair BJ (Eds) Manual of Afrotropical Diptera. Vol. 2. Nematocerous Diptera and Lower Brachycera. Suricata 5. South African National Biodiversity Institute, Pretoria, 427–477.
  • Edwards FW (1919) Results of an expedition to Korinchi Peak, Sumatra. Part III. Invertebrates. II. Diptera. Collected in Korinchi, West Sumatra, by Messrs. H. C. Robinson and C. Boden Kloss. Journal of the Federated Malay States Museums 8: 7–59.
  • Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3(5): 294–299.
  • Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16(2): 111–120. https://doi.org/10.1007/BF01731581
  • Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547–1549. https://doi.org/10.1093/molbev/msy096
  • Osten Sacken CR (1869) Monographs of the Diptera of North America. Part IV. Smithsonian Miscellaneous Collections 8(219): 1–345.
  • Podenas S, Seo HY, Kim T, Hur JM, Kim AY, Klein TA, Kim HC, Kang TH, Aukštikalnienė R (2019) Dicranomyia crane flies (Diptera: Limoniidae) of Korea. Zootaxa 4595(1): 1–110. https://doi.org/10.11646/zootaxa.4595.1.1
  • Podenas S, Park S, Byun H, Kim A, Klein TA, Kim H, Aukštikalnienė R (2020) New data on Limoniinae and Limnophilinae crane flies (Diptera: Limoniidae) of Korea. Journal of Species Research 9(4): 492–531. https://doi.org/10.12651/jsr.2020.9.4.492
  • Rogers JS (1930) The summer crane-fly fauna of the Cumberland Plateau in Tennessee. Occasional Papers of the Museum of Zoology 215: 1–50.
  • Savchenko EN, Krivolutskaya GO (1976) Limoniid flies (Diptera, Limoniidae) of the South Kuril and South Sakhalin. Akad. Nauk. Ukr. SSR, Kiev, 160 pp. [in Russian]
  • Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P (1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Annals of the Entomological Society of America 87(6): 651–701. https://doi.org/10.1093/aesa/87.6.651
  • Suh KI, Hwang JM, Bae YJ, Kang JH (2019) Comprehensive DNA barcodes for species identification and discovery of cryptic diversity in mayfly larvae from South Korea: Implications for freshwater ecosystem biomonitoring. Entomological Research 49(1): 46–54. https://doi.org/10.1111/1748-5967.12334
login to comment