﻿Description of two new species of Dicranomyia (Erostrata) crane fly (Diptera, Limoniidae) from Korea, with remarks on DNA barcoding and updated taxonomic key

﻿Abstract Two new crane fly species, Dicranomyia (Erostrata) jejuensissp. nov. and D. (E.) koreanasp. 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.


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. 2019Podenas et al. , 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, andPodenas 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 nongenitalic 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.

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).
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: KUEM -Korea University Entomological Museum, Seoul, Republic of Korea; NIBR -National Institute of Biological Resources, Incheon, Republic of Korea.

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.
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.
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).

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.
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; R 1 indistinct; R 2 ending distinctly beyond tip of R 1 ; 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.
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) 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.

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., 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).  ) 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.