Contribution to the Knowledge of Dicranoptychini (Diptera, Tipuloidea, Limoniidae) in China, with the First Mitochondrial Genome of the Tribe and Its Phylogenetic Implications

Simple Summary Limoniidae, the most speciose family in the superfamily Tipuloidea (crane flies), consists of four subfamilies (i.e., Chioneinae, Dactylolabidinae, Limnophilinae, and Limoniinae) and more than 11,000 species. However, its four-subfamily classification system has been controversial, among which the taxonomic position of the tribe Dicranoptychini (Limoniinae) is subject to debate. In addition, the knowledge of species diversity and geographical distribution of the tribe in China is seriously insufficient. We conducted investigations on Dicranoptychini in China and examined specimens from several Chinese localities to discover new species and new records and selected suitable materials for mitochondrial (mt) genome sequencing. In this study, two new and one newly recorded species from China have been found, which increases the number of Dicranoptychini species in the Chinese Mainland from four to seven, and adds three new provincial distribution records for the tribe in China. The first key for all Chinese Dicranoptycha species based on the type and non-type specimens and literature is also presented. The mt genome of one of the new species is sequenced and annotated, representing the first mt genome of the tribe Dicranoptychini. Phylogenetic analysis supports both new and traditional arrangements. This study will improve the knowledge of Chinese Dicranoptychini and provide genomics data and new horizons for the phylogeny of Limoniidae. Abstract Dicranoptychini is a tribe in the subfamily Limoniinae (Diptera, Tipuloidea, and Limoniidae) and includes only the genus Dicranoptycha Osten Sacken, 1860. However, the species diversity of the tribe in China was seriously underestimated, and the taxonomic status of Dicranoptycha has long been controversial. In this study, types of Chinese Dicranoptycha species and specimens collected from several localities in China were examined, and the first mitochondrial (mt) genome of the tribe Dicranoptychini is presented. Two Dicranoptycha species, D. jiufengshana sp. nov. and D. shandongensis sp. nov., from China, are described and illustrated as new to science. A Palaearctic species, D. prolongata Alexander, 1938, is recorded in China for the first time. In addition, the complete mt genome of D. shandongensis sp. nov. is sequenced and annotated, indicating that it is a typical circular DNA molecule with a length of 16,157 bp and shows a similar gene order, nucleotide composition, and codon usage to mt genomes of other Tipuloidea species. The two pairs of repeat elements are found in its control region. Phylogenetic results confirm the sister-group relationship between Cylindrotomidae and Tipulidae, question the position of the genus Epiphragma Osten Sacken, 1860 in Limoniidae, and indicate that Dicranoptychini may be a basal lineage within Limoniinae.

Dicranoptychini is one of the tribes in the subfamily Limoniinae and includes only the genus Dicranoptycha Osten Sacken, 1860 [7,8]. However, the taxonomic position of the genus Dicranoptycha has been subject to debate. Based on the reduced meron, the absence of tibial spurs and the absence of cell m 1 , Savchenko (1982) transferred Dicranoptycha to Gonomyini (Chioneinae) but considered this genus as an "aberrant representative of Eriopterinae" [9]. In a study by Savchenko (1989), Dicranoptycha was also listed among Gonomyini [10]. Later, Starý (1992) transferred Dicranoptycha to Limoniinae because of the two-branched radial sector of the wing, which is common in Limoniinae [1]. In most Chioneinae, the radial sector of the wing branches into two veins. The phylogeny of Tipuloidea based on characters of larvae and pupae indicated that Dicranoptycha strongly diverged from Limoniinae and Chioneinae [11], which was supported by Petersen et al. (2010) based on the combined morphological characters (adult, larvae, and pupae) and nuclear gene sequence data (28S rDNA and CAD) [3]. The recovery of Dicranoptycha as an independent lineage is similar to the placement of Dactylolabis Osten Sacken, 1860, so Petersen et al. (2010) proposed to treat Dicranoptycha as a separate subfamily, Dicranoptychinae, and defined this subfamily through a combination of characters, including completeness of larval head capsule, dorsal larval creeping welts absent, dorsal and ventral pupal creeping welts, wing venation, tibial spurs, antennal flagellomeres and male genetalic characters [3]. However, in a study by Starý (2021), Dicranoptycha was treated as a basal lineage within the Limoniinae [12].
Dicranoptycha is a cosmopolitan genus with 87 extant species from the Afrotropic (31 species), Palaearctic (24 species), Nearctic (23 species), Oriental (seven species), and Neotropic (three species) regions [2]. Nine fossil Dicranoptycha species have been found so far, among which the oldest representatives are from the Upper Cretaceous Burmese amber [13]. The most distinctive feature of the genus is the wing with a fold in the cell cu, which is not found in other genera of Limoniidae. The members of Dicranoptycha usually have one generation a year. Larvae are often found in relatively dry soil, such as dry woodland traversed by a small stream that is entirely dry [14], and open woodland far from running water [15]. Adults are usually found in open woodlands, often but not necessarily near water [14][15][16][17][18]. They often rest on the upper surface of the leaves of tall herbs and low shrubs [14], or in the undergrowth stratum of shrubs and herbs [18]. In addition, there are also some specialized dry-psammophilous species [19,20].
In the last half-century, taxonomic studies on the genus Dicranoptycha in the world have mainly focused on European and American species. A large number of studies have been carried out in Europe [17,[19][20][21][22][23]. Chen Wen Young made important contributions to the taxonomy of Dicranoptycha in North America [18]. Some taxonomic studies on Dicranoptycha in Asia have only been carried out in the past decade, mainly focusing on species of the Korean peninsula [24][25][26]. These studies have increased the number of Dicranoptycha species in the Korean peninsula from two to five. At present, Japan has the richest fauna of Dicranoptycha crane flies in Asia, with a total of nine species [2]. Only four Dicranoptycha species are known to occur in the Chinese Mainland, which spans the Palaearctic and Oriental regions: D. kwangtungensis Alexander, 1942, from Guangdong; D. pallidibasis Alexander, 1931, from Hebei; D. phallosomica Alexander, 1937, from Ningxia, Zhejiang, and Jiangxi; and D. vulpes Alexander, 1935, from Sichuan [2]. No new Dicranoptycha species from the Chinese Mainland have been described for more than 80 years, and the lack of systematic investigation for a long time has led to a serious underestimation of the species diversity of the genus.
To improve the understanding of the species diversity of Dicranoptycha crane flies in China, some investigations were initiated by the authors together with other entomologists. We examined specimens from several localities in China, resulting in the discovery of three Dicranoptycha species, of which D. jiufengshana sp. nov. from Inner Mongolia and D. shandongensis sp. nov. from Shandong are described as new to science, and D. prolongata Alexander, 1938, previously known from the Far East of Russia, North Korea, and South Korea, is recorded in China for the first time. The descriptions and illustrations of the males and females, as well as the first key to Chinese Dicranoptycha crane flies based on the type and non-type specimens and literature, are presented. Due to maternal inheritance, rapid evolution, and highly conserved gene content, the mitochondrial (mt) genome has been widely used in insect phylogenetic research . In 2016, we reported the first three mt genomes of Limoniidae [5]. After that, we and other researchers have successively published some mt genomes of Limoniidae [5,[55][56][57][58][59]. Prior to this study, 11 complete or nearly complete mt genomes of Limoniidae have been included in the GenBank. However, mt genomes that can be used for phylogenetic study are far from sufficient. To provide genomics data and new horizons for the phylogeny of Limoniidae, we sequenced and annotated the complete mt genome of D. shandongensis sp. nov., representing the first mt genome of the tribe Dicranoptychini. Based on this new sequence and previously published sequences, the phylogenetic relationships of Limonidae are also reconstructed. The monophyly of Limoniidae and the taxonomic position of Dicranoptychini will be mainly explored.

Specimen Collection, Observation, and Description
Specimens for this study were collected from several localities in China by different entomologists from 2013 to 2022. The type specimens of all Chinese Dicranoptycha species, deposited in the National Museum of Natural History, Smithsonian Institution, Washington, DC, USA (USNM) and the Institute of Zoology, Chinese Academy of Sciences, Beijing, China (IOZ), were examined during the study ( Table 1). The type specimens of D. jiufengshana sp. nov. are deposited in the Entomological Museum of China Agricultural University, Beijing, China (CAU). Other specimens including the type specimens of D. shandongensis sp. nov. are deposited in the Entomological Museum of Qingdao Agricultural University, Shandong, China (QAU). Genitalic preparations were made by macerating the apical portion of the abdomen in cold 10% sodium hydroxide for 12-15 h. The ZEISS Stemi 2000-C stereomicroscope was used for observations and illustrations. The Canon EOS 90D digital camera was used to take photos through a macro lens. The terminal abdomens were macerated in cold 10% sodium hydroxide for 12-15 h to better observe the genitalic structure. The specimens were soaked in 75% ethanol when examining the details of coloration. The morphological terminology mainly followed Cumming and Wood (2017) [60], while the terminologies for venation and terminalia followed de Jong (2017) [61] and Young (1987) [18], respectively. The following abbreviations in figures were used: aa = anterior apodeme; abc = aperture of bursa copulatrix; aed = aedeagus; aep = aedeagal process; cerc = cercus; gf = genital furca; goncx = gonocoxite; hyp vlv = hypogynial valve; i gonst = inner gonostylus; lp = lateral process; o gonst = outer gonostylus; st = sternite; tg = tergite; ve = vesica.

Phylogenetic Analysis
A total of 18 mt genomes were used for molecular analysis, including the newly sequenced mt genome in this study, 11 published mt genomes, and 6 mt genomes provided by the DNAmark Project conducted by F. Leerhoei in NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 11 April 2023) ( Table 2). All complete or nearly complete mt genomes of Limoniidae available in the GenBank of NCBI were used. Some mt genomes of Cylindro- tomidae and Tipulidae were also selected to verify the monophyly of Limoniidae. The mitochondrial genome of Pediciidae was used as an outgroup.
Individual gene alignments were checked manually in MEGA 7.0. Alignments of individual genes were concatenated using SequenceMatrix 1.8 [65] to generate the following four datasets: (1) PCGRNA matrix, including all three codon positions of 13 protein-coding genes (PCGs), and all available transfer RNA (tRNA) and ribosomal RNA (rRNA) genes (11, Phylogenetic trees inferred from the four datasets were constructed under Bayesian inference (BI) and maximum likelihood (ML) methods. MrBayes 3.2.7 [66] and RAxML 8.2.4 [67] were used for BI and ML analyses, respectively. The BIC criterion and greedy search algorithm in PartitionFinder 2.1.1 [68] were used to determine partitioning schemes and the best-fitting substitution models. In the BI analysis, four runs of 5-10 million generations were conducted simultaneously until the average standard deviation of split frequencies was below 0.01; samples were taken every 1000 generations and the initial 25% generations were discarded as burn-in. In the ML analysis, the GTRGAMMAI model was used, and the best ML tree was calculated with branch support estimated at 1000 bootstrap replicates. Diagnosis. Antenna with scape brownish yellow; pedicel yellow; first flagellomere brownish yellow with brown tip, remaining flagellomeres brown. Setae on flagellomeres just slightly exceeding the length of respective segments. Prescutum and presutural scutum brownish black. Pleuron dark brown. Wing with stigma indistinct, not darker than remaining wing area; Rs slightly longer than cell dm; cell dm about three times as long as wide; m-cu about its length beyond fork of M and approximately at two-fifth of cell dm. Outer gonostylus with smooth lower and upper margins. Aedeagal process umbrella-shaped, connected beneath the margin to a rod-like lower part. Aedeagus short and broad, concave at the tip, with two hook-like sclerites at the base. Aperture of bursa copulatrix in females with two sharp corners.
Description. Male. Body length 12.0 mm, wing length 10.5 mm. Head (Figure 1b), dark brown. Hairs on head dark brown. Antenna length 1.9 mm; scape brownish yellow; pedicel yellow; first flagellomere brownish yellow with brown tip, remaining flagellomeres brown. Scape long and cylindrical, 2.5 times as long as wide; pedicel oval, widened distally; flagellomeres subcylindrical, tapering apically and elongated. Setae on flagellomeres just slightly exceeding the length of respective segments. Mouthparts dark brown with dark brown hairs; palpus dark brown with dark brown hairs.  Thorax ( Figure 1c). Pronotum dark brown. Prescutum and presutural scutum uniformly brownish black. Postsutural scutum brownish black, paler medially and laterally. Scutellum brownish black, slightly paler medially. Mediotergite brownish black. Pleuron ( Figure 1a) dark brown, without stripes. Hairs on thorax white. Coxae and trochanters yellow; femora brownish yellow with wide dark brown tip; tibiae brownish yellow with wide brownish black tip; tarsi dark brown. Hairs on legs dark brown. Wing (Figure 1d) uniformly tinged with brownish yellow, except yellow costal field. Stigma indistinct, not darker than the remaining wing area. Veins brown, CuA and veins at wing base and in costal area brownish yellow. Venation: Sc long, ending conspicuously beyond fork of Rs; sc-r close to the tip of Sc; Rs slightly longer than cell dm; cell dm elongated, about three times as long as wide; m-cu about its length beyond fork of M, approximately at two-fifth of cell dm. Halter white, its length 1.5 mm.
Hypopygium (Figures 2 and 3a-c). Posterior margin of tergite 9 with a broad and shallow V-shaped emargination. Posterior margin of sternite 9 with a U-shaped emargination. Gonocoxite simple, elongate-oval. Outer gonostylus sclerotized distally and curved inwards with pointed apex, lower and upper margins smooth. Inner gonostylus nearly as long as the outer gonostylus, wide and fleshy, curved at two-thirds of its length, with a blunt apex and covered with sparse setae. Lateral process curved outwards, wide at the base and narrowed distally, apex pointed ( Figure 2). Anterior apodemes slender at the point of fusion with vesica, widening to broadly rounded apices. Aedeagal process umbrella-shaped, connected beneath the margin to a rod-like lower part. Aedeagus short and broad, concave at the tip, with two hook-like sclerites at the base (Figures 2 and 3a-c).   Remarks. This species is somewhat similar to D. buksubaeksaniana Podenas, Byun, and Kim, 2015, from North and South Korea with a similar male hypopygium (see Figure 11 in Podenas et al. 2015 [25]) but can be easily distinguished from the latter by the antenna with a pedicel lighter rather than the scape (Figure 1b), the uniform brownish black prescutum and presutural scutum (Figure 1c), the femora and the tibiae with wide darker tips (Figure 1a), and the vein Sc ending conspicuously beyond the fork of Rs (beyond the crossvein r-m). In D. buksubaeksaniana, the pedicel of the antenna is darker than the scape, the prescutum and presutural scutum have four dark brown longitudinal stripes, the femora, and tibiae have narrowly darkened distal ends [25], and the vein Sc ends slightly beyond the fork of Rs (before the crossvein r-m) (see Figure 10 in Podenas et al. 2015 [25]). The discovery of this species indicates that the tribe Dicranoptychini is recorded in Inner Mongolia, China, for the first time.  Female. Body length 11.5 mm, wing length 10.0 mm. Similar to male, but abdomen with segments 1-8 dark brown. Tergite 9 brownish yellow, posterior margin with a broad V-shaped emargination. Tergite 10 brownish yellow, base, and middle area paler. Cercus brownish yellow, caudal half widened and apex blunt. Hypogynial valve reaching approximately two-thirds of the cercus, setae on the dorsal margin long and distinct (Figure 3d-f). Vaginal apodeme as shown in Figure 3g,  Remarks. This species is somewhat similar to D. buksubaeksaniana Podenas, Byun, and Kim, 2015, from North and South Korea with a similar male hypopygium (see Figure  11 in Podenas et al. 2015 [25]) but can be easily distinguished from the latter by the antenna with a pedicel lighter rather than the scape (Figure 1b), the uniform brownish black

Dicranoptycha prolongata Alexander, 1938
Dicranoptycha prolongata Alexander, 1938: 139 [70]. Type locality: Ompo (North Korea). Diagnosis. Antenna with scape and pedicel dark yellow; first flagellomere dark yellow at base, remaining flagellomeres brown. Setae on flagellomeres just slightly exceeding the length of respective segments. Brown prescutum and presutural scutum with three broad dark brown longitudinal stripes, median stripe subdivided by a narrow line of brownish black. Pleuron dark brown with indistinct black longitudinal stripe. Wing wide; stigma indistinct, not darker than remaining wing area. Rs subequal to or slightly longer than cell dm Rs and dm; m-cu about its length beyond fork of M and approximately at two-fifth of cell dm. Outer gonostylus with outer and inner margins of the tip serrated. Aedeagal process with flattened upper part emarginates at the posterior margin, hook-like lower part bent downward across aedeagus. Aedeagus with a U-shaped emargination, each side with the apex pointed medially. Aperture of bursa copulatrix in females oval.
Description. Male. Body length 9.0-9.4 mm, wing length 9.5-10.0 mm. Head (Figure 4b), brown. Hairs on head dark brown. Antenna length 1.9-2.0 mm; scape and pedicel dark yellow, flagellomeres dark yellow at base, remaining flagellomeres brown. Scape long and cylindrical, 2.5 times as long as wide; pedicel globular, widened distally, nearly as wide as scape; flagellomeres subcylindrical, tapering apically. Setae on Hypopygium ( Figures 5 and 6a-c). Posterior margin of tergite 9 with a broad and shallow round emargination. Posterior margin of sternite 9 with two long, round-tipped medial lobes and deep narrow grooves between them. Gonocoxite simple, cylindrical. Outer gonostylus sclerotized distally and curved inwards, with pointed apex, outer and inner margins of the tip serrated. Inner gonostylus nearly as long as outer gonostylus, wide and fleshy, curved at two-thirds of its length, with a blunt apex and covered with sparse setae. Lateral process slender and straight, tip blunt and strongly curved dorsally ( Figure 5). Anterior apodemes fused, each slender at the point of fusion with vesica, widened at the tip. Aedeagal process with flattened upper part emarginates at the posterior margin, hook-like lower part bent downward across aedeagus. Aedeagus with a U-shaped emargination, each side with the apex pointed medially (Figures 5 and 6a-c). Thorax (Figure 4c). Prescutum and presutural scutum brown with three broad dark brown longitudinal stripes, median stripe subdivided by a narrow line of brownish black. Postsutural scutum dark brown with a brown median area. Scutellum brown. Mediotergite dark brown. Pleuron (Figure 4a) dark brown with indistinct black longitudinal stripe. Hairs on thorax white. Coxae and trochanters yellow; femora yellow with tips narrowly dark brown; tibiae yellow with tips narrowly dark brown; first tarsal segments dark yellow with tips narrowly dark brown, remaining tarsal segments dark brown. Hairs on legs brown. Wing (Figure 4d) wide, uniformly tinged with brownish yellow, except yellow costal field. Stigma indistinct, not darker than the remaining wing area. Veins brown, CuA and veins at wing base and in costal area yellow. Venation: Sc long, ending beyond fork of Rs; sc-r a short distance before the tip of Sc, a distal section of Sc twice as long as sc-r; Rs subequal to or slightly longer than cell dm; cell dm elongated, about three times as long as wide; m-cu about its length beyond fork of M, approximately at two-fifth of cell dm. Halter pale yellow, its length 1.3-1.4 mm.
Hypopygium (Figures 5 and 6a-c). Posterior margin of tergite 9 with a broad and shallow round emargination. Posterior margin of sternite 9 with two long, round-tipped medial lobes and deep narrow grooves between them. Gonocoxite simple, cylindrical. Outer gonostylus sclerotized distally and curved inwards, with pointed apex, outer and inner margins of the tip serrated. Inner gonostylus nearly as long as outer gonostylus, wide and fleshy, curved at two-thirds of its length, with a blunt apex and covered with sparse setae. Lateral process slender and straight, tip blunt and strongly curved dorsally ( Figure 5). Anterior apodemes fused, each slender at the point of fusion with vesica, widened at the tip. Aedeagal process with flattened upper part emarginates at the posterior margin, hook-like lower part bent downward across aedeagus. Aedeagus with a U-shaped emargination, each side with the apex pointed medially (Figures 5 and 6a-c).     Diagnosis. Antenna with scape and pedicel brownish yellow; first flagellomere brownish yellow with brownish black tip, flagellomeres 2-5 brownish black with bases brownish yellow, remaining flagellomeres brownish black. Setae on flagellomeres just slightly exceeding the length of respective segments. Prescutum and presutural scutum brownish black. Pleuron brownish black with anepimeron and dorsal half of katepisternum brownish yellow. Wing with stigma indistinct, not darker than the remaining wing area. Rs slightly longer than cell dm; cell dm about three times as long as wide; m-cu about two-thirds of its length beyond fork of M and approximately at one-third of cell dm. Outer gonostylus with outer margin serrated. Aedeagal process arrowhead-shaped, connected beneath the margin to a rod-like lower part. Aedeagus short and broad, bifurcated, each with a hook-like sclerite at the subbase. Aperture of bursa copulatrix in females with two rod-like branches.
Description. Male. Body length 10.6-11.0 mm, wing length 8.5-8.9 mm. Head (Figure 7b), brownish black. Hairs on head brownish black. Antenna length 1.6-1.8 mm; scape and pedicel brownish yellow; first flagellomere brownish yellow with brownish black tip, flagellomeres 2-5 brownish black with bases brownish yellow, remaining flagellomeres brownish black. Scape long and cylindrical, twice as long as wide; pedicel globular, widened distally, slightly wider than scape; flagellomeres subcylindrical, tapering apically, first flagellomere elongated. Setae on flagellomeres just slightly exceeding the length of respective segments. Mouthparts dark brown with brownish black hairs; palpus brownish black with first segment brown; hairs on palpus brownish black.
Thorax (Figure 7c). Pronotum dark brown. Prescutum and presutural scutum brownish black. Postsutural scutum brownish black, paler medially and laterally. Scutellum brownish black, slightly paler medially and posteriorly, with anterior margin black laterally. Mediotergite brownish black. Pleuron (Figure 7a) brownish black with anepimeron and dorsal half of katepisternum brownish yellow. Hairs on thorax white. Fore coxae dark brown with paler tip, middle and hind coxae yellow; trochanters yellow; femora and tibiae dark yellow; first tarsal segments dark yellow with narrowly brownish black tips, remaining tarsal segments brownish black. Hairs on legs brownish black. Wing (Figure 7d) uniformly tinged with brownish grey, except yellowish costal field. Stigma indistinct, not darker than the remaining wing area. Veins brown, CuA and veins at wing base and in costal area brownish yellow. Venation: Sc long, ending beyond fork of Rs, sc-r close to the tip of Sc; Rs slightly longer than cell dm; cell dm elongated, about three times as long as wide; m-cu about two-thirds of its length beyond fork of M, approximately at one-third of cell dm. Halter white, its length 1.3-1.4 mm.
Abdomen (Figure 7a). Tergites 1-2 brownish black; tergites 3-6 brownish yellow; sternites 1-6 dark yellow; segments 7-8 brownish black; segment 9 dark yellow with a dorsal side brownish black. Hairs on abdomen white.  Thorax (Figure 7c). Pronotum dark brown. Prescutum and presutural scutum brownish black. Postsutural scutum brownish black, paler medially and laterally. Scutellum brownish black, slightly paler medially and posteriorly, with anterior margin black laterally. Mediotergite brownish black. Pleuron (Figure 7a) brownish black with anepimeron and dorsal half of katepisternum brownish yellow. Hairs on thorax white. Fore coxae dark brown with paler tip, middle and hind coxae yellow; trochanters yellow; femora and tibiae dark yellow; first tarsal segments dark yellow with narrowly brownish black tips, remaining tarsal segments brownish black. Hairs on legs brownish black. Wing (Figure 7d) uniformly tinged with brownish grey, except yellowish costal field. Stigma indistinct, not darker than the remaining wing area. Veins brown, CuA and veins at wing base and in costal area brownish yellow. Venation: Sc long, ending beyond fork of Rs, sc-r close to the tip of Sc; Rs slightly longer than cell dm; cell dm elongated, about three times as long as wide; m-cu about two-thirds of its length beyond fork of M, approximately at one-third of cell dm. Halter white, its length 1.3-1.4 mm.
Hypopygium (Figures 8 and 9a-c). Posterior margin of tergite 9 with a broad and shallow V-shaped emargination. Posterior margin of sternite 9 with a U-shaped emargination. Gonocoxite simple, elongate-oval. Outer gonostylus sclerotized and curved inwards with pointed apex, outer margin serrated. Inner gonostylus longer than the outer gonostylus, wide and fleshy, curved at one-third of its length, with a blunt apex, and covered with sparse setae. Lateral process curved outwards, wide at the base, and narrowed distally, apex blunt ( Figure 8). Anterior apodemes wide at the point of fusion with vesica, narrowed at the tip. Aedeagal process arrowhead-shaped, connected beneath the margin to a rod-like lower part. Aedeagus short and broad, bifurcated, each with a hook-like sclerite at the subbase (Figures 8 and 9a-c). Hypopygium (Figures 8 and 9a-c). Posterior margin of tergite 9 with a broad and shallow V-shaped emargination. Posterior margin of sternite 9 with a U-shaped emargination. Gonocoxite simple, elongate-oval. Outer gonostylus sclerotized and curved inwards with pointed apex, outer margin serrated. Inner gonostylus longer than the outer gonostylus, wide and fleshy, curved at one-third of its length, with a blunt apex, and covered with sparse setae. Lateral process curved outwards, wide at the base, and narrowed distally, apex blunt ( Figure 8). Anterior apodemes wide at the point of fusion with vesica, narrowed at the tip. Aedeagal process arrowhead-shaped, connected beneath the margin to a rod-like lower part. Aedeagus short and broad, bifurcated, each with a hook-like sclerite at the subbase (Figures 8 and 9a-c).  Distribution. China (Shandong). Etymology. The species name shandongensis (adjective, feminine) refers to Shangdong, a province where the type locality is situated.
Habitat. Adults were collected in dryer places in forests and shrubs ( Figure 10). The species can be attracted by light.  Remarks. This species is somewhat similar to the D. gyebangsaniana presented by Podenas, Byun, and Kim, 2015, (see Figure 8 in Podenas et al. 2015 [25]) from South Korea and D. venosa by Alexander, 1924 (see Figure 14 in Podenas et al. 2015 [25]) from the Far East of Russia, South Korea, and Japan with a similar male hypopygium, but it can be easily distinguished from the latter two by the antenna with the scape and pedicel brownish yellow and the flagellomeres light brownish yellow at the base, turning darker (brownish black) distally (Figure 7b), the uniform brownish black prescutum and presutural scutum (Figure 7c), and dark yellow femora with the tips not darker or lighter than the remaining parts (Figure 7a). In D. gyebangsaniana, the flagellomeres are light reddish brown at the base and turn lighter (yellow) towards the distal end, the prescutum and presutural scutum are brown with indistinct longitudinal stripes, and the femora are obscure yellow and become dark brown in the distal third [25]. In D. venosa, the antenna has a black scape and obscure yellow pedicel (that is, the pedicel is conspicuously lighter than the scape), the prescutum and presutural scutum have four indistinct dark brown stripes, the fore femur is black with the basal third yellow and other femora obscure yellow with the basal third or shorter basal part black [71]. This species is also somewhat similar to D. fuscescens by Schummel, 1829, widely distributed in the Palaearctic region with a similar wing venation (see the only figure in Stubbs & Little 1974 [17]) and the male hypopygium (see Figure 3 in Starý 1972 [22]), but it can be distinguished from the latter by the uniformly brownish black prescutum and presutural scutum (Figure 7c), the dark yellow legs with tarsi brownish black (Figure 7a), and aperture of bursa copulatrix of female ovipositor with two rod-like branches (Figure 9g). In D. fuscescens, the prescutum and presutural scutum have dark longitudinal stripes, the legs are uniformly testaceous to brown with only the bases lighter, and the aperture of bursa copulatrix has no branches [22]. The discovery of this species indicates that the tribe Dicranoptychini is recorded in Shandong, China for the first time.  Rs nearly as long as cell dm, no more than 1.2 times the length of cell dm (Figures 1d, 4d and 7d)  Prescutum and presutural scutum with longitudinal stripes according to Alexander ( , 1931 [72,73] (Figure 4c) (Figures 4a and 7a In this study, we sequenced and annotated the complete mt genome of D. shandongensis (GenBank accession no. OR074105), which represents the first mt genome of the crane fly tribe Dicranoptychini. The mt genome is 16,157 bp in length and its gene composition and arrangement are identical to the presumed ancestral insect mt genomes [78] (Figure 11a). Thirteen protein-coding genes (PCGs), twenty-two transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control region (AT-rich region) are detected. For PCGs, COI, COII, COIII, CytB, ATP6, ATP8, ND2, ND3, and ND6 are coded in the majority strand, whereas ND4, ND4L, ND5, and ND1 are coded in the minority strand (Figure 11a). The lengths of tRNA genes range from 63 bp to 72 bp, whereas the lengths of large rRNA (lrRNA) and small rRNA (srRNA) genes are 1331 bp and 788 bp, respectively (Table S1). The control region is 1212 bp in length and located between the srRNA gene and tRNA Ile , including a total of 16 (TA)n regions, of which 10 are (TA)3, 3 are (TA)4, 1 is (TA)5, 1 is (TA)6, and 1 is (TA)7. In addition, there are two tandem repeat elements with a length of 59 bp and two 47 bp repeat elements with an interval of 329 bp in the control region (Figure 11b). The organization of the mt genome is generally compact. The intergenic spacers other than the control region are 19 in number and generally less than 26 bp, and the maximum intergenic spacer exists between ATP6 and COIII. The mt genome also has overlapping genes and most neighboring genes overlap by no more than 8 bp.
(0.04, 0.14, 0.32, and 0.22) ( Table 3). For this species, the whole mt genome and the five major partitions all had the same trends in the AT content, AT, and GC-skews, which display similar patterns and are consistent with the common base composition biases of mt genomes of Tipuloidea [5].  Gene map (a) and control region (b) of the mitochondrial genome of Dicranoptycha shandongensis sp. nov.
All PCGs of the mt genome show the typical ATN start codons (ATT/ATG) and the typical TAR (TAA/TAG) stop codons, except that TCG is the start codon for COI (Table  S1). The 62 codons of PCGs, with a total number of 3739, determine 22 amino acids, of which Leu (UUR) and Phe are the 2 most frequently used amino acids, and Arg and Cys are the least used amino acids (Table S2). Relative synonymous codon usage (RSCU) is also provided (Figure 12), which reflects how often a particular codon is used relative to the expected number of times that codon would be used in the absence of codon usage bias.    The whole mt genome and its five major genome partitions (i.e., PCGs, tRNA genes, lrRNA gene, srRNA gene, and the control region) are biased to a high content of AT bases (77.1%, 74.3%, 78.1%, 81.3%, 79.2%, and 92.9%) ( Table 3). The AT content of the control region is the highest, which is consistent with the trend of mt genomes in other crane flies [5]. This species shows positive AT-skew for the whole mt genome, tRNA genes, and the control region (0.00, 0.02, and 0.04); negative AT-skew for PCGs, lrRNA, and srRNA (−0.17, −0.05, and −0.01); negative GC-skew for the whole mt genome and the control region (−0.19 and −0.30); and positive GC-skew for PCGs, tRNA genes, lrRNA, and srRNA (0.04, 0.14, 0.32, and 0.22) ( Table 3). For this species, the whole mt genome and the five major partitions all had the same trends in the AT content, AT, and GC-skews, which display similar patterns and are consistent with the common base composition biases of mt genomes of Tipuloidea [5].

Phylogenetic Analyses
Eighteen representatives from all four families of Tipuloidea were included in the phylogenetic analysis. Eight phylogenetic trees inferred from the four datasets under BI and ML methods were constructed (Figures 13 and 14). In all BI and ML trees, Symplecta (Chioneinae) is a sister taxon to all non-pediciid crane flies (100% or 98% PP for all BI trees; 76%, 92%, 59%, and 78% BVs for ML trees). The monophyly of the remaining Limoniidae is supported in the trees inferred from the PCGRNA and PCG12RNA datasets under the BI method (96% and 89% PPs), as well as in the trees inferred from the PCG, PCGRNA, and PCG12RNA datasets under the ML method with low BVs (41%, 35%, and 19%). In the trees inferred from the PCG and PCG12 datasets under the BI method and the PCG12 dataset under the ML method, there is also a weakly supported arrangement; that is, the Limnophilinae and Chionea (Chioneinae) are a sister group to a clade of Cylindrotomidae and Tipulidae (51% and 58% PPs, 51% BV). However, Limoniidae is not supported as a monophyletic clade in all phylogenetic trees. The monophyly of Tipulidae is strongly supported (100% PP/BV for all trees). The sister relationship between Cylindrotomidae and Tipulidae is supported (100% or 97% PP for all BI trees; 53%, 64%, 54%, and 64% BVs for ML trees), which was accepted by Oosterbroek and Theowald (1991) [11], Ribeiro (2008)  Our Limoniidae species (excluding Symplecta) are strongly supported to be divided into two well-supported clades: a clade of the Limoniinae and Epiphragma (Limnophilinae) (100% PP for all BI trees; 82%, 85%, 89% and 91% BVs for ML trees) and a clade of the Limnophilinae and Chionea (100% or 97% PP for all BI trees; 57%, 59%, 48%, and 62% BVs for ML trees). Therefore, in this study, the monophyly of both Limoniinae and Limnophilinae with their current composition of genera is not supported, which was also proposed by Oosterbroek and Theowald (1991) based on 105 morphological characters of larvae and pupae [11], by Ribeiro (2008) based on 88 morphological characters [4], and by Petersen et al. (2010) based on the combined morphological characters and two nuclear genes [3]. However, it does not mean that the Limoniinae and Limnophilinae are artificial groups, and it is more likely that the position of some genera in the classification of Limoniidae is erroneous. Due to insufficient sampling and the problem of the long branch attraction, further studies with more representatives are needed to confidently define the monophyly of the Chioneinae.
Within the clade of Limoniinae and Epiphragma, the sister relationship between Dicranoptychini and Epiphragma (100% PP for all BI trees; 82%, 74%, 88%, and 87% BVs for ML trees) and the monophyly of the remaining Limoniinae (100% PP/BV for all trees) are strongly supported in all phylogenetic trees. This means that the position of Epiphragma in the classification of Limoniidae may be erroneous. Savchenko (1986) mentioned that Epiphragma differs significantly from all other genera of the Limnophilinae in several important characters [79]. This genus should possibly be transferred to Limoniinae, or it may be a possible transition to Limoniinae. Dicranoptychini is more likely to represent a basal lineage within the Limoniinae.

Phylogenetic Analyses
Eighteen representatives from all four families of Tipuloidea were included in the phylogenetic analysis. Eight phylogenetic trees inferred from the four datasets under BI and ML methods were constructed (Figures 13 and 14).   In all BI and ML trees, Symplecta (Chioneinae) is a sister taxon to all non-pediciid crane flies (100% or 98% PP for all BI trees; 76%, 92%, 59%, and 78% BVs for ML trees). The monophyly of the remaining Limoniidae is supported in the trees inferred from the PCGRNA and PCG12RNA datasets under the BI method (96% and 89% PPs), as well as in the trees inferred from the PCG, PCGRNA, and PCG12RNA datasets under the ML method with low BVs (41%, 35%, and 19%). In the trees inferred from the PCG and PCG12 datasets under the BI method and the PCG12 dataset under the ML method, there

Conclusions
The taxonomic study here is intended to improve the knowledge of Dicranoptychini in China. Two new species and a newly recorded species from China are found in this study, which increases the number of Dicranoptychini species in the Chinese Mainland from four to seven, and adds three new provincial distribution records for the tribe in China. This study also presented the first mt genome of the tribe, which is a typical circular DNA molecule with a length of 16,157 bp. The newly sequenced mt genome shows a similar gene order, nucleotide composition, and codon usage to mt genomes of other crane flies. It is worth noting that two tandem repeat elements with a length of 59 bp, and two 47 bp repeat elements with an interval of 329 bp are found in the control region. Phylogenetic analysis indicates that the position of the genus Epiphragma in the classification of Limoniidae needs to be revised, and Dicranoptychini may be a basal lineage within the Limoniinae. This study also reconfirms the traditional view that Cylindrotomidae and Tipulidae have a sister-group relationship. The phylogenetic results in this study are very preliminary, and a more complex history of independent lineages of Limoniidae needs to be revealed based on more data in the future.