﻿Two new species of Macropelopia (Diptera, Chironomidae) from Oriental China, delineated with morphology and COI sequences

﻿Abstract Two new species, Macropelopia (Macropelopia) excavata Xu & Fu, sp. nov. and Macropelopia (Macropelopia) quadrimacula Xu & Fu, sp. nov., are described as male adults. A key to identify the males of Macropelopia from China is provided. Furthermore, in order to ascertain the genetic distance between these species and their morphological characteristics, mitochondrial cytochrome c oxidase subunit I gene sequences were uploaded to the National Center for Biotechnology Information. These COI sequences were then utilized to infer the relationships between the species, employing the neighbor-joining method.


Introduction
established the genus Macropelopia, with Isoplastus bimaculatus Kieffer as the type species.Macropelopia is classified in the tribe Macropelopiini and is further divided into two subgenera: Bethbilbeckia Fittkau et Murray and Macropelopia s. str.(Cranston and Epler 2013).Previous studies described a total of 18 species within the subgenus Macropelopia s. str.and three species in the subgenus Bethbilbeckia.The main difference between the two subgenera in the adult stage is that Macropelopia s. str.has a tibial comb, while Bethbilbeckia does not.Among these, 11 species of the subgenus Macropelopia are found in the Palearctic region (Kieffer 1912(Kieffer , 1916;;Tokunaga 1937;Fittkau 1962;Lencioni and Marziali 2005), one in the New North region (Roback 1971), two in the Oriental region (Tang and Niitsuma 2020;Hazra and Chaudhuri 2001), three in the Neotropical region (Andersen 2018;Silva and Pinho 2020;Dantas et al. 2023), and one in the African region (Freeman 1955).
According to Wang (2000), only one species of Macropelopia, M. nebulosa (Meigen), has been described based on adult, and four other species have been recorded based on larvae.Wang et al. (2011)  M. rotunda Wang, Cheng & Wang, which was discovered in Fujian Province.However, recent examinations conducted by Tang and Niitsuma (2020) have resulted in significant taxonomic revisions.The species M. grandivolsella has been synonymized with Macropelopia paranebulosa Fittkau, while Macropelopia rotunda is now referred to as M. kibunensis (Tokunaga), with these two species considered synonymous.Additionally, M. galbina was transferred to the genus Brundiniella.Furthermore, Tang and Niitsuma (2020) have described a previously unknown species, Macropelopia (Macropelopia) pergrandis, originating from Yunnan Province.
In this study, we report the discovery of two new species within the subgenus Macropelopia s. str., sourced from the remarkable Dabie Mountain National Nature Reserve situated in Hubei Province.The discovery holds significant importance as it contributes to the diversity of the genus.In addition, we have also assembled a key for distinguishing and identifying the known adult males of the subgenus in China.Moreover, we have conducted an analysis utilizing the mitochondrial cytochrome c oxidase subunit I (COI) gene to infer genetic distance and determine the differences between the species within the genus Macropelopia,further enhancing our understanding of their morphological characteristics.

Material and methods
Specimens were collected using the light trap induction method and preserved in 85% alcohol.Subsequently, they were sent to the laboratory for preliminary species identification and assigned individual numbers under a microscope.Images of slide mounts were obtained using a Nexcope NE930 compound microscope equipped with Capture 2.1 software.Genomic DNA was extracted from the thorax and legs of the specimens using the Qiagen DNA Blood & Tissue Kit.PCR amplification of the COI gene was performed following the primers and temperature regimes described by Folmer et al. (1994).After DNA extraction, the transparent exoskeleton was rinsed with 96% ethanol and mounted in Euparal on microscope slides, along with the corresponding antennae, head, wings, and legs, following the protocol outlined by Saether (1969).Morphological nomenclature adheres to Saether (1980), and measurements include the minimum, maximum, and average values for at least three specimens.All specimens are currently housed at the College of Biology and Agricultural Resources, Huanggang Normal University, China.Evolutionary analyses were conducted using MEGA 11.
Diagnostic characters.The distinguishing characteristics of this new species are the presence of two prominent longitudinal thick spots positioned in the middle of tergites II to IV, and the wing with brown markings on the distal end of Cu 1 and basal part of cell an.Additionally, the tergites from V to IX display a distinctive brown hue, adding to their identification.The posterior edge of tergite IX is concave in shape, and the anal point is absent.The gonostylus is prominently curved at a right angle.
Coloration (Fig. 1D).The head and thorax are uniformly dark brown.The femur of the legs is also dark brown, while the remaining parts of the legs are yellow.The wings exhibit two significant gray spots positioned near the Cu 1 and An veins.There is a longitudinal color spot present in the middle of tergites II to IV, the tergites V to IX and hypopygium are all brown in coloration.
Legs.The fore tibia possesses a single spur measuring 85 µm in length and features 15 side teeth.The width of the fore tibia at its apex is 84 µm, and the fore tibial comb consists of 5 setae (Fig. 1G).Two spurs of mid tibia are observed, measuring 87 µm and 51 µm long, bearing 16 and 11 lateral teeth, respectively, and has a width at the apex of 73 µm.The hind tibia exhibits two spurs, measuring 75 µm and 45 µm, with 18 and 12 lateral teeth, respectively.The hind tibial comb composed of 10 setae, with the longest seta 73 µm and the shortest 45 µm.The width of the hind tibia at its apex is recorded as 80 µm.The lengths and proportions of each leg are shown in Table 1.Hypopygium (Fig. 1G, H).The anal point absent.The phallapodeme, although short, measures at a clearly discernible length of 69 µm.The gonocoxite showcases a cylindrical shape and spans 206 µm in length.The gonostylus (Fig. 1K) is 125 µm long and exhibits a curved inward shape at approximately two-thirds of its length.It displays protrusions on both the inside and outside, gradually narrowing towards the tip.The megaseta is 16 µm long.The inferior volsella unconspicuous, along the inside of gonocoxite, and contains concentrated long inner microhairs.HR: 1.64, HV: 3.74.
Remarks.This new species is similar to M. kibunensis (Tokunaga) because their anal point is absent and the inferior volella is undeveloped, but can be distinguished by AR 2.24, wing with brown markings on distal end of Cu 1 and basal part of cell an, otherwise unmarked, and a concave rear edge line of tergite IX, while AR 1.7-1.9,wing with brown markings on distal end of Cu 1 , M 3+4 and M 1+2 .The pairwise distance based on the COI sequence of M. kibunensis and M. excavata sp.nov. is 0.105-0.107,further distinguishing them from each other.
Distribution.Hubei Province, Oriental China.Etymology.The name of this new species is derived from the Latin words "quartri" and "macula", meaning "four" and "spot", "stain" or "mark", respectively.The name specifically pertains to the presence of four distinctive black spots found on the tergites of this species.

Macropelopia quadrimacula
Diagnostic characters.This species has two short longitudinal striped spots on each side of tergites II to V, as well as two elliptical spots in the center.Wing with brown markings on apical of Cu1, M3+4 and basal part of cell an.Additionally, tergite IX with a triangular anal point beyond the margin of tergite IX.Lastly, the gonostylus is curved inward at two-thirds of its length, and the apex is markedly tapered.
Coloration (Fig. 2D).The head and thorax of this species are uniformly brown.The femurs of all legs are also brown, while the other sections display a yellow coloration.The wings are adorned with various color spots.Notably, there is a longitudinal color band on both sides of tergites II to V, accompanied by two elliptical spots at the center.Tergites VI to VII exhibit a dark brown hue, while tergites VIII to IX and the hypopygium are brown in color.
Remarks.This new species can be identified by the presence of two short longitudinal color bands on the sides of tergites II to V, along with two elliptical spots in the middle.These distinctive characteristics set it apart from other species within the genus.However, the abdominal spots of this species may sometimes be indistinct and appear blurry.In tergites III to V, these spots may be partially obscured by brown spots, but tergite II consistently displays four clearly visible elliptical spots.The overall shape of this new species is similar to that of M. kibunensis, and it shares the same gonostylus morphology.However, M. kibunensis lacks an inferior volsella and anal point, while this new species possesses a protrusion on the inferior volsella.The shape of the inferior volsella is comparable to that of M. excavata sp.nov., but this new species can still be differentiated by the presence of color spots on the tergites, the presence of the anal point, and a higher HR value (1.91-2.09)compared to M. excavata sp.nov.Based on COI sequences, the pairwise distances between M. quadrimacula and M. kibunensis, and between M. quadrimacula and M. excavata, are 0.119-0.125 and 0.131, respectively, further setting it apart from them.

Discussion
Based on the statistical data presented in this study, it has been revealed that there are currently seven species of the genus Macropelopia known to be distributed in China.However, it is important to note that the species Macropelopia (M.) notata and M. (M.) decedens, as reported by Wang et al. (2020), lack sufficient morphological characteristics for definitive identification.While we have included these species in the key provided in this study to reflect the current research records, further investigations are necessary to verify their distribution with more certainty.We successfully obtained eight COI sequences for two new species and downloaded an additional eight sequences for seven species from the National Center for Biotechnology Information (NCBI).Their taxonomic names and Gen-Bank accession numbers can be found in Fig. 3.By utilizing the neighbor-joining method (Tamura et al. 2021) for constructing a phylogenetic tree, our analysis revealed that M. excavata sp.nov.and M. kibunensis are closely related, as they appeared on the same branch of the tree.This finding is consistent with their shared morphological characteristics, such as the similar coloration of the tergites, anal point absent, and thorax features.Macropelopia quadrimacula sp.nov. is shown to be the sister group to (M. excavata sp.nov.+ M. kibunensis); the primary distinguishing features between M. quadrimacula sp.nov.and M. excavata sp.nov.lie in their hypopygium and the presence or absence of a dorsal stripe on their tergites.These results exemplify a strong congruence between the molecular and morphological data.Interestingly, our findings contrast with the description of M. kibunensis, which includes yellow femora and the wing with dense setae.In contrast, the two new species possess brown femora and a dorsal stripe on the tergites.Thus, within the genus Macropelopia, key criteria for morphological classification encompass the characteristics of the hypopygium and dorsal stripe patterns on the tergites, followed by the markings and macrotrichia of the wing, and the color and features of the legs.
Chironomids offer the advantage of having three distinct stages (larvae, pupae, and adults); the morphology of the larvae and pupae of Macropelopia also plays an important role in species delineation (Fittkau 1962, Roback 1978, Fittkau and Murray 1986, Tang and Niitsuma 2020).This study primarily focuses on morphological differences in the adult stage; collecting specimens from multiple life stages simultaneously remains a challenge.However,previous studies have demonstrated that COI is suitable for summarizing sequence diversity and detecting taxonomically challenging species within Macropelopia (Silva and Pinho 2020).Therefore, we anticipate further studies on species delineation using the COI gene segment to enhance the reliability of new species establishment.The analysis of partial DNA barcode sequences supports Macropelopia excavata sp.nov.and Macropelopia quadrimacula sp.nov.as valid species.

Figure 1 .
Figure 1.Macropelopia (Macropelopia) excavata Xu & Fu, sp.nov., male imago A head B antenna C wing D whole body of male adult E spotted shape of the tergites F thorax G fore tibial apex H hypopygium, dorsal view I photo of hypopygium, dorsal view J hypopygium, ventral view K gonostylus.

Figure 2 .
Figure 2. Macropelopia (Macropelopia) quadrimacula Xu & Fu, sp.nov., male imago A head B antenna C wing D whole body of male adult E spotted shape of the tergites F thorax G fore tibial apex H hypopygium, dorsal view I photo of hypopygium, ventral view J hypopygium, ventral view K gonostylus.

Figure 3 .
Figure 3. Neighbor-joining tree based on cytochrome c oxidase subunit I (COI) of sixteen Macropelopia specimens.Numbers on branches refer to the evolutionary distances computed using the Kimura 2-parameter method (Kimura 1980) and represent number of base substitutions per site.Taxa names include scientific names and GenBank accession numbers of corresponding COI gene fragments.