A New Species of Ascodipteron (Diptera: Hippoboscidae) from China Based on Morphology and DNA Barcodes

Simple Summary Ascodipteron Adensamer, 1896 is a small genus of bat flies distributed in tropical and subtropical areas of the Eastern Hemisphere, with 16 valid species currently known. Ascodipteron has not been taxonomically well-studied in China, where only four species have been reported. Here, a new species, Ascodipteron guoliangi sp. nov. from Fujian Province, is described, based on dealate neosomic females and supported by molecular data from the cytochrome B (Cytb) and cytochrome oxidase subunit I (COI) genes. Abstract A new species of the genus Ascodipteron Adensamer, 1896 (Diptera: Hippoboscidae) is described from Fujian, namely A. guoliangi sp. nov. Habitus and diagnostic details, as well as the attachment sites on the host, are documented with photographs. A detailed comparison of the new species with related species is provided and the new species is accommodated in the most recent key to the world species of Ascodipteron.


Introduction
Ascodipteron Adensamer, 1896 is a small genus belonging to the strebline grade of nycteribiine Hippoboscidae [1,2] and currently comprising 16 described species from tropical and subtropical areas of the Eastern Hemisphere [3,4]. Ascodipterine bat flies are highly specialized subcutaneous endoparasites, easily distinguishable from other streblines by their unusual mode of parasitic life and strong polymorphism in the adult stage, where females lose halteres, wings, and legs at the trochanter immediately after attachment to a host bat and transform into neosomes [3][4][5]. The neosome produces a considerable swelling in the bat's skin, but the only part visible externally is the posterior, knob-like tip of the abdomen [6,7]. Thus, these bat parasites have received attention from but a few entomologists.
South and Southeast China belong to the Oriental region and is an area known for its high biodiversity [8], but very few studies have been undertaken on the dipterous parasites of bats. According to present knowledge, only four species of Ascodipteron have been recorded in China [3,4], and in this study we describe a new species of Ascodipteron from Fujian, Ascodipteron guoliangi sp. nov., with extensive documentation of its dealate neosomic females, and it is incorporated in a key to the known species of the genus. Table 1. Specimens with GenBank accession numbers and host and locality. Superscript numbers behind binomial names indicate different specimens of the same species, from different hosts and/or habitats. Sequences downloaded from GenBank marked by asterisks. "-" indicates no data.

Species GenBank Accession Numbers Host Locality
Cytb COI Measurements and terminology follow Hastriter and Bush (2006) [5], except that the term "genital aperture" has been replaced with "terminalia". This term refers to the knob-like tip of the neosome abdomen that protrudes from the pouch or warble formed by the host (Figure 1D,E), and which is morphologically distinct from the remaining part of the abdomen (Figure 2A), and as such it conforms to contemporary terminology [9]. The terminalia are comprised of segments V-VII with the often diagnostic circular arrangements of setae plus the cerci, anus, and vaginal orifice. seum of Beijing Forestry University, Beijing, China (MBFU).

Specimen Imaging, Measurements, and Terminology
Z-stack photographs were acquired with a Zeiss Axio Zoom.V16 microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a PlanApo Z 1.0×/0.25 FWD 60 objective and an AxioCam 503 color camera. Images were processed with the software Zen 2 (Carl Zeiss AG, Oberkochen, Germany) and Adobe Photoshop 2021 (Adobe Systems Incorporated, San Jose, USA) by cropping, contrast enhancement, and removal of the background.
Photographs were taken with EF 100 mm f/2.8L IS USM and MP-E 65 mm f/2.8 1-5X lenses attached to a Canon 5D Mark IV SLR camera. Images and plates were processed on a standard Windows 10 platform using Adobe Photoshop 2021 (Adobe Systems, Inc., San Jose, CA, USA).
Measurements and terminology follow Hastriter and Bush (2006) [5], except that the term "genital aperture" has been replaced with "terminalia". This term refers to the knoblike tip of the neosome abdomen that protrudes from the pouch or warble formed by the host ( Figure 1D,E), and which is morphologically distinct from the remaining part of the abdomen (Figure 2A), and as such it conforms to contemporary terminology [9]. The terminalia are comprised of segments V-VII with the often diagnostic circular arrangements of setae plus the cerci, anus, and vaginal orifice.

DNA Extraction, Amplification, Sequencing and Sequence Editing
Two specimens (BFU-2435, BFU-2436) of Ascodipteron guoliangi sp. nov., four specimens of A. sp2, six of A. sanmingense Sun et al., and two of A. speiserianum Muir were dissected to extract total genomic DNA using the TIANamp Genomic DNA Kit (Tiangen, Beijing, China). Head and thorax were used for all specimens, except for two of A. sanmingense, which hatched from a puparium, and for which the whole body was used. After extraction, the head and thorax or whole body were cleaned with demineralized water and retained with the remaining body parts as vouchers, deposited in the Museum of Beijing Forestry University, Beijing, China (MBFU). The mitochondrial cytochrome B (Cytb) gene was amplified using the primer pair A5 (forward: 5 -AGG RCA AAT ATC ATT TTG AG-3 ) and B1.1 (reverse: 5 -AAA TAT CAT TCT GGT TGA ATA TG-3 ) [10]. The mitochondrial cytochrome oxidase subunit I (COI) gene was amplified and sequenced using primers LCO1490: GGT CAA CAA ATC ATA AAG ATA TTG G and HCO2198: TAA ACT TCA GGG TGA CCA AAA AAT CA [11]. PCR reactions were conducted as described in Zhang et al. (2016) [12] and Yan et al. (2019) [13], and amplification conditions were as described by Dittmar and Whiting (2003) [10] and Zhang et al. (2013) [14]. The PCR products were purified and sequenced bidirectionally by BGI Inc., Beijing, China.
SeqMan Pro v. 7.1.0 (DNASTAR Inc., Maddison, WI, USA) was used to edit and assemble the forward and reverse sequences.

DNA Extraction, Amplification, Sequencing and Sequence Editing
Two specimens (BFU-2435, BFU-2436) of Ascodipteron guoliangi sp. nov., four specimens of A. sp2, six of A. sanmingense Sun et al., and two of A. speiserianum Muir were dissected to extract total genomic DNA using the TIANamp Genomic DNA Kit (Tiangen, Beijing, China). Head and thorax were used for all specimens, except for two of A. sanmingense, which hatched from a puparium, and for which the whole body was used. After extraction, the head and thorax or whole body were cleaned with demineralized water and retained with the remaining body parts as vouchers, deposited in the Museum of Beijing Forestry University, Beijing, China (MBFU). The mitochondrial cytochrome B (Cytb) gene was amplified using the primer pair A5 (forward: 5′-AGG RCA AAT ATC ATT TTG AG-3′) and B1.1 (reverse: 5′-AAA TAT CAT TCT GGT TGA ATA TG-3′) [10]. The mitochondrial cytochrome oxidase subunit I (COI) gene was amplified and sequenced using primers LCO1490: GGT CAA CAA ATC ATA AAG ATA TTG G and HCO2198: TAA ACT TCA GGG TGA CCA AAA AAT CA [11]. PCR reactions were conducted as described in Zhang et al. (2016) [12] and Yan et al. (2019) [13], and amplification conditions were as described by Dittmar and Whiting (2003) [10] and Zhang et al. (2013) [14]. The PCR products were purified and sequenced bidirectionally by BGI Inc., Beijing, China.
SeqMan Pro v. 7.1.0 (DNASTAR Inc., Maddison, WI, USA) was used to edit and assemble the forward and reverse sequences.

DNA Sequence Analysis
The only three cytochrome b gene (Cytb) sequences of the genus Ascodipteron were downloaded from GenBank. The COI and Cytb sequences in this study, together with the Cytb sequences from GenBank, were aligned using Muscle as implemented in Mega X [15,16]. Subsequently, nucleotide sequence divergences were calculated, using the Kimura 2-parameter (K2P) model in Mega X.
Diagnosis. Ascodipteron guoliangi sp. nov. is most similar to A. phyllorhinae Adensamer and A. sanmingense. It can be separated from the former by the setae on the labial theca: ca. 50+, peg-like, spiniform setae dorsally and 100+ uniform setae ventrally in A. guoliangi sp. nov., versus 18-20 peg-like, spiniform setae dorsally and 46-50 uniform setae ventrally in A. phyllorhinae, and from both by R4 absent (present in A. phyllorhinae and A. sanmingense).
Description. Female. Anterior part of abdomen pyriform, the posterior part mushroomshaped. Head. Length and width of labial theca nearly equal ( Figure 2B,D and Figure 3B,C); posterior margin concave dorsally, convex ventrally. Labial theca dorsally with ca. 50+ peg-like, spiniform setae and ventrally with ca. 100+ similar setae. Peg-like setae identical to those on gena. Gena with ca. 40 irregularly scattered peg-like setae on dorsal half, anterior margin convex and posterior margin concave, approach triangular ( Figure 3A).

Etymology.
The new species is named in honor of Mr. Guo Liang (Fuzhou, China), an enthusiastic amateur entomologist who discovered the cave and thereby was instrumental to affording the first author the possibility to collect valuable specimens. The eponym is also a tribute to Mr. Guo Liang's decades of dedication to collecting and investigating the insects of Fujian Province.

Etymology.
The new species is named in honor of Mr. Guo Liang (Fuzhou, China), an enthusiastic amateur entomologist who discovered the cave and thereby was instrumental to affording the first author the possibility to collect valuable specimens. The eponym is also a tribute to Mr. Guo Liang's decades of dedication to collecting and investigating the insects of Fujian Province. Distribution.

Oriental-China (Fujian).
Remarks. Ascodipteron guoliangi sp. nov. is only known from China (Fujian). Its known host, C. frithiii, is considered to be relatively widespread in South Asia, but it is listed as 'near threatened' in the IUCN Red List [17].
Ascodipteron guoliangi sp. nov. will run to couplet 14 in the identification key to dealate ascodipterine females proposed by Hastriter (2007) [3], and it can be incorporated in the key together with the recently described A. sanmingense as follows, with host data given in square brackets: [Couplets 1-12 as in Hastriter (2007)

Biology
Muir (1912) [6], Jobling (1939) [7], Dick and Patterson (2006) [1], and Hastriter (2007) [3] provided extensive biological information for ascodipterines, with differences between species mainly including host and attachment sites. Ascodipteron guoliangi sp. nov. has so far only been recovered embedded in the elbow pit where the upper arm meets the forearm of Coelops frithii ( Figure 1A-C). A position in the elbow pit may reduce the risk of host grooming and facilitate access to blood vessels suitable for feeding ( Figure 1F), while allowing unimpeded breathing when the bat rests in the roost ( Figure 1A). Each neosome supports a thin layer of host skin, forming a bulge of the same shape as the neosome. Its flask-shaped body is smooth, with a very distinct constriction between the terminalia and the major, pyriform part of the abdomen, which lies inside the skin of the bat (Figure 1D,E). Ascodipteron guoliangi sp. nov. is the only Ascodipteron known to parasitize Coelops frithii. Five neosomes on a single host is the most we have ever seen, three on one forearm and two on the other, packed close together ( Figure 1A-C).
The cave where C. frithii was found is an abandoned bomb shelter located in a hilly area at the outskirts of Sanming, Fujian Province, China. The cave was dug into the slope along a gravel road, and it has two entrances to two short tunnels, which meet and then branch out again, the cave thereby taking the shape of an X. Each branch is about 1.3 m high and 1 m wide and with a depth of 3-4 m ( Figure 5A,B).  The cave is slightly damp, but there is no water seepage. The soil is a mixture of quartzite fragments and sandstone, and the cave floor is covered with accumulated bat droppings. During the present study, the cave was a habitat for 4-5 species of bats with about fifty individuals, of which only five or six were C. frithii, roosting together on the roof of the deepest parts of the cave ( Figure 5C). The cave is slightly damp, but there is no water seepage. The soil is a mixture of quartzite fragments and sandstone, and the cave floor is covered with accumulated bat droppings. During the present study, the cave was a habitat for 4-5 species of bats with about fifty individuals, of which only five or six were C. frithii, roosting together on the roof of the deepest parts of the cave ( Figure 5C).

Molecular Results
A 384 bp fragment of Cytb was sequenced from 11 Ascodipteron specimens containing five species. Pairwise comparison of the fragments gave an average genetic divergence of 4.90% between A. guoliangi sp. nov. and A. sp2, 6.31% between A. guoliangi sp. nov. and A. phyllorhinae, 9.77% between A. guoliangi sp. nov. and A. sanmingense, 13.20% between A. guoliangi sp. nov. and A. speiserianum, and 12.29% between A. guoliangi sp. nov. and A. sp2 ( Table 2). Table 2. Pairwise differences of mitochondrial cytochrome b gene (Cytb) sequences between species, based on Kimura 2-parameter. Numbers in column headers refer to species and populations listed in their respective rows. Superscript numbers behind binomial names indicate different specimens of the same species, from different hosts and/or habitats. Sequences downloaded from GenBank marked by asterisks. All specimens are neosomes obtained from a host except number 7, which is a male that emerged from puparium. A 672 bp fragment of COI was sequenced from 13 Ascodipteron specimens containing four species. Pairwise comparison of the fragments gave an average genetic divergence of 5.00% between A. guoliangi sp. nov. and A. sp2, 6.61% between A. guoliangi sp. nov. and A. sanmingense, and 8.42% between A. guoliangi sp. nov. and A. speiserianum (Table 3).
The intraspecific variation of all known ascodipterine Cytb and COI sequences are 0.00-0.79% (Table 2) and 0.00-1.45% (Table 3), respectively. Nucleotide sequence data were deposited in the GenBank database under the accession numbers indicated in Table 1. Table 3. Pairwise differences of cytochrome oxidase subunit I gene (COI) sequences between species, based on Kimura 2-parameter. Numbers in column headers refer to species and populations listed in their respective rows. Superscript numbers behind binomial names indicate different specimens of the same species, from different hosts and/or habitats. All specimens are neosomes obtained from host except number 9, which emerged from puparium.

Discussion
The present record of A. guoliangi sp. nov. is the first documentation of a bat fly from the Southeast Asian genus Coelops Blyth, 1848. Coelops belongs to the bat family Hipposideridae and contains two species, the widely distributed C. frithii (East Asian tailless leaf-nosed bat) and the more narrowly distributed C. robinsoni (Bonhote, 1908) (Malayan tailless leaf-nosed bat), from which no bat flies have been recorded. Of the six genera included in the family Hipposideridae, species of Ascodipteron have previously been recorded exclusively from Hipposideros Gray, 1831, although at least the genus Aselliscus Tate, 1941 has been examined for ectoparasites [5]. Current evidence indicates that bat flies are highly host specific [18], and species of Ascodipteron appear to be rather specific in the selection of attachment site [3,18,19]. The specimens of A. guoliangi sp. nov. obtained in the present study were all embedded in the elbow pit, and although several species of Ascodipteron are recorded for an attachment site on the wing, there are no records specifically from the elbow [3].

Data Availability Statement:
The data generated in this study are provided here, and they are also available upon request from the corresponding author.