Ascodipteron sanmingensis sp. nov., a new bat fly (Hippoboscidae: streblid grade) from Fujian, China

Abstract Background The bat fly genus Ascodipteron Adensamer, 1896 currently contains 15 species, all of which occur in tropical and subtropical areas of the Eastern Hemisphere. A new species of endoparasitic bat fly, Ascodipteron sanmingensis sp. nov., was collected from the Great Himalayan Leaf-nosed Bat, Hipposideros armiger (Hodgson, 1853), during ecological studies on bats in Fujian, China. New information A new species, Ascodipteron sanmingensis sp. nov., is described, based on dealate neosomic females and is supported by molecular data from a 368 bp fragment of the cytochrome B (Cytb) gene. 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
Bats are parasitised by some 500 species of hippoboscoid bat flies (Dick and Patterson 2006). These have traditionally been classified in the families Streblidae and Nycteribiidae, but increasing phylogenetic evidence suggests that the Streblidae are paraphyletic, with the monophyletic Nycteribiidae (which is the older name) nested inside an Old World 'streblid grade' (Petersen et al. 2007, Dittmar et al. 2015, Dittmar et al. 2006, Pape et al. 2011. Within this streblid grade, the Ascodipterine bat flies are highly specialised endoparasites, easily distinguishable from other streblids 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 (Hastriter andBush 2006, Hastriter 2007). Apart from the monotypic genus Maabella Hastriter & Bush, 2006, all other ascodipterines are assigned to the genus Ascodipteron Adensamer, 1896 and the distribution of diagnostic features would appear to support that the two taxa most likely are sister taxa (Hastriter andBush 2006, Hastriter 2007).
Early taxonomists, such as Adensamer (1896), Muir (1912), Monticelli (1898), Speiser (1908), Jobling (1939), Jobling (1940), Jobling (1952), Jobling (1956), Jobling (1958) and Lavoipierre (1946), studied Ascodipteron spp. from the Afrotropical Region, which formed the basis for further research on this genus and Maa (1965) revised the African species for the first time. Hastriter and Bush (2006) erected the genus Maabella and suggested Paraascodipteron Advani and Vazirani, 1981 should be placed in a different subfamily. Following these advances, Hastriter (2007) presented a worldwide species list, a key to all Ascodipterinae and a taxonomic review of the Oriental and Australasian species including what is known on their biology.
During the course of examining parasites collected from bats in Fujian, China, we found dealate females (neosomes) of one further undescribed species of Ascodipteron, which is morphologically similar to A. phyllorhinae Adensamer, 1896 andA. speiserianum Muir, 1912. Despite the flood of molecular data in the phylogenomic era (Misof et al. 2014, Kutty et al. 2019, Yan et al. 2020, morphological characters are still essential in phylogenetic and evolutionary studies (Giribet 2015). Therefore, the purpose of our study is to describe the new species and report on the host species and site of attachment and compare the new species with its most similar congeners, which will extend our knowledge of the taxonomy, distribution and biology of these parasitic flies.
Entire female ascodipterine bat flies (neosomes) were removed with forceps without hurting the host. Three specimens were preserved in 75% ethanol and seven in 95% ethanol, all deposited at the Museum 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 colour camera. Images were processed with the software Zen 2 and Adobe Photoshop CS6 (Adobe Systems Incorporated, San Jose, USA) by cropping, contrast enhancement and removal of the background.
Ecological 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 CS6 (Adobe Systems, Inc., San Jose, CA, USA).
Measurements and terminology follow Hastriter and Bush (2006).

DNA extraction, amplification, sequencing and sequence editing
One specimen (BFU-2227) of Ascodipteron sanmingensis sp. nov. was selected for DNA extraction. The specimen was dissected and abdominal muscle tissue was used to extract the total genomic DNA, using the DNeasy Blood & Tissue kit (Qiagen, Dusseldorf, Germany). The remaining body parts were retained as vouchers and deposited in the entomological collection of Beijing Forestry University. A fragment of 368 bp of the cytochrome B (Cytb) gene was amplified using the primer pairs 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') (Dittmar and Whiting 2003). PCR reactions were conducted as described in Zhang et al. (2016) and Yan et al. (2019) and amplification conditions as described by Dittmar and Whiting (2003). The PCR products were purified and sequenced bidirectionally by BGI Inc., Beijing, China.
SeqMan Pro v. 7.1.0 (DNASTAR Inc., USA) was used to edit and assemble the forward and reverse sequences.

DNA sequence analysis
We downloaded the only two mitochondrial cytochrome b gene (Cytb) sequences of the genus Ascodipteron from GenBank. The sequences, together with the Cytb sequenced in this study, were aligned using Muscle as implemented in Mega X (Kimura 1980, Kumar et al. 2018). Subsequently, nucleotide sequence divergences were calculated, using Kimura 2-parameter (K2P) model in Mega X.

Diagnosis
Labial theca slightly longer than wide ( Fig. 2B and D; Fig. 3B and C), R1, R2, R4 as minute, thick setae and R3, R5 as long setae. Most similar to A. phyllorhinae and A. speiserianum, but separable from the former by having 50＋ spiniform setae dorsally on the labial theca and a lateral fold on the lateral vertex and from the latter by the setose lateral vertex (Fig. 4A).

Etymology
The new species is named after its type locality Sanming.

Biology
Ascodipteron sanmingensis sp. nov. is embedded at the base of an ear or on the lower jaw area of Hipposideros armiger (Fig. 1A-D). The neosomes are not inserted perpendicular to the skin, but at a slight angle.

Molecular results
The genetic distance between Ascodipteron sanmingensis sp. nov. and A. phyllorhinae is 7.75% and between A. sanmingensis sp. nov. and an unidentified A. sp. is 11.86% (Table 1). The Cytb sequence generated in this study was deposited in GenBank (Accession: MW822598).

Remarks:
Ascodipteron sanmingensis sp. nov. will run to couplet 14 in the identification key to dealate ascodipterine females proposed by Hastriter (2007) and it can be incorporated in the key as follows, with host data given in square brackets:

Discussion
Hastriter (2007) revised the species of Ascodipteron from the Oriental and Australasian regions and provided a detailed review of what is known of their biology. He studied extensive material of A. phyllorhinae from across its extensive range and he mentioned that the specimens "may prove to represent more than one species" (p. 13). Given the very small morphological differences between A. phyllorhinae and A. sanmingensis sp. nov., a closer examination of the lectotype of A. emballoneurae Banks from Borneo would seem justified.
Only two other species of Ascodipteron are, so far, known to use Hipposideros armiger as host: A. longiascus Hastriter, 2007, known in four specimens obtained from a single specimen of H. armiger in China (Yunnan) and A. phyllorhinae, which appears to favour H. diadema Geoffroy, 1813 as its host (Hastriter 2007).
Ascodipteron sanmingensis sp. nov. is only known from China (Fujian), although its known host has a much wider distribution over most of South-East Asia (Bates et al. 2020). Hastriter (2007 concluded that "Of the hundreds of specimens studied across the entire Oriental and Australasian regions … there are only two previously described sympatric Ascodipteron species, A. phyllorhinae and A. speiserianum". Both A. phyllorhinae and A. speiserianum have broad distributional ranges, but are, in general, of a more southern distribution, with A. phyllorhinae reaching north into southern China (Guangxi) and A.
speiserianum reaching further north into China (Hainan, Taiwan) and southern Japan (Hastriter 2007). Further sampling is needed to fully assess the degree of sympatry for these three species.
Liu et al. 2018 evaluated the potential use of Cytb for insect species identification and found it almost as efficient as COI with a success rate of 95% correct identifications. (Gilarriortua et al. 2013, Liu et al. 2018). Dittmar et al. 2006 provided molecular data from two species of Ascodipteron from a single locality in Malaysia, Pahang. These were identified as A. phyllorhinae and a species given as new to science, but not described. Comparing data from the present material with Cytb sequences provided by Dittmar et al. 2006 reveals a difference of 7.75% with their A. phyllorhinae and of 11.86% with the species considered to be undescribed ( Table 1). The intraspecific variation of Cytb for insects has been estimated to be 0.66 ± 0.81% by Liu et al. (2018) in able 2, indicating that A. sanmingensis sp. nov. is a different species from A. phyllorhinae and A. sp.