Phylogenetic implications of the complete mitochondrial genome of Ogcogastersegmentator (Westwood, 1847) and first record of the genus Ogcogaster Westwood, 1847 from China (Neuroptera, Myrmeleontidae, Ascalaphinae)

Abstract Background The genus Ogcogaster Westwood, 1847, which is endemic to the Oriental Region and contains only five species, has been recorded in India and Pakistan, but not in China. The genus was not sampled in any previous study on the phylogeny of Neuroptera and its affinity within Ascalaphinae is unclear. New information The owlfly species Ogcogastersegmentator (Westwood, 1847) is firstly recorded from China, based on a female specimen collected from Yunnan Province, which represents the first record of the genus Ogcogaster from China. The complete mitochondrial genome of this species is first sequenced and described. The phylogenetic analysis, based on all 13 PCGs and two rRNA genes of the owlfly mitogenomes determined so far, assigned O.segmentator into a monophyletic group with Libelloidesmacaronius (Scopoli, 1763) and Ascalohybrissubjacens (Walker, 1853).

The genus Ogcogaster Westwood, 1847, which belongs to the tribe Ascalaphini, is endemic to the Oriental Region, currently including five species (Machado et al. 2019, Oswald 2022. The adults of Ogcogaster are characterised by the wings with particular marking pattern: crossveins in costal area of both wings each with a dark brown marking and brownish marking posteriad pterostigma in hind-wings, the short abdomen and the long male ectoprocts extending ventrally. Ogcogaster segmentator (Westwood, 1847) is a remarkable species with spectacular colouration and markings as for the genus. So far, O. segmentator has been recorded in India and Pakistan (Ghosh and Sen 1977, Mészáros and Ábrahám 2003, Hassan and Liu 2021, Oswald 2022. The phylogenetic status of this genus in Ascalaphinae is unknown.
In this study, we first record O. segmentator from China and sequenced the complete mitogenome of this species. The characterisation of the O. segmentator mitogenome is described in detail and a phylogenetic analysis based on the mitogenome data of owlflies is presented.

Dissection, photos and description
The classification system follows Machado et al. (2019). The terminology mainly follows Jones (2014) for body morphology, Aspöck and Aspöck (2008) for genitalia,  for wing venation and Machado and Oswald (2020) for wing fields. Genitalia were prepared by clearing the apex of the abdomen with saturated potassium hydroxide (KOH) solution in 135? for seven minutes. After rinsing the KOH with distilled water, the apex of the abdomen was stained with Chlorazol Black and then transferred to glycerine for further examination. The genitalia of the specimen is preserved in microvial with glycerine and pinned beneath the specimen.
Habitus photos were taken by using Nikon D850 digital camera with AF-S Micro Nikkor 105 mm 1/2.8G ED lens. Head, thorax and female genitalia were photographed by a Nikon D850 digital camera with Laowa 25 mm F/2.8 2.5-5.0X Ultra Macro lens. Photographs were adjusted and organised with Helicon Focus v.7.7.6 (method C) and Adobe Photoshop CS 6.0.

DNA extraction, PCR amplification and sequencing
Total genomic DNA was extracted and purified from the mid-leg muscle using TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China). For mitochondrial genome sequencing, we constructed a 150 bp paired-end sequencing library for the specimen and used Illumina NovaSeq platform to obtain 4 Gb of sequence data. Raw data were trimmed of adapters using Trimmomatic (Bolger et al. 2014). The sample was assembled by mapping the mitogenome of L. macaronius (GenBank accession: FR669150) using GENEIOUS v.9.0 (Kearse et al. 2012).

Annotation and bioinformatic analysis
Mitogenome sequence was uploaded to MITOS Web Server (http://mitos.bioinf.unileipzig.de/index.py) for initial mitochondrial genome annotation (Bernt et al. 2013). The tRNA genes were detected using invertebrate genetic codes. Secondary structures of the tRNAs were also predicted by MITOS. The annotation of protein coding genes (PCGs) and rRNA genes of this species was verified by hand alignment according to those of L. macaronius (GenBank accession: FR669150). The control region was identified by the ends of rrnS gene and the start of trnI gene. The AT and GC skews were measured using the following formulae: AT-skew = (A-T) / (A+T) and GC-skew = (G-C) / (G+C) (Perna and Kocher 1995). Nucleotide substitution rates, base composition and codon usage were analysed with MEGA 7.0 (Kumar et al. 2016). The circular map of the mitogenome was generated via CG view Server (http://cgview.ca/) (Grant and Stothard 2008).

Diagnosis
Forewing length: 25-40 mm, hind-wing length: 21-35 mm. Antenna nearly 3/4 as long as forewing. Gena wide. Crossveins in costal area of both wings each with a dark brown marking. Brownish marking posteriad pterostigma in hind-wings. Poststigmal area generally with two rows of cells. Abdomen short, nearly half as long as hind-wing. Male ectoproct elongated, slightly curved, distally swollened with some stout setae, extending ventrally.
Head. Vertex yellowish-brown, with brown setae. Scape, pedicel and basal flagellum generally yellow, remaining part of flagellum generally dark brown. Compound eye     Thorax. Yellow, with obvious black stripes. Pronotum medially with a transversal black marking, laterally with dense pale setae. Meso-and metathorax with dense long seate, most pale, but some brown. Mesoprescutum and mesoscutellum each with a T-shaped black marking; mesonotum laterally with a pair of discontinuous reversed U-shaped black marking. Metanotum with a black marking.
Wing. Forewing subcostal area yellow; crossveins near subcostal area each with a dark brown marking; pterostigma yellow; basal mediocubital crossveins each with a dark brown marking. Five presectoral crossveins present, with a biareolate cell; RP with five branches. Hind-wing marking similar to forewing, some indistinct dark brown markings posteriad pterostigma.
Abdomen. Yellow with many black stripes. Male genitalia. Tergite 9 very large, rhomboid-shaped with yellow and brownish pattern and medium-long stiff black hairs on margin caudally. In lateral view, tergite 9 with an acute apex caudo-ventrally. Ectropocts short, stiff, black setae, stronger inside than outside; in ventral views ectoprocts short, stiff, black seate, stronger inside than outside; in ventral view ectoprocts slightly curved downwards. Gonarcus (gonocoxites 11) hood-like, hairless, fused with parameres (gonocoxites 9), anterior side of parameres with tooth. Pelta present with seate, pulvini bag-like their hairs long and brownish. Setimere black, a bit longer than hairs on pulvini (Mészáros and Ábrahám 2003). Female genitalia. Gonocoxites 8 present as a pair of ovoid structures, with sparse short setae; gonapophyses 8 small, with sparse short setae. Gonocoxites 9 base separated, apex close, with sparse short setae. Ectoproct narrowed in lateral view, distally rounded.

Diagnosis
Body yellow with black markings. Scape, pedicel and basal flagellum generally yellow, but remaining part of flagellum generally dark brown. Meso-and metathorax with dense long seate, most pale, but some brown. Femur medially with a black marking. Subcostal area yellow; crossveins near subcostal area each with a dark brown marking; pterostigma yellow.

Mitogenomic characterisation of Ogcogaster segmentator
The complete mitochondrial genome of O. segmentator is a typical circular, doubledstranded molecule of 15,916 bp, with 37 genes (13 PCGs, 22 tRNAs and two rRNAs) and 15 non-coding regions. The tRNA rearrangement of trnC-trnW-trnY ( Wang et al. 2016) occurs in the mitogenome this species. Amongst all mitochondrial genes, 14 genes (including two rRNAs, four PCGs and eight tRNAs) are situated on the light strand (L) and the rest 23 genes (including nine PCGs and 14 tRNAs) are situated on the heavy strand (H) (Fig. 5, Table 2). Four gene overlaps were detected in the mitogenome of O. segmentator and the length of the largest one, which is present between atp8 and atp6, is only 7 bp.  The nucleotide composition of the mitogenome of O. segmentator is as follows: A = 40.3%, T = 34.4%, C = 14.9%, G = 10.4%, suggesting an obvious bias towards A and T. Its ATskew and GC-skew are 0.079 and -0.178, respectively and are similar to those of L. macaronius (Negrisolo et al. 2011).
Protein coding genes and codon usages. The total length of the 13 PCGs of the O. segmentator mitogenome is 12,186 bp. The A+T nucleotide composition of all PCGs is 73.4%. All 13 PCGs show a positive AT-skew (0.0736) and a negative GC-skew (-0.1804). The start/stop codons of the 13 PCGs are listed in Table 2. ATN (N represents A, T, C or G) is the most commonly used start codon for PCGs in the mitogenome of O. segmentator. Nonetheless, the start codon of cox1 in O. segmentator is ACG, which also can be found in L. macaronius (Negrisolo et al. 2011) and Suhpalacsa sp. (Song et al. 2019). The other PCGs, except nad1, use complete TAA or incomplete TA/T as their stop codons. Currently, the stop codon of nad1 in most owlfly species with the mitogenomes determined is known as TAG (Beckenbach and Stewart 2009, Negrisolo et al. 2011, Cheng et al. 2013, Gao et al. 2018), but TAT for Suhpalacsa sp. (Song et al. 2019).
The relative synonymous codon usage (RSCU) of the mitochondrial genes of O. segmentator was analysed (Fig. 6). The result suggests that the most frequently used codon is AAA-Lys (7.09%) and the least frequently used codon is CGG-Arg (0.08%). UUA-Leu2 has the highest relative synonymous codon usage (2.70), which indicates that UUA is the most preferred codon in the mitogenome of O. segmentator. The result also shows a preference in using A or T in the 3rd codon position of the PCGs in this species.  Transfer RNA genes. Twenty-two complete tRNA genes were detected in the mitogenome of O. segmentator and their secondary structures are shown in Fig. 7 respectively. The total length of the tRNAs is 1,471 bp, with length of each tRNA gene ranging from 63 bp (trnC) to 72 bp (trnV). The A+T content of the total tRNAs is 75.2%, but it shows negative AT-skew (-0.0079) and positive GC-skew (0.1566). Most tRNA genes, except trnS1, are folded as classical cloverleaf secondary structure. The absence of the dihydrouridine (DHU) arm in trnS1 is a common phenomenon in insects (Wolstenholme 1992). The T?C loop is absent in trnC of O. segmentator and in trnF and trnL1 of M. umbrosus (Gao et al. 2018). Most base pairs conform to the classical A-U and G-C pattern matching, but there are 25 G-U mismatched base pairs in the tRNA genes of the mitogenome of O. segmentator. Furthermore, infrequent U-U base pairs were observed in trnW of O. segmentator and in trnW of M. umbrosus (Gao et al. 2018). Ribosomal RNA genes and non-coding regions. The rrnL gene is located between trnL1 and trnV, with the length of 1,316 bp. The rrnS gene is located between trnV and A+T-rich region, with the length of 782 bp. The total length of two rRNA genes is 2,098 bp with an average AT content of 77.1%. The AT-skew and GC-skew of all rRNAs are 0.1413 and -0.2807 respectively, suggesting the positive AT-skew and negative GC-skew of this species.
Most of non-coding regions in arthropod mitogenomes are shorter than 20 bp (Cook 2005). The mitogenome of O. segmentator contains 15 non-coding regions, from 1 to 1,053 bp. Two of these intervals (S1 and the A+T-rich region) are larger than 20 bp ( Table 2). The intergenic spacer 1 (S1) is located between trnI and trnQ, with the length of 55 bp. This spacer is also present at the same position in the mitogenomes of other five owlfly species, from 42 to 55 bp (Beckenbach and Stewart 2009, Negrisolo et al. 2011, Cheng et al. 2013, Gao et al. 2018. The length of the A+T-rich region of O. segmentator is 1,053 bp and its AT and GC content are 83.8% and 16.3%, respectively. The A+T-rich region of this species shows a preference in using A over T and C over G, with the AT-skew of 0.0501 and GCskew of -0.0184.

Phylogeny
The BI and ML analyses generated trees with the same topology (Fig. 8). First, we found that Maezous umbrosus and Suhpalacsa sp. share a very long branch and have the interspecific divergence of the cox1 between each other to be just 0.002. Therefore, the sequenced specimens probably belong to same species and, considering the synonymy between Maezous and Suhpalacsa, the two mitogenomes (GenBank accession numbers KC758703 and MK301247) should be assigned to Maezous umbrosus (Esben-Petersen, 1913). All the owlflies herein sampled constitute a monophyletic clade, which is sister to the clade comprising two antlion genera of the subfamily Dendroleontinae, which, however, is a preliminary result, based on incomplete sampling. Within Ascalaphinae, Ascalop. appendiculata was assigned to be the sister group of the remaining owlfly species.
Ascaloh. subjacens was recovered to be the sister group to L. macaronius and both species constitute a sister group of O. segmentator. Maezous umbrosus was considered to be the sister group to the lineage of O. segmentator + ( L. macaronius + Ascaloh. subjacens).

Discussion
The phylogeny of owlflies, especially their relationship with the antlions, has always been controversial amongst recent studies using different sets of molecular data (Wang et al. 2016, Michel et al. 2017 (Vasilikopoulos et al. 2020) and the phylogenetic analysis, based on multi-loci data (Michel et al. 2017), recovered the monophyletic Ascalaphidae and Myrmeleontidae, respectively, although the sampling of ascalaphids in the latter study is much less than that of antlions. Based on both morphological and molecular genetic data,  also recovered the monophyly of traditional Ascalaphidae. Moreover, Jones (2019) presented another classification different from that in Machado et al. (2019) based on the multi-loci and morphological dataset. In Jones (2019), the monophyly of Ascalaphidae was corroborated and its family rank was restored. Besides, Jones (2019) treated the aforementioned tribes Palparini and Stilbopterygini as families, i.e. Palparidae and Stilbopterygidae. Thus, the molecular phylogenetic studies, which focus on owlflies and related antlions, are still needed by greater numbers of samples.
The relationships amongst major lineages of Ascalaphinae (or previous Ascalaphidae) is also debatable. According to the traditional classification, Ascalaphidae is divided into three subfamilies (Tjeder 1992): Albardiinae, with relatively short antennae; Ascalaphinae, with the split compound eyes; and Haplogleniinae, with the non-split compound eyes. However, neither split-eyed owlflies (Ascalaphinae or Ascalaphini) nor entire-eyed owlflies (Haplogleniinae or Haplogleniini) were recovered to be monophyletic, either based solely on the anchored hybrid enrichment (AHE) data (Machado et al. 2019) or the mixed data with a combination of morphological characters and molecular data (Jones 2014, Jones 2019). Notably, according to Machado et al. (2019), the split-eyed Ululodini (Ululodinae in Jones 2019) is distantly related to the other split-eyed Ascalaphini (Ascalaphinae in Jones 2019), which is sister group to the entire-eyed Haplogleniini. In addition, the entire-eyed owlfy genus Protidricerus was assigned into Ascalaphinae (Ascalaphini in Machado et al. (2019). He also emphasised that the spilt compound eye that was used as a key character of owlfly taxonomy should be removed, re-evaluated and re-interpreted.

2019) in Jones
So far, there have been very few phylogenetic studies focusing on intergeneric relationships of owlflies. The genus Ogcogaster was not sampled in any previous studies (Jones 2019, Machado et al. 2019). In Jones (2019), Ascaloptynx was assigned in a monophyletic group with several genera of Haplogleniini, but not all entire-eyed owlflies and this group was recovered to be sister group to the clade including many split-eyed genera, such as Ascalohybris, Libelloides, Maezous and Suhpalacsa. Thus, this phylogenetic framework is supported by the present result. Jones (2019) also recovered two subclades within the aforementioned clade of split-eye genera and Ascalohybris and Libelloides belong to a same subclade, while Maezous and Suhpalacsa belong to the other. Generally, our result is consistent with the result of Jones (2019). Concerning the generally concordant phylogeny recovered from a different dataset, our result suggests that the mitogenomes should be a helpful source of data for inferring the phylogeny of owlflies. In our result, Ogcogaster was recovered to be closer to the genera Ascalohybris and Libelloides. The similarity of morphological characteristics, especially the genital characters, amongst the three genera could also provide more support for this conclusion. A comprehensive worldwide phylogenetic analysis is needed for understanding the evolutionary history of Ascalaphinae.