The Complete Mitochondrial Genome of Glischropus bucephalus (Vespertilionidae; Chiroptera) Provides New Evidence for Pipistrellus Paraphyly

: The Indochinese thick-thumbed bat, Glischropus bucephalus , was described in 2011


Introduction
The name Glischropus was established by G. Dobson as a subgenus of Vesperugo for two species: African Vesperugo nanus (now-Afronycteris nanus) and Asian V. tylopus [1].Later, it was raised to a full genus as its distribution is restricted to Southeast Asia (see Tate: [2]).Tate considered three species within the genus: the relatively widespread G. tylopus; G. batjanus Matschie, 1901; and G. javanus Chasen, 1929, with the two latter species known only from their type localities.Glischropus batjanus was then considered a partial synonym of G. tylopus, and a new species, G. bucephalus, was described as ranging from the mainland Asian north of the Isthmus of Kra [3].Later, one extra species, G. aquillus, was described in Sumatra [4], and more recently, another new species, G. meghalayanus, was revealed in Meghalaya, India [5].
When separated as a genus, Glischropus was differentiated from Pipistrellus by its developed thumb pads (similar to that of Tylonycteris) and by the outer upper incisor, which is outwardly displaced from the row of teeth; otherwise, it was considered quite similar to Pipistrellus per se [2,6].Chromosomal and genetic data support a close relationship between Glischropus and Pipistrellus [7,8], and it was placed within the tribe Pipistrellini [9].Recent genetic data suggest that Glischropus represents one of the taxa that make Pipistrellus paraphyletic ( [10]).However, whole mitogenomes, which seem to be useful tools for phylogenetic research (e.g., ref. [11]), were not yet studied for this genus.
Glischropus bucephalus is a small bat species (forearm length 31.7-35.7 mm) of pipistrellelike appearance.Characteristic thickened pads on their thumb bases and plantar surface distinguish it from the similarly sized Pipistrellus.This feature is considered to have evolved as an adaptation for roosting in shelters with smooth surfaces like bamboo stems; however, this is still unconfirmed for this particular species [3].This bat is sporadically found in forested areas at low and middle elevations across the mainland of Southeast Asia, from the north to the Isthmus of Kra.Its flight pattern and echolocation signals resemble those of Pipistrellus [12].
Although not ideal, the full mitochondrial DNA sequence is a functional tool for taxonomic studies at different levels, e.g., refs.[13,14], giving more reliable results than single gene sequences.Despite the fact that Glischropus bucephalus was described more than ten years ago, it was apparently involved in molecular genetic studies only twice [4,10], and its mitochondrion was not described.Moreover, not a single published mitochondrion of representatives of the genus Glischropus was known (although during the course of our research it was found that this was not entirely true).In order to fill the gap and provide additional clarity on the taxonomic position of Glischropus, we have isolated the complete mitochondrial DNA sequence of G. bucephalus.
The four DNA fragments (~1800-2500 bp) that were the result of the amplification of the whole mitochondrial genomes from G. bucephalus were used for library preparation with an SQK-LSK110 ligation sequencing kit (1D) and a Native Barcoding Sequencing Kit, EXP-NBD (Oxford Nanopore Technologies, Oxford, UK).The sequencing library preparation procedure was performed as follows: the concentration of amplicons was measured by fluorometry using the Qubit 4.0 dsDNA BR assay, and a ~50-60 fmol mixture containing each of the four amplicons was prepared (in sum, 200-240 fmol).End repair and dA-tailing were performed on extracted DNA using the NEBNext Ultra II End Repair/dA-Tailing Module (NEB, E7546) and NEBNext FFPE Repair Mix (NEB, M6630).Next, the sample was purified at a ratio of 1:1 using MGIEasy DNA Clean Beads (MGI, 1000005278).Barcode ligation was performed using NEB Blunt/TA Ligase Master Mix (NEB, M0367) and a Native Barcoding Sequencing Kit, EXP-NBD104 (Oxford Nanopore Technologies).The barcode-ligated DNA was cleaned up by adding 1× volume of (MGI, 1000005278).Adapter ligation was performed using the Adapter Mix II from SQK-LSK110 (1D) and the NEBNext Quick Ligation Module (NEB, E6056).The adaptor-ligated DNA was cleaned up by adding 1× the volume (MGI, 1000005278) and sequenced using the workflow recommended by the manufacturer.The PromethION device (Oxford Nanopore Technologies, UK) was used for sequencing with flow cells FLO-PRO002 (R9.4.1 chemistry).Sequencing runs were controlled using MinKNOW software (version 22.03.4).The high-accuracy basecalling program integrated into the MinKNOW software was used for basecalling the raw signal, with a minimum Q-score of 9.

Mitogenome Assembling and Annotation
The raw data obtained in this work was used to assemble mitochondrial genomes.The draft assembly of the mtDNA reads was performed using CLC Genomic Workbench 8.5 [QIAGEN Aarhus A/S, US].Reads that were presumably assigned to reference mtDNA were selected and used for assembly (10046 raw reads).The mitogenome attributed to Pipistrellus coromandra (GenBank:NC_0291919.1) was used as a reference sequence for the search.10,046 raw reads were used as references for assembling the consensus sequences.The method of consensus polishing by raw ONT reads was used to improve and update the information obtained during the primary assembly of the mitochondrial genome.

Phylogenetic Reconstruction
To elucidate the molecular phylogeny of the species under study, we reconstructed the phylogenetic trees under the maximum likelihood (ML) criterion based on concatenated sequences of all the mtDNA PCGs for 19 Vespertilioninae species, with 3 Myotis species used as outgroups.Sequences were aligned using the Clustal W algorithm, implemented in MEGA v.11.0.13 [20], and adjusted manually.The ML reconstructions were conducted in IQTree version 1.6 [25].Each gene in the alignment was partitioned into three codon positions; the best-fitting substitution models were determined using the ModelFinder implementation [26] in IQTree (Table 1).Clade stability was inferred using Ultrafast Bootstrap [27] with 10,000 replicates.Phylogenetic trees for this dataset were also reconstructed with third codon positions omitted or with third codon positions and the Nd6 gene both omitted to avoid possible saturation bias.
An additional ML phylogenetic reconstruction was performed based on the 54 cytb gene sequences of 26 Vespertilionid species using the same approach.The following substitution models were assigned for each codon position: TIM2e+G4 for the first, TIM3+F+I for the second, and TN+F+I+G4 for the third position.
GenBank accession numbers for specimens used in both analyses are provided in Table 2.

Results
The G. bucephalus complete mitogenome contains a total of 17,023 bp in length, which consists of 13 complete protein-coding genes (PCGs), 22 tRNA genes and 2 rRNA genes (Figure 1, Table 3).The mean depth of coverage is 9216.Eight tRNAs and Nd6 are transcribed from the light strand, while 11 PCGs, 14 tRNAs, and 2 rRNAs are located on the heavy strand.The H-strand base composition is 33.2%A, 29.7% T, 13.6% G, and 23.5% C, which differs only slightly from the Pipistrellus species.AT content is distinctly higher than GC content, which is common for Vespertilioninae.The gene order and orientation of the Glischropus mitochondrial genome were the same as those of "P.coromandra" (NC029191.1)and other Vespertilioninae.The total length of the 13 mitochondrial PCGs in G. bucephalus is 11,379 bp, which could be translated into 3793 amino acids.ATG is the most common start codon and is used in 10 PCGs.However, the start codon ATT is used twice in both Nd2 and Nd3, and ATA is used only once in Nd5.TAA is the most common stop codon, which is used for the termination of 7 PCGs (Cox1, Cox2, ATP8, ATP6, Nd4L, Nd5, and Nd6).The incomplete stop codons are used for the termination of 5 PCGs (TA-for Nd1, Cox3, and Nd3; T-for Nd2 and Nd4).The replication origin (O R ) is 35 bp in size and located between tRNA Asn and tRNA Cys within the WANCY tRNA cluster, as seen in most vertebrates [16].
Both the tribe Pipistrellini (with the genera Pipistrellus, Nyctalus, and Glischropus) and its association with Vespertilionini have maximum support regardless of the data type.Similar to data published previously [10], Pipistrellini includes two well-supported clades, conditionally "western" and "eastern", each of which includes species now assigned to Pipistrellus.Our G. bucephalus clearly belongs to the "eastern" clade, in which it occupies a sister position to NC029191.1 with maximum support (Figure 2).The total length of the 13 mitochondrial PCGs in G. bucephalus is 11,379 bp, which could be translated into 3793 amino acids.ATG is the most common start codon and is used in 10 PCGs.However, the start codon ATT is used twice in both Nd2 and Nd3, and ATA is used only once in Nd5.TAA is the most common stop codon, which is used for the termination of 7 PCGs (Cox1, Cox2, ATP8, ATP6, Nd4L, Nd5, and Nd6).The incomplete stop codons are used for the termination of 5 PCGs (TA-for Nd1, Cox3, and Nd3; T-for Nd2 and Nd4).The replication origin (OR) is 35 bp in size and located between tRNA Asn and tRNA Cys within the WANCY tRNA cluster, as seen in most vertebrates [16].
Both the tribe Pipistrellini (with the genera Pipistrellus, Nyctalus, and Glischropus) and its association with Vespertilionini have maximum support regardless of the data type.Similar to data published previously [10], Pipistrellini includes two well-supported clades, conditionally "western" and "eastern", each of which includes species now assigned to Pipistrellus.Our G. bucephalus clearly belongs to the "eastern" clade, in which it occupies a sister position to NC029191.1 with maximum support (Figure 2).

Discussion
In general, the obtained mitogenome of G. bucephalus in its length and structure is similar to the published mitogenomes of Pipistrellus spp.and Nyctalus spp., and especially to the mitogenome NC029191.1 attributed to P. coromandra.Moreover, the nucleotide composition has some differences: NC029191.1 has 1% more T and almost 1% less C (see [16]).Differences in the nucleotide composition with P. abramus (which is also very close on the phylogenetic tree) are less pronounced [28].

Discussion
In general, the obtained mitogenome of G. bucephalus in its length and structure is similar to the published mitogenomes of Pipistrellus spp.and Nyctalus spp., and especially to the mitogenome NC029191.1 attributed to P. coromandra.Moreover, the nucleotide composition has some differences: NC029191.1 has 1% more T and almost 1% less C (see [16]).Differences in the nucleotide composition with P. abramus (which is also very close on the phylogenetic tree) are less pronounced [28].
As expected, according to our data, the genus Glischropus undoubtedly belongs to the tribe Pipistrellini and is very closely related to the "eastern" clade of the genus Pipistrellus in its current recognition.In addition, both its position and the position of Nyctalus (forming a well-supported sister clade to the "western" Pipistrellus) obviously make Pipistrellus a paraphyletic taxon, which indicates its need for further revision.However, the position of G. bucephalus, sister to NC029191.1 and attributed to P. coromandra, is naturally surprising, because if this is correct, then Glischropus makes not only the whole Pipistrellus genus paraphyletic but also its "eastern" branch.This, on the one hand, could mean a special taxonomic status for P. abramus, but on the other hand, it contradicts all previously obtained results [10,29].
We included the cytb gene sequences taken from the mitochondrions of G. bucephalus and NC029191.1 in an expanded set of sequences of the same gene from different members of the Pipistrellini tribe (Figure 3).The tree, obtained as a result of the analysis, gives a somewhat different topology in which Glischropus stands in a sister position next to the other members of the tribe.However, the association of the "eastern" and "western" clades has very low support; hence, we could talk about trichotomy.These differences obviously indicate various resolution possibilities for an individual gene and genomic sequence for the phylogenetic constructions.Specimen NC029191.1 occupies a position within the G. tylopus clade, strongly suggesting its initial misattribution as P. coromandra.Thus, according to both the data of an individual gene and the mitochondrial genome, G. bucephalus forms a common clade with its congeneric, G. tylopus, while demonstrating undoubted differences in the species level from the latter.As expected, according to our data, the genus Glischropus undoubtedly belongs to the tribe Pipistrellini and is very closely related to the "eastern" clade of the genus Pipistrellus in its current recognition.In addition, both its position and the position of Nyctalus (forming a well-supported sister clade to the "western" Pipistrellus) obviously make Pipistrellus a paraphyletic taxon, which indicates its need for further revision.However, the position of G. bucephalus, sister to NC029191.1 and attributed to P. coromandra, is naturally surprising, because if this is correct, then Glischropus makes not only the whole Pipistrellus genus paraphyletic but also its "eastern" branch.This, on the one hand, could mean a special taxonomic status for P. abramus, but on the other hand, it contradicts all previously obtained results [10,29].
We included the cytb gene sequences taken from the mitochondrions of G. bucephalus and NC029191.1 in an expanded set of sequences of the same gene from different members of the Pipistrellini tribe (Figure 3).The tree, obtained as a result of the analysis, gives a somewhat different topology in which Glischropus stands in a sister position next to the other members of the tribe.However, the association of the "eastern" and "western" clades has very low support; hence, we could talk about trichotomy.These differences obviously indicate various resolution possibilities for an individual gene and genomic sequence for the phylogenetic constructions.Specimen NC029191.1 occupies a position within the G. tylopus clade, strongly suggesting its initial misattribution as P. coromandra.Thus, according to both the data of an individual gene and the mitochondrial genome, G. bucephalus forms a common clade with its congeneric, G. tylopus, while demonstrating undoubted differences in the species level from the latter.This example demonstrates the need to carefully handle the published data in addition to checking it when there is doubt.In general, our work confirms the assumption that This example demonstrates the need to carefully handle the published data in addition to checking it when there is doubt.In general, our work confirms the assumption that there is a close relationship between Glischropus and Pipistrellus based on new data and again indicates a probable generic status for the "eastern" clade of the latter genus.

Figure 1 .
Figure 1.Map of the G. bucephalus mitogenome.Gray color indicates the PCG regions; red color-tRNAs; yellow color-rRNAs.The heavy strand in the outer circle encodes 28 genes, whereas 9 genes are encoded in light strand in the inner circle.

Figure 1 .
Figure 1.Map of the G. bucephalus mitogenome.Gray color indicates the PCG regions; red color-tRNAs; yellow color-rRNAs.The heavy strand in the outer circle encodes 28 genes, whereas 9 genes are encoded in the light strand in the inner circle.

Figure 2 .
Figure 2. The phylogenetic relationship between G. bucephalus and the other Vespertilioninae species is inferred by the maximum likelihood analysis based on the concatenated protein-coding gene sequences.The bootstrap values (indicated by the slashes on the branches) correspond to the trees constructed on full sequences (three codon positions), the first two codon positions (third positions omitted), and two positions with the exclusion of the Nd6 gene.The asterisks mark branches that in the second or third case have a different topology than shown.Myotis species are used as outgroups.

Figure 2 .
Figure 2. The phylogenetic relationship between G. bucephalus and the other Vespertilioninae species is inferred by the maximum likelihood analysis based on the concatenated protein-coding gene sequences.The bootstrap values (indicated by the slashes on the branches) correspond to the trees constructed on full sequences (three codon positions), the first two codon positions (third positions omitted), and two positions with the exclusion of the Nd6 gene.The asterisks mark branches that in the second or third case have a different topology than shown.Myotis species are used as outgroups.

Figure 3 .
Figure 3.The phylogenetic relationship between G. bucephalus and the other Pipistrellini species is inferred by maximum likelihood analysis based on cytb sequences.The numbers in the branches show the bootstrap values.Vespertilio species are used as outgroups.

Figure 3 .
Figure 3.The phylogenetic relationship between G. bucephalus and the other Pipistrellini species is inferred by maximum likelihood analysis based on cytb sequences.The numbers in the branches show the bootstrap values.Vespertilio species are used as outgroups.

Table 1 .
Model types for protein-coding gene analysis configured by IQtree ModelFinder through ultrafast bootstrap (10,000 replicates) for the phylogenetic tree with 3 codons.

Table 2 .
GenBank accession numbers for mitochondrion and cytb sequences used in analysis.

Table 3 .
Gene organization and characterization of the G. bucephalus mitogenome.