Characterization of complete mitogenome data of two flies (Diptera) as orchid pollinators from China

Abstract

The Diptera insects have important ecological functions. Many plants rely on Diptera insects for pollination, and they play an important role in Co-evolution with plants. We described the detailed characteristics across the complete mitogenome sequences of Desmometopa sabroskyi Brake, 2003 (Diptera: Milichiidae) and an unidentified species of Gampsocera (Diptera: Chloropidae), which are pollinators of orchid species. Sequences were assembled and annotated using the reference genomes of Phyllomyza sp. (OP612805) and Elachiptera insignis (OP612812) available in Genbank. The complete mitogenomes of D. sabroskyi and Gampsocera sp. are 15,841 bp and 16,036 bp in length, respectively. Both mitogenomes include 37 genes consisting of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and one noncoding region (NCR). The mitogenome data would better contribute to species identification, taxonomy, phylogenetics, and evolutionary analysis of Diptera insects. 

Specifications Table

Subject	Biological Sciences: Biodiversity; Entomology and Insect Science
Specific subject area	Diptera, Chloropidae, Milichiidae, Mitogenomics
Type of data	Table: gene annotations, base composition. Figure: two flies, mitogenomic circular map, AT-rich region sequence, phylogenetic tree Fasta: mitogenome data Fastq: DNA sequence reads Data format: Raw and analyzed
Data collection	DNA extraction and sequencing: The total DNA was extracted using DNeasy Kit. The Illumina paired-end DNA library was constructed and sequenced by the Illumina NovaSeq 6000 with 150 paired-end mode (PE150). Mitogenome assembly and annotation: The raw data were filtered using the Trimmomaticv0.30, and the final high-quality data was obtained and uploaded to supercomputing, assembled using the Getorganelle, and the results were examined using the Bandage. Both protein-coding genes (PCGs) and rRNA genes were predicted by MITOS tools, and the tRNA genes were identified through tRNAscan-SE webserver. A mitogenome map was drawn using the OGDRAW web server. Phylogenetic analyses: IQ-tree and MrBayes programs were used to construct the phylogenetic tree using Maximum-Likelihood (ML) and Bayesian (BI) methods.
Data source location	The samples for adult flies were collected in 2023 from Xishuangbanna, Yunnan, China. Desmometopa sabroskyi was collected at 100°33′ 5.41′′ E, 22°5′ 7.56′′ N, and Gampsocera sp. was collected at 101°38′E, 21°8′N. Specimens were deposited at Center for Integrative Conservation, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences (contact: Yan Luo, luoyan@xtbg.org.cn) with voucher number HB20230225 and SM20230621.
Data accessibility	Repository name: NCBI BioProject Data identification number: PRJNA1045338 and PRJNA1070834 Direct URL to data: https://www.ncbi.nlm.nih.gov/bioproject/1045338 and https://www.ncbi.nlm.nih.gov/bioproject/1070834 Repository name: NCBI BioSample Data identification number: SAMN38441133 and SAMN39663932 Direct URL to data: https://www.ncbi.nlm.nih.gov/biosample/38441133 and https://www.ncbi.nlm.nih.gov/biosample/39663932 Repository name: NCBI SRA Data identification number: SRR27065045 and SRR27833488 Direct URL to data: https://www.ncbi.nlm.nih.gov/sra/SRX22754512 and https://www.ncbi.nlm.nih.gov/sra/SRX23496836 Repository name: NCBI Genbank Data identification number: OR854638 and PP232099 Direct URL to data: https://www.ncbi.nlm.nih.gov/nuccore/OR854638 and https://www.ncbi.nlm.nih.gov/nuccore/PP232099

1. Value of Data

• •The Diptera is one of the most diverse insect groups in the world [1]. As an important group of pollinators, they play a significant role in the diversification of flowering plants [2]. The taxonomic research on Diptera insects is very challenging, and morphological characteristics are inadequate for resolving species delimitation and phylogenetic relationships [3,4]. However, there is a lack of genetic information about Diptera insects, which makes it difficult to understand their diversity, as well as hinders the study of flowering plant diversification and pollinator-plant interactions.
• •The data represent the first complete mitogenome for Desmometopa sabroskyi (Milichiidae) and Gampsocera sp. (Chloropidae), respectively, which served as effective pollinators of Bulbophyllum orchids in Yunnan, southwestern China.
• •The complete mitochondrial references for the Diptera species can provide accurate identification of the pollinator at the molecular level, clarify its taxonomic status, and increase knowledge of the diversity of pollinators for orchid species.
• •The complete mitogenome might serve as a valuable resource for phylogenetic and evolutionary analysis for Milichiidae and Chloropidae of the Diptera.

2. Background

As an important group of pollinators, the Diptera play a significant role in the diversification of flowering plants [5]. It is estimated that over 70 families of Diptera are pollinators of at least 555 flowering plant species [6,7]. In the course of observation of pollinators of Bulbophyllum orchids, we found two flies pollinating Bulbophyllum nigripetalum (Fig. 1A) and B. andersonii (Fig. 1B), respectively. By critical examinations of morphological characters and evaluation of literature, the fly pollinating B. nigripetalum was identified as Desmometopa sabroskyi Brake, 2003 (Diptera: Milichiidae) (Fig. 1C), and the fly pollinating B. andersonii was an undescribed species belonging to the genus Gampsocera (Diptera: Chloropidae) (Fig. 1D).

Fig. 1.

Desmometopa sabroskyi pollinates Bulbophyllum nigripetalum and Gampsocera sp. pollinates Bulbophyllum andersonii. A: Desmometopa sabroskyi (white arrow) pollinating a Bulbophyllum nigripetalum flower; B: Gampsocera sp. (white arrow) pollinating a Bulbophyllum andersonii flower; C: Desmometopa sabroskyi carrying pollinia of Bulbophyllum nigripetalum: D: Gampsocera sp. carrying pollinia of Bulbophyllum andersonii.

3. Description of Data

The complete mitogenomes of D. sabroskyi and Gampsocera sp. are 15,841 bp and 16,036 bp in length, respectively (Table 1). The mitogenome nucleotide composition of D. sabroskyi is 40.9 % of A, 36.7 % of T, 9 % of G, 13.3 % of C, and AT content was 77.6 % (Table 1). The length of tRNAs ranges from 60 to 72 bp. 16S rRNA is 1301 bp in length and 12S rRNA is 786 bp in length. The CR is 999 bp in length and has rich AT content (90.7 %). The sequence shows weakly positive AT-skew (0.0545) and negative GC-skew (−0.1936). The mitogenome nucleotide composition of Gampsocera sp. is 40.9 % of A, 38.3 % of T, 8.6 % of G, 12.2 % of C, AT content was 79.2 % (Table 1). The length of tRNAs ranges from 62 to 72 bp. 16S rRNA is 1,302 bp in length and 12S rRNA is 790 bp in length. The CR is 1,148 bp in length and has rich AT content (83.7 %). The sequence shows weakly positive AT-skew (0.0333) and negative GC-skew (−0.1684).

Table 1.

Base composition and skewness of mitogenomes of Desmometopa sabroskyi and Gampsocera sp.

Species	Gene	size (bp)	A%	G%	T%	C%	A + T%	AT skew	GC skew
Desmometopa sabroskyi	Mitogenome	15,841	40.9	9	36.7	13.3	77.6	0.0545	−0.1936
	PCGs	11,202	32.3	12.5	43.3	11.8	75.6	−0.1460	0.0289
	tRNAs	1453	39	12.9	38	10.2	77	0.0126	0.1164
	rRNAs	2087	37.8	12	44.2	6	82	−0.0789	0.3297
	CR	999	48.6	3.6	42.1	5.6	90.7	0.0716	−0.2173
Gampsocera sp.	Mitogenome	16,036	40.9	8.6	38.3	12.2	79.2	0.0333	−0.1684
	PCGs	11,227	33.8	11.8	43.7	10.7	77.5	−0.1282	0.0479
	tRNAs	1478	39	12.7	38.5	9.8	77.5	0.0069	0.1265
	rRNAs	2092	39.8	11.4	43.1	5.7	82.9	−0.0392	0.3351
	CR	1148	49.1	3.8	40.4	6.6	89.5	0.0972	−0.2666

Both mitogenomes include 37 genes consisting of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a control region (CR) (Table 2, Fig. 2). In total, 16 intergenic spacer regions with an average size of 7 bp and a total length of 115 bp were identified in D. sabroskyi and 17 intergenic spacer regions with an average size of 7 bp and a total length of 122 bp were identified Gampsocera sp. (Table 2).

Table 2.

Mitogenome organization of Desmometopa sabroskyi and Gampsocera sp.

Strand	Gene	Desmometopa sabroskyi						Gampsocera sp.
		Position	Size	Int	Start	Stop	Anti	Position	Size	Int	Start	Stop	Anti
H	tRNA-Ile	1–65	65	−3			GAT	1–66	66	6			GAT
L	tRNA-Gln	63–131	69	10			TTG	73–141	69	−1			TTG
H	tRNA-Met	142–210	69	0			CAT	141–209	69	0			CAT
H	ND2	211–1233	1023	−2	ATT	TAA		210–1247	346	7	ATT	TAG
H	tRNA-Trp	1232–1299	68	−8			TCA	1255–1323	69	−8			TCA
L	tRNA-Cys	1292–1354	63	0			GCA	1316–1380	65	3			GCA
L	tRNA-Tyr	1355–1419	65	1			GTA	1384–1448	65	−2			GTA
H	COX1	1421–2956	1536	−5	CGA	TAA		1447–2980	1534	−1	TCG	T–
H	tRNA-Leu	2952–3017	66	0			TAA	2980–3047	68	−1			TAG
H	COX2	3018–3705	679	0	ATG	T–		3047–3734	688	−1	ATG	T–
H	tRNA-Lys	3706–3776	71	−1			CTT	3734–3805	72	1			CTT
H	tRNA-Asp	3776–3843	68	0			GTC	3807–3872	66	0			GTC
H	ATP8	3844–4005	162	−7	ATC	TAA		3873–4034	162	−7	ATT	TAA
H	ATP6	3999–4676	678	7	ATG	TAA		4028–4705	678	−1	ATG	TAA
H	COX3	4684–5472	789	12	ATG	TAA		4705–5496	792	20	ATG	TAA
H	tRNA-Gly	5485–5550	66	0			TCC	5517–5582	66	0			TCC
H	ND3	5551–5904	354	7	ATT	TAA		5583–5936	354	4	ATT	TAA
H	tRNA-Ala	5912–5976	65	−1			TGC	5941–6007	67	4			TGC
H	tRNA-Arg	5976–6039	64	7			CGG	6012–6073	62	7			TCG
H	tRNA-Asn	6047–6110	64	0			GTT	6081–6146	66	0			GTT
H	tRNA-Ser	6111–6176	66	10			GCT	6147–6213	67	0			TGA
H	tRNA-Glu	6187–6251	65	18			TTC	6214–6279	66	17			TTC
L	tRNA-Phe	6270–6335	66	0			GAA	6297–6365	69	−2			GAA
L	ND5	6336–8070	1735	0	ATT	T–		6364–8099	1736	0	ATC	TA-
L	tRNA-His	8071–8134	64	−1			GTG	8100–8165	66	0			GTG
L	ND4	8134–9474	1341	−7	ATG	TAA		8166–9504	1339	−1	ATG	T–
L	ND4L	9468–9764	297	2	ATG	TAA		9504–9794	291	2	ATG	TAA
H	tRNA-Thr	9767–9831	65	0			TGT	9797–9862	66	0			TGT
L	tRNA-Pro	9832–9896	65	2			TGG	9863–9930	68	2			TGG
H	ND6	9899–10,423	525	3	ATT	TAA		9933–10,457	525	3	ATT	TAA
H	CYTB	10,427–11,563	1137	1	ATG	TAA		10,461–11,597	1137	−1	ATG	TAA
H	tRNA-Ser	11,565–11,631	67	6			TGA	11,597–11,664	68	16			GCT
L	ND1	11,638–12,595	958	10	TTG	T–		11,681–12,628	948	1	TTG	TAA
L	tRNA-Leu	12,606–12,665	60	10			TAG	12,630–12,696	67	5			TAG
L	16S rRNA	12,676–13,976	1301	9				12,702–14,003	1302	24
L	tRNA-Val	13,986–14,057	72	−1			TAC	14,028–14,098	71	0			TAC
L	12S rRNA	14,057–14,842	786	0				14,099–14,888	790	0
H	Control region	14,843–15,841	999	0				14,889–16,036	1148	0

Fig. 2.

Circular map of the assembled Gampsocera sp. and Desmometopa sabroskyi mitogenome, consisting of 13 protein-coding genes, 22 transfer RNA, and two ribosomal RNA genes. Different colors indicate different gene families and the outer and inner rings represent heavy and light chains, respectively. The darker and lighter gray area in the inner circle represent the GC and AT contents.

Phylogenetic analysis showed that the taxa of Milichiidae and Chloropidae formed a well-supported clade, D. sabroskyi formed an independent lineage with strong support, and Gampsocera sp. is closely related to Cadrema minor within Chloropidae with strong support. (Fig. 3). The mitogenome data indicate Milichiidae to be paraphyletic, as suggested by previous researchers [8].

Fig. 3.

Molecular phylogeny based on the concatenated nucleotide sequences of protein-coding genes (PCGs) of 19 species in Ephydroide or Carnoidea and 2 outgroups. Phylogenetic tree conducted using Maximum Likelihood (ML) and Bayesian Inference (BI) methods and the numbers above branches represent bootstrap percentage (BP) of ML /posterior probability (PP) of BI. Genbank accession numbers are located after the species' scientific name. The position of Gampsocera sp. and Desmometopa sabroskyi are marked with a solid square.

4. Experimental Design, Materials And Methods

4.1. Samples collection

The samples for adult flies were collected in June 2023 from two sites of Xishuangbanna, Yunnan, China (100°33′ 5.41′′ E, 22°5′ 7.56′′ N; 101°38′E, 21°8′N). Specimens were deposited at Center for Integrative Conservation, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences (contact: Yan Luo, luoyan@xtbg.org.cn) with voucher HB20230225 and SM20230621.

4.2. DNA extraction and sequencing

The total DNA was extracted using DNeasy Kit (Qiagen, Germany) according to the manufacturer's instructions. The Illumina paired-end DNA libraries were constructed using the standard library building procedure of Illumina's Truseq DNA PCR-Free Prep kit (San Diego, CA, USA) following the manufacturer's instructions. The DNA libraries were subjected to next-generation sequencing using the high-throughput sequencing platform Illumina NovaSeq6000 of Shanghai Personal Biotechnology Company (Shanghai, China).

4.3. Mitogenome assembly and annotation

The sequencing runs produced 28,787,148 reads (total bases: 4.3 G, size: 2.2 GB) in D. sabroskyi with the values of Q20 (97.24 %) and Q30 (92.51 %) over 90 % and 25,863,920 reads (total bases: 3.9 G, size: 1.7 GB) in Gampsocera sp. with the values of Q20 (97.09 %) and Q30 (94.75 %) over 90 %, respectively.

The raw data was filtered using the Trimmomaticv0.30 [9], and the final high-quality data of 28,722,578 reads (total bases: 4.2 G, size: 1.9 GB) in D. sabroskyi and 25,602,128 reads (total bases: 3.7 G, size: 1.5 GB) in Gampsocera sp., respectively, were obtained and uploaded to supercomputing, assembled using the Getorganelle pipeline [10], and the results were examined using the Bandage. Both protein-coding genes (PCGs) and rRNA genes were predicted by MITOS tools (http://mitos2.bioinf.uni-leipzig.de/index.py) [11], and the tRNA genes was identified through tRNAscan-SE webserver [12]. A mitogenome map was drawn using the OGDRAW web server (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html).

4.4. Phylogenetic analysis

To validate the position of these flies, the PCGs sequences of mitogenome from 23 Diptera species were used in phylogenetic analysis, including 11 species in Carnoidea, 10 species in Ephydroide, and Chironomus tepperi Skuse, 1889 and Dixella aestivalis (Meigen, 1818) as outgroups, were clustered together to construct phylogenetic tree using maximum-likelihood (ML) and Bayesian (BI) analysis. The ML tree with 1000 bootstrap replicates was performed using IQ-tree [13]. The BI tree was generated using MrBayes 3.2.4 [14] with Markov chain Monte Carlo (MCMC) algorithm running for 2 × 10⁶ generations, sampled once every 100 generations. The first 25 % of all generations were excluded, and the remaining samples were used to generate the majority consensus trees and estimate the posterior probabilities.

Limitations

Not applicable.

Ethics Statement

This study did not involve humans or animals. No approval or permission was necessary in this study for the sample collection.

CRediT authorship contribution statement

Jinrui He: Investigation, Data curation, Writing – original draft. Xiong Zhang: Investigation, Data curation, Writing – original draft. Xiaoyan Liu: Methodology, Writing – review & editing. Qingqing Li: Data curation, Writing – review & editing. Yinling Luo: Conceptualization, Methodology, Supervision, Writing – review & editing. Yan Luo: Conceptualization, Methodology, Supervision, Writing – review & editing.

Data Availability

• SAMN38441133 (Original data) (NCBI BioSample)

• SAMN39663932 (Original data) (NCBI BioSample)

• PRJNA1045338 (Original data) (NCBI BioProject)

• PRJNA1070834 (Original data) (NCBI BioProject)

• SRR27065045 (Original data) (NCBI SRA)

• SRR27833488 (Original data) (NCBI SRA)

• OR854638 (Original data) (NCBI Genbank)

• PP232099 (Original data) (NCBI Genbank)