The Complete Mitochondrial Genome of a Newly Recorded Chinese Species of Diglyphus sabulosus (Hymenoptera: Eulophidae) and Insights into Its Phylogenetic Position

doi:10.21203/rs.3.rs-8308075/v1

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The Complete Mitochondrial Genome of a Newly Recorded Chinese Species of Diglyphus sabulosus (Hymenoptera: Eulophidae) and Insights into Its Phylogenetic Position

https://doi.org/10.21203/rs.3.rs-8308075/v1

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Diglyphus Walker, 1844 is an economically important genus which many species acting as biocontrol agents against agromyzid leafminer pests, but there is a lack of mitogenomic data on the evolutionary relationships within this genus, hindering a comprehensive understanding of its evolutionary history. we used traditional morphological methods to identify species, and present the first complete mitochondrial genome sequence and characterization of features of Diglyphus sabulosus and further infer its phylogenetic position based on the complete mitochondrial genome sequence. The complete mitochondrial genome of Diglyphus sabulosus is 15,690 bp in length, including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 1 control region. The AT content of the whole genome sequence was 81.0%, indicating a significant AT bias. All 13 protein-coding genes have the typical ATN as the start codon and TAA as the stop codon. Phylogenetic analysis inferred from the complete mitogenome revealed that all species within the family Eulophidae constituted a monophyletic clade, supporting the monophyly of this family. Diglyphus sabulosus and Diglyphus poppoea form a well-supported sister group, representing the species with the closest phylogenetic relationship within the analyzed taxa. This inferred close phylogenetic affinity is highly consistent with their morphological characteristics, which exhibit the highest degree of similarity among the studied species. In this study, the mitogenome structure was analyzed and the taxonomic status of Diglyphus sabulosus was clarified, thus providing a theoretical basis for understanding the phylogenetic relationships of Diglyphus.

Diglyphus sabulosus

Mitochondrial genome

Phylogenetic relationship

Newly recorded species from China

Diglyphus sabulosus belongs to the family Eulophidae, subfamily Eulophinae, genus Diglyphus. Diglyphus currently includes 41 species, and 17 of them are recorded from China (Gordh and Hendrickson; 1979; Gauthier et al. 2000; Zhu et al. 2000; Yefremova et al. 2011; Ye et al. 2018; UCD 2023)^[1–6]. Diglyphus is an economically important genus containing species that attack Agromyzidae (Diptera) leafminers and occasionally Lepidoptera pests (Gelechiidae, Gracillariidae, Lyonetiidae, and Nepticulidae) (Zhu et al. 2000; Yefremova et al. 2011; Hansson and Navone 2017; UCD 2023). Agromyzidae leafminers such as Chromatomyia horticola (Goureau) and Liriomyza spp. are pests of vegetables and ornamental plants worldwide (Bader et al. 2006; Kaspi and Pawella 2006; Foba et al. 2016; Kumar and Sharma 2016). Most species of the genus Diglyphus can serve as biological control agents, providing protection for important cash crops.

Eulophidae is a large and biologically varied family of parasitoid wasps, traditionally split into four subfamilies (Gauthier et al. 2000). Identification of Eulophidae species mainly depends on morphological data. However, combining morphological and molecular analyses for species identification is essential owing to the morphological similarities among species. The rapid advancement of molecular biology techniques has led to an increasing focus among researchers on taxonomic and genetic studies of Eulophidae based on molecular markers such as mitochondrial gene sequences. The cytochrome c oxidase I (COI) gene of the mitochondrial DNA and internal transcribed spacer II (ITS2) ribosomal DNA genes have previously been applied to enhance species identification (Hebert et al. 2003; Bernardo et al. 2008; Gebiola et al. 2012; Song et al. 2020; Du et al. 2021; Huang et al. 2022). Although 28S ribosomal DNA (28S rDNA) has mostly been used for phylogenetic studies at the genus level and above, it has also been used for species identification (Burks et al. 2011; Hansson and Schmidt 2020). However, due to the limited amount of genetic information carried by individual genes, polygenic association studies have become increasingly prevalent in Eulophidae research. For example, Gebiola et al. (2009) used a combined molecular approach involving 28S rDNA and COI to determine the taxonomic status of two species from the Pygalio genus that had previously been the subject of morphological identification disputes. This approach provided molecular evidence to clarify their taxonomic positions. Gebiola et al. (2015) employed an integrated taxonomic approach to revise the classification of the genus Necremnus. Molecular data from the mitochondrial cytochrome oxidase c subunit I and the nuclear D2 expansion region of the 28S ribosomal subunit and internal transcribed spacer 2, the discovery of new morphological features, and study of type material resulted in the delineation of three species groups, the Necremnus artynes, Necremnus cosconius and Necremnus tidius groups, the discovery of four new species, and the resurrection of three taxa from synonymy. Wan et al. (2023) discovered one new species of Diglyphus based on morphological characteristics and molecular analyses of COI, ITS2 and 28S rDNA genes.

With the advancement of high-throughput sequencing technology, obtaining mitogenome sequences has become increasingly straightforward. The typical insect mitogenome is circular, being 14–20 kb in length and normally containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes and 1 A + T-rich region (control region, CR) (Dowton et al. 2002; Cameron 2014). The mitogenomes of insects are characterised by a high degree of conservation and compactness. Compared to those of other eukaryotes, insect mitogenomes exhibit a higher gene density, with genes often lacking non-coding sequences between them (Liu et al. 2016). Insect mitochondrial DNA is maternally inherited, non-recombining, and has an elevated mutation rate compared to nuclear DNA, making it the most popular marker in phylogenetic analysis of insects (Zheng et al. 2018; Liu et al. 2019; Tang et al. 2019; Nie et al. 2020). In recent years, with the maturation of sequencing technologies and the application of universal primers for mitochondrial genes (Simon et al. 1994; Simon et al. 2006), the number of insect mitogenomes that have been sequenced has increased rapidly. The study of insect mitogenomes is significant in fields such as insect taxonomy, evolutionary studies and population genetics. These genomes may also serve as crucial tools for identifying insect species and reconstructing their phylogeny (Boore 1999; Ballard and Rand 2005; Ma et al. 2012; Nelson et al. 2012). However, the availability of Diglyphus mitogenomes remains extremely limited. So far, merely three mitogenomes of Diglyphus species have been deposited in the GenBank database (as of August 30, 2025), with Diglyphus sabulosus not among these sequenced species.

In this study, we determined the first complete mitogenome sequence of the genus Diglyphus, Diglyphus sabulosus, along with the details of its mitogenomic structure, providing a valuable data resource with which to address the lack of mitogenomes in this genus. Based on mitogenome data, we reconstructed the phylogenetic relationships of Eulophidae and discussed the phylogenetic position of Diglyphus, contributing novel molecular evidence for understanding the evolution of this group.

2.1 Sample Collection and Species Identification

All specimens in this study were collected in accordance with Chinese laws. The collection and sampling of the specimens were reviewed and approved by the Animal Ethics Committee of Xinjiang University. All the experiments were conducted with respect to animal welfare and care. We obtained specimens of Diglyphus sabulosus from Aktao County Aoyi Take Glacier Park, Xinjiang Uygur Autonomous Region, China (38°53′54″N, 75°12′5″E). Specimens were promptly preserved in anhydrous ethanol during field collection. Subsequently, all specimens were transferred to and preserved at − 20℃ in the Entomology Collection at the College of Life Science and Technology, Xinjiang University, Urumqi, Xinjiang, China (ICXU).

The specimens were examined under a stereomicroscope (Nikon, SMZ25, Japan). Morphological photographs were captured and morphometric measurements were taken using the Nikon DS-U3 imaging system that accompanies the microscope. Image processing and layout were performed using Adobe Photoshop. The morphological terminology and measurement methods follow Gibson (1989), Hansson (1990), and Yefremova et al. (2011), and the following abbreviations were used:

F1-2: maximum length of Funicle 1–2.

OOL: Ocular ocellar line: shortest distance between the lateral ocelli and eyes.

POL: Posterior ocellar line: shortest distance between lateral ocelli.

2.2 DNA Extraction and Genome sequencing

Genomic DNA was extracted from ten female adult specimens of Diglyphus sabulosus. The extraction process was carried out by Sangon Biotech (Shanghai) Co., Ltd. The mitogenome sequencing of Diglyphus sabulosus was conducted using the Illumina Nova Xplus platform (Sangong Biotech (Shanghai) Co., Ltd.). A genomic DNA library with insert fragments of approximately 350 bp was constructed and sequenced using a PE150 strategy, yielding approximately 6 Gb of raw data in FASTQ format. Subsequently, Fastp v0.23.4 (Chen et al. 2018) was used to filter the raw reads for quality control, removing those containing adapter sequences, high read redundancy, a high N content and low quality. This produced high-quality data for subsequent assembly.

2.3 Genome assembly, annotation and validation

Multiple strategies are employed in the assembly and validation of mitogenomes to ensure the accuracy of the results. The raw sequencing data were processed using MitoZ v3.6 (Meng et al. 2019), including quality control, de novo assembly, and preliminary annotation. We compared the two assembly results and manually annotated in MEGAX v10.2.6 (Kumar et al. 2018), yielding a consistent, high-quality mitogenome sequence. The sequence was submitted to the MITOS2 (2.1.9 + galaxy0) (Donath et al. 2019) online server for systematic annotation. Annotation results from MitoZ and MITOS2 were manually verified and confirmed in Geneious Prime, yielding the final annotation. Protein-coding genes (PCGs) were further validated using ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 10 August 2025). The secondary structures of tRNAs were predicted and visualized using MITOS2 and VARNA v3-93 (Darty et al. 2009), respectively. The annotated mitogenome sequence has been submitted to NCBI GenBank under accession number PX395427. The mitogenomic structure was visualized through Proksee (https://proksee.ca/, accessed on 10 August 2025) (Grant et al. 2023).

2.4 Sequence Analysis

To analyze the full-sequence characteristics of the mitogenome, we used Geneious Prime v2025.1.2, MEGAX v10.2.6, and PhyloSuite v1.2.3pre3 for comprehensive analysis, including: (1) Genome-wide base composition analysis to determine its distribution; (2) Investigation of codon usage patterns and relative synonymous codon usage (RSCU) in protein-coding genes (PCGs) to assess codon preference; (3) Calculation of mitogenome composition skew using formulae: AT skew=(A − T)/(A + T) and GC skew=(G − C)/(G + C) (Perna and Kocher 1995).

2.5 Phylogenetic Analysis

We selected mitogenome sequences from 8 species of Eulophidae and 1 outgroup species (Pteromalus puparum), in addition to the complete mitogenome sequence of Diglyphus sabulosus determined in this study. All species used for phylogenetic analysis, along with their mitogenome GenBank accession numbers, are listed in Table 1. Multiple sequence alignments were performed with MAFFT v7.505 (Katoh and Standley 2013), and the alignments were then optimized using MACSE (Ranwez et al. 2011). The aligned sequences of PCGs and RNAs were trimmed using Gblocks 0.91b (Talavera and Castresana 2007), respectively. Subsequently, we concatenated all 37 genes with a total of 15,690 bp using PhyloSuite v1.2.3pre3. We selected the best-fit model based on the BIC criterion using ModelFinder (Kalyaanamoorthy et al. 2017), then performed phylogenetic analyses on each dataset via Bayesian inference (BI) and maximum likelihood (ML), respectively. The BI tree was constructed in MrBayes v3.2.7 (Ronquist et al. 2012), using four Markov chains run for 50,000,000 generations, with a sampling frequency of 1,000 generations and a relative burn-in of 25%. Majority consensus trees were then generated, and node support was assessed via posterior probabilities. Maximum likelihood analysis was performed in IQ-TREE v2.2.2.7 (Nguyen et al. 2015), with branch support assessed using 5,000 ultrafast bootstrap replicates to generate bootstrap values. The phylogenetic tree was edited in Figtree v1.4.4 and further embellished in iTOL (https://itol.embl.de/, accessed on 20 September 2025) (Letunic and Bork 2024).

Table 1

Taxonomic information and GenBank accession numbers of mitochondrial genomes used in the study.
Family	Species	GenBank Accession no.	Size(bp)
	Chouioia cunea	NC_060368	14,930
	Chouioia cunea	MW192646	14,930
	Citrostichus phyllocnistoides	PQ059861	14,669
	Tetrastichus howardi	NC_079567	14,791
Eulophidae	Tetrastichus sp.	PP735728	14,910
	Necremnus tutae	NC_053857	15,252
	Diglyphus begini	OQ863035	14,478
	Diglyphus sabulosus	PX395427	15,690
	Diglyphus poppoea	OQ863037	13,193
	Diglyphus isaea	OQ863031	14,178
Pteromalidae (Outgroup)	Pteromalus puparum	NC_039656	18,217

3.1 Diglyphus sabulosus Erdӧs, 1951(Newly Recorded Species from China)

Diglyphus sabulosus Erdös, 1951:197.

Diglyphus sabulosus Thuróczy, 1992: 167.

Specimens examined. 15♀♀3♂♂, CHINA, Xinjiang Uygur Autonomous Region, Akto County Oyatake Glacier Park, 38°53′54″N, 75°12′5″E, 2764.51 m, 17. Ⅶ. 2022. Hongying Hu et al., sweeping.

Diagnosis. Female. Body length 1.30–1.41 mm (n = 15). Body (Fig. 1F) with a dark green metallic sheen. Antenna scape white, pedicel and funicle light brown (Fig. 1C); proximal 1/2 of femur brown, tibia pale yellow, entire tarsus yellowish-brown; wing veins pale yellow. F1 1.18× as long as F2. Malar sulcus prominent, POL 2.56× that of OOL (Fig. 1A). Thorax (Fig. 1D) with reticulated engraving pattern. Foring veins slightly thickened (Fig. 1E), with postmarginal vein subequal in length to stigmal vein which possesses a stalk; speculum minute. Gaster (Fig. 1F) approximately oblong-ovate, ovipositor extends slightly from the end of the gaster.

Male. Veins of both forewings and hindwings are distinctly thickened (Fig. 1H); all other characters silimar with those of the female.

Hosts. Agromyzidae (Yefremova et al.2011; UCD 2023).

Distribution. China (Xinjiang); Czech Republic, Hungary, Romania, Slovakia, Sweden, Turkey (UCD 2023).

Comments. This species is closely similar to Diglyphus poppoea, with the main difference being the color of the hind tibia. In Diglyphus poppoea, the distal two-thirds of the hind tibia are dark green (Jafarlu et al. 2023), whereas in this species, they are entirely yellow. Additionally, the male wings of this species have distinctly thickened, yellow veins, a feature that differs from those of other congeneric species.

3.2 Mitogenome Organization

The complete mitogenome of Diglyphus sabulosus is a circular molecule of 15,690 bp, encoding 13 PCGs, 22 transfer RNA genes (tRNAs), and 2 ribosomal RNA genes (rRNAs). The organization of 37 genes and one non-coding region, together with the GC content and GC skew along the mitogenome, is presented in Fig. 2. A total of 10 PCGs, 15 tRNA genes, and 2 rRNA genes are located on the major coding strand (H-strand); 3 PCGs (cytb, nad2, and nad6) and 7 tRNA genes (trnS2, trnT, trnE, trnK, trnN, trnW, and trnQ) are situated on the minor coding strand (L-strand).

There is one long non-coding region in the Diglyphus sabulosus mitogenome. The control region is located between trnI and trnM. Basic information on all the genes and non-coding region is listed in Table 2, including strand positions, lengths, tRNA anticodons, the start and stop codons of the PCGs, and intergenic spacer lengths. The mitochondrial adjacent genes of Diglyphus sabulosus exhibit variable-length intergenic spacers and gene overlap. In this study, 19 intergenic gaps and 15 gene overlaps were identified in the mitogenome of Diglyphus sabulosus. Among these gaps, the longest (99 bp) was detected between nad2 and trnI, and the shortest (2 bp) was present between two gene pairs: trnT-trnP and trnC-trnN. For gene overlaps, the longest spanned 21 bp and was located between rrnL and trnL1.

Table 2

Positions and basic features of genes and non-coding region of the *Diglyphus sabulosus* mitogenome
Gene	From	To	Length/bp	Intergenic nucleotide/bp	Anticodon	Start codon	Stop codon	Coding strand
trnA	1	64	64		TGC			H
rrnL	80	1394	1315	15				H
trnL1	1374	1441	68	-21	TAG			H
nad1	1457	2389	933	15		ATT	TAA	H
trnS2	2388	2454	67	-2	TGA			L
cytb	2453	3592	1140	-2		ATG	TAA	L
nad6	3592	4113	522	-1		ATT	TAA	L
trnP	4170	4236	67	56	TGG			H
trnT	4239	4306	68	2	TGT			L
nad4L	4324	4593	270	17		ATG	TAA	H
nad4	4608	5939	1332	14		ATG	TAA	H
trnH	5923	5989	67	-17	GTG			H
nad5	5993	7690	1698	3		ATT	TAA	H
trnF	7671	7734	64	-20	GAA			H
trnE	7734	7798	65	-1	TTC			L
cox1	7802	9337	1536	3		ATG	TAA	H
trnL2	9333	9399	67	-5	TAA			H
cox2	9425	10102	678	25		ATT	TAA	H
trnK	10112	10180	69	9	TTT			L
trnD	10188	10255	68	7	GTC			H
atp8	10280	10441	162	24		ATT	TAA	H
atp6	10435	11109	675	-7		ATG	TAA	H
cox3	11109	11897	789	-1		ATG	TAA	H
trnG	11908	11973	66	10	TCC			H
nad3	11971	12324	354	-3		ATA	TAA	H
trnR	12328	12386	59	3	TCG			H
trnC	12402	12467	66	15	GCA			H
trnN	12470	12536	67	2	GTT			L
trnY	12536	12602	67	-1	GTA			H
trnS1	12602	12663	62	-1	TCT			H
trnW	12663	12732	70	-1	TCA			L
nad2	12731	13723	993	-2		ATT	TAA	L
trnI	13823	13890	68	99	GAT			H
Control region	13891	14692	802	0
trnM	14693	14757	65	0	CAT			H
trnV	14758	14833	76	0	TAC			H
rrnS	14841	15600	760	7				H
trnQ	15608	15676	69	7	TTG			L

3.3 Nucleotide Composition

The whole mitogenome of Diglyphus sabulosus consists of 37.5% A, 43.5% T, 7.9% C, and 11.1% G. It shows an obvious bias towards A + T (81.0%), with a negative AT skew of -0.074 and a positive GC skew of 0.171 (Table 3), similar to what is observed in other Eulophid species (Tang et al. 2021; Tian et al. 2021). The AT content of the four datasets comprising the complete mitogenome, protein-coding genes, tRNA genes, and rRNA genes of Diglyphus sabulosus were 81.0%, 79.8%, 86.6%, and 86.0%, respectively. The AT content of 13 protein-coding genes ranged from 73.7% to 86.0%, with cox1 exhibiting the lowest value at 73.7% and nad2 the highest at 86.0%. Analysis of AT skew in each protein-coding gene revealed that, with the exception of the atp8 gene, 12 remaining protein-coding genes exhibited T bias. In contrast, in GC skew analysis, 4 protein-coding genes (atp8, cytb, nad2, and nad6) showed C bias, while the remaining 9 protein-coding genes exhibited G bias.

Table 3

Nucleotide composition and skewness of the *Diglyphus sabulosus* mitogenome.
Regions	Size(bp)	T	C	A	G	AT(%)	GC(%)	AT skewness	GC skewness
Full genome	15690	43.5	7.9	37.5	11.1	81.0	19.0	-0.074	0.171
PCGs	11082	45.7	9.4	34.1	10.8	79.8	20.2	-0.146	0.073
tRNAs	1469	42.0	5.2	44.6	8.2	86.6	13.4	0.030	0.218
rRNAs	2075	43.6	4.8	42.4	9.2	86.0	14.0	-0.013	0.313
Control region	802	39.0	12.0	31.3	17.7	70.3	29.7	-0.110	0.190
atp6	675	44.6	10.5	34.1	10.8	78.7	21.3	-0.134	0.014
atp8	162	37.7	8.6	47.5	6.2	85.2	14.8	0.116	-0.167
cox1	1536	43.0	12.0	30.7	14.3	73.7	26.3	-0.166	0.089
cox2	678	42.2	9.7	34.4	13.7	76.6	23.4	-0.102	0.170
cox3	789	46.4	9.9	30.7	13.1	77.1	23.0	-0.204	0.138
cytb	1140	42.1	13.1	33.1	11.8	75.2	24.9	-0.120	-0.053
nad1	933	46.0	8.0	33.7	12.3	79.7	20.3	-0.155	0.211
nad2	993	47.3	9.3	38.7	4.7	86.0	14.0	-0.101	-0.324
nad3	354	47.5	7.3	35.6	9.6	83.1	16.9	-0.143	0.133
nad4	1332	47.7	7.4	33	11.8	80.7	19.2	-0.182	0.227
nad4L	270	50.7	4.4	35.2	9.6	85.9	14.0	-0.181	0.368
nad5	1698	48.8	7.2	34.4	9.6	83.2	16.8	-0.173	0.144
nad6	522	46.4	9.6	39.1	5.0	85.5	14.6	-0.085	-0.316

3.4 Protein-Coding Genes

The total tandem length of 13 protein-coding genes in Diglyphus sabulosus is 11,082 bp, accounting for 70.63% of the entire mitogenome and encoding 3,681 amino acids. The amino acids were ranked by content in descending order as follows: Leu > Ile > Phe > Ser > Met > Asn > Val > Tyr > Gly > Lys > Pro = Thr > Ala > Trp > Glu > His > Gln > Asp > Arg > Cys, with Pro and Thr having identical percentages. Among these, Leu exhibited the highest relative content at 13.69%, whereas Cys showed the lowest, accounting for only 0.08%. RSCU values were calculated to measure the codon usage bias (Fig. 3). The codons with an RSCU value > 1.0 were defined as abundant codons. Relative codon usage analysis was performed on Diglyphus sabulosus, revealing 64 distinct codons in its coding sequences. A total of 29 codons showed RSCU > 1.0, of which 14 terminated in U and 15 in A (Table 4). Among these preferred codons, UUA had the highest usage frequency (n = 393, RSCU = 4.68). Notably, all amino acids exhibited distinct codon usage biases, with preferences specifically for codons terminating in A or U (A/U at the third position). As illustrated in Fig. 3, notable examples include: Leu preferring the codon UUA, Ile preferring AUU, Phe preferring UUU, and Met preferring AUA, among others. All PCGs start with typical ATN initiation codons, including one ATA (nad3), six ATTs (nad1, nad6, nad5, cox2, atp8, and nad2), and six ATGs (cytb, nad4L, nad4, cox1, atp6, and cox3). All PCGs terminate with conventional stop TAA codon (Table 2).

Table 4

Relative synonymous codon usage (RSCU) in the mitochondrial genome of *Diglyphus sabulosus*
Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU
UUU(F)	388	1.87	UCU(S)	110	2.29	UAU(Y)	161	1.85	UGU(C)	29	2.00
UUC(F)	26	0.13	UCC(S)	8	0.17	UAC(Y)	13	0.15	UGC(C)	0	0.00
UUA(L)	393	4.68	UCA(S)	128	2.66	UAA(*)	13	2.00	UGA(W)	76	1.85
UUG(L)	22	0.26	UCG(S)	3	0.06	UAG(*)	0	0.00	UGG(W)	6	0.15
CUU(L)	57	0.68	CCU(P)	65	2.39	CAU(H)	53	1.86	CGU(R)	17	1.48
CUC(L)	6	0.07	CCC(P)	4	0.15	CAC(H)	4	0.14	CGC(R)	0	0.00
CUA(L)	24	0.29	CCA(P)	36	1.32	CAA(Q)	44	1.57	CGA(R)	24	2.09
CUG(L)	2	0.02	CCG(P)	4	0.15	CAG(Q)	12	0.43	CGG(R)	5	0.43
AUU(I)	421	1.90	ACU(T)	59	2.17	AAU(N)	201	1.83	AGU(S)	21	0.44
AUC(I)	22	0.10	ACC(T)	8	0.29	AAC(N)	19	0.17	AGC(S)	1	0.02
AUA(M)	322	1.86	ACA(T)	41	1.50	AAA(K)	129	1.80	AGA(S)	101	2.10
AUG(M)	24	0.14	ACG(T)	1	0.04	AAG(K)	14	0.20	AGG(S)	13	0.27
GUU(V)	77	1.74	GCU(A)	47	2.16	GAU(D)	46	1.74	GGU(G)	53	1.25
GUC(V)	5	0.11	GCC(A)	0	0.00	GAC(D)	7	0.26	GGC(G)	4	0.09
GUA(V)	84	1.90	GCA(A)	38	1.75	GAA(E)	61	1.58	GGA(G)	74	1.74
GUG(V)	11	0.25	GCG(A)	2	0.09	GAG(E)	16	0.42	GGG(G)	39	0.92

3.5 Transfer RNAs and Ribosomal RNAs

Diglyphus sabulosus has 22 tRNAs, ranging from 59 to 76 bp in length. Fifteen of these tRNAs are located on the major coding strand (H-strand), and seven on the minor coding strand (L-strand). These tRNAs exhibit an AT bias (86.6%), similar to the overall mitogenomic composition. The AT and GC skew values are 0.030 and 0.218, respectively (Table 3). The predicted secondary structure of all tRNAs in the Diglyphus sabulosus mitogenome is presented in Fig. 4. With the exception of trnR and trnS1, which lack both the dihydrouracil arm (DHU arm) and dihydrouracil ring (DHU ring), all the tRNAs are folded into typical clover-leaf secondary structures, consisting of four domains and a variable loop. trnS1 lacks the DHU arm, which is almost ubiquitous in insect mitogenomes (Sun et al. 2010; Jühling et al. 2012; Cameron 2014). In the 22 tRNAs of the Diglyphus sabulosus mitogenome, a total of 11 G-U base mismatches were detected: one mismatch in each of trnD, trnL1, trnL2, and trnP; two mismatches in each of trnF and trnY; and three mismatches in trnA (Fig. 4). The mitogenome of Diglyphus sabulosus also contains 2 rRNAs. rrnL is 1,315 bp long and positioned between trnA and trnL1, while rrnS is 760 bp long and located between trnV and trnQ (Table 2).

3.6 Phylogenetic Relationships

In the current study, we analyzed phylogenetic relationships using complete mitogenome sequences. The primary objective was to gain insights into interrelationships within the Eulophidae clade, focusing on Diglyphus sabulosus. The phylogenetic relationships of Eulophidae were subsequently inferred via the ML and BI methods, based on the complete mitogenomes from Diglyphus sabulosus and other selected species. The two trees exhibited nearly congruent topologies. Therefore, we present the consensus tree, along with both Bayesian posterior probabilities and maximum likelihood bootstrap values, in Fig. 5. The phylogenetic results revealed that all species of the family Eulophidae form a single well-supported clade, confirming that Eulophidae is a monophyletic group with strong statistical support. Within Eulophidae, the two tribes Eulophinae and Tetrastichinae were also recovered as monophyletic. Diglyphus and Necremnus form a tightly clustered subclade, with the four Diglyphus species coalescing into a single clade. Within this clade, Diglyphus sabulosus and Diglyphus poppoea are sister groups, representing the closest relatives (posterior probability of 1; bootstrap value of 100%). Rao et al. (2025) reconstructed a ML phylogenetic tree using 13 protein-coding genes derived from four species within the family Eulophidae. The phylogenetic results revealed that all species belonging to the family Eulophidae form a single well-supported clade, confirming that Eulophidae is a monophyletic group with strong statistical support. The phylogenetic relationships within the family Eulophidae inferred in this study are generally consistent with previous findings ^[54–55] and congruent with conclusions from traditional taxonomy.

We obtained the first complete mitogenome sequence of Diglyphus sabulosus through next-generation sequencing. We characterized the mitogenomic architecture of Diglyphus sabulosus and found that it shares many significant features with other Eulophidae species, including gene order, nucleotide composition, codon usage bias, and one control region between trnI and trnM. Phylogenetic trees constructed using all 37 mitochondrial genes, which revealed the phylogenetic relationships of Eulophidae, aligning with previous studies in this respect. The phylogenetic results revealed that all species belonging to the family Eulophidae form a single well-supported clade, confirming that Eulophidae is a monophyletic group with strong statistical support. Diglyphus sabulosus and Diglyphus poppoea form a well-supported sister group, representing the species with the closest phylogenetic relationship within the analyzed taxa. This inferred close phylogenetic affinity is highly consistent with their morphological characteristics. The newly determined mitogenome sequence of Diglyphus sabulosus enables us to view the genus Diglyphus from a perspective differing from that of morphology-based taxonomy. There is a pressing need to acquire more mitogenomic data from Eulophidae species to improve our understanding of the phylogenetic relationships and evolutionary history of the family Eulophidae.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This project was supported by National Natural Science Foundation of China (32070472) and National Animal Collection Resource Center, China (No. 202406120003).

Author Contribution

F: Formal analysis, Writing – original draft, Writing – review & editing. G: Data curation, Formal analysis, Writing – review & editing. X: Methodology, Writing – review & editing. M: Writing – review & editing. H: Funding acquisition, Writing – review & editing.

Data Availability

The data presented in this study were deposited in the NCBI repository (accession numbers PX395427).

Bader AE, Heinz KM, Wharton RA, Bográn CE (2006) Assessment of interspecific interactions among parasitoids on the outcome of inoculative biological control of leafminers attacking chrysanthemum. Biol Control 39(3):441–452. https://doi.org/10.1016/j.biocontrol.2006.06.010
Ballard JWO, Rand DM (2025) The population biology of mitochondrial DNA and its phylogenetic implications. Annu Rev Ecol Evol Syst 36(1):621–642. https://doi.org/10.1146/annurev.ecolsys.36.091704.175513
Bernardo U, Monti MM, Nappo AG, Nappo AG, Gebiola M, Russo A, Pedata PA, Viggiani G (2008) Species status of two populations of Pnigalio soemius (Hymenoptera: Eulophidae) reared from two different hosts: An integrative approach. Biol Control 46(3):293–303. https://doi.org/10.1016/j.biocontrol.2008.05.009
Boore JL (1999) Animal mitochondrial genomes. Nucleic Acids Res 27(8):1767–1780. https://doi.org/10.1093/nar/27.8.1767
Burks RA, Heraty JM, Gebiola M, Hansson C (2011) Combined molecular and morphological phylogeny of Eulophidae (Hymenoptera: Chalcidoidea), with focus on the subfamily Entedoninae. Cladistics 27(6):581–605. https://doi.org/10.1111/j.1096-0031.2011.00358.x
Cameron SL (2014) Insect mitochondrial genomics: implications for evolution and phylogeny. Ann Rev Entomol 59(1):95–117. https://doi.org/10.1146/annurev-ento-011613-162007
Chen S, Zhou Y, Chen Y, Gu J (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34(17):i884–i890. https://doi.org/10.1093/bioinformatics/bty560
Darty K, Denise A, Ponty Y (2009) VARNA: Interactive drawing and editing of the RNA secondary structure. Bioinformatics 25(15):1974–1975. https://doi.org/10.1093/bioinformatics/btp250
Donath A, Jühling F, Al-Arab M, Bernhart SH, Reinhardt F, Stadler PF, Middendorf M, Bernt M (2019) Improved annotation of protein-coding genes boundaries in metazoan mitochondrial genomes. Nucleic Acids Res 47(20):10543–10552. https://doi.org/10.1093/nar/gkz833
Dowton M, Castro LR, Austin AD (2002) Mitochondrial gene rearrangements as phylogenetic characters in the invertebrates: the examination of genome'morphology'. Invertebrate Syst 16(3):345–356. https://doi.org/10.1071/IS02003
Du SJ, Yefremova Z, Ye FY, Zhu CD, Guo JY, Liu WX (2021) Morphological and molecular identification of arrhenotokous strain of Diglyphus wani (Hymenoptera, Eulophidae) found in China as a control agent against agromyzid leafminers. Zookeys 1071:109–126. https://doi.org/10.3897/zookeys.1071.72433
Foba CN, Salifu D, Lagat ZO, Gitonga LM, Akutse KS, Fiaboe KKM (2016) Liriomyza leafminer (Diptera: Agromyzidae) parasitoid complex in different agroecological zones, seasons, and host plants in Kenya. Environ Entomol 45(2):357–366. https://doi.org/10.1093/ee/nvv218
Gauthier N, LaSalle J, Quicke DLJ, Godfray HCJ (2000) Phylogeny of Eulophidae (Hymenoptera: Chalcidoidea), with a reclassification of Eulophinae and the recognition that Elasmidae are derived eulophids. Syst Entomol 25(4):521–539. https://doi.org/10.1046/j.1365-3113.2000.00134.x
Gebiola M, Bernardo U, Monti MM, Navone P, Viggiani G (2009) Pnigalio agraules (Walker) and Pnigalio mediterraneus Ferrière & Delucchi (Hymenoptera: Eulophidae): two closely related valid species. J Nat Hist 43(39):2465–2480. https://doi.org/10.1080/00222930903105088
Gebiola M, Bernardo U, Ribes A, Gibson GA (2015) An integrative study of Necremnus Thomson (Hymenoptera: Eulophidae) associated with invasive pests in Europe and North America: taxonomic and ecological implications. Zool J Linn Soc 173(2):352–423. https://doi.org/10.1111/zoj.12210
Gebiola M, Gómez-Zurita J, Monti MM, Navone P, Bernardo U (2012) Integration of molecular, ecological, morphological and endosymbiont data for species delimitation within the Pnigalio soemius complex (Hymenoptera: Eulophidae). Mol Ecol 21(5):1190–1208. https://doi.org/10.1111/j.1365-294x.2011.05428.x
Gibson GAP (1989) Phylogeny and classification of Eupelmidae, with a revision of the world genera of Calosotinae and Metapelmatinae (Hymenoptera: Chalcidoidea). Mem Entomol Soc Can 121(S149):3–121. https://doi.org/10.4039/entm121149fv
Gordh G, Hendrickson RJ (1979) New species of Diglyphus, a world list of the species, taxonomic notes, and a key to New World species of Diglyphus and Diaulinopsis (Hymenoptera: Eulophidae). Proceedings of the Entomological Society of Washington 81(4): 666–684
Grant JR, Enns E, Marinier E, Mandal A, Herman EK, Chen CY, Graham M, Van Domselaar G, Stothard P (2023) Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res 51(W1):W484–W492. https://doi.org/10.1093/nar/gkad326
Hansson C (1990) A taxonomic study on the Palearctic species of Chrysonotomyia Ashmead and Neochrysocharis Kurdjumov (Hymenoptera: Eulophidae). Insect Syst Evol 20:29–52
Hansson C, Navone P (2017) Review of the European species of Diglyphus Walker (Hymenoptera: Eulophidae) including the description of a new species. Zootaxa 4269(2):197–229. https://doi.org/10.11646/zootaxa.4269.2.2
Hansson C, Schmidt S (2020) A revision of European species of the genus Tetrastichus Haliday (Hymenoptera: Eulophidae) using integrative taxonomy. Biodivers data J 8:e59177. https://doi.org/10.3897/BDJ.8.e59177
Hebert PD, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society of London. Series B: Biological Sciences 270(1512): 313–321. https://doi.org/10.1098/rspb.2002.2218
Huang FN, Cao HX, Zhu CD (2022) Notes on the Genus Aceratoneuromyia Girault (Hymenoptera: Eulophidae). Insects 13(5):450. https://doi.org/10.3390/insects13050450
Jafarlu M, Karimpour Y, Lotfalizadeh H (2023) Fauna of the genus Diglyphus (Hymenoptera: Eulophidae) in the alfalfa fields of Iran. J Entomol Soc Iran 42(3):213–221. https://doi.org/10.52547/jesi.42.3.5
Jühling F, Pütz J, Bernt M, Donath A, Middendorf M, Florentz C, Stadler PF (2012) Improved systematic tRNA gene annotation allows new insights into the evolution of mitochondrial tRNA structures and into the mechanisms of mitochondrial genome rearrangements. Nucleic Acids Res 40(7):2833–2845. https://doi.org/10.1093/nar/gkr1131
Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods 14(6):587–589. https://doi.org/10.1038/nmeth.4285
Kaspi R, Parrella MP (2006) Improving the biological control of leafminers (Diptera: Agromyzidae) using the sterile insect technique. J Econ Entomol 99(4):1168–1175. https://doi.org/10.1603/0022-0493-99.4.1168
Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30(4):772–780. https://doi.org/10.1093/molbev/mst010
Kumar R, Sharma PL (2016) Studies on diversity and abundance of parasitoids of Chromatomyia horticola (Goureau)(Agromyzidae: Diptera) in north-western Himalayas, India. J Appl Nat Sci 8(4):2256–2261. https://doi.org/10.31018/jans.v8i4.1121
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35(6):1547–1549. https://doi.org/10.1093/molbev/msy096
Letunic I, Bork P (2024) Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res 52(W1). https://doi.org/10.1093/nar/gkae268. W78-W82
Liu QN, Xin ZZ, Bian DD, Chai XY, Zhou CL, Tang BP (2016) The first complete mitochondrial genome for the subfamily Limacodidae and implications for the higher phylogeny of Lepidoptera. Sci Rep 6:35878. https://doi.org/10.1038/srep35878
Liu Y, Li H, Song F, Zhao Y, Wilson JJ, Cai W (2019) Higher-level phylogeny and evolutionary history of Pentatomomorpha (Hemiptera: Heteroptera) inferred from mitochondrial genome sequences. Syst Entomol 44(4):810–819. https://doi.org/10.1111/syen.1235
Ma C, Yang P, Jiang F, Chapuis MP, Shali Y (2012) Mitochondrial genomes reveal the global phylogeography and dispersal routes of the migratory locust. Mol Ecol 21(17):4344–4358. https://doi.org/10.1111/j.1365-294X.2012.05684.x
Meng G, Li Y, Yang C, Liu S (2019) MitoZ: a toolkit for animal mitochondrial genome assembly, annotation and visualization. Nucleic Acids Res 47(11):e63. https://doi.org/10.1093/nar/gkz173
Nelson LA, Lambkin CL, Batterham P, Wallman JF, Dowton M, Whiting MF, Yeates DK, Cameron SL (2012) Beyond barcoding: a mitochondrial genomics approach to molecular phylogenetics and diagnostics of blowflies (Diptera: Calliphoridae). Gene 511(2):131–142. https://doi.org/10.1016/j.gene.2012.09.103
Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32(1):268–274. https://doi.org/10.1093/molbev/msu300
Nie R, Andújar C, Gómez-Rodríguez C, Bai M, Xue HJ, Tang M, Yang CT, Tang P, Yang XK, Vogler A (2020) The phylogeny of leaf beetles (Chrysomelidae) inferred from mitochondrial genomes. Syst Entomol 45(1):188–204. https://doi.org/10.1111/syen.12387
Perna NT, Kocher TD (1995) Patterns of Nucleotide Composition at Fourfold Degenerate Sites of Animal Mitochondrial Genomes. J Mol Evol 41(3):353–358. https://doi.org/10.1007/bf00186547
Ranwez V, Harispe S, Delsuc F, Douzery EJ (2011) MACSE: Multiple Alignment of Coding SEquences accounting for frameshifts and stop codons. PLoS ONE 6(9):e22594. https://doi.org/10.1371/journal.pone.0022594
Rao HY, Li XL, Wei XY, Sun JH, Wang X, Huang YX (2025) Sequencing and Analysis of Mitochondrial Genome of Chouioia cunea. J Zhejiang Forestry Sci Technol 45(01):87–95. https://doi.org/10.3969/j.issn.1001-3776.2025.01.012
Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61(3):539–542. https://doi.org/10.1093/sysbio/sys029
Simon C, Buckley TR, Frati F, Stewart JB, Beckenbach AT (2006) Incorporating molecular evolution into phylogenetic analysis, and a new compilation of conserved polymerase chain reaction primers for animal mitochondrial DNA. Annu Rev Ecol Evol Syst 37:545–579. https://doi.org/10.1146/annurev.ecolsys.37.091305.110018
Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P (1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am 87(6):651–701. https://doi.org/10.1093/aesa/87.6.651
Song HT, Fei MH, Li BP, Zhu CD, Cao HX (2020) A new species of Oomyzus Rondani (Hymenoptera, Eulophidae) reared from the pupae of Coccinella septempunctata (Coleoptera, Coccinellidae) in China. ZooKeys 953:49–60. https://doi.org/10.3897/zookeys.953.53175
Sun Z, Zhang J, Wang R, Xu YJ, Zhang DQ (2010) Progress of insect mitochondrial genome. J Inspection Quarantine 3:69–73
Talavera G, Castresana J (2007) Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol 56(4):564–577. https://doi.org/10.1080/10635150701472164
Tang P, Zhu JC, Zheng BY, Wei SJ, Sharkey M, Chen XX, Vogler AP (2019) Mitochondrial phylogenomics of the Hymenoptera. Mol Phylogenet Evol 131:8–18. https://doi.org/10.1016/j.ympev.2018.10.040
Tang X, Lyu B, Lu H, Tang J, Meng R, Cai B (2021) The mitochondrial genome of a parasitic wasp, Chouioia cunea Yang (Hymenoptera: Chalcidoidea: Eulophidae) and phylogenetic analysis. Mitochondrial DNA Part B 6(3):872–874. https://doi.org/10.1080/23802359.2021.1886008
Tian XC, Xian XQ, Zhang GF, Castañé C, Romeis J, Wan FH, Zhang YB (2021) Complete mitochondrial genome of a predominant parasitoid, Necremnus tutae (Hymenoptera: Eulophidae) of the South American tomato leafminer Tuta absoluta (Lepidoptera: Gelechiidae). Mitochondrial DNA Part B 6(2):562–563. https://doi.org/10.1080/23802359.2021.1875902
UCD Community (2023) Universal Chalcidoidea Database (UCD) curated in Taxon Works [DB/OL]. [2025-06-16]. https://sfg.taxonworks.org/api/v1/
Wan WJ, Du SJ, Hansson C, Liu WX (2023) A new species of Diglyphus Walker (Hymenoptera, Eulophidae) from China, with morphological characterizations and molecular analysis. ZooKeys 1148:65–78. https://doi.org/10.3897/zookeys.1148.98853
Ye FY, Zhu CD, Yefremova Z, Liu WX, Guo JY, Wan FH (2018) Life history and biocontrol potential of the first female-producing parthenogenetic species of Diglyphus (Hymenoptera: Eulophidae) against agromyzid leafminers. Sci Rep 8(1):3222. https://doi.org/10.1038/s41598-018-20972-3
Yefremova Z, Civelek HS, Boyadzhiyev P, Dursun O, Eskin A (2011) A review of Turkish Diglyphus Walker (Hymenoptera: Eulophidae), with description of a new species [J]. Annales de la Société Entomologique de France (N.S.) 47(3–4): 273–279
Zheng BY, Cao LJ, Tang P, van Achterberg K, Hoffmann AA, Chen HY, Chen XX, Wei SJ (2018) Gene arrangement and sequence of mitochondrial genomes yield insights into the phylogeny and evolution of bees and sphecid wasps (Hymenoptera: Apoidea). Mol Phylogenet Evol 124:1–9. https://doi.org/10.1016/j.ympev.2018.02.028
Zhu CD, Lasalle J, Huang DW (2000) A review of the Chinese Diglyphus Walker (Hymenoptera: Eulophidae). Orient insects 34(1):263–288. https://doi.org/10.1080/00305316.2000.10417266

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The Complete Mitochondrial Genome of a Newly Recorded Chinese Species of Diglyphus sabulosus (Hymenoptera: Eulophidae) and Insights into Its Phylogenetic Position

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Abstract

Figures

1 Introduction

2 Materials and Methods

2.1 Sample Collection and Species Identification

2.2 DNA Extraction and Genome sequencing

2.3 Genome assembly, annotation and validation

2.4 Sequence Analysis

2.5 Phylogenetic Analysis

3 Results and Discussion

3.1 Diglyphus sabulosus Erdӧs, 1951(Newly Recorded Species from China)

3.2 Mitogenome Organization

3.3 Nucleotide Composition

3.4 Protein-Coding Genes

3.5 Transfer RNAs and Ribosomal RNAs

3.6 Phylogenetic Relationships

4 Conclusion

Declarations

Conflict of interest

Funding

Author Contribution

Data Availability

References

Additional Declarations

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