Five mitochondrial genomes of black fungus gnats (Sciaridae) and their phylogenetic implications

Journal Pre-proof Five mitochondrial genomes of black fungus gnats (Sciaridae) and their phylogenetic implications

Xiaoqian Miao, Junhao Huang, Frank Menzel, Qingyun Wang, Qiaoyu Wei, Xiao-Long Lin, Hong Wu PII:

S0141-8130(19)38697-0

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.271

Reference:

BIOMAC 14576

To appear in:

International Journal of Biological Macromolecules

Received date:

26 October 2019

Revised date:

26 January 2020

Accepted date:

27 January 2020

Please cite this article as: X. Miao, J. Huang, F. Menzel, et al., Five mitochondrial genomes of black fungus gnats (Sciaridae) and their phylogenetic implications, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/ j.ijbiomac.2020.01.271

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© 2018 Published by Elsevier.

Journal Pre-proof Five mitochondrial genomes of Black Fungus Gnats (Sciaridae) and their phylogenetic implications Xiaoqian Miaoa, Junhao Huanga*, Frank Menzelb, Qingyun Wanga, Qiaoyu Weia, Xiao-Long Linc, Hong Wua

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a Department of Forestry Protection, School of Forestry and Biotechnology, Zhejiang A&F University, 666 Wusu street, Linan, Hangzhou, Zhejiang 311300, China b Senckenberg Deutsches Entomologisches Institut, Eberswalder Straße 90, 15374 Müncheberg, Germany. E-mail: [email protected] c College of Life Sciences, Nankai University, Tianjin 300071, China. E-mail: [email protected] * Corresponding author: E-mail: [email protected], Tel: 86-571-63732758, Fax: 86-571-63740898

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Abstract: Sciaridae is a family of great species diversity, distributed worldwide,

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that includes important agricultural pests of cultivated mushrooms and plants produced in greenhouses. Here we sequenced five nearly complete

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mitochondiral genomes representing three subfamilies of Sciaridae. The lengths of these mitogenomes range from 13,849 bp to 16,923 bp with 13

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protein-coding genes (PCGs), 20–22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control region (CR). Compared with other dipteran species, rearrangements in Sciaridae are more common. Inversion or transition is observed frequently of trnL2, and in the tRNA clusters trnI-trnQ-trnM,

trnW-trnC-trnY,

and

trnA-trnR-trnN-trnS1-trnE-trnF.

Phylogenetic relationships within the family were reconstructed based on these newly sequenced species, combined with the published mitogenomes of related

families,

and

recovered

the

topology

within

Sciaroidea

as

Cecidomyiidae + (Sciaridae + Keroplatidae). Relationships recovered within Sciaridae were Sciarinae + (‗Pseudolycoriella group‘ + Megalosphyinae). Keywords: Sciariodea; molecular phylogeny; Diptera

Journal Pre-proof 1. Introduction The adults of black fungus gnats (Diptera: Sciaroidea: Sciaridae) are mostly tiny flies with a slender dark-colored body, long legs and simple wings [1, 2]. They are widely distributed across the world, with more than 2,800 species [3]. Sciarids are found in various terrestrial habitats ranging from caves to high altitude mountains, but primarily in forests and other moist shady areas [4]. Larvae feed on fungi, detritus, rotten wood and organic matter in soil, playing a role as decomposers [1, 5, 6]. Nevertheless, several sciarid species

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are also known as economically significant pests. The larvae of Bradysia

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cause serious damage to onions, carrots, and edible mushrooms [1]. In addition, adult flies serve as vectors for the pathogen Fusarium foetens on the

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ornamental plant begonias, causing serious economic loss [7, 8].

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Phylogenetic relationships within Sciaridae are still controversial despite more than two-hundred years of research [9, 10]. Menzel & Mohrig (2000)

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used 160 characters to construct a morphological phylogenetic tree of Sciaridae, suggesting that this family include Sciarinae, Megalosphyinae,

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Cratyninae and a clade composed of the Pseudolycoriella group plus the Corynoptera sensu lato group. However, all species included in Menzel &

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Mohrig (2000) were confined to the Palearctic region. Although Vilkamaa & Hippa (2004) and Hippa & Vilkamaa (2005, 2006) reconstructed morphological trees for Cratyna senso lato and of fossil Sciaroidea, phylogenetic relationships with the broader family remain unresolved because of limited taxon

representation

and

homoplastic

characters.

Recent

molecular

phylogenetic trees [15, 16] based on a few loci supported major clades of Sciarinae, Cratyninae, Megalosphyinae and the Pseudolycoriella group that were found in the morphological work by Menzel & Mohrig (2000), plus a Chaetosciara group, which was later recognized as a new subfamily Chaetosciarinae [17]. Topological relationships within Sciaridae based on either morphological characters or a few molecular loci were incongruent, and were unstable when additional taxa or genes were included [16].

Journal Pre-proof In general, the mitochondrial genome of most insects is a closed circular DNA molecule ranging in size from 14 to 20 kb, that encodes a common set of 37 genes (13 protein-coding genes, 22 tRNA genes, and two rRNA genes) and an A+T-rich region [18, 19, 20]. The mitochondrial genome is small-sized, with high coding content conservation, high evolutionary rates, maternal inheritance and rare recombination, and as such has been widely used for species identification and molecular evolutionary studies of Diptera [21, 22, 23, 24, 25]. Due to the rapid progress of sequencing technology, studies of Diptera

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mitogenomes have covered most families, especially medically significant taxa

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such as mosquitoes (Culicidae) [27, 28, 29], or economically important species from leaf-miners (Agromyzidae) and fruitflies (Tephritidae) [23, 30, 31, 32].

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Additionally, many common families have been sequenced as well [e.g. 33, 34,

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35]. However, the study on the mitochondrial genome of Sciaridae is still

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limited with only one species been published, that is incomplete with ten tRNA genes missing [24].

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In this study, we sequenced nearly complete mitochondrial genome sequences from five species representing three sciarid subfamilies using

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next-generation sequencing. We compared mitogenomes within Sciaridae and with other families of Sciaroidea, and analyzed phylogenetic relationships among the Sciaroidea based on nine mitochondrial genomes.

2. Materials and methods

2.1 DNA extraction and species identification Representative specimens were identified based on morphology: Bradysia sp. (representative of B. pallipes species group); Dolichosciara megumiae; Sciara ruficauda (representative of S. ruficauda species group); Trichosia lengersdorfi (representative of subgenus Trichosia Winnertz s. str.); Pseudolycoriella sp. (representative of Ps. bruckii species group) [11, 36, 37, 38, 39]. The unnamed species from genera Bradysia Winnertz and Pseudolycoriella Menzel & Mohrig are new to science and will be described

Journal Pre-proof elsewhere in a taxonomic paper. Information on the specimens studied (e.g. collecting sites, GenBank accession numbers) is summarized in Table 1. All specimens were preserved in absolute alcohol and stored at −20°C before DNA extraction. Total genomic DNA was extracted was from single sample using the DNeasy Blood & Tissue kit (Qiagen Hilden, Germany) following manufacturer instructions. Concentration of extracted genomic DNA was quantified by Qubit 3.0 (Invitrogen, Life Technologies, Carlsbad, CA, USA). Voucher specimens are deposited in the School of Forestry and Biotechnology,

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Zhejiang A&F University, China (Specimen number: SCMI1–SCMI5).

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2.2 Mitochondrial genome sequencing, assembly and annotation Genomic DNA libraries were constructed for each sample using the

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Illumina® VAHTSTM Universal DNA Library. Indexed libaries were directly

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sequenced using Illumina NovaSeq 150bp paired-end reads of Novogene (Tianjin, China).

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FastQC version 0.11.3 was used to evaluate read quality [40], with low quality reads and sites filtered by Trimmomatic version 3.2.57 [41]. To simplify

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de novo assembly, target mitochondrial genome sequences were extracted by BLAST version 3.6 [42] run against a local database composed of GenBank

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accessions of Sciaroidea mitochondrial genomes. Whole mitochondrial genomes were assembled by Spades version 3.13.1 with default settings [43, 44]. Mitochondrial genome annotations were predicted by the Mitos webserver [45] using the invertebrate mito genetic code. tRNA genes were confirmed by tRNA scan-SE [46]. The 5′ and 3′ ends of each protein-coding or rRNA gene were checked with Geneious-prime against homologous genes from Diptera. All mitochondrial genomes were submitted to GenBank (accession numbers: MN161585–MN161589).

Table 1. Information on the mitochondrial genomes used in this study. Note: -, represents an unknow collecting site. Species

Family

Subfamily

GenBank accession number

Collecting sites

Reference

Journal Pre-proof Bradysia sp.

Sciaridae

Megalosphyinae

MN161585

Zhejiang, China

This study

Dolichosciara megumiae

Sciaridae

Megalosphyinae

MN161588

Zhejiang, China

This study

Undescribed

subfamily

(named ‘Pseudolycoriella Pseudolycoriella sp.

Sciaridae

group’)

MN161587

Sichuan, China

This study

Sciara ruficauda

Sciaridae

Sciarinae

MN161586

Zhejiang, China

This study

Trichosia lengersdorfi

Sciaridae

Sciarinae

MN161589

Zhejiang, China

This study

Providence, RI,

Beckenbach

USA

and Joy [47]

-

Beckenbach

Bradysia tilicola

Sciaridae

Megalosphyinae

GQ387651

Arachnocampa flava

Keroplatidae

Arachnocampinae

JN861748

Cecidomyiidae

KM888183

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Rhopalomyia pomum

Cecidomyiinae Cecidomyiinae

2.3 Genome feature analysis

GQ387649

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Cecidomyiidae

Hyderabad,

Atray et al.

India

[49]

Kamloops,

BC,

Canada

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Orseolia oryzae

[48]

Base composition and codon usage of PCGs was calculated in MEGA7

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[50]. Compositional skew analysis was calculated according to the formulas:

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AT-skew = (A-T) / (A+T) and GC-skew = (G-C) / (G+C) [51]. Rates of synonymous (Ks) and non-synonymous (Ka) substitutions for each PCG were

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calculated in DnaSP 5.0 [52].

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2.4 Phylogenetic inference

A total of 9 species were analyzed, including five species of Sciaridae sequenced in this study and four additional species, Bradysia amoena (Winnertz, 1867) [= Bradysia tilicola (Loew, 1850)] (Sciaridae), Arachnocampa flava (Harrison, 1966) (Keroplatidae), Rhopalomyia pomum (Gagné, 1975) and Orseolia oryzae (Wood-Mason, 1889) (both Cecidomyiidae, as outgroup) were retrieved from GenBank (Table 1). MAFFT version 7.205 was used to align each protein-coding gene separately [47, 53]. Nucleotide sequences of PCGs were aligned by codon using the G-INS-I algorithm [53, 54]. Aliscore version 2.2 and Alicut version 3.2 were applied to identify and mask sites with ambiguous alignment to reduce noise [55].. FASconCAT-G version 1.0 was used to concatenate genes into data matrices [56]. Partitioning scheme and substitution models were selected using PartitionFinder version 1.1.1 [57].

Beckenbach and Joy [47]

Journal Pre-proof Initial partitions were by codon position and gene for each PCG (39 partitions), and by gene for amino acids analyses (13 partitions) (Supplement Table S1). Two inference methods, maximum likelihood (ML) and Bayesian inference (BI)

were

performed

using

the

IQ-Tree

online

sever

[58]

(http://iqtree.cibiv.univie.ac.at.) and MrBayes version 3.2.6 [59, 60] respectively. Two data matrices, DNA and amino acid sequences from all protein-coding genes were used for phylogenetic analysis. 3 Results

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3.1 Genome structure and organization

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Mitochondrial genome lengthes range from 15,167 bp to 16,170 bp in Sciaridae (Table 2). Each mitogenome contains the typical set of 37 genes,

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including 13 PCGs, two rRNAs, and 22 tRNAs (except Bradysia sp. and

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Dolichosciara megumiae are missing trnL2, and Bradysia sp. is missing trnI).

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These genomes were sequenced near completely, with the CRs of all species failing to completely assemble. A + T content is lower than 80% in all species

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of Sciaridae. A comparison of nucleotide composition suggests that the AT skew values are positive (from 0.0165 to 0.1050), indicating bias towards A

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content rather than T content in all species, except for Pseudolycoriella sp. (-0.0101) and Arachnocampa flava (-0.0341), while the GC skew values are negative (from -0.11 to -0.2233), indicating bias towards C content in all species.

Table 2. Base composition of the mitochondrial genomes in Sciaroidea. Note: AT-skew and GC-skew were calculated to analyze the A + T and G + C bias of PCGs, with the formula AT-skew= [A−T] / [A+T] and GC-skew= [G−C] / [G+C], respectively. Species Bradysia sp. Dolichosciara megumiae Pseudolycoriella sp. Sciara ruficauda Trichosia lengersdorfi Rhopalomyia pomum Bradysia tilicola

Assembled genome T(U)％ 35.7 37.9 39.9 38.8 38.5 40.6 38.5

C％

A％

G％

AT-skew

GC-skew

Length

14.6 12.9 11.6 12.4 12.6 8.3 12.1

39.4 39.5 39.1 40.1 40.9 44.6 39.8

10.3 9.7 9.3 8.7 8.0 6.5 9.5

0.0493 0.0207 -0.0101 0.0165 0.0302 0.0469 0.0166

-0.1727 -0.1416 -0.1100 -0.1754 -0.2233 -0.1216 -0.1204

15512.0 15931.0 15981.0 15167.0 16170.0 14503.0 13849.0

Journal Pre-proof Arachnocampa flava Orseolia oryzae

42.4 38.3

10.6 8.1

39.6 47.4

7.3 6.2

-0.0341 0.1050

-0.1844 -0.1329

16923.0 15286.0

The mitochondrial genome of all five Sciaridae species included 13 PCGs. Nine genes are located in J strand, nd2, cox1, cox2, atp8, atp6, cox3, nd3, nd6 and cytb, while the others nd1, nd5, nd4, and nd4l are located in the N strand. Total PCG lengthes range from 11,161 to 11,268 bp (Table 4). A + T content across PCGs are 73.5% in Bradysia sp., 74.3% in Dolichosciara megumiae,

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76.8% in Trichosia lengersdorfi, 76.5% in Pseudolycoriella sp. and 76.8% in

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Sciara ruficauda, with an average of 75.6% (Table 3).

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Table 3. Base composition of Protein-Coding Genes in the mitochondrial genomes of Sciaridae. Note: AT-skew and GC-skew were calculated to analyze the A + T and G + C bias of PCGs, with the formula AT-skew= [A−T] / [A+T] and GC-skew= [G−C] / [G+C], respectively. All Protein-Coding Genes (PCGs) T% 41.1 42.6 44.4 44.4 44.2

14.1 12.9 11.7 11.7 11.8

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Bradysia sp. Dolichosciara megumiae Pseudolycoriella sp. Sciara ruficauda Trichosia lengersdorfi

C%

A%

G%

AT-skew

GC-skew

Length

30.8 31.7 32.1 32.4 32.6

14.0 12.8 11.9 11.5 11.4

-0.1433 -0.1467 -0.1608 -0.1563 -0.1510

-0.0036 -0.0039 0.0085 -0.0086 -0.0172

11249.0 11215.0 11268.0 11161.0 11201.0

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Species

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3.2 Gene arrangement

Compared to the ancestral gene arrangement of insects, only Sciara ruficauda retains the typical gene positions and orientation (Figure 1). Each of the other five Sciaridae species showed rearrangement of tRNAs. In Dolichosciara megumiae and Bradysia sp., trnL2 was all missed, trnC-trnY was translocated into the tRNA cluster between nad3 and nad5, while trnR-trnN was translocated between nad6 and cob. trnI was not found in either Bradysia sp. or Bradysia tilicola [= B. amoena]. Differences were also found in Pseudolycoriella sp. and Trichosia lengersdorfi. In Pseudolycoriella sp. trnC moved from its typical location to a position between trnQ and trnM, while trnY and trnL2 transposed to the position between nad2 and CR. trnC transposed from the typical position between nad2 and cox1 to the block of tRNA genes

Journal Pre-proof between the CR and nad2, while the trnA and trnR genes were switched in Trichosia lengersdorfi. Rearrangements in sciarid mostly occurred in trnL2 and the

tRNA

clusters

of

the

trnI-trnQ-trnM,

trnW-trnC-trnY,

and

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trnA-trnR-trnN-trnS1-trnE-trnF.

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Figure.1 Mitochondrial gene arrangement pattern of Sciaridae. Note: the blue rectangles represented 13 PCGs (cox1-cox3: cytochrome oxidase subunits; ctyb: cytochrome b; nad1-nad6: nadh dehydrogenase com-ponents; atp6, atp8: ATP synthase subunit 6 and 8 genes); the green rectangles represented tRNAs and the red rectangles represented rrnL and rrnS (ribosomal RNAs). Single letters identify the transfer RNA genes. The broken lines represent unsequenced regions in the mitochondrial genome and the arrow indicated the tRNA gene transfer orientation and location.

3.3 Phylogenetic relationships Phylogenetic trees (Figure 2) were reconstructed based on nine near complete mitochondrial genomes of Sciaroidea, representing three families. Similar phylogenetic topologies were found from both data matrixes (AA-PCGs, NU-PCGs) and both inference methods (BI and ML method). All analyses support the monophyly of Sciaridae (AA-PCGs: PP (posterior probability)=1, BS (boot-straps)=100; NU-PCGs: PP=1, BS=100). Within Sciaridae, the relationships of three subfamilies are Sciarinae + (‗Pseudolycoriella group‘ + Megalosphyinae). Sciara ruficauda and Trichosia lengersdorfi form Sciarinae with strongsupport (PP=1, BS>70), while Dolichosciara megumiae, Bradysia tilicola and Bradysia sp. form the subfamily Megalosphyinae with strong

Journal Pre-proof support (AA-PCGs: PP=1/1, BS=84/100; NU-PCGs: PP=1/1, BS=79/100) and

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Pseudolycoriella sp. is sister to Megalosphinae.

Figure 2．Phylogenetic relationships of Sciaroidea inferred from mitochondrial genomes. Note: The numbers separated by ―/‖ near the nodes represent support values of BI and ML analyses based on matrixes of AA-PCG (in purple) and NU-PCG (in yellow) respectively, while ‖*‖ represents the support values PP=1 and BS=100.

4. Discussion The monophyly of Sciaridae has been demonstrated in recent studies [15, 16, 61] and was well supported in this study. The topology of Sciaridae was recovered as (Sciarinae + (‗Pseudolycoriella group‘ + Megalosphyinae)), consistent with previous morphological and molecular studies [11, 15, 16]. The

Journal Pre-proof Sciarinae and Megalosphyinae are highly supported, consistent with previous research [15, 16]. The present phylogenetic system of Diptera is composed of five major clades including the ―lower‖ Diptera groups, Tipulomorpha, Culicomorpha, Psychodomorpha and Bibionomorpha, and the ―higher‖ Diptera, the Brachycera [24, 62]. Sciaroidea is usually regarded as the most speciose superfamily

of

the

Bibionomorpha

[63,

64].

Most

of

mitogenome

rearrangements found in Diptera are duplications or inversions of tRNA genes 66].

For

example,

in

Paracladura

trichoptera

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[65,

(Tipulomorpha:

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Trichoceridae), rearrangements involved both tRNA and protein-coding genes [48]; trnS is inverted in Anopheles quadrimaculatus and A. gambiae

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(Culicomorpha: Culicidae) [27, 28]. Novel gene orders were found in

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Chrysomya (Brachycera: Calliphoridae with a trnI duplications on either side of

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the CR [67, 68, 69]. Compared with other dipteran species, rearrangements in Sciaridae are more common in that inversion or transition of trnL2 frequent,

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as is rearrangements in the tRNA clusters of trnI-trnQ-trnM, trnW-trnC-trnY, and trnA-trnR-trnN-trnS1-trnE-trnF.

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Although the composition of the Sciaroidea is still controversial [13, 14, 61, 70], studies based on mitochondrial and nuclear loci show Cecidomyiidae nested within Sciaroidea [47, 61, 62]. Within Sciaroidea, PCGs are not translocated within the mitogenome. Rearrangements within Cecidomyiidae mitochondrial genome are notably complicated for Diptera, including losses of tRNA genes and inversions of trnI, trnC, trnY, trnE, trnN, trnP and trnT [47]. Rearrangements in Sciaridae are comparatively less complicated involving trnL2, trnC, trnY, trnR and trnN, while Keroplatidae has only a trnE inversion recorded in Arachnocampa flava [48].

5. Conclusion In this study, we sequenced five nearly complete mitochondrial genomes by next generation sequencing technologies. Our phylogenetic results support

Journal Pre-proof previous studies based on molecular data [15] and morphological characters [11], however, the taxon selection is still limited. Whole mitogenomes will serve as a useful dataset for further study of the genetics, systematics and phylogeny of Sciaridae.

Acknowledgments We are grateful to Dr. Pu Tang and Ms. Bo-Ying Zheng (Zhejiang

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University, China) for their assistance on data analysis and manuscript

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improvement. We also thank Andrew Liston (Senckenberg Deutsches Entomologisches Institut, Müncheberg, Germany) for checking the English and

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Mr. Xue Yang and Mr. Zuluan Chen (Zhejiang A&F University, China) for their

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help with the identification. This study was supported by the National Natural

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Science Foundation of China (NSFC, grant no. 31872270).

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Journal Pre-proof Author statement Xiaoqian Miao: Writing, Investigation, Formal analysis Junhao Huang: Conceptualization, Writing-Review, Funding acquisition Frank Menzel: Reviewing Qingyun Wang: Visualization Qiaoyu Wei: Data curation, Software, Validation Xiao-Long Lin: Methodology, Reviewing

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Hong Wu: Supervision