First mitogenome for the subfamily Miltogramminae (Diptera: Sarcophagidae) and its phylogenetic implications

The mitochondrial genome of Mesomelena mesomelaena (Loew, 1848) is the first to be sequenced in the flesh fly subfamily Miltogramminae (Diptera: Sarcophagidae). The 14,559 bp mitogenome contains 37 typical metazoan mitochondrial genes: 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes, with the same locations as in the insect ground plan. All the protein-coding genes have the start codon ATN, except for cox1 (TCG). Eight protein-coding genes have the stop codon TAA, while the remaining five have the stop codon T (cox1, cox2, nad5, and nad4) or TAG (cytb). Synonymous and non-synonymous substitution rates (Ks and Ka) for each protein-coding gene indicate that these genes evolved primarily under negative (or purifying) selection (Ka < Ks). Phylogeny of Sarcophagidae is proposed based on all the sarcophagid mitogenomes in GenBank, and the subfamily topology is reconstructed as (Sarcophaginae (Paramacronychiinae, Miltogramminae)).

Nineteen mitogenomes of Oestroidea (Table 2) were downloaded from GenBank to reconstruct the phylogeny of Sarcophagidae, with Lucilia cuprina as an outgroup.All 15 genes were aligned separately with MAFFT v 7.3.1 (Katoh & Standley, 2016), following Kutty et al. (2014).After alignment, all 13 PCGs and two rRNAs were concatenated using SequenceMatrix v 1.7.8 (Meier et al., 2006).Subsequently, PartitionFinder v 2.1.1 (Lanfear et al., 2016) was used to evaluate the best partitioning schemes based on BIC (Bayesian Information Criterion) using the greedy option with branch lengths estimated as "unlinked", with the dataset partitioned by genes.Phylogenetic analyses were conducted using Maximum Likelihood (ML) and Bayesian Inference (BI) as described in Zhang et al. (2016b).
Synonymous and non-synonymous substitution rates (Ks and Ka) for each species of each protein-coding gene (PCG) alignment were calculated using DnaSP version 5.10.1 (Librado & Rozas, 2009), with stop codons and codons with alignment gaps excluded.A plot of pairwise Ka versus Ks for each gene was then constructed using Sigmaplot v 12.5 (Systat Software Inc. California, USA).

RESULTS AND DISCUSSION
As the control region of M. mesomelaena could not be amplifi ed, the small ribosomal RNA gene is incomplete at the 5' end.The nearly-complete mitogenome of M. mesomelaena (14,559 bp) was submitted to GenBank (Accession Number: KY003227).
All PCGs share the start codon of ATT or ATG, except for cox1 (TCG), nad3 and nad1 (ATA).Four genes (cox1, cox2, nad5, and nad4) use the incomplete stop codon T, and cytb uses TAG.The remaining eight PCGs have the typical stop codon of TAA.
Th e selection acting on a gene may be detected by comparing the synonymous (Ks) and non-synonymous (Ka) nucleotide distances between gene sequences (Hurst, 2002).Higher Ka than Ks indicates positive selection, and lower Ka than Ks indicates negative selection.The Ka value is obviously lower than the Ks value for all sarcophagid * Primer pair designed using Primer Premier 5.0 (Lalitha, 2000).mitochondrial genes (Fig. 4), except atp8, for which only non-synonymous substitutions between Sarcophaga africa (locus: NC_025944) (Fu et al., 2016)  ML and BI analyses give an identical result, with Sarcophagidae being monophyletic and Sarcophaginae as the sister group to (Par amacronychiinae + Miltogramminae) (Fig. 5).According to previous research the relationship of these three subfamilies is still unresolved.Pape (1996: 9) proposed a probable sister-group relationship between the Paramacronychiinae and Sarcophaginae, based on "the shared possession of a ventrally displaced acrophallus, lack of epiphallus and perhaps spherical female accessory glands", which was corroborated by Giroux et al. (2010)  Within the Sarcophaginae, Boettcheria is located as a basal branch, forming a sister group to (Ravinia + Sarcophaga).This is in confl ict with morphology-based phylogenies of this subfamily, where Ravinia is subordinate to Boettcheria and Sarcophaga (Pape, 1994;Giroux et al., 2010).The monophyly of Sarcophaga is well supported (ML bootstrap = 100; BI posterior probability = 1), but the few taxa sampled and inclusion of only Old World species do not allow the testing of any of the biogeographic hypotheses proposed by Buenaventura et al. (2016) and Buenaventura & Pape (2017).
This study documents the fi rst mitogenome of Miltogramminae, which contains 37 typical metazoan mitochondrial genes and retains the organization of the ancestral insect mitogenome.The subfamily relationship within Sarcophagidae is reconstructed for the fi rst time as (Sarcophaginae (Paramacronychiinae, Miltogramminae)) based on mitogenomic data.
and S. similis (locus: NC_025573) (Yan et al., 2016) were detected.The lower values of Ka than Ks indicate negative selection on sarcophagid mitochondrial genes.Specifi cally, the cox1 gene has undergone strong negative selection, indicated by the extremely low Ka value compared to Ks value, which also implies the conservativeness of cox1 gene.
in an explicit morphology-based phylogenetic analysis.This relationship was corroborated by Kutty et al. (2010) using a combination of mitochondrial and nuclear genes, although with low support.However, based on a large-scale (in terms of taxa) molecular phylogeny , Piwczyński et al. (2014) suggest a sister-group relationship of Sarcophaginae to (Paramacronychiinae + Miltogramminae), with Paramacronychiinae being paraphyletic, and Piwczyński et al.'s (2017) results are fully concordant with the above, with each subfamily being monophyletic.

Fig. 4 .
Fig. 4. Plot of pairwise Ka versus Ks of the mitochondrial genes of Sarcophagidae.

Table 2 .
Taxa included in the phylogenetic analyses in this study.