Mitochondrial genomic investigation reveals a clear association between species and genotypes of Lucilia and geographic origin in Australia

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Young, Ying Ting Yang, Philip Batterham, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2914299/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Aug, 2023 Read the published version in Parasites & Vectors → Version 1 posted 7 You are reading this latest preprint version Abstract Background Lucilia cuprina and L. sericata (family Calliphoridae) are globally significant ectoparasites of sheep. Current literature suggests that only one of these blowfly subspecies, L. cuprina dorsalis , is a primary parasite causing myiasis (flystrike) in sheep in Australia. These species and subspecies are difficult to distinguish using morphological features. Hence, being able to accurately identify blowflies is critical for diagnosis and for understanding their relationships with their hosts and environment. Methods In this study, adult blowflies (5 pools of 17 flies; n = 85) were collected from five locations in different states [New South Wales (NSW), Queensland (QLD), Tasmania (TAS), Victoria (VIC) and Western Australia (WA)] of Australia and their mitochondrial (mt) genomes were assembled. Results Each mt genome assembled was ~ 15 kb in size and encoded 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs and a control region. The Lucilia species mt genomes were conserved in structure and the genes retained the same order and direction. The overall nucleotide composition was heavily biased towards As and Ts − 77.7% of the whole genomes. Pairwise nucleotide diversity suggested divergence between L. cuprina cuprina, L. c. dorsalis and L. sericata . Comparative analyses of these mt genomes with published data demonstrated that the blowflies collected from sheep farm in TAS clustered within a clade with L. sericata . The flies collected from an urban location in QLD were more closely related to L. sericata and represented the subspecies L. c. cuprina , whereas the flies collected from sheep farms in NSW, VIC and WA represented the subspecies L. c. dorsalis. Conclusions Phylogenetic analyses of the mt genomes representing Lucilia from the five geographic locations in Australia supported the previously demonstrated paraphyly of L. cuprina with respect to L. sericata and revealed that L. c. cuprina is distinct from L. c. dorsalis , and that L. c. cuprina is more closely related to L. sericata than L. c. dorsalis . The mt genomes reported here provide an important molecular resource to develop tools for species- and subspecies-level identification of Lucilia from different geographical regions across Australia. Australian sheep blowfly Lucilia cuprina dorsalis Lucilia cuprina cuprina Lucilia sericata mt genome phylogenetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Background In Australasia, blowflies of the genus Lucilia (Insecta, Diptera, Calliphoridae) are recognised as facultative ectoparasites of domesticated sheep but can severely affect various domestic and wild animals [ 1 – 3 ]. The Australian sheep blowfly, Lucilia cuprina (Wiedemann, 1830), causes cutaneous myiasis – a serious skin disease of sheep [ 4 – 6 ]. This disease results in an estimated loss of $ 324 million per annum to the Australian wool industry alone [ 7 ]. The common green bottle fly, Lucilia sericata (Meigen, 1826), is another closely related blowfly that does not cause myiasis in sheep in Australia, as it does not infest live animals as L. cuprina does [ 8 ]. However, L. sericata is the primary initiator of myiasis in sheep in Europe and is also a saprophagous species in other parts of the world [ 9 – 12 ]. Although both species are considered to have a cosmopolitan distribution [ 13 , 14 ], L. cuprina is typically found more frequently in temperate subtropical areas and L. sericata is more prevalent in cool to temperate climes [ 3 ]. Phylogenetic studies conducted using mitochondrial (mt) gene and whole mt genome data sets have demonstrated that L. cuprina is paraphyletic with respect to L. sericata [ 15 – 17 ]. Populations of these two species were phylogenetically distinct, except for L. cuprina from Hawaii [ 15 ]. L. cuprina and L . sericata can hybridise, with hybrids displaying L. cuprina -like morphology [ 8 ]. A previous study using a combination of complementary nuclear − 28S subunit ribosomal RNA (28S rRNA) and mt cytochrome c oxidase subunits I and II ( cox1 and cox2 ) demonstrated that these hybrids possessed L. sericata -type mt DNA and L. cuprina -type nuclear DNA [ 15 ]. Such hybrid flies have also been reported in other parts of the world, such as Asia [ 18 ], Australia [ 9 , 17 ], North America [ 16 ] and South Africa [ 19 ]. Two distinct subspecies of L. cuprina have been described: Lucilia cuprina cuprina (Wiedemann, 1830) and Lucilia cuprina dorsalis (Robineau-Desvoidy, 1830) [ 8 , 20 , 21 ]. L. c. cuprina has been identified in Australia, China, Fiji, Hawaii, India, Indonesia, Japan, Java, Malaysia, North America, South America and Timor, whereas L. c. dorsalis is distributed throughout Africa, Australia, India and New Zealand [ 8 , 20 , 22 ]. These two species as well as their subspecies are morphologically very similar and can be very challenging to identify and distinguish. L. c. cuprina and L. c. dorsalis interbreed in Eastern parts of Australia [ 20 ] and produce hybrids that have overlapping morphological features complicating identification. The purpose of this study was to gain important insights into systematic relationships of Lucilia species from different states of Australia using mt genomic data sets. In this study, a molecular approach was used to identify and characterise species and subspecies of Lucilia employing mt DNA with the aim of establishing high-quality genomic resources for these parasites. Little is known about the current distribution and genetics of these species/subspecies in Australia, which has resulted in significant bottlenecks in efforts to characterise and control these pests. Our work on the mt genomes of Lucilia species provides data that can be used to develop fast and reliable molecular tools that could identify and differentiate between individuals from wild populations of Lucilia across Australia. Methods Fly collection, DNA extraction and sequencing Blowflies were collected from five Australian states, namely four mainland states: New South Wales (NSW), Queensland (QLD), Victoria (VIC) and Western Australia (WA) and one island state: Tasmania (TAS) (see Table 1 ), and were stored in RNAlater (Thermo Fisher Scientific). Flies from five unique locations were identified as L. cuprina dorsalis, L. cuprina cuprina or L. sericata based on morphological characteristics [ 9 , 23 – 25 ]. Total genomic DNA (gDNA) was extracted from the head of each blowfly (n = 85) using the established approach [ 26 , 27 ]. DNA quality was assessed using a 1% agarose gel and DNA quantity was measured using a Qubit Fluorometer (Invitrogen). Five genomic DNA samples, each containing equal amounts of DNA from 17 individual flies from each of the five locations in Australia, were prepared. Libraries were constructed for each of these five samples using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina and paired-end sequenced using 2 × 150 cycles on an Illumina NovaSeq 6000 platform. Thus, DNA sequence data obtained from each library represented each of the five geographic locations. Table 1 Australian regions and Lucilia species/subspecies from which isolates were derived for mitochondrial (mt) genome sequencing. Sample Location Latitude Longitude Lucilia cuprina dorsalis Carwarp, VIC 36.7167°S 142.1997°E Lucilia cuprina dorsalis McMahons Reef, NSW 34.6595°S 148.4504°E Lucilia cuprina dorsalis Mount Romance, WA 34.7685°S 117.1358°E Lucilia cuprina cuprina Brisbane, QLD 27.4698°S 153.0251°E Lucilia sericata Campbell Town, TAS 41.9287°S 41.9287°S Mt genome assembly and annotation The consensus mt genomes representing Lucilia from each of the five geographic locations in Australia were assembled (Table 1 ). To do this, the Trimmomatic v.0.39 program [ 28 ] was used to remove adapters, contaminants, low quality (Phred scores < 30) and short (< 50 bp) sequencing reads. Read quality post-filtering was evaluated using the FastQC program v.0.11.9 [ 29 ] and remaining high-quality sequencing reads were used to perform a de novo assembly using NOVOPlasty v.4.2 [ 30 ]. The assembled mt genomes were annotated using the MITOS2 [ 31 ] web server based on translation Table 5 (Invertebrate Mitochondrial) of NCBI and transfer RNA (tRNA) genes were further annotated using the software ARWEN [ 32 ]. The protein-coding genes (PCGs) for each mt genome were conceptually translated based on translation table 5 (Invertebrate Mitochondrial) of NCBI and manually curated to ensure each PCG encoded a functional protein. The mt genomes were visualised using the Geneious Prime v.2019.2.3 software [ 33 ]. The mt genomes of individual Lucilia samples from the five individual geographic locations were drawn using OrganellarGenomeDRAW (OGDRAW) v.1.3.1 [ 34 ]. The nucleotide composition and nucleotide similarity percentages were determined using Geneious Prime v.2019.2.3 software [ 33 ]. The nucleotide composition skewness was calculated using the following formula: AT skew = (A − T)/(A + T) and GC skew = (G–C)/(G + C) [ 35 ]. Genetic analyses Quality-filtered FASTQ reads for each sample were mapped to the L. c. dorsalis mt reference genome (GenBank accession number: MW255536; VIC Australia) using Bowtie2 v2.4.5 [ 36 ]. Nucleotide polymorphisms were predicted and extracted using Annotate and Predict: Find Variations/SNPs function in Geneious Prime v.2019.2.3 software [ 33 ]. The minimum coverage and minimum variant allelic frequency was set to 5 and 0.25, respectively. Nucleotide variation was assessed within and among samples. The single nucleotide polymorphisms (SNPs) with allelic frequency > 99% within the 17 flies for each sample were used for calculating Nucleotide diversity (π) for each PCG in a pairwise comparison using DNASP 6.12.03 [ 37 ]. Evolutionary rates including synonymous, non-synonymous substitution rates and their ratio for each species of each PCG alignment were calculated using KaKsCalculator 2.0 [ 38 , 39 ]. Relationships among Lucilia species/subspecies A phylogeny was constructed using cytochrome c oxidase subunit I ( cox1 ) sequence data for L. cuprina and L. sericata available in GenBank (NCBI; Additional file 1: Table S1 ). An isolate of Lucilia porphyrina (QLD, Australia; NC_019637; [ 17 ]) was used as an outgroup in the analysis. Nucleotide sequences were aligned using MAFFT v7.450 [ 40 ]. Substitution model selection for the cox1 data set was carried out using jModeltest v2.1.10 [ 41 , 42 ] and the best-fitting model was chosen using the Akaike Information Criterion. Aligned sequences were then subjected to phylogenetic analysis using Bayesian inference (BI) employing Monte Carlo Markov Chain analysis in the program MrBayes v.3.2.6 [ 43 ]. Posterior probabilities (pp) were calculated using the GTR + I + G model, generating 1,000,000 trees and sampling every 200th tree until potential scale reduction factors for each parameter approached one. The final 75% of trees were used to construct a majority rule tree. The phylogenetic tree was visualised and annotated using FigTree v1.4.4 ( http://tree.bio.ed.ac.uk/software/figtree/ ). To investigate the relationship of the five Lucilia samples and the dipteran species (Additional file 2: Table S2 ) available on GenBank ( www.ncbi.nlm.nih.gov ), a phylogenetic tree was constructed based on the combined mt gene set (13 PCGs) + 2 ribosomal RNAs (rRNAs). The phylogenetic positions of the sequenced Lucilia species whole mt genomes among other species of dipteran flies were examined (Additional file 2: Table S2 ). The nucleotide sequences of 13 PCGs and 2 rRNAs were aligned separately with MAFFT v 7.450 [ 40 ]. The buffalo fly, Haematobia irritans irritans (Muscidae) was used as the outgroup. After alignment, all 13 PCGs and 2 rRNAs were concatenated using Geneious Prime v.2019.2.3 [ 33 ]. Subsequently, PartitionFinder v 2.1.1 [ 44 ] was run prior to phylogenetic analysis to select the best-fit partitioning schemes and substitution models based on the Bayesian Information Criterion (BIC). Phylogenetic analysis was conducted using the program MrBayes v.3.2.6 [ 43 ]. Posterior probabilities (pp) were calculated using the GTR + I + G model, generating 1,000,000 trees and sampling every 200th tree until potential scale reduction factors for each parameter approached one. The initial 25% of sample trees were discarded as burn-in, and the others were used to construct a majority rule tree. The phylogenetic tree was visualised and annotated using FigTree v1.4.4 ( http://tree.bio.ed.ac.uk/software/figtree/ ). Results Mt genome structure, organisation, and composition The five mt genomes representing Lucilia from distinct locations in Australia were circular and 15,941 bp ( L. c dorsalis ; NSW), 15,941 bp ( L. c. dorsalis ; VIC), 15,944 bp ( L. c. dorsalis ; WA), 15,952 bp ( L. c. cuprina ; QLD) and 15,946 bp ( L. sericata; TAS) in length/size (see Table 2 ). The L. cuprina mt genomes included here are in the size range of previously published mt genomes: L. cuprina strain DI213.5 (GenBank accession number: JX913753) 15,226 bp, L. cuprina strain DI213.2 (JX913750) 15,310 bp, L. cuprina strain DI190.5 (JX913748) 15,946 bp, and L. cuprina strain DI190.1 (JX913744) 15,952 bp [ 17 ]). The lengths of previously sequenced L. sericata mt genomes are as follows: L. sericata strain DI257 (JX913757) 15,380 bp, L. sericata strain DI245 (JX913756) 15,214 bp, L. sericata strain DI220 (JX913755) 15,300 bp, L. sericata strain DI246 (JX913754) 15,243 bp [ 17 ], L. sericata (NPY120886) 15938 bp [ 45 ] and L. sericata (AJ422212) 15,945 bp [ 46 ]. Based on the predicted annotation, each complete mt genome characterised herein included 37 genes consisting of 13 PCGs, 2 rRNAs [small (rrnS) and large (rrnL)], 22 tRNAs and a control region (Additional file 3: Table S3 , Fig. 1 ). The gene organisation for the five Lucilia mt genomes is identical. These genomes are presumably homologous without any genome rearrangements. The longest gene was nad5 , with a length of 1719 bp − 1725 bp, and the shortest was the atp8 gene, with a consistent length of only 165 bp. The total length of all the genes in these mt sequences represents approximately 92% of the length of the mt genomes (equivalent to 14,708 bp − 14,715 bp: PCGs = 11,160 bp − 11,166 bp; rRNAs = 2,080 bp; tRNAs = 1,468 bp − 1,469 bp), and the non-coding regions were 1,231 bp − 1,243 bp. The start codon for most of the PCGs was ATG or ATT (Additional file 3: Table S3 ). For the cox2 and nad5 genes, incomplete stop codons were found. The most commonly observed codon was TAA as the termination codon with most initiation codons also being AT-rich. Table 2 Size and skewness of the mitochondrial (mt) genomes of five Lucilia species/subspecies in Australia. Species/sub species Size (bp) A + T% AT-skew GC-skew Lucilia cuprina dorsalis (WA) 15,944 77.70 0.016 -0.165 Lucilia cuprina dorsalis (NSW) 15,941 77.70 0.016 -0.165 Lucilia cuprina dorsalis (VIC) 15,941 77.70 0.016 -0.165 Lucilia cuprina cuprina (QLD) 15,952 77.60 0.015 -0.166 Lucilia sericata (TAS) 15,946 77.70 0.015 -0.169 Average 15944.8 77.68 0.016 -0.166 The base composition of the mt sequences of all the species/subspecies was similar, with an average AT content of 77.7% (Table 2 ). The average AT skew was 0.016, and GC skew was − 0.166 (Table 2 ). The frequency of nucleotide A was higher than that of T and the frequency of nucleotide C was higher than that of G. Pairwise comparison of the mt genomes revealed sequence identities of 97.8% − 99.9% (Table 3 ). The number of nucleotide differences between the five mt genomes of Lucilia species/subspecies ranged between 12–354 nucleotides. The minimum nucleotide differences (n = 12) were between L. c. dorsalis from NSW and VIC and the maximum nucleotide differences (n = 354) were between L. c. dorsalis from VIC and L. c. cuprina from QLD, respectively (Table 3 ). Table 3 Nucleotide similarity percentages (number of nucleotide differences) between the five mitochondrial (mt) genomes of Australian Lucilia species/subspecies. Lucilia cuprina dorsalis (WA) Lucilia cuprina dorsalis (NSW) Lucilia cuprina dorsalis (VIC) Lucilia cuprina cuprina (QLD) Lucilia sericata (TAS) Lucilia cuprina dorsalis (WA) 99.9 (27) 99.91 (20) 97.83 (352) 97.89 (347) Lucilia cuprina dorsalis (NSW) 99.9 (27) 99.93 (12) 97.84 (352) 97.91 (345) Lucilia cuprina dorsalis (VIC) 99.91 (20) 99.93 (12) 97.83 (354) 97.89 (349) Lucilia cuprina cuprina (QLD) 97.83 (352) 97.84 (352) 97.83 (354) 99.21 (133) Lucilia sericata (TAS) 97.89 (347) 97.91 (345) 97.89 (349) 99.21 (133) The mt genomes are accessible in GenBank under the accession numbers MW255536 - MW255540 with the NCBI BioProject accession number PRJNA419080. Genetic analyses The nucleotide polymorphisms within each of the five DNA samples were recorded with respect to the L. c. dorsalis mt reference genome from VIC (GenBank accession number: MW255536). The number of SNPs with allelic frequency > 99% in L. sericata (TAS) and L. c. cuprina (QLD) were 198 and 78, respectively. The number of SNPs with allelic frequency < 99% in L. sericata (TAS), L. c. cuprina (QLD) and L. c dorsalis from NSW and WA were 26, 202, 2 and 3, respectively. There were 143 species-specific nucleotide polymorphisms in L. sericata (Fig. 1 , Additional file 4: Table S4 ), with most of these markers found in the nad5 (n = 27) gene followed by cox1 (n = 26) and nad1 (n = 17) gene (Additional file 4: Table S4 ). A total of 23 nucleotide polymorphisms were identified in the L. c. cuprina genome which can be used to identify these blowflies from other Lucilia species (Fig. 1 , Additional file 5: Table S5 ). These markers were present in the cox1, nad1, nad2, nad4, nad5, nad6 genes and the non-coding region. Pairwise nucleotide diversity (π) analysis showed high nucleotide diversities (π: 0.015–0.036) for all the PCGs between L. c. cuprina (QLD) and L. c. dorsalis (VIC, NSW and WA) samples and suggested a close relationship between L. c. dorsalis samples (VIC, NSW and WA). Pairwise nucleotide diversity comparison for L. c. cuprina (QLD) and L. c. dorsalis (VIC, NSW and WA) showed that genetic differentiation was lowest for nad1 (π = 0.014) and highest for cob (π = 0.036), cox1 (π = 0.027), nad5 (π = 0.027) and nad6 (π = 0.026), respectively. Pairwise comparison of nucleotide diversity (π: 0.010–0.036) also showed differentiation between L. sericata (TAS) and L. c. dorsalis (VIC, NSW and WA) populations (Fig. 2 A). The gene atp8 showed no nucleotide diversity (π = 0) between L. sericata and L. c. dorsalis samples. The nucleotide diversity was highest for genes cob (π = 0.036), cox1 (π = 0.026) and nad6 (π = 0.026), respectively. The pairwise comparison between L. c. cuprina and L. sericata showed low nucleotide diversities (π: 0.002–0.017) for all the 13 PCGs. No nucleotide diversity was seen (π = 0) between the L. c. dorsalis pairwise comparisons. All the nonsynonymous to synonymous substitution (Ka/Ks) ratios were found to be less than 1, suggestive of purifying selection acting on all PCGs within this group (Fig. 2 B). Among all 13 PCGs, the average Ka/Ks of nad6 is the highest (0.09) for the pairwise comparison of L. c. cuprina and L. sericata , followed by atp8 , nad4 and nad4l (Ka/Ks: 0.04 for atp8 , 0.03 for nad4 and nad4l ). Overall, the cox2 gene showed a relatively low value of Ka/Ks ratios (0.001) for all the pairwise comparisons. Phylogenetic analysis Bayesian inference (BI) analysis of the cox1 data set revealed that all L. sericata samples collected from different locations around the world [JX913754 (Canberra, ACT, Australia), JX913755 (QLD, Australia), JX913756 (WA, Australia), JX913757 (Utah, USA), AJ417713 (New Zealand), AJ422214 (UK), AJ417715 (WA, Australia) and NC_009733 (UK)] clustered together in a single clade with the newly sequenced L. sericata (MW255540) collected from TAS, Australia (Fig. 3 ). One node supporting L. sericata sequences JX913757 (Utah, USA) and JX913754 (Canberra, ACT, Australia) was inferred. L. cuprina formed two distinct clades in the phylogenetic tree. The L. c. dorsalis sequenced from VIC (MW255536), NSW (MW255537), and WA (MW255539) clustered in a separate clade with other L. cuprina sequences [JX913745 (Melbourne, Australia), JX913746 (Melbourne, Australia), JX913747 (Melbourne, Australia), JX913749 (QLD, Australia), AJ417708 (Senegal, Dakar), AJ417710 (QLD, Australia) and AJ417711 (Uganda)]. Interestingly, L. c. cuprina (QLD) (MW255538) formed a clade with the L. cuprina flies collected from QLD, Australia (JX913750, JX913751, JX913752, and JX913753), Hawaii, USA (AJ417704, AJ417705, DQ453495 and DQ453496) and Taipei City, Taiwan (AY097335) and shared a common ancestor with L. sericata (Fig. 3 ). Phylogenetic analysis of PCGs and rRNA genes based on BI showed stronger support for species and subspecies level relationships, with L. sericata and L. cuprina , separating to form species groupings with full nodal support (Fig. 4 ). Flies collected from TAS (MW255540) clustered together with the L. sericata clade containing specimens from ACT, Australia (JX913754), Utah, USA (JX913757), UK (AJ422212), QLD, Australia (JX913755) and WA, Australia (JX913756) (Fig. 4 ). Comparative analyses showed that L. cuprina formed two sister clades. One of the L. cuprina clade members represented the subspecies L. c. dorsalis collected from different locations in Australia [Melbourne, Australia (JX913744 - JX913748), QLD, Australia (JX913749) and newly sequenced specimens from NSW, Australia (MW255537), VIC, Australia (MW255536) and WA, Australia (MW255539)]. The other sister clade represented the subspecies L. c. cuprina collected from QLD, Australia (JX913750 - JX913753 and MW255538). L. c. cuprina specimens from QLD shared a common ancestor and were more closely related to L. sericata. There was also strong support for the sister grouping of Calliphorinae and Chrysomyinae with Luciliinae. The branch connecting Sarcophaga impatiens is less than half the length of the branches found within the Oestridae and Tachinidae clade (between D. hominis, H. lineatum, E. sorbilans and R. goerlingiana ). The molecular phylogeny based on the cox1 gene (Fig. 3 ) and 13 PCGs and 2 rRNA genes (Fig. 4 ) confirmed the paraphyly between L. cuprina and L. sericata. Discussion Lucilia cuprina is an economically important pest for the sheep industry in Australia. It is difficult to accurately identify Lucilia species and subspecies that parasitise sheep and differentiate them from those that do not affect sheep based on morphological characteristics, such as the colour of the frontocyopeal membrane and the fore femur, the position and number of hairs on the posterior slope of humeral callus and the number of paravertical setulae in the central occipital area [ 23 – 25 ]. A further challenge is when these morphological features are not always available/accurate due to damage/degradation during collection and/or due to intraspecific phenotypic variation. Mt genomes can provide molecular markers for the accurate identification of poorly understood species and subspecies [ 47 ]. Blowflies were collected from single sites across five Australian states, identified using morphological characteristics and sequenced to assemble their complete mt genomes. A total of five Lucilia mt genomes were characterised, pairwise nucleotide diversities and the Ka/Ks ratios were calculated, and phylogenetic trees were constructed to examine the phylogenetic position of these Lucilia species/subspecies with respect to other Calliphoridae. All five mt genomes of Lucilia characterised in this study are in the size range of previously published mt genomes, with the L. cuprina mt genome size between 15,226 bp − 15,952 bp [ 17 ] and the L. sericata mt genome size between 15,214 bp − 15,945 bp [ 17 , 46 ]. The L. sericata genome reported here is longer than the previously sequenced genome for L. sericata (i.e.,15,946 bp) and contains additional nucleotides (1–732 bp) in the non-coding regions. The length of the nad5 gene in the newly sequenced L. sericata in this study was 1725 bp which is similar to the length of the nad5 gene in L. sericata strain DI257 (GenBank accession number: JX913757), DI245 (JX913756), DI246 (JX913754) and DI220 (JX913755) [ 17 ] from Australia but 6 bp longer than the nad5 gene (1719 bp) in L. sericata (AJ422212) collected from Somerset, UK [ 46 ]. For all the Lucilia species/subspecies, the cox1 gene start codon (TCG) encodes a methionine, which does not conform to the standard invertebrate mt code (i.e., TCG normally encodes serine). This result is not unusual within the Diptera, and this non-standard initiation codon has previously been reported in a range of Calliphoridae [ 46 , 48 ]. For the cox2 and nad5 genes, incomplete stop codons were found, which has also been reported previously for members of the Calliphoridae [ 17 ], and it is assumed that the termination codon is completed by polyadenylation [ 48 , 49 ]. In the mt genomes, the gene order was identical to other Lucilia species sequenced to date [ 16 , 17 , 49 ] and is the same as that first identified in the fly Drosophila yakuba [ 50 ]. This mt genome arrangement is highly conserved in a wide range of different organisms with some exceptions within the class Insecta, indicating an ancestral gene order for this group [ 17 , 51 – 53 ]. The overall nucleotide composition was heavily A + T biased, accounting for 77.7% of the whole mt genome. This is typical of members of the Calliphoridae [ 9 , 15 , 54 – 56 ], including blowflies [ 15 , 57 , 58 ]. One of the possible biological reasons for an AT-bias is due to energy efficacy trade-offs [ 59 ]. The synthesis of A and T nucleotides consumes less energy and nitrogen than G and C nucleotides [ 59 ]. The average AT skew for all five whole mt genomes was positive (0.016) and the GC skew was negative (-0.166) indicating the bias against the use of Gs which is characteristic of the metazoan mt genome [ 60 ]. The AT and GC skews calculated for the whole mt genomes of Lucilia species followed the same pattern of bias as previously shown for dipteran species such as Chrysomya chloropyga (AF352790, AT skew: 0.020; GC skew: -0.170) [ 54 ], Cochliomyia hominivorax (AF260826, AT skew: 0.034; GC skew: -0.207) [ 48 ], Bactrocera oleae (AY210702 and AY210703, AT skew: 0.088; GC skew: -0.280) [ 61 ], Ceratitis capitata (AJ242872, AT skew: 0.021; GC skew: -0.185), D. melanogaster (U37541, AT skew: 0.017; GC skew: -0.150) [ 62 ], D. yakuba (X03240, AT skew: 0.005; GC skew: -0.136) [ 63 ], Anopheles gambiae (L20934, AT skew: 0.032; GC skew: -0.154) [ 64 ], A. quadrimaculatus (L04272, AT skew: 0.041; GC skew: -0.181) [ 65 ] and Exorista sorbillans (HQ322500, AT skew: 0.021; GC skew: -0.171) [ 66 ]. The sequencing of mt genomes in the present study provided additional species-specific molecular markers to differentiate among L. c. dorsalis , L. c. cuprina and L. sericata; 143 and 23 species-specific markers were characterised for identifying L. sericata and L. c. cuprina , respectively. The mt genes cox1 , cox2 , rrnS , nad4 and nad4l had been used previously for identifying members of the Calliphoridae [ 2 , 9 , 15 , 16 , 18 , 19 , 67 ] but a short gene sequence may not be sufficient to differentiate two species with greater than 95% bootstrap output [ 16 ]. Complete mt genomes provide many genetic markers that can be used to discriminate between Lucilia species/subspecies. To estimate the variation in 13 PCGs, nucleotide diversity was calculated. Nucleotide diversity was identified between different Lucilia species. The mean percentage of divergence between the L. cuprina and L. sericata clade was highest for cob (π = 2.10) followed by cox1 (π = 1.63) [ 17 ] which is similar to results presented here, where nucleotide diversity for cox1 was 0.026 between L. cuprina and L. sericata . Previously, a nucleotide diversity of π = 0.020 ± 0.003 had been observed among nine haplotypes amongst 24 cox1 sequences of L. cuprina and L. sericata from South Africa [ 19 ]. L. sericata and L. cuprina were clearly differentiated (by 2.8%) from each other based on the cox1 gene sequence [ 18 ]. The pairwise comparison of nucleotide diversity based on 13 PCGs provides a better differentiation between these sister species than using a single gene. The Ka/Ks ratio which is used to detect selective pressure and molecular adaptation [ 39 ] was used to estimate the evolutionary rate in Lucilia species. A higher Ka/Ks ratio indicates that the gene has evolved at a faster rate than the other PCGs. The gene nad6 (in pairwise comparison of L. sericata with other species/subspecies sequenced in this study) overall exhibited the highest rate of Ka/Ks ratio which can be a result of positive selection. The gene cox2 had the smallest Ka/Ks ratio, which indicates a strong purifying selection [ 58 , 68 ]. In a similar study, the evolutionary pressure among 13 PCGs of 34 species within the Calliphoridae was investigated and showed that Ka/Ks ratio was highest for atp8 (0.280) followed by nad5 (0.228) and lowest for cox1 (0.075) and cox2 (0.090) genes, respectively [ 69 ]. The Ka/Ks ratio for the 13 PCGs of mt genomes in flesh flies (Diptera: Sarcophagidae) exhibited the highest rate (0.26) for the gene atp8 indicating positive or relaxed selection and showed the lowest Ka/Ks ratio (0.06) for the cox1 gene indicating strong purifying selection [ 70 ]. The cox1 barcoding gene has been widely used worldwide for many different taxonomic groups including Lucilia [ 15 , 19 , 67 , 71 ]. A comparison of the cox1 gene in our sequenced Lucilia species with the cox1 sequences in the GenBank database provided insights into the evolutionary relationship between these flies but this information was limited to a few informative sites. The phylogenetic relationship between L. cuprina and L. sericata has been analysed in the past due to their economic and welfare significance in sheep husbandry [ 2 , 9 , 15 , 17 , 18 , 24 , 72 ]. Phylogenetic analyses (Fig. 3 & Fig. 4 ) based on the cox1 gene, 13 PCGs and 2 rRNAs [small (rrnS) and large (rrnL)] indicated that L. cuprina flies formed two different clades that were together paraphyletic to L. sericata. This is consistent with previously demonstrated paraphyly of L. cuprina with respect to L. sericata [ 9 , 15 – 19 , 21 , 24 , 67 ]. The L. sericata collected from a sheep farm in TAS, Australia grouped with the other specimens of L. sericata that had been previously shown to group together in the L. sericata clade [ 15 – 17 , 46 ]. Based on the phylogenetic trees, L. sericata from Australia and the UK grouped together in a clade and had similar mt DNA sequences, although there are clear differences reported in their ability to cause myiasis in sheep [ 10 , 21 ]; L. sericata is the primary cause of flystrike in Europe and a saprophagous species in other parts of the world [ 9 – 12 ], whereas it plays a secondary role in flystrike in Australia [ 73 ]. This is consistent with the hypothesis that parasitism arose relatively recently and independently in geographically isolated populations of Lucilia blowflies [ 21 , 74 ]. In the present study, the L. c. dorsalis flies collected from sheep farms in NSW, VIC and WA (Australia) clustered with the L. cuprina sequences obtained from GenBank (AJ417708, AJ417711, AJ417710 and JX913744 to JX913749), which had been previously suggested to belong to the subspecies L. c. dorsalis [ 15 , 17 ]. The flies which were collected from urban area of Brisbane, QLD (Australia) and identified as L. c. cuprina formed a clade with previously proposed subspecies L. c. cuprina (JX913750 - JX913753, DQ453495, DQ453496, AJ417704, AJ417705 and AY097335) and formed a sister clade to L. c. dorsalis . The results of our phylogenetic analysis indicate that L. c. cuprina is a hybrid of L. c. dorsalis and L. sericata. The presence of L. c. cuprina in QLD was previously reported and it has been proposed that these flies hybridise with L. c. dorsalis in eastern Australia and are not known to interbreed elsewhere [ 17 , 20 ]. Originally, L. c. cuprina was reported from Hawaii [ 3 , 15 , 21 ] and its presence has been reported in southeast Asia [ 18 ], South Africa [ 19 ], North America [ 16 ] and Australia [ 17 ]. L. c. cuprina has a synanthropic behaviour, like L. sericata , and is concentrated in urban areas [ 2 , 9 , 17 , 21 , 75 ]. The other taxon, which is not closely related to L. sericata is L. c. dorsalis . It is found predominantly on sheep farms and is recognised as a primary initiator of flystrike [ 9 , 17 , 20 ]. The present results are consistent with previous studies, showing that L. cuprina from QLD is closely related to L. sericata and likely represents the subspecies L. c. cuprina [ 9 , 17 ]. Lucilia from QLD was identified as L. c. cuprina based on the mt genome analysis [ 17 ], and another study proposed that a blowfly specimen from Townsville, QLD, appeared to be L. c. dorsalis [ 18 ] based on an analysis of cox1 sequence data [ 18 ]. In addition, it has been proposed that the L. cuprina from WA are L. c. dorsalis , and those from NSW and QLD represent L. c. cuprina [ 9 ]. A phylogenetic analysis including more specimens of L. c. cuprina flies from all Australian states and other parts of the world would be beneficial to determine the geographic distribution of these subspecies, given the tendency of the species to interbreed. The classification of Lucilia species to subspecies is somewhat debatable [ 17 , 19 , 21 ]. The results of the phylogenetic analyses of mt DNA sequence data have indicated that L. c. cuprina is genetically closer to L. sericata than L. c. dorsalis [ 2 , 9 , 21 ]. However, the concept of two separate subspecies is not supported because the blowflies that were morphologically consistent with L. c. cuprina were not monophyletic in previous studies [ 2 , 21 ]. Similarly, Nelson et al. [ 17 ] concluded that the two L. cuprina sister clades did not represent the two subspecies ( L. c. cuprina and L. c. dorsalis ) as the five flies collected in their study were collected at the same time and location (Petrie Terrace, Brisbane, QLD) and were morphologically consistent with L. c. dorsalis (JX913749 and JX913750 - JX913753). Tourle et al. [ 19 ] proposed that the flies that are closely related to L. sericata contained nuclear pseudogenes (NUMTs), however, the hypothesis was dismissed due to the absence of false stop codons in the cox1 sequences. A plausible explanation for the paraphyly of L. cuprina with respect to L. sericata is that there has been a hybridisation event between these two species, prior to the last common ancestor of the “modern” L. sericata populations [ 17 , 21 , 71 ]. Conclusions This study provides important insights into systematic relationships of Lucilia species from different states of Australia using mt genomic data sets. Pairwise nucleotide diversity suggests divergence between L. c. cuprina, L. c. dorsalis and L. sericata . Phylogenetic analyses reveal that L. c. cuprina collected from an urban location in QLD is distinct from L. c. dorsalis collected from sheep farms in NSW, VIC and WA, and that L. c. cuprina is more closely related to L. sericata collected from TAS than L. c. dorsalis . The species-specific genetic markers will aid in not only identifying and analysing Lucilia species population structure but also detecting the presence of hybridisation between these flies, particularly given there are many regions where their habitats overlap. The present study underpins the identification and distinction of Lucilia species and subspecies in the absence of reliable morphological features. Abbreviations mt mitochondrial NSW New South Wales QLD Queensland TAS Tasmania VIC Victoria WA Western Australia kb kilobase bp base pairs PCGs protein-coding genes rRNA ribosomal RNA tRNA transfer RNA SNPs single nucleotide polymorphisms BI Bayesian inference Declarations Acknowledgements The authors would like to acknowledge Australian Wool Innovation (AWI) for assistance with obtaining blowfly samples. The authors would like to thank Dr Peter James and Dr Geoff Brown for providing the L. c. cuprina samples (QLD) and the sheep growers who provided other samples used in this study. Funding Funding from Australian Wool Innovation (AWI ON-00624; to VMB, CAA and TP) is gratefully acknowledged. AWI is grateful for its funding, which is primarily provided by Australian woolgrowers through a wool levy, and by the Australian Government which provides a matching contribution for eligible R&D activities. Availability of data and materials The datasets supporting the conclusions of this article are included within the article and its Additional files. Any additional data are available from the corresponding author upon request. The mt genomes are deposited on GenBank (accession numbers MW255536 - MW255540) with the NCBI BioProject accession number PRJNA419080. Authors’ Contributions SK, CAA, and TP planned the experimental design. SK and YTY extracted the genomic DNA. SK analysed the data and interpreted the results with major contributions in computational analysis from CAA and NDY. Drafting of the manuscript was performed by SK with editing assistance from NDY, PB, RBG, VMB, TP and CAA. VMB, CAA and TP secured funding. All authors read and approved the final manuscript. 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Priority list of endemic diseases for the red meat industries. Project B. AHE. 0010. Meat and Livestock Australia. 2015. Additional Declarations No competing interests reported. Supplementary Files R0Additionalfile1TableS1.docx Additional file 1: Table S1 Collection details and cox1 gene sequences for Lucilia species/subspecies used in the present study. R0Additionalfile2TableS2.docx Additional file 2: Table S2 Collection details and mitochondrial (mt) genomic sequences for dipterans used in the present study. R0Additionalfile3TableS3.docx Additional file 3: Table S3 Mitochondrial (mt) genome architecture of Lucilia species/subspecies collected from different locations in Australia. R0Additionalfile4TableS4.docx Additional file 4: Table S4 Species-specific nucleotide polymorphisms in Lucilia sericata (TAS). R0Additionalfile5TableS5.docx Additional file 5: Table S5 Species-specific nucleotide polymorphisms in Lucilia cuprina cuprina (QLD). 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Young","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Neil","middleName":"D.","lastName":"Young","suffix":""},{"id":198841674,"identity":"8ae4efbb-be3d-43cb-ae52-5896326f5ec0","order_by":2,"name":"Ying Ting Yang","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ying","middleName":"Ting","lastName":"Yang","suffix":""},{"id":198841675,"identity":"3320ff83-f75c-4c83-be47-a6cff772bd8b","order_by":3,"name":"Philip Batterham","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Philip","middleName":"","lastName":"Batterham","suffix":""},{"id":198841676,"identity":"50df421e-7140-4b0e-898c-8a60d562674d","order_by":4,"name":"Robin B. Gasser","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Robin","middleName":"B.","lastName":"Gasser","suffix":""},{"id":198841677,"identity":"d824ffb5-2245-4a92-b511-fa6469cbd3a1","order_by":5,"name":"Vernon M. Bowles","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Vernon","middleName":"M.","lastName":"Bowles","suffix":""},{"id":198841679,"identity":"c0b93b35-094e-45b0-9b55-0fb758f5a5a1","order_by":6,"name":"Clare A. Anstead","email":"data:image/png;base64,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","orcid":"","institution":"University of Melbourne","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Clare","middleName":"A.","lastName":"Anstead","suffix":""},{"id":198841681,"identity":"b97eb386-7c05-4be6-b781-e8d11987b1c5","order_by":7,"name":"Trent Perry","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Trent","middleName":"","lastName":"Perry","suffix":""}],"badges":[],"createdAt":"2023-05-10 04:44:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2914299/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2914299/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-023-05902-1","type":"published","date":"2023-08-13T21:56:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":36958592,"identity":"fb00e335-b4b7-4ec2-a54c-ba94db952926","added_by":"auto","created_at":"2023-05-12 14:51:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":125088,"visible":true,"origin":"","legend":"\u003cp\u003eLinear maps of the circular mitochondrial (mt) genomes of \u003cem\u003eLucilia\u003c/em\u003e species/subspecies reported herein.\u003c/p\u003e\n\u003cp\u003eThe name of \u003cem\u003eLucilia \u003c/em\u003especies with their collection region is indicated above plots of gene order. Large bars situated on the mt genome maps indicate the position of protein‐coding genes and ribosomal RNA genes. Dark blue bars with annotated labels demarcate the positions of transfer RNA genes. Features of mt genomes are colour coded as indicated at the bottom of the figure. Protein‐coding genes are colour coded to mt complexes and the other features are coloured by type. Colours of protein‐coding, ribosomal RNA and transfer RNA genes are as follows: pink, cytochrome \u003cem\u003ec\u003c/em\u003e oxidase (\u003cem\u003ecox\u003c/em\u003e genes); dark green, ATP synthase\u003cem\u003e \u003c/em\u003e(\u003cem\u003eatp\u003c/em\u003e genes); yellow, NADH dehydrogenase (\u003cem\u003enad\u003c/em\u003e genes); light green, cytochrome \u003cem\u003eb\u003c/em\u003egene (\u003cem\u003ecob\u003c/em\u003e); red, ribosomal RNAs and dark blue, transfer RNAs. For protein‐coding and ribosomal RNA genes, those sitting above the black line are on the positive strand, and those below the line are on the negative strand. Standard nomenclature was applied for protein-coding genes and ribosomal RNA genes, whereas for transfer RNA genes, single-letter abbreviations were used. The number of species-specific single nucleotide polymorphisms (SNPs) present in the genes were marked in brown text. Detailed annotations of the mt genomes are provided in Additional file 3: Table S3.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2914299/v1/54b4fa1567aa40867fb51eff.jpg"},{"id":36958596,"identity":"ce20fc76-82f4-4925-a3db-7ddc5eed09e0","added_by":"auto","created_at":"2023-05-12 14:51:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1738350,"visible":true,"origin":"","legend":"\u003cp\u003eAnalyses of protein-coding genes in \u003cem\u003eLucilia\u003c/em\u003e species/subspecies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Nucleotide diversity (π) of individual protein-coding genes for pairs of \u003cem\u003eLucilia\u003c/em\u003e species/subspecies in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e The rates of nonsynonymous substitutions to the rate of synonymous substitutions (Ka/Ks) of individual protein-coding genes for pairs of \u003cem\u003eLucilia\u003c/em\u003e species/subspecies.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2914299/v1/767b3073a416e47a64f53105.jpg"},{"id":36958593,"identity":"06958255-4b83-4ac9-b9b7-54d598eadaa9","added_by":"auto","created_at":"2023-05-12 14:51:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":751224,"visible":true,"origin":"","legend":"\u003cp\u003eA summary of the molecular phylogeny of the \u003cem\u003eLucilia \u003c/em\u003especies/subspecies based on the cytochrome \u003cem\u003ec\u003c/em\u003e oxidase subunit I \u003cem\u003e(cox1)\u003c/em\u003e gene sequences.\u003c/p\u003e\n\u003cp\u003eThe phylogenetic tree was created using Bayesian inference (BI) implemented in MrBayes v.3.2.6. The numbers above the branches represents the posterior probabilities. Each specimen is labelled with the species name and location. Mitochondrial (mt) genomes sequenced in this study are colour coded: \u003cem\u003eLucilia cuprina cuprina\u003c/em\u003e (QLD) in pink, \u003cem\u003eLucilia sericata\u003c/em\u003e (TAS) in green, \u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (NSW) in brown, \u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (VIC) in purple and \u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (WA) in blue. The phylogram provided is presented to scale (scale bar = 0.006 estimated number of substitutions per site) with the species \u003cem\u003eLucilia porphyrina \u003c/em\u003eused as the outgroup.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2914299/v1/86da5c3a8eb78f322f6f6c7a.jpg"},{"id":36958600,"identity":"36ea9f43-5cd5-4357-b4eb-f6e1b97bab36","added_by":"auto","created_at":"2023-05-12 14:51:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":756738,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular phylogeny of dipteran flies.\u003c/p\u003e\n\u003cp\u003eThe phylogenetic tree was created using Bayesian inference (BI) implemented in MrBayes v.3.2.6. Numbers above the branches represent the posterior probabilities. \u003cem\u003eHaematobia irritans irritans \u003c/em\u003efrom the family Muscidae was used as the outgroup. Mitochondrial (mt\u003cbr\u003e\n) genomes sequenced in this study are colour-coded: \u003cem\u003eLucilia cuprina cuprina\u003c/em\u003e(QLD) in pink, \u003cem\u003eLucilia sericata\u003c/em\u003e (TAS) in green, \u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (NSW) in brown, \u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (VIC) in purple and \u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (WA) in blue. To the right of the species names family names (Muscidae, Oestridae, Tachnidae, Calliphoridae and Sarcophagidae) are reported. The subfamilies Luciliinae (yellow colour), Calliphorinae and Chrysominae (peach colour) of the Calliphoridae family are reported. The phylogram provided is presented to scale (scale bar = 0.04 estimated number of substitutions per site).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2914299/v1/51c32795ce73b619cbaee7c1.jpg"},{"id":44735281,"identity":"9e346dac-50b7-4b71-851e-3cce5cf2523f","added_by":"auto","created_at":"2023-10-16 22:24:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":842313,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2914299/v1/da05c21d-a426-433f-97bd-bb0b2cfd45c7.pdf"},{"id":36959786,"identity":"def29c74-b83b-4890-9bff-75e4e14d990f","added_by":"auto","created_at":"2023-05-12 14:59:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18531,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: Table S1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollection details and \u003cem\u003ecox1\u003c/em\u003e gene sequences for \u003cem\u003eLucilia\u003c/em\u003e species/subspecies used in the present study.\u003c/p\u003e","description":"","filename":"R0Additionalfile1TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-2914299/v1/7ddf331da2a53141217da698.docx"},{"id":36959788,"identity":"dfa14898-33bd-412d-a0cb-6c19f0efeaa1","added_by":"auto","created_at":"2023-05-12 14:59:26","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23330,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: Table S2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollection details and mitochondrial (mt) genomic sequences for dipterans used in the present study.\u003c/p\u003e","description":"","filename":"R0Additionalfile2TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-2914299/v1/a98fa2b8dc300314abc4babd.docx"},{"id":36960507,"identity":"a4b70874-c96d-4a9d-98d2-ffcf86aa427a","added_by":"auto","created_at":"2023-05-12 15:07:26","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":21997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 3: Table S3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondrial (mt) genome architecture of\u003cem\u003e Lucilia\u003c/em\u003e species/subspecies collected from different locations in Australia.\u003c/p\u003e","description":"","filename":"R0Additionalfile3TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-2914299/v1/b12f20500004db06e43b4391.docx"},{"id":36960508,"identity":"42801689-9bd9-48a6-849d-0ea1a8f5bf73","added_by":"auto","created_at":"2023-05-12 15:07:26","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":29589,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 4: Table S4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecies-specific nucleotide polymorphisms in \u003cem\u003eLucilia sericata \u003c/em\u003e(TAS).\u003c/p\u003e","description":"","filename":"R0Additionalfile4TableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-2914299/v1/9ab5e5072e3191edd3cd4dba.docx"},{"id":36958597,"identity":"99d04848-0bd6-4b0d-b7d2-d0604d1224c2","added_by":"auto","created_at":"2023-05-12 14:51:26","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 5: Table S5\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecies-specific nucleotide polymorphisms in \u003cem\u003eLucilia cuprina cuprina \u003c/em\u003e(QLD).\u003c/p\u003e","description":"","filename":"R0Additionalfile5TableS5.docx","url":"https://assets-eu.researchsquare.com/files/rs-2914299/v1/c5dfc4f1d23dafc0ef5078b2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mitochondrial genomic investigation reveals a clear association between species and genotypes of Lucilia and geographic origin in Australia","fulltext":[{"header":"Background","content":"\u003cp\u003eIn Australasia, blowflies of the genus \u003cem\u003eLucilia\u003c/em\u003e (Insecta, Diptera, Calliphoridae) are recognised as facultative ectoparasites of domesticated sheep but can severely affect various domestic and wild animals [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The Australian sheep blowfly, \u003cem\u003eLucilia cuprina\u003c/em\u003e (Wiedemann, 1830), causes cutaneous myiasis \u0026ndash; a serious skin disease of sheep [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This disease results in an estimated loss of \u003cspan\u003e$\u003c/span\u003e324\u0026nbsp;million per annum to the Australian wool industry alone [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The common green bottle fly, \u003cem\u003eLucilia sericata\u003c/em\u003e (Meigen, 1826), is another closely related blowfly that does not cause myiasis in sheep in Australia, as it does not infest live animals as \u003cem\u003eL. cuprina\u003c/em\u003e does [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, \u003cem\u003eL. sericata\u003c/em\u003e is the primary initiator of myiasis in sheep in Europe and is also a saprophagous species in other parts of the world [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Although both species are considered to have a cosmopolitan distribution [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], \u003cem\u003eL. cuprina\u003c/em\u003e is typically found more frequently in temperate subtropical areas and \u003cem\u003eL. sericata\u003c/em\u003e is more prevalent in cool to temperate climes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Phylogenetic studies conducted using mitochondrial (mt) gene and whole mt genome data sets have demonstrated that \u003cem\u003eL. cuprina\u003c/em\u003e is paraphyletic with respect to \u003cem\u003eL. sericata\u003c/em\u003e [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Populations of these two species were phylogenetically distinct, except for \u003cem\u003eL. cuprina\u003c/em\u003e from Hawaii [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eL. cuprina\u003c/em\u003e and \u003cem\u003eL\u003c/em\u003e. \u003cem\u003esericata\u003c/em\u003e can hybridise, with hybrids displaying \u003cem\u003eL. cuprina\u003c/em\u003e-like morphology [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A previous study using a combination of complementary nuclear \u0026minus;\u0026thinsp;28S subunit ribosomal RNA (28S rRNA) and mt cytochrome \u003cem\u003ec\u003c/em\u003e oxidase subunits I and II (\u003cem\u003ecox1\u003c/em\u003e and \u003cem\u003ecox2\u003c/em\u003e) demonstrated that these hybrids possessed \u003cem\u003eL. sericata\u003c/em\u003e-type mt DNA and \u003cem\u003eL. cuprina\u003c/em\u003e-type nuclear DNA [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Such hybrid flies have also been reported in other parts of the world, such as Asia [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], Australia [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], North America [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and South Africa [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTwo distinct subspecies of \u003cem\u003eL. cuprina\u003c/em\u003e have been described: \u003cem\u003eLucilia cuprina cuprina\u003c/em\u003e (Wiedemann, 1830) and \u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (Robineau-Desvoidy, 1830) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. \u003cem\u003eL. c. cuprina\u003c/em\u003e has been identified in Australia, China, Fiji, Hawaii, India, Indonesia, Japan, Java, Malaysia, North America, South America and Timor, whereas \u003cem\u003eL. c. dorsalis\u003c/em\u003e is distributed throughout Africa, Australia, India and New Zealand [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These two species as well as their subspecies are morphologically very similar and can be very challenging to identify and distinguish. \u003cem\u003eL. c. cuprina\u003c/em\u003e and \u003cem\u003eL. c. dorsalis\u003c/em\u003e interbreed in Eastern parts of Australia [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and produce hybrids that have overlapping morphological features complicating identification.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to gain important insights into systematic relationships of \u003cem\u003eLucilia\u003c/em\u003e species from different states of Australia using mt genomic data sets. In this study, a molecular approach was used to identify and characterise species and subspecies of \u003cem\u003eLucilia\u003c/em\u003e employing mt DNA with the aim of establishing high-quality genomic resources for these parasites. Little is known about the current distribution and genetics of these species/subspecies in Australia, which has resulted in significant bottlenecks in efforts to characterise and control these pests. Our work on the mt genomes of \u003cem\u003eLucilia\u003c/em\u003e species provides data that can be used to develop fast and reliable molecular tools that could identify and differentiate between individuals from wild populations of \u003cem\u003eLucilia\u003c/em\u003e across Australia.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFly collection, DNA extraction and sequencing\u003c/h2\u003e \u003cp\u003eBlowflies were collected from five Australian states, namely four mainland states: New South Wales (NSW), Queensland (QLD), Victoria (VIC) and Western Australia (WA) and one island state: Tasmania (TAS) (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and were stored in RNAlater (Thermo Fisher Scientific). Flies from five unique locations were identified as \u003cem\u003eL. cuprina dorsalis, L. cuprina cuprina\u003c/em\u003e or \u003cem\u003eL. sericata\u003c/em\u003e based on morphological characteristics [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Total genomic DNA (gDNA) was extracted from the head of each blowfly (n\u0026thinsp;=\u0026thinsp;85) using the established approach [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. DNA quality was assessed using a 1% agarose gel and DNA quantity was measured using a Qubit Fluorometer (Invitrogen). Five genomic DNA samples, each containing equal amounts of DNA from 17 individual flies from each of the five locations in Australia, were prepared. Libraries were constructed for each of these five samples using NEBNext\u0026reg; Ultra\u0026trade; II DNA Library Prep Kit for Illumina and paired-end sequenced using 2 \u0026times; 150 cycles on an Illumina NovaSeq 6000 platform. Thus, DNA sequence data obtained from each library represented each of the five geographic locations.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAustralian regions and \u003cem\u003eLucilia\u003c/em\u003e species/subspecies from which isolates were derived for mitochondrial (mt) genome sequencing.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLatitude\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLongitude\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarwarp, VIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.7167\u0026deg;S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e142.1997\u0026deg;E\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMcMahons Reef, NSW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.6595\u0026deg;S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e148.4504\u0026deg;E\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMount Romance, WA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.7685\u0026deg;S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e117.1358\u0026deg;E\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina cuprina\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrisbane, QLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.4698\u0026deg;S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e153.0251\u0026deg;E\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia sericata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCampbell Town, TAS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.9287\u0026deg;S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.9287\u0026deg;S\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMt genome assembly and annotation\u003c/h2\u003e \u003cp\u003eThe consensus mt genomes representing \u003cem\u003eLucilia\u003c/em\u003e from each of the five geographic locations in Australia were assembled (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To do this, the Trimmomatic v.0.39 program [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] was used to remove adapters, contaminants, low quality (Phred scores\u0026thinsp;\u0026lt;\u0026thinsp;30) and short (\u0026lt;\u0026thinsp;50 bp) sequencing reads. Read quality post-filtering was evaluated using the FastQC program v.0.11.9 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and remaining high-quality sequencing reads were used to perform a \u003cem\u003ede novo\u003c/em\u003e assembly using NOVOPlasty v.4.2 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The assembled mt genomes were annotated using the MITOS2 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] web server based on translation Table\u0026nbsp;5 (Invertebrate Mitochondrial) of NCBI and transfer RNA (tRNA) genes were further annotated using the software ARWEN [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The protein-coding genes (PCGs) for each mt genome were conceptually translated based on translation table 5 (Invertebrate Mitochondrial) of NCBI and manually curated to ensure each PCG encoded a functional protein. The mt genomes were visualised using the Geneious Prime v.2019.2.3 software [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The mt genomes of individual \u003cem\u003eLucilia\u003c/em\u003e samples from the five individual geographic locations were drawn using OrganellarGenomeDRAW (OGDRAW) v.1.3.1 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The nucleotide composition and nucleotide similarity percentages were determined using Geneious Prime v.2019.2.3 software [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The nucleotide composition skewness was calculated using the following formula: AT skew = (A\u0026thinsp;\u0026minus;\u0026thinsp;T)/(A\u0026thinsp;+\u0026thinsp;T) and GC skew = (G\u0026ndash;C)/(G\u0026thinsp;+\u0026thinsp;C) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGenetic analyses\u003c/h2\u003e \u003cp\u003eQuality-filtered FASTQ reads for each sample were mapped to the \u003cem\u003eL. c. dorsalis\u003c/em\u003e mt reference genome (GenBank accession number: MW255536; VIC Australia) using Bowtie2 v2.4.5 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Nucleotide polymorphisms were predicted and extracted using Annotate and Predict: Find Variations/SNPs function in Geneious Prime v.2019.2.3 software [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The minimum coverage and minimum variant allelic frequency was set to 5 and 0.25, respectively. Nucleotide variation was assessed within and among samples. The single nucleotide polymorphisms (SNPs) with allelic frequency\u0026thinsp;\u0026gt;\u0026thinsp;99% within the 17 flies for each sample were used for calculating Nucleotide diversity (π) for each PCG in a pairwise comparison using DNASP 6.12.03 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Evolutionary rates including synonymous, non-synonymous substitution rates and their ratio for each species of each PCG alignment were calculated using KaKsCalculator 2.0 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eRelationships among\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eLucilia\u003c/span\u003e \u003cb\u003especies/subspecies\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA phylogeny was constructed using cytochrome \u003cem\u003ec\u003c/em\u003e oxidase subunit I (\u003cem\u003ecox1\u003c/em\u003e) sequence data for \u003cem\u003eL. cuprina\u003c/em\u003e and \u003cem\u003eL. sericata\u003c/em\u003e available in GenBank (NCBI; Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). An isolate of \u003cem\u003eLucilia porphyrina\u003c/em\u003e (QLD, Australia; NC_019637; [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]) was used as an outgroup in the analysis. Nucleotide sequences were aligned using MAFFT v7.450 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Substitution model selection for the \u003cem\u003ecox1\u003c/em\u003e data set was carried out using jModeltest v2.1.10 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and the best-fitting model was chosen using the Akaike Information Criterion. Aligned sequences were then subjected to phylogenetic analysis using Bayesian inference (BI) employing Monte Carlo Markov Chain analysis in the program MrBayes v.3.2.6 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Posterior probabilities (pp) were calculated using the GTR\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G model, generating 1,000,000 trees and sampling every 200th tree until potential scale reduction factors for each parameter approached one. The final 75% of trees were used to construct a majority rule tree. The phylogenetic tree was visualised and annotated using FigTree v1.4.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tree.bio.ed.ac.uk/software/figtree/\u003c/span\u003e\u003cspan address=\"http://tree.bio.ed.ac.uk/software/figtree/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo investigate the relationship of the five \u003cem\u003eLucilia\u003c/em\u003e samples and the dipteran species (Additional file 2: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) available on GenBank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.ncbi.nlm.nih.gov\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), a phylogenetic tree was constructed based on the combined mt gene set (13 PCGs)\u0026thinsp;+\u0026thinsp;2 ribosomal RNAs (rRNAs). The phylogenetic positions of the sequenced \u003cem\u003eLucilia\u003c/em\u003e species whole mt genomes among other species of dipteran flies were examined (Additional file 2: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The nucleotide sequences of 13 PCGs and 2 rRNAs were aligned separately with MAFFT v 7.450 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The buffalo fly, \u003cem\u003eHaematobia irritans irritans\u003c/em\u003e (Muscidae) was used as the outgroup. After alignment, all 13 PCGs and 2 rRNAs were concatenated using Geneious Prime v.2019.2.3 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Subsequently, PartitionFinder v 2.1.1 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] was run prior to phylogenetic analysis to select the best-fit partitioning schemes and substitution models based on the Bayesian Information Criterion (BIC). Phylogenetic analysis was conducted using the program MrBayes v.3.2.6 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Posterior probabilities (pp) were calculated using the GTR\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G model, generating 1,000,000 trees and sampling every 200th tree until potential scale reduction factors for each parameter approached one. The initial 25% of sample trees were discarded as burn-in, and the others were used to construct a majority rule tree. The phylogenetic tree was visualised and annotated using FigTree v1.4.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tree.bio.ed.ac.uk/software/figtree/\u003c/span\u003e\u003cspan address=\"http://tree.bio.ed.ac.uk/software/figtree/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMt genome structure, organisation, and composition\u003c/h2\u003e \u003cp\u003eThe five mt genomes representing \u003cem\u003eLucilia\u003c/em\u003e from distinct locations in Australia were circular and 15,941 bp (\u003cem\u003eL. c dorsalis\u003c/em\u003e; NSW), 15,941 bp (\u003cem\u003eL. c. dorsalis\u003c/em\u003e; VIC), 15,944 bp (\u003cem\u003eL. c. dorsalis\u003c/em\u003e; WA), 15,952 bp (\u003cem\u003eL. c. cuprina\u003c/em\u003e; QLD) and 15,946 bp (\u003cem\u003eL. sericata;\u003c/em\u003e TAS) in length/size (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The \u003cem\u003eL. cuprina\u003c/em\u003e mt genomes included here are in the size range of previously published mt genomes: \u003cem\u003eL. cuprina\u003c/em\u003e strain DI213.5 (GenBank accession number: JX913753) 15,226 bp, \u003cem\u003eL. cuprina\u003c/em\u003e strain DI213.2 (JX913750) 15,310 bp, \u003cem\u003eL. cuprina\u003c/em\u003e strain DI190.5 (JX913748) 15,946 bp, and \u003cem\u003eL. cuprina\u003c/em\u003e strain DI190.1 (JX913744) 15,952 bp [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]). The lengths of previously sequenced \u003cem\u003eL. sericata\u003c/em\u003e mt genomes are as follows: \u003cem\u003eL. sericata\u003c/em\u003e strain DI257 (JX913757) 15,380 bp, \u003cem\u003eL. sericata\u003c/em\u003e strain DI245 (JX913756) 15,214 bp, \u003cem\u003eL. sericata\u003c/em\u003e strain DI220 (JX913755) 15,300 bp, \u003cem\u003eL. sericata\u003c/em\u003e strain DI246 (JX913754) 15,243 bp [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], \u003cem\u003eL. sericata\u003c/em\u003e (NPY120886) 15938 bp [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and \u003cem\u003eL. sericata\u003c/em\u003e (AJ422212) 15,945 bp [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Based on the predicted annotation, each complete mt genome characterised herein included 37 genes consisting of 13 PCGs, 2 rRNAs [small (rrnS) and large (rrnL)], 22 tRNAs and a control region (Additional file 3: Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The gene organisation for the five \u003cem\u003eLucilia\u003c/em\u003e mt genomes is identical. These genomes are presumably homologous without any genome rearrangements. The longest gene was \u003cem\u003enad5\u003c/em\u003e, with a length of 1719 bp \u0026minus;\u0026thinsp;1725 bp, and the shortest was the \u003cem\u003eatp8\u003c/em\u003e gene, with a consistent length of only 165 bp. The total length of all the genes in these mt sequences represents approximately 92% of the length of the mt genomes (equivalent to 14,708 bp \u0026minus;\u0026thinsp;14,715 bp: PCGs\u0026thinsp;=\u0026thinsp;11,160 bp \u0026minus;\u0026thinsp;11,166 bp; rRNAs\u0026thinsp;=\u0026thinsp;2,080 bp; tRNAs\u0026thinsp;=\u0026thinsp;1,468 bp \u0026minus;\u0026thinsp;1,469 bp), and the non-coding regions were 1,231 bp \u0026minus;\u0026thinsp;1,243 bp. The start codon for most of the PCGs was ATG or ATT (Additional file 3: Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). For the \u003cem\u003ecox2\u003c/em\u003e and \u003cem\u003enad5\u003c/em\u003e genes, incomplete stop codons were found. The most commonly observed codon was TAA as the termination codon with most initiation codons also being AT-rich.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSize and skewness of the mitochondrial (mt) genomes of five \u003cem\u003eLucilia\u003c/em\u003e species/subspecies in Australia.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies/sub species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSize (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u0026thinsp;+\u0026thinsp;T%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAT-skew\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGC-skew\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (WA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15,944\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e77.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.165\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (NSW)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15,941\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e77.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.165\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (VIC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15,941\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e77.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.165\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina cuprina\u003c/em\u003e (QLD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15,952\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e77.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.166\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia sericata\u003c/em\u003e (TAS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15,946\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e77.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.169\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15944.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e77.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.166\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe base composition of the mt sequences of all the species/subspecies was similar, with an average AT content of 77.7% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The average AT skew was 0.016, and GC skew was \u0026minus;\u0026thinsp;0.166 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The frequency of nucleotide A was higher than that of T and the frequency of nucleotide C was higher than that of G. Pairwise comparison of the mt genomes revealed sequence identities of 97.8% \u0026minus;\u0026thinsp;99.9% (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The number of nucleotide differences between the five mt genomes of \u003cem\u003eLucilia\u003c/em\u003e species/subspecies ranged between 12\u0026ndash;354 nucleotides. The minimum nucleotide differences (n\u0026thinsp;=\u0026thinsp;12) were between \u003cem\u003eL. c. dorsalis\u003c/em\u003e from NSW and VIC and the maximum nucleotide differences (n\u0026thinsp;=\u0026thinsp;354) were between \u003cem\u003eL. c. dorsalis\u003c/em\u003e from VIC and \u003cem\u003eL. c. cuprina\u003c/em\u003e from QLD, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNucleotide similarity percentages (number of nucleotide differences) between the five mitochondrial (mt) genomes of Australian \u003cem\u003eLucilia\u003c/em\u003e species/subspecies.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (WA)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (NSW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (VIC)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina cuprina\u003c/em\u003e (QLD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eLucilia sericata\u003c/em\u003e (TAS)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (WA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.9\u003c/p\u003e \u003cp\u003e(27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99.91\u003c/p\u003e \u003cp\u003e(20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97.83\u003c/p\u003e \u003cp\u003e(352)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e97.89\u003c/p\u003e \u003cp\u003e(347)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (NSW)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.9\u003c/p\u003e \u003cp\u003e(27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99.93\u003c/p\u003e \u003cp\u003e(12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97.84\u003c/p\u003e \u003cp\u003e(352)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e97.91\u003c/p\u003e \u003cp\u003e(345)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina dorsalis\u003c/em\u003e (VIC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.91\u003c/p\u003e \u003cp\u003e(20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.93\u003c/p\u003e \u003cp\u003e(12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97.83\u003c/p\u003e \u003cp\u003e(354)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e97.89\u003c/p\u003e \u003cp\u003e(349)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia cuprina cuprina\u003c/em\u003e (QLD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.83 (352)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97.84\u003c/p\u003e \u003cp\u003e(352)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e97.83\u003c/p\u003e \u003cp\u003e(354)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.21\u003c/p\u003e \u003cp\u003e(133)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLucilia sericata\u003c/em\u003e (TAS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97.89\u003c/p\u003e \u003cp\u003e(347)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97.91\u003c/p\u003e \u003cp\u003e(345)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e97.89\u003c/p\u003e \u003cp\u003e(349)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.21\u003c/p\u003e \u003cp\u003e(133)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe mt genomes are accessible in GenBank under the accession numbers MW255536 - MW255540 with the NCBI BioProject accession number PRJNA419080.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGenetic analyses\u003c/h2\u003e \u003cp\u003eThe nucleotide polymorphisms within each of the five DNA samples were recorded with respect to the \u003cem\u003eL. c. dorsalis\u003c/em\u003e mt reference genome from VIC (GenBank accession number: MW255536). The number of SNPs with allelic frequency\u0026thinsp;\u0026gt;\u0026thinsp;99% in \u003cem\u003eL. sericata\u003c/em\u003e (TAS) and \u003cem\u003eL. c. cuprina\u003c/em\u003e (QLD) were 198 and 78, respectively. The number of SNPs with allelic frequency\u0026thinsp;\u0026lt;\u0026thinsp;99% in \u003cem\u003eL. sericata\u003c/em\u003e (TAS), \u003cem\u003eL. c. cuprina\u003c/em\u003e (QLD) and \u003cem\u003eL. c dorsalis\u003c/em\u003e from NSW and WA were 26, 202, 2 and 3, respectively. There were 143 species-specific nucleotide polymorphisms in \u003cem\u003eL. sericata\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Additional file 4: Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), with most of these markers found in the \u003cem\u003enad5\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;27) gene followed by \u003cem\u003ecox1\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;26) and \u003cem\u003enad1\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;17) gene (Additional file 4: Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). A total of 23 nucleotide polymorphisms were identified in the \u003cem\u003eL. c. cuprina\u003c/em\u003e genome which can be used to identify these blowflies from other \u003cem\u003eLucilia\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Additional file 5: Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). These markers were present in the \u003cem\u003ecox1, nad1, nad2, nad4, nad5, nad6\u003c/em\u003e genes and the non-coding region.\u003c/p\u003e \u003cp\u003ePairwise nucleotide diversity (π) analysis showed high nucleotide diversities (π: 0.015\u0026ndash;0.036) for all the PCGs between \u003cem\u003eL. c. cuprina\u003c/em\u003e (QLD) and \u003cem\u003eL. c. dorsalis\u003c/em\u003e (VIC, NSW and WA) samples and suggested a close relationship between \u003cem\u003eL. c. dorsalis\u003c/em\u003e samples (VIC, NSW and WA). Pairwise nucleotide diversity comparison for \u003cem\u003eL. c. cuprina\u003c/em\u003e (QLD) and \u003cem\u003eL. c. dorsalis\u003c/em\u003e (VIC, NSW and WA) showed that genetic differentiation was lowest for \u003cem\u003enad1\u003c/em\u003e (π\u0026thinsp;=\u0026thinsp;0.014) and highest for \u003cem\u003ecob\u003c/em\u003e (π\u0026thinsp;=\u0026thinsp;0.036), \u003cem\u003ecox1\u003c/em\u003e (π\u0026thinsp;=\u0026thinsp;0.027), \u003cem\u003enad5\u003c/em\u003e (π\u0026thinsp;=\u0026thinsp;0.027) and \u003cem\u003enad6\u003c/em\u003e (π\u0026thinsp;=\u0026thinsp;0.026), respectively. Pairwise comparison of nucleotide diversity (π: 0.010\u0026ndash;0.036) also showed differentiation between \u003cem\u003eL. sericata\u003c/em\u003e (TAS) and \u003cem\u003eL. c. dorsalis\u003c/em\u003e (VIC, NSW and WA) populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The gene \u003cem\u003eatp8\u003c/em\u003e showed no nucleotide diversity (π\u0026thinsp;=\u0026thinsp;0) between \u003cem\u003eL. sericata\u003c/em\u003e and \u003cem\u003eL. c. dorsalis\u003c/em\u003e samples. The nucleotide diversity was highest for genes \u003cem\u003ecob\u003c/em\u003e (π\u0026thinsp;=\u0026thinsp;0.036), \u003cem\u003ecox1\u003c/em\u003e (π\u0026thinsp;=\u0026thinsp;0.026) and \u003cem\u003enad6\u003c/em\u003e (π\u0026thinsp;=\u0026thinsp;0.026), respectively. The pairwise comparison between \u003cem\u003eL. c. cuprina\u003c/em\u003e and \u003cem\u003eL. sericata\u003c/em\u003e showed low nucleotide diversities (π: 0.002\u0026ndash;0.017) for all the 13 PCGs. No nucleotide diversity was seen (π\u0026thinsp;=\u0026thinsp;0) between the \u003cem\u003eL. c. dorsalis\u003c/em\u003e pairwise comparisons. All the nonsynonymous to synonymous substitution (Ka/Ks) ratios were found to be less than 1, suggestive of purifying selection acting on all PCGs within this group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Among all 13 PCGs, the average Ka/Ks of \u003cem\u003enad6\u003c/em\u003e is the highest (0.09) for the pairwise comparison of \u003cem\u003eL. c. cuprina\u003c/em\u003e and \u003cem\u003eL. sericata\u003c/em\u003e, followed by \u003cem\u003eatp8\u003c/em\u003e, \u003cem\u003enad4\u003c/em\u003e and \u003cem\u003enad4l\u003c/em\u003e (Ka/Ks: 0.04 for \u003cem\u003eatp8\u003c/em\u003e, 0.03 for \u003cem\u003enad4\u003c/em\u003e and \u003cem\u003enad4l\u003c/em\u003e). Overall, the \u003cem\u003ecox2\u003c/em\u003e gene showed a relatively low value of Ka/Ks ratios (0.001) for all the pairwise comparisons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eBayesian inference (BI) analysis of the \u003cem\u003ecox1\u003c/em\u003e data set revealed that all \u003cem\u003eL. sericata\u003c/em\u003e samples collected from different locations around the world [JX913754 (Canberra, ACT, Australia), JX913755 (QLD, Australia), JX913756 (WA, Australia), JX913757 (Utah, USA), AJ417713 (New Zealand), AJ422214 (UK), AJ417715 (WA, Australia) and NC_009733 (UK)] clustered together in a single clade with the newly sequenced \u003cem\u003eL. sericata\u003c/em\u003e (MW255540) collected from TAS, Australia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). One node supporting \u003cem\u003eL. sericata\u003c/em\u003e sequences JX913757 (Utah, USA) and JX913754 (Canberra, ACT, Australia) was inferred. \u003cem\u003eL. cuprina\u003c/em\u003e formed two distinct clades in the phylogenetic tree. The \u003cem\u003eL. c. dorsalis\u003c/em\u003e sequenced from VIC (MW255536), NSW (MW255537), and WA (MW255539) clustered in a separate clade with other \u003cem\u003eL. cuprina\u003c/em\u003e sequences [JX913745 (Melbourne, Australia), JX913746 (Melbourne, Australia), JX913747 (Melbourne, Australia), JX913749 (QLD, Australia), AJ417708 (Senegal, Dakar), AJ417710 (QLD, Australia) and AJ417711 (Uganda)]. Interestingly, \u003cem\u003eL. c. cuprina\u003c/em\u003e (QLD) (MW255538) formed a clade with the \u003cem\u003eL. cuprina\u003c/em\u003e flies collected from QLD, Australia (JX913750, JX913751, JX913752, and JX913753), Hawaii, USA (AJ417704, AJ417705, DQ453495 and DQ453496) and Taipei City, Taiwan (AY097335) and shared a common ancestor with \u003cem\u003eL. sericata\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePhylogenetic analysis of PCGs and rRNA genes based on BI showed stronger support for species and subspecies level relationships, with \u003cem\u003eL. sericata\u003c/em\u003e and \u003cem\u003eL. cuprina\u003c/em\u003e, separating to form species groupings with full nodal support (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Flies collected from TAS (MW255540) clustered together with the \u003cem\u003eL. sericata\u003c/em\u003e clade containing specimens from ACT, Australia (JX913754), Utah, USA (JX913757), UK (AJ422212), QLD, Australia (JX913755) and WA, Australia (JX913756) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Comparative analyses showed that \u003cem\u003eL. cuprina\u003c/em\u003e formed two sister clades. One of the \u003cem\u003eL. cuprina\u003c/em\u003e clade members represented the subspecies \u003cem\u003eL. c. dorsalis\u003c/em\u003e collected from different locations in Australia [Melbourne, Australia (JX913744 - JX913748), QLD, Australia (JX913749) and newly sequenced specimens from NSW, Australia (MW255537), VIC, Australia (MW255536) and WA, Australia (MW255539)]. The other sister clade represented the subspecies \u003cem\u003eL. c. cuprina\u003c/em\u003e collected from QLD, Australia (JX913750 - JX913753 and MW255538). \u003cem\u003eL. c. cuprina\u003c/em\u003e specimens from QLD shared a common ancestor and were more closely related to \u003cem\u003eL. sericata.\u003c/em\u003e There was also strong support for the sister grouping of Calliphorinae and Chrysomyinae with Luciliinae. The branch connecting \u003cem\u003eSarcophaga impatiens\u003c/em\u003e is less than half the length of the branches found within the Oestridae and Tachinidae clade (between \u003cem\u003eD. hominis, H. lineatum, E. sorbilans\u003c/em\u003e and \u003cem\u003eR. goerlingiana\u003c/em\u003e). The molecular phylogeny based on the \u003cem\u003ecox1\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and 13 PCGs and 2 rRNA genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) confirmed the paraphyly between \u003cem\u003eL. cuprina\u003c/em\u003e and \u003cem\u003eL. sericata.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eLucilia cuprina\u003c/em\u003e is an economically important pest for the sheep industry in Australia. It is difficult to accurately identify \u003cem\u003eLucilia\u003c/em\u003e species and subspecies that parasitise sheep and differentiate them from those that do not affect sheep based on morphological characteristics, such as the colour of the frontocyopeal membrane and the fore femur, the position and number of hairs on the posterior slope of humeral callus and the number of paravertical setulae in the central occipital area [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A further challenge is when these morphological features are not always available/accurate due to damage/degradation during collection and/or due to intraspecific phenotypic variation. Mt genomes can provide molecular markers for the accurate identification of poorly understood species and subspecies [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBlowflies were collected from single sites across five Australian states, identified using morphological characteristics and sequenced to assemble their complete mt genomes. A total of five \u003cem\u003eLucilia\u003c/em\u003e mt genomes were characterised, pairwise nucleotide diversities and the Ka/Ks ratios were calculated, and phylogenetic trees were constructed to examine the phylogenetic position of these \u003cem\u003eLucilia\u003c/em\u003e species/subspecies with respect to other Calliphoridae. All five mt genomes of \u003cem\u003eLucilia\u003c/em\u003e characterised in this study are in the size range of previously published mt genomes, with the \u003cem\u003eL. cuprina\u003c/em\u003e mt genome size between 15,226 bp \u0026minus;\u0026thinsp;15,952 bp [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and the \u003cem\u003eL. sericata\u003c/em\u003e mt genome size between 15,214 bp \u0026minus;\u0026thinsp;15,945 bp [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The \u003cem\u003eL. sericata\u003c/em\u003e genome reported here is longer than the previously sequenced genome for \u003cem\u003eL. sericata\u003c/em\u003e (i.e.,15,946 bp) and contains additional nucleotides (1\u0026ndash;732 bp) in the non-coding regions. The length of the \u003cem\u003enad5\u003c/em\u003e gene in the newly sequenced \u003cem\u003eL. sericata\u003c/em\u003e in this study was 1725 bp which is similar to the length of the \u003cem\u003enad5\u003c/em\u003e gene in \u003cem\u003eL. sericata\u003c/em\u003e strain DI257 (GenBank accession number: JX913757), DI245 (JX913756), DI246 (JX913754) and DI220 (JX913755) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] from Australia but 6 bp longer than the \u003cem\u003enad5\u003c/em\u003e gene (1719 bp) in \u003cem\u003eL. sericata\u003c/em\u003e (AJ422212) collected from Somerset, UK [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. For all the \u003cem\u003eLucilia\u003c/em\u003e species/subspecies, the \u003cem\u003ecox1\u003c/em\u003e gene start codon (TCG) encodes a methionine, which does not conform to the standard invertebrate mt code (i.e., TCG normally encodes serine). This result is not unusual within the Diptera, and this non-standard initiation codon has previously been reported in a range of Calliphoridae [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. For the \u003cem\u003ecox2\u003c/em\u003e and \u003cem\u003enad5\u003c/em\u003e genes, incomplete stop codons were found, which has also been reported previously for members of the Calliphoridae [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and it is assumed that the termination codon is completed by polyadenylation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the mt genomes, the gene order was identical to other \u003cem\u003eLucilia\u003c/em\u003e species sequenced to date [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and is the same as that first identified in the fly \u003cem\u003eDrosophila yakuba\u003c/em\u003e [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This mt genome arrangement is highly conserved in a wide range of different organisms with some exceptions within the class Insecta, indicating an ancestral gene order for this group [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The overall nucleotide composition was heavily A\u0026thinsp;+\u0026thinsp;T biased, accounting for 77.7% of the whole mt genome. This is typical of members of the Calliphoridae [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], including blowflies [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. One of the possible biological reasons for an AT-bias is due to energy efficacy trade-offs [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The synthesis of A and T nucleotides consumes less energy and nitrogen than G and C nucleotides [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The average AT skew for all five whole mt genomes was positive (0.016) and the GC skew was negative (-0.166) indicating the bias against the use of Gs which is characteristic of the metazoan mt genome [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The AT and GC skews calculated for the whole mt genomes of \u003cem\u003eLucilia\u003c/em\u003e species followed the same pattern of bias as previously shown for dipteran species such as \u003cem\u003eChrysomya chloropyga\u003c/em\u003e (AF352790, AT skew: 0.020; GC skew: -0.170) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], \u003cem\u003eCochliomyia hominivorax\u003c/em\u003e (AF260826, AT skew: 0.034; GC skew: -0.207) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], \u003cem\u003eBactrocera oleae\u003c/em\u003e (AY210702 and AY210703, AT skew: 0.088; GC skew: -0.280) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], \u003cem\u003eCeratitis capitata\u003c/em\u003e (AJ242872, AT skew: 0.021; GC skew: -0.185), \u003cem\u003eD. melanogaster\u003c/em\u003e (U37541, AT skew: 0.017; GC skew: -0.150) [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], \u003cem\u003eD. yakuba\u003c/em\u003e (X03240, AT skew: 0.005; GC skew: -0.136) [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], \u003cem\u003eAnopheles gambiae\u003c/em\u003e (L20934, AT skew: 0.032; GC skew: -0.154) [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], \u003cem\u003eA. quadrimaculatus\u003c/em\u003e (L04272, AT skew: 0.041; GC skew: -0.181) [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] and \u003cem\u003eExorista sorbillans\u003c/em\u003e (HQ322500, AT skew: 0.021; GC skew: -0.171) [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe sequencing of mt genomes in the present study provided additional species-specific molecular markers to differentiate among \u003cem\u003eL. c. dorsalis\u003c/em\u003e, \u003cem\u003eL. c. cuprina\u003c/em\u003e and \u003cem\u003eL. sericata;\u003c/em\u003e 143 and 23 species-specific markers were characterised for identifying \u003cem\u003eL. sericata\u003c/em\u003e and \u003cem\u003eL. c. cuprina\u003c/em\u003e, respectively. The mt genes \u003cem\u003ecox1\u003c/em\u003e, \u003cem\u003ecox2\u003c/em\u003e, \u003cem\u003errnS\u003c/em\u003e, \u003cem\u003enad4\u003c/em\u003e and \u003cem\u003enad4l\u003c/em\u003e had been used previously for identifying members of the Calliphoridae [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] but a short gene sequence may not be sufficient to differentiate two species with greater than 95% bootstrap output [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Complete mt genomes provide many genetic markers that can be used to discriminate between \u003cem\u003eLucilia\u003c/em\u003e species/subspecies. To estimate the variation in 13 PCGs, nucleotide diversity was calculated. Nucleotide diversity was identified between different \u003cem\u003eLucilia\u003c/em\u003e species. The mean percentage of divergence between the \u003cem\u003eL. cuprina\u003c/em\u003e and \u003cem\u003eL. sericata\u003c/em\u003e clade was highest for \u003cem\u003ecob\u003c/em\u003e (π\u0026thinsp;=\u0026thinsp;2.10) followed by \u003cem\u003ecox1\u003c/em\u003e (π\u0026thinsp;=\u0026thinsp;1.63) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] which is similar to results presented here, where nucleotide diversity for \u003cem\u003ecox1\u003c/em\u003e was 0.026 between \u003cem\u003eL. cuprina\u003c/em\u003e and \u003cem\u003eL. sericata\u003c/em\u003e. Previously, a nucleotide diversity of π\u0026thinsp;=\u0026thinsp;0.020\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 had been observed among nine haplotypes amongst 24 \u003cem\u003ecox1\u003c/em\u003e sequences of \u003cem\u003eL. cuprina\u003c/em\u003e and \u003cem\u003eL. sericata\u003c/em\u003e from South Africa [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. \u003cem\u003eL. sericata\u003c/em\u003e and \u003cem\u003eL. cuprina\u003c/em\u003e were clearly differentiated (by 2.8%) from each other based on the \u003cem\u003ecox1\u003c/em\u003e gene sequence [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The pairwise comparison of nucleotide diversity based on 13 PCGs provides a better differentiation between these sister species than using a single gene.\u003c/p\u003e \u003cp\u003eThe Ka/Ks ratio which is used to detect selective pressure and molecular adaptation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] was used to estimate the evolutionary rate in \u003cem\u003eLucilia\u003c/em\u003e species. A higher Ka/Ks ratio indicates that the gene has evolved at a faster rate than the other PCGs. The gene \u003cem\u003enad6\u003c/em\u003e (in pairwise comparison of \u003cem\u003eL. sericata\u003c/em\u003e with other species/subspecies sequenced in this study) overall exhibited the highest rate of Ka/Ks ratio which can be a result of positive selection. The gene \u003cem\u003ecox2\u003c/em\u003e had the smallest Ka/Ks ratio, which indicates a strong purifying selection [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. In a similar study, the evolutionary pressure among 13 PCGs of 34 species within the Calliphoridae was investigated and showed that Ka/Ks ratio was highest for \u003cem\u003eatp8\u003c/em\u003e (0.280) followed by \u003cem\u003enad5\u003c/em\u003e (0.228) and lowest for \u003cem\u003ecox1\u003c/em\u003e (0.075) and \u003cem\u003ecox2\u003c/em\u003e (0.090) genes, respectively [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. The Ka/Ks ratio for the 13 PCGs of mt genomes in flesh flies (Diptera: Sarcophagidae) exhibited the highest rate (0.26) for the gene \u003cem\u003eatp8\u003c/em\u003e indicating positive or relaxed selection and showed the lowest Ka/Ks ratio (0.06) for the \u003cem\u003ecox1\u003c/em\u003e gene indicating strong purifying selection [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The \u003cem\u003ecox1\u003c/em\u003e barcoding gene has been widely used worldwide for many different taxonomic groups including \u003cem\u003eLucilia\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. A comparison of the \u003cem\u003ecox1\u003c/em\u003e gene in our sequenced \u003cem\u003eLucilia\u003c/em\u003e species with the \u003cem\u003ecox1\u003c/em\u003e sequences in the GenBank database provided insights into the evolutionary relationship between these flies but this information was limited to a few informative sites.\u003c/p\u003e \u003cp\u003eThe phylogenetic relationship between \u003cem\u003eL. cuprina\u003c/em\u003e and \u003cem\u003eL. sericata\u003c/em\u003e has been analysed in the past due to their economic and welfare significance in sheep husbandry [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Phylogenetic analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u0026amp; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) based on the \u003cem\u003ecox1\u003c/em\u003e gene, 13 PCGs and 2 rRNAs [small (rrnS) and large (rrnL)] indicated that \u003cem\u003eL. cuprina\u003c/em\u003e flies formed two different clades that were together paraphyletic to \u003cem\u003eL. sericata.\u003c/em\u003e This is consistent with previously demonstrated paraphyly of \u003cem\u003eL. cuprina\u003c/em\u003e with respect to \u003cem\u003eL. sericata\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. The \u003cem\u003eL. sericata\u003c/em\u003e collected from a sheep farm in TAS, Australia grouped with the other specimens of \u003cem\u003eL. sericata\u003c/em\u003e that had been previously shown to group together in the \u003cem\u003eL. sericata\u003c/em\u003e clade [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Based on the phylogenetic trees, \u003cem\u003eL. sericata\u003c/em\u003e from Australia and the UK grouped together in a clade and had similar mt DNA sequences, although there are clear differences reported in their ability to cause myiasis in sheep [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]; \u003cem\u003eL. sericata\u003c/em\u003e is the primary cause of flystrike in Europe and a saprophagous species in other parts of the world [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], whereas it plays a secondary role in flystrike in Australia [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. This is consistent with the hypothesis that parasitism arose relatively recently and independently in geographically isolated populations of \u003cem\u003eLucilia\u003c/em\u003e blowflies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, the \u003cem\u003eL. c. dorsalis\u003c/em\u003e flies collected from sheep farms in NSW, VIC and WA (Australia) clustered with the \u003cem\u003eL. cuprina\u003c/em\u003e sequences obtained from GenBank (AJ417708, AJ417711, AJ417710 and JX913744 to JX913749), which had been previously suggested to belong to the subspecies \u003cem\u003eL. c. dorsalis\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The flies which were collected from urban area of Brisbane, QLD (Australia) and identified as \u003cem\u003eL. c. cuprina\u003c/em\u003e formed a clade with previously proposed subspecies \u003cem\u003eL. c. cuprina\u003c/em\u003e (JX913750 - JX913753, DQ453495, DQ453496, AJ417704, AJ417705 and AY097335) and formed a sister clade to \u003cem\u003eL. c. dorsalis\u003c/em\u003e. The results of our phylogenetic analysis indicate that \u003cem\u003eL. c. cuprina\u003c/em\u003e is a hybrid of \u003cem\u003eL. c. dorsalis\u003c/em\u003e and \u003cem\u003eL. sericata.\u003c/em\u003e The presence of \u003cem\u003eL. c. cuprina\u003c/em\u003e in QLD was previously reported and it has been proposed that these flies hybridise with \u003cem\u003eL. c. dorsalis\u003c/em\u003e in eastern Australia and are not known to interbreed elsewhere [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Originally, \u003cem\u003eL. c. cuprina\u003c/em\u003e was reported from Hawaii [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and its presence has been reported in southeast Asia [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], South Africa [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], North America [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and Australia [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. \u003cem\u003eL. c. cuprina\u003c/em\u003e has a synanthropic behaviour, like \u003cem\u003eL. sericata\u003c/em\u003e, and is concentrated in urban areas [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. The other taxon, which is not closely related to \u003cem\u003eL. sericata\u003c/em\u003e is \u003cem\u003eL. c. dorsalis\u003c/em\u003e. It is found predominantly on sheep farms and is recognised as a primary initiator of flystrike [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The present results are consistent with previous studies, showing that \u003cem\u003eL. cuprina\u003c/em\u003e from QLD is closely related to \u003cem\u003eL. sericata\u003c/em\u003e and likely represents the subspecies \u003cem\u003eL. c. cuprina\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. \u003cem\u003eLucilia\u003c/em\u003e from QLD was identified as \u003cem\u003eL. c. cuprina\u003c/em\u003e based on the mt genome analysis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and another study proposed that a blowfly specimen from Townsville, QLD, appeared to be \u003cem\u003eL. c. dorsalis\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] based on an analysis of \u003cem\u003ecox1\u003c/em\u003e sequence data [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, it has been proposed that the \u003cem\u003eL. cuprina\u003c/em\u003e from WA are \u003cem\u003eL. c. dorsalis\u003c/em\u003e, and those from NSW and QLD represent \u003cem\u003eL. c. cuprina\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A phylogenetic analysis including more specimens of \u003cem\u003eL. c. cuprina\u003c/em\u003e flies from all Australian states and other parts of the world would be beneficial to determine the geographic distribution of these subspecies, given the tendency of the species to interbreed.\u003c/p\u003e \u003cp\u003eThe classification of \u003cem\u003eLucilia\u003c/em\u003e species to subspecies is somewhat debatable [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The results of the phylogenetic analyses of mt DNA sequence data have indicated that \u003cem\u003eL. c. cuprina\u003c/em\u003e is genetically closer to \u003cem\u003eL. sericata\u003c/em\u003e than \u003cem\u003eL. c. dorsalis\u003c/em\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the concept of two separate subspecies is not supported because the blowflies that were morphologically consistent with \u003cem\u003eL. c. cuprina\u003c/em\u003e were not monophyletic in previous studies [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Similarly, Nelson et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] concluded that the two \u003cem\u003eL. cuprina\u003c/em\u003e sister clades did not represent the two subspecies (\u003cem\u003eL. c. cuprina\u003c/em\u003e and \u003cem\u003eL. c. dorsalis\u003c/em\u003e) as the five flies collected in their study were collected at the same time and location (Petrie Terrace, Brisbane, QLD) and were morphologically consistent with \u003cem\u003eL. c. dorsalis\u003c/em\u003e (JX913749 and JX913750 - JX913753). Tourle et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] proposed that the flies that are closely related to \u003cem\u003eL. sericata\u003c/em\u003e contained nuclear pseudogenes (NUMTs), however, the hypothesis was dismissed due to the absence of false stop codons in the \u003cem\u003ecox1\u003c/em\u003e sequences. A plausible explanation for the paraphyly of \u003cem\u003eL. cuprina\u003c/em\u003e with respect to \u003cem\u003eL. sericata\u003c/em\u003e is that there has been a hybridisation event between these two species, prior to the last common ancestor of the \u0026ldquo;modern\u0026rdquo; \u003cem\u003eL. sericata\u003c/em\u003e populations [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides important insights into systematic relationships of \u003cem\u003eLucilia\u003c/em\u003e species from different states of Australia using mt genomic data sets. Pairwise nucleotide diversity suggests divergence between \u003cem\u003eL. c. cuprina, L. c. dorsalis\u003c/em\u003e and \u003cem\u003eL. sericata\u003c/em\u003e. Phylogenetic analyses reveal that \u003cem\u003eL. c. cuprina\u003c/em\u003e collected from an urban location in QLD is distinct from \u003cem\u003eL. c. dorsalis\u003c/em\u003e collected from sheep farms in NSW, VIC and WA, and that \u003cem\u003eL. c. cuprina\u003c/em\u003e is more closely related to \u003cem\u003eL. sericata\u003c/em\u003e collected from TAS than \u003cem\u003eL. c. dorsalis\u003c/em\u003e. The species-specific genetic markers will aid in not only identifying and analysing \u003cem\u003eLucilia\u003c/em\u003e species population structure but also detecting the presence of hybridisation between these flies, particularly given there are many regions where their habitats overlap. The present study underpins the identification and distinction of \u003cem\u003eLucilia\u003c/em\u003e species and subspecies in the absence of reliable morphological features.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emt\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emitochondrial\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNSW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNew South Wales\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eQLD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQueensland\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTAS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTasmania\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVictoria\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWestern Australia\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ekb\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ekilobase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ebp\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebase pairs\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCGs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprotein-coding genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003erRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eribosomal RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003etRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etransfer RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSNPs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esingle nucleotide polymorphisms\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBayesian inference\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge Australian Wool Innovation (AWI) for assistance with obtaining blowfly samples. The authors would like to thank Dr Peter James and Dr Geoff Brown for providing the \u003cem\u003eL. c. cuprina\u003c/em\u003e samples (QLD) and the sheep growers who provided other samples used in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding from Australian Wool Innovation (AWI ON-00624; to VMB, CAA and TP) is gratefully acknowledged. AWI is grateful for its funding, which is primarily provided by Australian woolgrowers through a wool levy, and by the Australian Government which provides a matching contribution for eligible R\u0026amp;D activities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are included within the article and its Additional files. Any additional data are available from the corresponding author upon request. The mt genomes are deposited on GenBank (accession numbers MW255536 - MW255540) with the NCBI BioProject accession number PRJNA419080.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSK, CAA, and TP planned the experimental design. \u0026nbsp;SK and YTY extracted the genomic DNA. SK analysed the data and interpreted the results with major contributions in computational analysis from CAA and NDY. Drafting of the manuscript was performed by SK with editing assistance from NDY, PB, RBG, VMB, TP and CAA. VMB, CAA and TP secured funding. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting details\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZumpt F. Myiasis in man and animals in the old world. A textbook for physicians, veterinarians and zoologists. London: Butterworth; 1965.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStevens JR, Wall R. Genetic variation in populations of the blowflies \u003cem\u003eLucilia cuprina\u003c/em\u003e and \u003cem\u003eLucilia sericata\u003c/em\u003e (Diptera: Calliphoridae). Random amplified polymorphic DNA analysis and mitochondrial DNA sequences. Biochem. Syst. Ecol. 1997;25(2):81\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStevens JR, Wall R. The evolution of ectoparasitism in the genus \u003cem\u003eLucilia\u003c/em\u003e (Diptera: Calliphoridae). Int. J. Parasitol. 1997;27(1):51\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMackerras IM, Fuller ME. A survey of the Australian sheep blowflies. J. CSIR. 1937;10(4):261\u0026ndash;270.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGleeson DM, Sarre S. Mitochondrial DNA variability and geographic origin of the sheep blowfly, \u003cem\u003eLucilia cuprina\u003c/em\u003e (Diptera: Calliphoridae), in New Zealand. Bull. Entomol. Res. 1997;87(3):265\u0026ndash;272.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnstead CA, Korhonen PK, Young ND, Hall RS, Jex AR, Murali SC, et al. \u003cem\u003eLucilia cuprina\u003c/em\u003e genome unlocks parasitic fly biology to underpin future interventions. Nat. Commun. 2015;6(1):7344.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShephard R, Ware JW, Blomfield B, Niethe G. Priority list of endemic diseases for the red meat industry-2022 update. Project B.AHE.0327. Meat and Livestock Australia Limited. 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaterhouse DF, Paramonovo SJ. The status of the two species of \u003cem\u003eLucilia\u003c/em\u003e (Diptera, Calliphoridae) attacking sheep in Austhalia. Aust. J. Biol. Sci. 1950;3(3):310\u0026ndash;336.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWallman JF, Leys R, Hogendoorn K. 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Meat and Livestock Australia. 2015.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Australian sheep blowfly, Lucilia cuprina dorsalis, Lucilia cuprina cuprina, Lucilia sericata, mt genome, phylogenetics","lastPublishedDoi":"10.21203/rs.3.rs-2914299/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2914299/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eLucilia cuprina\u003c/em\u003e and \u003cem\u003eL. sericata\u003c/em\u003e (family Calliphoridae) are globally significant ectoparasites of sheep. Current literature suggests that only one of these blowfly subspecies, \u003cem\u003eL. cuprina dorsalis\u003c/em\u003e, is a primary parasite causing myiasis (flystrike) in sheep in Australia. These species and subspecies are difficult to distinguish using morphological features. Hence, being able to accurately identify blowflies is critical for diagnosis and for understanding their relationships with their hosts and environment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study, adult blowflies (5 pools of 17 flies; n\u0026thinsp;=\u0026thinsp;85) were collected from five locations in different states [New South Wales (NSW), Queensland (QLD), Tasmania (TAS), Victoria (VIC) and Western Australia (WA)] of Australia and their mitochondrial (mt) genomes were assembled.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEach mt genome assembled was ~\u0026thinsp;15 kb in size and encoded 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs and a control region. The \u003cem\u003eLucilia\u003c/em\u003e species mt genomes were conserved in structure and the genes retained the same order and direction. The overall nucleotide composition was heavily biased towards As and Ts \u0026minus;\u0026thinsp;77.7% of the whole genomes. Pairwise nucleotide diversity suggested divergence between \u003cem\u003eL. cuprina cuprina, L. c. dorsalis\u003c/em\u003e and \u003cem\u003eL. sericata\u003c/em\u003e. Comparative analyses of these mt genomes with published data demonstrated that the blowflies collected from sheep farm in TAS clustered within a clade with \u003cem\u003eL. sericata\u003c/em\u003e. The flies collected from an urban location in QLD were more closely related to \u003cem\u003eL. sericata\u003c/em\u003e and represented the subspecies \u003cem\u003eL. c. cuprina\u003c/em\u003e, whereas the flies collected from sheep farms in NSW, VIC and WA represented the subspecies \u003cem\u003eL. c. dorsalis.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePhylogenetic analyses of the mt genomes representing \u003cem\u003eLucilia\u003c/em\u003e from the five geographic locations in Australia supported the previously demonstrated paraphyly of \u003cem\u003eL. cuprina\u003c/em\u003e with respect to \u003cem\u003eL. sericata\u003c/em\u003e and revealed that \u003cem\u003eL. c. cuprina\u003c/em\u003e is distinct from \u003cem\u003eL. c. dorsalis\u003c/em\u003e, and that \u003cem\u003eL. c. cuprina\u003c/em\u003e is more closely related to \u003cem\u003eL. sericata\u003c/em\u003e than \u003cem\u003eL. c. dorsalis\u003c/em\u003e. The mt genomes reported here provide an important molecular resource to develop tools for species- and subspecies-level identification of \u003cem\u003eLucilia\u003c/em\u003e from different geographical regions across Australia.\u003c/p\u003e","manuscriptTitle":"Mitochondrial genomic investigation reveals a clear association between species and genotypes of Lucilia and geographic origin in Australia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-12 14:51:21","doi":"10.21203/rs.3.rs-2914299/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-05-21T03:40:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-05-15T14:08:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"e65ccdf4-f71e-4cdb-906b-bf6f9d31b5ce","date":"2023-05-12T13:45:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-05-12T03:47:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-05-10T11:25:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-05-10T11:20:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2023-05-10T04:36:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"30c21291-f503-449b-b73d-dcde294fa93b","owner":[],"postedDate":"May 12th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T22:08:16+00:00","versionOfRecord":{"articleIdentity":"rs-2914299","link":"https://doi.org/10.1186/s13071-023-05902-1","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2023-08-13 21:56:32","publishedOnDateReadable":"August 13th, 2023"},"versionCreatedAt":"2023-05-12 14:51:21","video":"","vorDoi":"10.1186/s13071-023-05902-1","vorDoiUrl":"https://doi.org/10.1186/s13071-023-05902-1","workflowStages":[]},"version":"v1","identity":"rs-2914299","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2914299","identity":"rs-2914299","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}