Mitochondrial genome of Leocrates chinensis Kinberg, 1866 (Annelida: Hesionidae)

Abstract We report the complete mitochondrial genome of Leocrates chinensis Kinberg, 1866 – the type species of the genus. It is 15061 bp long, and contains 13 protein-coding genes (PCGs), 22 tRNA genes (tRNAs), and 2 rRNA genes (rRNAs), and 1 putative control region. Phylogenetic analysis indicated that L. chinensis was placed as sister to Sirsoe methanicola (BS = 100) of the same family Hesionidae.


Introduction
Leocrates chinensis Kinberg et al. 1866 is a hesionid widely distributed in southeastern Chinese waters. The holotype of L. chinensis was collected from Victoria Harbor, Hong Kong during the Swedish naval frigate Eugenie's around-the-world cruise during 1851-1853, and was later briefly described by Kinberg et al. (1866) as the type species of the newly established hesionid genus Leocrates. A recent study redescribed L. chinensis with morphological details based on specimens from the type locality (Wang et al. 2018). Nevertheless, since the specimens were preserved in formalin, no attempt was made to extract DNA and determine the placements of this species and Hesionidae in tree of life. Therefore, we conducted genome skimming (Zhang et al. 2018) of Leocrates chinensis to help clarify the species status of Leocrates specimens that have been identified as L. chinensis based on cox1 (accession number: OL763897) and several other loci (i.e. 16S, 18S, and 28S, with accession numbers: OL764386, OP104345, and OP104348, respectively) that have been used in species classification of Hesionidae Wang et al. 2020), and to provide resources for studies of mitochondrial genome evolution in the family that currently has only one sequenced mitochondrial genome (Sirsoe methanicola, accession no.: OM914591; Lim et al. 2022).

Materials
The single Leocrates chinensis specimen used in this study was collected from the subtidal muddy sediment (Site M(4), 22 14.160 0 N, 114 11.115 0 E, water depth 9.1 m) of the Hong Kong, China on 10 June 2020. The specimen was fixed and preserved in 100% ethanol, and both the specimen and its genomic DNA are now deposited in the Biology of Marine Benthic Invertebrates Group, College of Ocean and Earth Sciences, Xiamen University (https://www.xmu.edu.cn/, Zhi Wang, zhiwang00kxy@xmu.edu.cn) under the voucher number XMU-Pol-2021-354 ( Figure 1).

Methods
Whole genomic DNA was extracted by using TIANamp Genomic DNA Kit (TIANGEN, Beijing, China). The sequencing library was produced by using the Illumina Truseq TM DNA Sample Preparation Kit (Illumina, San Diego, USA) according to the manufacturer's recommendations. The prepared library was loaded on the Illumina Novaseq 6000 platform for PE 2 Â 150 bp sequencing at Novogene Company (Beijing, China). Sequence quality of raw genomic data was assessed using FastQC v.0.11.5 software (http://www.bioinformatics. babraham.ac.uk/projects/fastqc). Quality trimming and filtering of data was performed using fastp v.0.23.2 , reads containing more than 5% unknown nucleotides, and low-quality reads (reads containing more than 50% bases with Q-value 20) and all unpaired reads were discarded. To ensure the consistency in assembly result, the genome assembly was conducted using two methods, i.e. the filtered data were used to assemble the complete mitochondrial genome using the GetOrganelle v.1.7.6.1 pipeline with 'animal_mt' in the default database as seed reads (Jin et al. 2020) and NovoPlasty v.4.3.1 with the cox1 (OL763897) sequence as the seed reads (Dierckxsens et al. 2017). To check the coverage depth of the assembled mitogenome, "samtools depth" command in Samtools v.1.6 (Danecek et al. 2021) was used to calculate the coverage depth, and Circos v.0.69 (Krzywinski et al. 2009) was used to draw the coverage plot (as shown in Figure 2). The annotation of the mitogenome was made with the MITOS2 webserver (Donath et al. 2019) and the Mitoz annotation module (Meng et al. 2019). Additionally, we used maximum-likehood method with the software Phylosuite to reconstruct the phylogenetic tree  among Errantia polychaete families. For the three Nereidiformia families, i.e. Hesionidae, Syllidae, and Nereididae, sequences of two genera in each family were included in the analyses; and for the other families including the outgroups (i.e. Capitellidae, Urechidae, Lumbricidae), only one genera was used for each family. All the mitogenomic  data included in the analysis came from those published papers (Table 1), and concatenated protein-coding genes (PCGs) of these mitogemones were used in the analysis with each coding gene aligned individually using Mafft v.7.313 (Katoh and Standley 2013) under Codon alignment mode. Ambiguously aligned regions were removed with Gblocks v.0.91 with default settings (Castresana 2000). The best-fitting partition models (GTR þ F þ I þ G4) for ML analysis were selected by the software ModelFinder (Kalyaanamoorthy et al. 2017).

Results
The two assembly pipelines (i.e. GetOrganelle and NovoPlasty) gave an exact identical circular mitogenome. The size of the complete mitochondrial genome was 15,061 bp, and the data were submitted to the NCBI (accession no.: OP104125). The genome consisted of 60.3% A þ T, with 28.8% of A, 31.5% of T, 14.8% of G, and 24.9% of C. The genome contains 13 PCGs, i.e. 2 rRNA genes, 22 tRNA genes, and 1 putative control region consisting of 620 bp (Figure 2). Six Table 1. Mitochondrial sequences used in the phylogenetic analysis as shown in Figure 3.  PCGs started with ATG codon, two PCGs started with ATT codon, one PCG started with ATC and four PCGs started with ATA. All 13 PCGs terminated with TAA stop codon except nad4 and nad2, which with T stop codon. The real phylogenetic relationships among available Errantia polychaetous families are exhibited in Figure 3. Results showed that L. chinensis was sister taxon of Sirsoe methanicola (BS ¼ 100), which was the only one sequenced mitochondrial genome in the same family Hesionidae.

Discussion and conclusion
The first complete mitochondrial genome of the polychaetous species Leocrates chinensis is reported in this study, which is also the first mitogenome reported in the genus Leocrates. We used two different assembly methods (i.e. GetOrganelle v.1.7.6.1 and NovoPlasty v.4.3.1) to ensure the consistency of the assembly result. We noted that there was slight negative coverage anomaly in the control region (CR). It might be due to compositional bias affecting sequencing. As we all know, the control region has high A þ T content, and this region always facing sequencing problems. Based on our experiences in mitogenomic studies of polychaetes, the CR is difficult to assemble and it always has a slight negative coverage anomaly. This study provides a valuable data to studying the phylogenetic and evolutionary history of annelid polychaetes. And to make better understanding of the phylogeny within Hesionidae, mitogenomic studies on more genera of this family, such as Oxydromus, Podarkeopsis, Micropodarke, etc., should be put into agenda in future studies.