Comparative Analysis Reveals Recent Mitochondrial Introgression and Genome Size Variation in the Yellow Goosefish Lophius litulon

A specimen of the yellow goosefish was sampled from the coastal waters of Taizhou, China (28.65N, 121.94E) in November, 2023. The examined specimen was preserved at Fisheries Ecology and Biodiversity Laboratory in Zhejiang Ocean University under specimen accession ZJOU-09517. The specimen was morphologically identified, then a piece of muscle tissue was collected and stored in 95% ethanol at -80 °C.

Total genomic DNA was extracted using a standard phenol-chloroform method. A 350-bp paired-end genome resequencing library was constructed and sequenced using an Illumina NovaSeq6000 platform (Illumina, Inc., San Diego, CA, USA). The library construction and sequencing were performed at Novogene Co., Ltd. (Beijing, China).

After quality control of raw sequencing data, the retained clean data were used for mitogenome assembly. The MEANGS software (Song et al., 2022) was employed to de novo assemble the mitogenome sequence based on genome sequencing data. The assembled sequence was then annotated using the online Mitochondrial Genome Database of Fish server (Iwasaki et al., 2013).

Together with the publically available mitogenome sequence (KJ020931), the mitogenome sequences were aligned and compared using the Unipro UGENE v44.0 software (Okonechnikov et al., 2012). Unaligned fragments were blasted against the GenBank database using the online NCBI BLAST software (Johnson et al., 2008). Phylogenetic analysis was conducted using the mitogenome sequences of seven goosefishes and the anglerfish (Haplophryne mollis; outgroup). The nucleotide and amino acid sequences of protein-coding genes (excluding stop codons) were concatenated manually and aligned using the MUSCLE algorithm implemented in the UGENE software. Maximum likelihood (ML) algorithm was run using MEGA v 7.0 software (Kumar et al., 2016) for 1000 replicates to construct phylogenetic topologies.

The newly assembled mitogenome sequence in this study was a circularized DNA molecule with a length of 16,468 bp (Fig. 1). This mitogenome was deposited in the GenBank with accession number PP187730. The size of the newly assembled mitogenome was 37-bp larger than that of the published mitogenome in the GenBank database (KJ020931). Nucleotide frequencies of the newly assembled sequence were A: 28.64%, T: 25.53%, G: 16.80% and C: 29.02% with a slight bias to AT content (54.18%). The mitogenome possessed typical gene content, including two rRNAs (12S and 16s rRNAs), 22 tRNAs, 13 protein-coding genes (PCGs) and one noncoding control region. A total of 28 genes (2 rRNAs, 12 PCGs, and 14 tRNAs) were encoded on the heavy strand, while the remaining 9 genes (1 PCG and 8 tRNAs) were located on the light strand (Fig. 1).

Compared with the published mitogenome (KJ020931), an unaligned fragment (a 40-bp insertion) in the control region was observed. The observed insertion fragment should be the major contributor to mitogenome size variation of the yellow goosefish. We then investigated the origin of this insertion fragment using the BLAST program. Unexpectedly, the blast results against the GenBank database indicated that the insertion fragment matched with L. piscatorius, showing the highest identity of 98.44% (Fig. 2). Additionally, the MEANGS assembly for L. litulon was further confirmed through Polymerase Chain Reaction amplification and Sanger sequencing, indicating the accuracy of the MEANGS assembly. Considering that the publicly available mitogenome (KJ020931) was published ten years ago, and the sampling localities of these two mitogenomes were neighboring-sympatric, our result suggested a recent mitochondrial introgression event between L. litulon and L. piscatorius. Similarly, mitochondrial introgression events between closely related species have been detected in several taxa including Nasonia spp. (Lin et al., 2021), Crotaphytus spp. (McGuire et al., 2007), freshwater fish species (i.e., Kwan et al., 2019), and among others. For the first time, the mitochondrial introgression was observed in the goosefish species. However, the origin and underlying mechanisms of

mtDNA introgression between these two goosefish species remain puzzled and further studies are needed to resolve these questions.

Phylogenetic analyses based on concatenated nucleotide and amino acid sequences of the PCGs revealed our assembled sequence was first clustered with L. litulon (KJ020931), confirming the accuracy of species identification (Fig. 3). Besides, species in genus Lophius formed a monophyletic group, and then grouped with species in genus Lophiodes, suggesting a close relationship between these two genera. Our topology was inconsistent with the results in Miya et al. (2010), in which genus Lophius was first clustered with Lophiomus, and then grouped with Lophiodes. The phylogenetic inconsistency of family Lophiidae might be due to the different genetic datasets. Therefore, more genomic data are warranted for further comprehensive phylogenetic inferences of goosefishes and anglerfishes.

We sincerely thank Prof. Pengfei Li for helping in sample collection and species identification.

This work was supported by the National Students Platform for Innovation and Entrepreneurship Training Program (grant number 202210340022) and the National Natural Science Foundation of China (grant number 32200413).

The sequencing data in this study have been deposited in Sequence Read Archive (SRA) database under accession number PRJNA1034949. The complete mitogenome sequence assembled in this study has been deposited in GenBank database under accession number PP187730.

The authors have declared no conflict of interest.