The Complete Mitochondrial Genome of Entemnotrochus rumphii, a Living Fossil for Vetigastropoda (Mollusca: Gastropoda)

Pleurotomarioidea represents a truly isolated and basally diverging lineage in Vetigastropoda (Mollusca: Gastropoda) whose fossil record can date back to the late Cambrian, thus providing rare insights into the evolutionary history of molluscs. Here, we sequenced and assembled the complete mitochondrial genome of one representative species from Pleurotomarioidea—Entemnotrochus rumphii (Schepman, 1879)—of which the mitogenome is 15,795 bp in length, including 13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes. The nucleotide composition was biased toward AT, and A + T content reached 65.2%. E. rumphii was recovered as sister to all other living vetigastropods according to mitogenome-based phylogenetic analysis. The mitochondrial gene order was consistent with major vetigastropods and the hypothetical ancestral gastropoda, suggesting the deep conservation of mitogenome arrangement in Vetigastropoda.


Introduction
Entemnotrochus rumphii (Schepman, 1879) belongs to the superfamily Pleurotomarioidea, subclass Vetigastropoda, which is regarded as one of the most deeply diverging lineages among Gastropoda [1,2]. Pleurotomarioids can be distinguished from other gastropods based on their special morphological characteristics such as the coiled, conispiral shells and a slit along outer lips [3]. Pleurotomarioids are distributed in tropical and temperate latitudes [4]. Two species of Entemnotrochus are widely distributed, and one is E. rumphii, which is known from the southern islands of Japan to the central Philippines and Indonesia [3]. According to museum records, the bathymetric ranges of living E. rumphii range from about 80-280 m, and they inhabit sandy, coarse bottoms [5]. Tan [6] reported that the gut content and feces of E. rumphii consisted of sponge spicules, which indicated that this species lived on a diet of sponges.
Pleurotomarioidea is the only Gastropoda superfamily with fossil records from the Cambrian to the present and they are called "living fossils" [7][8][9][10]. The family Pleurotomariidae originated in the lower Triassic and is the only family of Pleurotomarioidea that survives now. Of 1500 described species of Pleurotomarioidea, only 25 are living today [4]. Their symmetrical organs and asymmetrical shells are regarded as transitional between symmetrical ancestors and asymmetrical modern gastropods [11,12]. Overall, these species have significance for understanding the early evolution of Gastropoda.
The mitochondrion is an important organelle in eukaryotes, and the mitochondrial genome has nearly the same structure in all animals, that is, a circular double-stranded DNA molecule, almost always including 13 protein-coding genes (PCGs), two ribosomal RNAs, and 22 transfer RNAs [13]. The conservation of its composition and structure makes the mitochondrial genome a good material for studying evolution and phylogenetic relationships [14,15]. Even as mitochondrial genome data are increasing for Gastropoda, few have been published yet for Vetigastropoda, especially within Pleurotomariida. To provide more molecular information on this order, we described a complete mitochondrial genome of E. rumphii, a representative species of Pleurotomarioidea. We also conducted a phylogenetic analysis and compared gene arrangements within Vetigastropoda.

Sample Collection and DNA Extraction
A specimen of E. rumphii was collected in 2020 from the East China Sea, near Japan (30.9 • N, 129.9 • E). The entire individual was preserved at −80 • C in the laboratory at the Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China. Total genomic DNA was extracted from its foot using CTAB methods.

Mitogenome Sequencing and Assembly
The complete mitochondrial genome was sequenced and assembled by Huitong Biotechnology, Shenzhen. Genomic libraries with an average length of 350 bp were constructed using the NexteraXT DNA Library Preparation Kit and then sequenced as 150 bp paired-end runs on the Illumina Novaseq platform (Total Genomics Solution Limited, SZHT) (San Diego, CA, USA). Raw data were filtered using the NGS QC Toolkit v2.3.3 [16] for quality control. De novo assembling was performed with SPAdes 3.11.0 [17].
To explore the phylogenetic relationship and gene arrangement of Vetigastropoda, 21 complete mitochondrial genomes were obtained from the NCBI database (Table 1). Of these, 19 species belong to the subclass Vetigastropoda, while the other two species from the subclass Neritimorpha were viewed as outgroups.
The nucleotide composition was 35.21% for A, 29.98% for T, 20.39% for C, and 14.43% for G, indicating it was biased toward A+T content at 65.19%, which was similar to other vetigastropods ( Table 3). The AT skew of E. rumphii was positive, while the GC skew was negative. Among 20 vetigastropods analyzed in our study, the AT skews of eight species were positive, and the GC skews of 13 species were negative, not showing any apparent consistency.

Protein-Coding Genes
The total length of all PCGs was 11,310 bp, accounting for 71.6% of the complete mitochondrial genome. The size of the 13 PCGs ranged from 165 (atp8) to 1731 bp (nad5). All PCGs used ATG as the start codon. Among the 20 vetigastropods we studied, ATG was the most frequently used start codon and 11 species used it for all PCGs. Two different kinds of stop codons were found in the 13 PCGs; TAA in nine PCGs (cox1, cox2, cox3, cytb, nad2, nad3, nad4, nad5, atp8) and TAG in the remaining four PCGs (nad1, nad4l, nad6, atp6). The other 19 vetigastropods also used TAA and TAG as stop codons.
The base composition of the PCGs was 25.2% for A, 40% for T, 15.4% for C, and 19.4% for G. The A + T content of each individual PCG and the concatenated data all exceeded 60%, which showed that the PCGs preferred AT. Except for atp8, all PCGs had a negative AT skew, suggesting they were biased toward T. As for GC skew, eight PCGs had a positive GC skew, while the other five PCGs had a negative one.
Genes 2022, 13, x FOR PEER REVIEW 4 of 12 eight species were positive, and the GC skews of 13 species were negative, not showing any apparent consistency. Figure 1. The mitochondrial genome map of Entemnotrochus. rumphii. From outside to inside, the first circle represents gene arrangement, the second represents the GC content, the third represents the GC skew, and the innermost circle represents the scale. The GC content circus was centered at 50% and the GC skew was centered at zero.  Figure 1. The mitochondrial genome map of Entemnotrochus. rumphii. From outside to inside, the first circle represents gene arrangement, the second represents the GC content, the third represents the GC skew, and the innermost circle represents the scale. The GC content circus was centered at 50% and the GC skew was centered at zero.   The relative synonymous codon usage (RSCU) of E. rumphii mitogenome was presented in Figure 2, indicating Leu, Phe, and Val were the three most frequently used amino acids. UUA-Leu, UUU-Phe, and AUU-Ile were the three most frequently utilized codons. Among 22 amino acids, nine (Ala, Arg, Gly, Leu1, Pro, Ser1, Ser2, Thr, and Val) had four codons, and others had two codons.

Transfer RNAs and Ribosomal RNAs
The size of 22 transfer RNA genes ranged from 64 (tRNA-Cys) to 70 bp (tRNA-Gly). The AT content of total tRNAs was 62.6%, also showing an AT bias. Almost all tRNAs of E. rumphii had typical clover-leaf secondary structures with four arms, except for trnS1 ( Figure 3). This phenomenon where this specific tRNA does not form a four-arms cloverleaf structure can be observed in most metazoan mitogenomes [25].
The 12S ribosomal RNA (rrnS) had a length of 894 bp, and the 16S ribosomal RNA (rrnL) had a length of 1386 bp. The 12S ribosomal RNA was located between tRNA-Val and tRNA-Met, while the 16S ribosomal RNA gene was located between tRNA-Leu CUA and tRNA-Val. For rrnL, the AT skew and GC skew values were −0.118 and 0.321; for rrnS, they were −0.054 and 0.321. Both of these rRNAs were biased toward T and G.

Transfer RNAs and Ribosomal RNAs
The size of 22 transfer RNA genes ranged from 64 (tRNA-Cys) to 70 bp (tRNA-Gly). The AT content of total tRNAs was 62.6%, also showing an AT bias. Almost all tRNAs of E. rumphii had typical clover-leaf secondary structures with four arms, except for trnS1 ( Figure 3). This phenomenon where this specific tRNA does not form a four-arms clover-leaf structure can be observed in most metazoan mitogenomes [25].
The 12S ribosomal RNA (rrnS) had a length of 894 bp, and the 16S ribosomal RNA (rrnL) had a length of 1386 bp. The 12S ribosomal RNA was located between tRNA-Val and tRNA-Met, while the 16S ribosomal RNA gene was located between tRNA-Leu CUA and tRNA-Val. For rrnL, the AT skew and GC skew values were −0.118 and 0.321; for rrnS, they were −0.054 and 0.321. Both of these rRNAs were biased toward T and G.

Phylogenetic Analysis
In our study, phylogenetic analysis of Vetigastropoda was conducted based on protein-coding genes ( Figure 4A). Based on two Neritimorpha species Clithon retropi and Nerita albicilla as outgroups, the vetigastropods grouped together in a single cla Within Vetigastropoda, E. rumphii was recovered as sister to all other vetigastropods = 100). Other vetigastropods were divided into three major clades. Fissurella volcano Diodora graeca, representing Fissurelloidea, formed the first clade with full supp Lepetodriloidea, Haliotidea, and Seguenzioidea formed the second clade, though w out significant support (BS = 50). Five families of Trochidea formed the third clade (B 98). However, the relationship of these clades remained unresolved because all b strap support values at deep nodes were lower than 95. Among Trochidea, all fami were recovered as monophyletic groups with full support. Phasianellidae was resol as the sister group to the remaining Trochidea species (BS = 98). However, phylogen

Phylogenetic Analysis
In our study, phylogenetic analysis of Vetigastropoda was conducted based on 13 proteincoding genes ( Figure 4A). Based on two Neritimorpha species Clithon retropictus and Nerita albicilla as outgroups, the vetigastropods grouped together in a single clade. Within Vetigastropoda, E. rumphii was recovered as sister to all other vetigastropods (BS = 100). Other vetigastropods were divided into three major clades. Fissurella volcano and Diodora graeca, representing Fissurelloidea, formed the first clade with full support. Lepetodriloidea, Haliotidea, and Seguenzioidea formed the second clade, though without significant support (BS = 50). Five families of Trochidea formed the third clade (BS = 98). However, the relationship of these clades remained unresolved because all bootstrap support values at deep nodes were lower than 95. Among Trochidea, all families were recovered as monophyletic groups with full support. Phasianellidae was resolved as the sister group to the remaining Trochidea species (BS = 98). However, phylogenetic relationships among the other four families were unstable. Turbinidae, Tegulidae, and Trochidae grouped together with weak support (BS = 66). This clade grouped with Angariidae, also without strong support (BS = 83).
Major phylogenetic relationships recovered in this study were consistent with previous studies based on Vetigastropoda mitogenomes [26,27]. However, bootstrap support values at some nodes were not high. The current matrix may not have enough phylogenetic information to resolve these nodes, so more data and research are needed for phylogenetic analyses about Vetigastropoda. E. rumphii was recovered as sister clade to all other vetigastropods, indicating that Pleurotomarioidea may be the most deeply diverging lineage of Vetigastropoda. This view was supported by some phylogenetic analyses based on morphology [28,29], or short molecular sequences [12,30], but first was proved by the mitochondrial genome data, as mitogenomes of Pleurotomarioidea were not included in other mitogenome-based studies. Moreover, a phylogenetic study of Vetigastropoda based on transcriptomes has been published recently [31]. Pleurotomarioidea was also recovered as sister group to all other vetigastropods in all their analyses.

Gene Arrangement
Nine vetigastropods of nine different families were chosen to explore Vetigastropoda mitochondrial gene arrangements ( Figure 4B).
Mitochondrial gene arrangements of Vetigastropoda are mostly conserved. Five species of the order Trochida, Granata lyrata (Choristellidae), and Haliotis rufescens (Haliotidae) shared the same gene order except for the position of a very few tRNAs. The or- Major phylogenetic relationships recovered in this study were consistent with previous studies based on Vetigastropoda mitogenomes [26,27]. However, bootstrap support values at some nodes were not high. The current matrix may not have enough phylogenetic information to resolve these nodes, so more data and research are needed for phylogenetic analyses about Vetigastropoda. E. rumphii was recovered as sister clade to all other vetigastropods, indicating that Pleurotomarioidea may be the most deeply diverging lineage of Vetigastropoda. This view was supported by some phylogenetic analyses based on morphology [28,29], or short molecular sequences [12,30], but first was proved by the mitochondrial genome data, as mitogenomes of Pleurotomarioidea were not included in other mitogenome-based studies. Moreover, a phylogenetic study of Vetigastropoda based on transcriptomes has been published recently [31]. Pleurotomarioidea was also recovered as sister group to all other vetigastropods in all their analyses.

Gene Arrangement
Nine vetigastropods of nine different families were chosen to explore Vetigastropoda mitochondrial gene arrangements ( Figure 4B).
Stöger and Schrödl [32] compared mitogenome arrangements of different mollusca taxa. They found that most vetigastropods had the same gene order as Octopus and Katharina, so this arrangement may be plesiomorphic for gastropods. Gene arrangement of E. rumphii was completely consistent with the hypothetical ancestral gastropoda mitochondrial gene order, indicating its deep conservation.
Within Vetigastropoda, most species have the same gene order, except for Lepetodrilidae and Fissurellidae. Uribe [26] indicated that the high number of gene rearrangements is correlated with the high evolutionary rates of mitogenomes, and this correlation has been found in many molluscs [33,34]. This may have a negative effect on phylogenetic analyses. Therefore, mitogenomes may not be appropriate for phylogenetic reconstruction at deep nodes, compared with genome and transcriptome data.

Conclusions
The complete mitochondrial genome of E. rumphii was 15,795 bp in length, containing 13 PCGs, two rRNAs, and 22 tRNAs. Phylogenetic analysis based on complete mitogenomes recovered E. rumphii as sister group to all other vetigastropods. The arrangement of mitochondrial genes was the same as most vetigastropods as well as the inferred ancestral gastropoda. This suggested that E. rumphii was a deeply diverging lineage of Vetigastropoda, and its mitogenome arrangement was very conserved. This study provided a mitochondrial genome of Pleurotomarioidea, which could be helpful for understanding phylogenetic relationships and the early evolution of Vetigastropoda.
Author Contributions: H.W. and H.S. designed this experiment. P.M., Z.Z., Y.L., Y.C., C.L. and J.W. provided technical and sample assistance. The first draft of the manuscript was written by Y.W. All authors commented on previous versions of the manuscript. All authors have read and agreed to the published version of the manuscript.