The complete mitochondrial genome of the largest amphipod, Alicella gigantea: Insight into its phylogenetic relationships and deep sea adaptive characters

International Journal of Biological Macromolecules 141 (2019) 570–577

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

The complete mitochondrial genome of the largest amphipod, Alicella gigantea: Insight into its phylogenetic relationships and deep sea adaptive characters Jun-yuan Li, Zeng-lei Song, Guo-yong Yan, Li-sheng He ⁎ Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, Sanya, Hainan 572000, PR China

a r t i c l e

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Article history: Received 1 July 2019 Received in revised form 23 August 2019 Accepted 6 September 2019 Available online 07 September 2019 Keywords: Deepsea amphipods Alicella gigantea Mitochondrial genome

a b s t r a c t Alicella gigantea (Alicelloidae) is a scavenger with the largest body size among amphipods. It is a participant in the foodweb of deepsea ecosystem and distributed with vast bathymetric and geographic ranges. In this study, the mitochondrial genome of A. gigantea was completely assembled and characterized. The complete sequence has a total length of 16,851 bp, comprising the usual eukaryotic components, with 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and 2 noncoding control regions (CRs). The gene rearrangement and reverse nucleotide strand bias of its mitochondrial genome are similar to those observed in the deepsea amphipod Eurythenes maldoror (Eurytheneidae), but different from the characters of Halice sp. MT-2017 (Dexaminoidea), an inhabitant of a deeper environment. Phylogenetic analysis indicates that A. gigantea occupies the basal branch of deepsea species—E. maldoror and Hirondellea gigas. This phylogeny supports the hypothesis that the evolution of hadal amphipods has undergone a transition from the abyssal depth. Compared to 41 available shallow water equivalents, the four accessible mitochondrial genomes from the deep sea, including the one produced in this study, show significantly fewer charged amino acids in the 13 PCGs, which suggests an adaption to the deepsea environment. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Amphipods are a kind of macrofauna that have a widespread distribution, including almost all aquatic environments, some subterranean habitats and some terrestrial habitats [1,2]. In the deep sea, scavenging amphipods play a critical role in the hadal foodweb [3]. Although much attention has been paid to the phylogenetic relationships and classifications of this group [4], amphipods have a historically taxonomic instability in regard of their higher ranks [5,6]. Family, superfamily or higher level relationships in Amphipoda have generally been so uncertain that they were simply listed alphabetically [6,7]. Alicella gigantea is a deepsea amphipod with the longest recorded body size up to 340 mm [8]. Its extraordinary body size overshadows all other large amphipods, such as Eurythenes gryllus (up to ~90 mm) [9] and Parargissa galatheae (~50 mm long) [9]. Although A. gigantea is a cosmopolitan species distributed over a large vertical range from 1720 m to 8233 m [8,10], these animals are captured at a low frequency [8], possibly because of their small populations. During a cruise to the Kermadec Trench, only an estimated nine individuals of A. gigantea were observed among 1500 photographs taken in situ by baited camera ⁎ Corresponding author. E-mail address: [email protected] (L. He).

https://doi.org/10.1016/j.ijbiomac.2019.09.050 0141-8130/© 2019 Elsevier B.V. All rights reserved.

[8]. In contrast to other deep sea amphipods (e.g., Hirondellea gigas and Eurythenes maldoror), which have been acknowledged to be members of the superfamily Lysianassoidea [11–14], the inclusion of A. gigantea within the lysianassoid assemblage has been questioned and its removal from this superfamily has been suggested [15]. In 2018, A. gigantea was separated from Lysianassoidea and regarded as an independent superfamily—Alicelloidea—in the World Amphipoda Database [16]. On the other hand, the phylogenetic position of A. gigantea has been inferred from molecular information, however, the relative relationships of Alicelloidae (the family A. gigantea belongs to) with other closely related lysianassoid families were inconsistent when based on different molecular markers in the analysis. Specifically, according to cox1-16S rDNA-18S rDNA concatenated sequences, Alicelloidae was found to be a sister clade to Eurytheneidae, and Hirondelleidae occupied the basal position of them. The topology deduced from individual 16S rDNA loci showed a close relationship between Alicelloidae and Hirondelleidae, but with Eurytheneidae as a basal group [17]. Even when inferred from five concatenated genetic markers (18S rDNA-28S rDNA-cox1-H3-16S rDNA), Alicelloidae was still nested within the Lysianassoidea clade [18]. The mitochondrial (MT) genome carries useful evolutionary information and can be utilized as a potent molecular marker for phylogenetic analysis because of its simple structure [19], conserved gene content [20], relatively high evolutionary rate [21] and

J. Li et al. / International Journal of Biological Macromolecules 141 (2019) 570–577

rare recombination events [22]. Therefore, analysis based on the entire MT genome, which is expected to produce a more credible result than single or several genetic genes [23], is required to clarify the phylogenetic relationships among these taxa. With the development of high-throughput sequencing, information on MT genomes of Amphipoda has been accumulating in public databases [24,25]. To date (August 2019), the MT genome sequences of 47 amphipods in 20 families from nine superfamilies have been deposited in GenBank. However, the representation of deep sea amphipods is limited. Only two hadal endemic species (inhabiting deeper than 6000 m) —Hirondellea gigas (Hirondelleidae) [26] and Halice sp. MT-2017 (Pardaliscidae) [27]—and one abyssal species—Eurythenes maldoror (Eurytheneidae) (from approximately 3000 m to 6000 m) are informatively accessible [11–13]. Furthermore, the MT genomes of hadal amphipods have some unique characteristics compared to those of shallow water amphipods, including a high proportion of nonpolar amino acids and special gene rearrangements [27]. In order to clarify the phylogenetic relationships between A. gigantea (Alicelloidae) and other deep sea lysianassoid families and draw a more general conclusion on the characteristics of deep sea MT genomes, the complete MT genome of A. gigantea collected at 7034 m in the Mariana Trench is presented and compared with that of other taxa in terms of their phylogeny, gene rearrangement, amino acid preference, and base composition bias in the present study. Our research not only contributes the MT DNA of A. gigantea to the amphipod genetic information database but also sheds further light upon the adaptive mechanisms and evolutionary relationships of deep sea amphipods. 2. Materials and methods 2.1. Sampling and DNA extraction A. gigantea were collected from ~5000 m to approximately 7500 m in the Mariana Trench using baited traps during the TS-01 and TS-03 cruises, which were conducted by the Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences in 2016 and 2017, respectively. Twenty-eight A. gigantea were collected in total, with body lengths ranging from 5.3 cm to 19.2 cm (Table S1). One individual of A. gigantea is shown in Fig. 1. Specimens were frozen in liquid nitrogen and then stored at −80 °C for later use. Total DNA was prepared from the leg muscle tissue of one individual using the salting-out method with the aid of an E.Z.N.A.® Tissue DNA Kit (OMEGA, Wuhan, China). The concentration of total isolated DNA was determined with a Qubit Fluorometer (Thermo Scientific, USA), and the quality of the extracted DNA was visualized by electrophoresis on a 1% agarose gel stained with SYBR® Safe DNA gel stain (Thermo Scientific, USA). 2.2. Genome sequencing A paired-end library with an insert size of 300 bp was prepared with total genomic DNA using a TruSeq DNA Sample Prep Kit (Illumina, USA) following the manufacturer's instructions. The library was sequenced by the Illumina HiSeq 2000 platform (2 × 150 bp paired-end reads)

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(Illumina, USA) at Novogene Bioinformatics Technology Co., Ltd. (TianJin, China). 2.3. Assembly of the mitochondrial genome Adapters and sequences with a quality score below 15 (Phred33 format) were removed from raw reads using Trimmomatic 0.36 [28]. Clean reads were assembled using the SPAdes 3.11.0 assembler [29] with default parameters. Using the BLAST tool, the assembled contigs were searched against the MT genome of Metacrangonyx longipes (GenBank No. AM944817) to extract the top hit sequence [30]. The average coverage depth for the obtained MT genome sequence was calculated using Bowtie2 2.2.4 [31] by mapping clean reads to the extracted contig. Visualization of the alignment file was achieved using Tablet 1.17.08.17 [32]. 2.4. Genome sequence annotation and analysis The assembled MT genome was annotated with the MITOS webserver [33]. Boundaries of the 13 protein-coding genes (PCGs) and 2 ribosomal RNA genes (rRNAs) were determined by alignment with the homologous genes of amphipods in Lysianassoidea, and LocARNA software in LocARNA-P probabilistic mode was used to further confirm rRNA boundaries [34]. Transfer RNA genes (tRNAs) and their secondary structures were predicted using three programs: the MiTFi model [35] in the MITOS pipeline [33], ARWEN 1.2.3.c [36] and tRNAscan-SE 1.21 [37]. CGView [38] was used to generate a circular display of the MT genome, which was then modified manually. The gene array of A. gigantea was compared to that of all available amphipod MT genomes with determined gene arrangements in GenBank as well as the putative ancestral pancrustacean gene order [39]. Gene rearrangement processes were deduced by detecting strong interval trees on the CREx webserver with common interval metrics. A larger common interval distance indicated a more similar gene arrangement in the analysis [40]. Nucleotide composition was computed using the DNAMAN program (Lynnon BioSoft, Vaudreuil, Canada). AT and GC skew values were calculated according to the following formulae: AT skew = (A − T) / (A + T) and GC skew = (G − C) / (G + C), where A, T, G, and C denoted the percentages of the four bases [41]. The control regions (CRs) of the MT genome were screened based on certain typical features discovered in insects and amphipods [24,42,43]. Only regions with high A + T contents, hairpin structures and conserved motifs were considered CR candidates, and the secondary structures of the putative CRs were predicted with Mfold 3.6 through the DNA Mfold server [44] using default parameters. Codon usage was analyzed using Sequence Manipulation Suite [45]. The content of each amino acid encoded by MT PCGs was calculated by summing the proportions of its corresponding codons. Amino acid compositions of groups with different properties (nonpolar, polar uncharged, and charged) were compared among hadal (Halice sp. MT-2017 and H. gigas), abyssal/cosmopolitan (A. gigantea and E. maldoror) and 41 shallow water species with available complete MT protein-coding regions. A two-tailed “t-test” was applied in R software (3.5.1) to calculate pairwise differences and significance levels (p-value) for the groups being compared. 2.5. Phylogenetic analysis

Fig. 1. Alicella gigantea specimens collected from 7125 m in the Mariana Trench.

For the phylogenetic analysis, one or two taxa were selected from each of the 18 amphipod families with MT genome information in GenBank. Because PCGs were incomplete for the only representative (Crypturopus tuberculatus) of Crypturopodinae, it was not included in our analysis. Ultimately, 30 taxa in Amphipoda, which included the A. gigantea collected in this study and the other 29 taxa covering 18 families in nine superfamilies, were used (Table S2). Three species in Isopoda were employed as outgroups [46,47] (Table S2). The 13 PCG amino acid sequences were aligned separately with MAFFT 7.0 [48]. The removal of poorly aligned regions was performed by Gblocks

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0.91b [49] with stringent default parameters. The resulting alignments were concatenated into a single matrix (3170 amino acids in size) to infer phylogenetic relationships. The PhyloSuite pipeline [50] was used for subsequent sequence component partitioning and tree construction with the help of plug-in programs. In the pipeline, the best partitioning schemes and evolutionary models were selected using PartitionFinder2 [51], with the greedy algorithm and corrected Akaike information criterion (AICc). The “all” and “Mrbayes” modes were applied to obtain the partitioning schemes and models for maximum likelihood analysis and Bayesian analysis, respectively, whereby 4 partitioned regions were schemed for the maximum likelihood analysis and 8 for the Bayesian method. The best model for each partition was listed in Table S3. Next, tree constructions were performed via maximum likelihood and Bayesian inference. The maximum likelihood phylogeny was inferred using IQ-TREE software [52] under the models selected for each identified partition with 20,000 ultrafast bootstraps [53]. The Bayesian tree was constructed using MrBayes 3.2.6 software [54], and the model for each component region was also selected according to the recommendations of PartitionFinder2. Four independent runs of four Markov chain Monte Carlo (MCMC) chains were performed; chains were run for five million generations, and the first 25% of generations were discarded as burn-in. 3. Results 3.1. Assembly of the mitochondrial genome A total of 63,121,690 clean reads (9.38 Gb) were generated by Illumina HiSeq sequencing. After assembly, the complete MT genome

of A. gigantea was obtained with a coverage depth of ~96.9×. The total length of the assembled MT genome is 16,851 bp (GenBank ID: MK215211), comparable to the lengths of available MT genomes from other amphipods ranging from 14,113 to 18,424 bp [25]. The MT genome of A. gigantea is a circular molecule containing 37 genes, including 13 PCGs, 22 tRNAs, and 2 rRNAs (Fig. 2). Among these genes, 23 (9 PCGs, 14 tRNAs) are located on the light strand; the remaining genes (8 tRNAs, 2 rRNAs, and 4 PCGs) are on the heavy strand (Fig. 2). Two CRs are present in the MT genome, located between rrnS and trnY and between trnV and trnM, respectively. 3.2. Gene rearrangement Incomplete and duplicated gene constitutions hindered pairwise comparisons of gene arrangements by CREx [40]. After exclusion of the above, 21 different MT gene arrangements of the 47 amphipod MT genomes available in GenBank remained. Measured by common interval distances, the maximum similarity distance to A. gigantea was found to be from the deepsea species E. maldoror, with a distance value of 552, indicating that these two species have a more similar MT gene arrangement (Table S4). In both A. gigantea and E. maldoror, 14 genes exhibit altered locations in comparison with the hypothetical ancestral Pancrustacea (hexapods and crustaceans) [55], including 2 PCGs (nad6 and cytb), 2 rRNAs and 10 tRNAs (Fig. 3). Changes in nad6 and cytb locations are commonly observed in amphipods, such as in Onisimus nanseni of Lysianassoidea, Caprella mutica of Caprelloidea, and Pseudoniphargus sorbasiensis of Allocrangonyctoidea (Fig. 3). Nonetheless, the altered placement of the two rRNAs in the MT genomes of A. gigantea and E. maldoror has seldom been observed (Fig. 3). Only

Fig. 2. Graph of the complete MT genome of Alicella gigantea. Different genes are represented by different boxes in different colors. tRNAs are displayed according to the one-letter code. Genes encoded by the light strand are shown outside the circle, and those encoded by the heavy strand are shown inside.

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after which A. gigantea experienced two complex tandem duplications with subsequent random gene loss (TDRLs) to achieve its current gene order (Fig. S1a). In contrast, E. maldoror underwent one transposition of the gene block trnL1, rrnL, trnV, and rrnS and one TDRL to reach its current status (Fig. S1b). The large-scale gene reversions involving as many as 20 genes in the hadal amphipod Halice sp. MT-2017 (reversal 1 in Fig. S1c) discovered in our previous study [27] were not found in these two deep sea species. In summary, the MT genomes of A. gigantea and E. maldoror display similar MT gene arrangements. Moreover, the characteristics of MT gene arrangements differ among the deep sea amphipod lineages. 3.3. Base composition and base bias The nucleotide composition of A. gigantea MT DNA exhibits an A and T bias, with 68.44% A + T content (Table S5), comparable to the AT richness typical in many other amphipod species [27]. The AT content of the third codon position (74.58% on average) was higher than those of the first (61.36%) and second positions (62.17%) (Table S5), indicating the strong effect of mutational pressure on the nucleotide at the third codon position. Positive AT skew (0.071) and negative GC skew (−0.301) were observed in the A. gigantea MT genome (Table S5), with the positive AT skew value being opposite to that of most other amphipod MT genomes [24,25,27]. In addition, the PCGs encoded on the light strand are slightly T skewed (−0.057) and moderately C skewed (−0.241), whereas those on the heavy strand are moderately T skewed (−0.226) and G skewed (0.431) (Table S5). The observed T-skewed pattern in the PCGs encoded on both strands might be explained by DNA damage occurring in a transiently single-stranded state during replication and transcription (i.e., the exposed single-stranded DNA has a high probability of deamination of C and A nucleotides, which results in a high frequency of C and A content on the complementary strand) [56,57]. The AT skewness with a positive value for the entire MT genome indicates more damage to PCGs encoded by the heavy strand than those encoded by the light strand, thus resulting in reversal of the overall AT skewness. Similar to A. gigantea, the other deepsea species E. maldoror also exhibited a complete MT genome with marginally positive AT skew and moderately negative GC skew (Table S5). The consistent patterns of skewness in most parts of the A. gigantea and E. maldoror MT genomes reveal the analogical architectures of their MT DNAs. Fig. 3. Comparison of gene arrangements among different superfamilies in Amphipoda. Genes with rearranged locations compared to the locations in the hypothetical Pancrustacea are highlighted in gray. The genes above the line are encoded by the light (or plus) strand, whereas those below the line are encoded by the heavy (or minus) strand. The CRs of Halice sp. MT-2017 and Metacrangonyctidae are placed in the middle of the line to denote the uncertainty of the replication origin. Segments with no explicit sequences are shown by dotted lines in some of the central bold lines. tRNAs are labeled as their corresponding single-letter amino acid code, apart from L1, L2, S1, and S2 for trnL (CUN), trnL (UUR), trnS (AGN), and trnS (UCN), respectively. The name of the superfamily is in bold and bracketed. Representative species of each superfamily are listed below each sequence.

the deep sea species Halice sp. MT-2017 also displays changed rRNA positions in our analysis (Fig. 3). However, unlike A. gigantea and E. maldoror with rRNAs encoded by the heavy strand, the rRNAs in the MT genome of Halice sp. MT-2017 were on the light strand. The tRNA strings—trnA, trnS2, trnN, trnE, and trnR—of A. gigantea and E. maldoror show conserved gene synteny with those of most other amphipods, but there were no or very few conserved arrangements among different superfamilies for the remaining tRNAs (Fig. 3). Compared with the putative ancestral pancrustacean gene order, the formations of gene arrangements in A. gigantea and E. maldoror MT genomes include five evolutionary steps, as deduced by CREx [40] (Fig. S1). During their transformation processes, both A. gigantea and E. maldoror underwent three transpositions (trnN, trnR, and trnG),

3.4. Protein-coding genes The amino acid compositions of the PCGs in the amphipod MT genomes were found to be relatively stable (Table S6), with Leu and Ser being the most predominant amino acids, accounting for nearly a quarter of the total number in PCGs. Cys and Arg are the most seldom used, accounting for b3% of the total. However, in terms of the proportion of each amino acid among the different species, small differences were still observed. Inhabitants dwelling in different depth ranges would suffer to different extents from the harmful effects exerted by high pressure. To investigate how mitochondria adapted to such pressure, comparisons of amino acid constitutions of the proteins encoded by MT PCGs were performed among amphipods from different depths. H. gigas and Halice sp. MT-2017 are common residents at a depth of 11,000 m in the Mariana Trench [27]. The nonpolar amino acids in the PCGs of these hadal MT genomes (64.50 ± 0.39%) were significantly more abundant than those from the shallow water (62.82 ± 0.58%) (Fig. 4a); correspondingly, the proportions of polar uncharged amino acids and charged amino acids were significantly lower in the hadal group (Fig. 4b, c). A. gigantea and E. maldoror are representatives of species widely spread in abyssal and hadal zones, but no records thus far have indicated that they might extend to depths below 10,000 m, regions where H. gigas and Halice sp. MT-2017 dominate [58]. Our results indicate that the A. gigantea and E. maldoror MT genomes contain a

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Fig. 4. Statistical data for amino acid contents of the MT PCGs of amphipods from different habitats. Significance tests were performed between hadal (Hirondellea gigas and Halice sp. MT2017), abyssal/cosmopolitan (Alicella gigantea and Eurythenes maldoror), and shallow water amphipods (others) regarding the percentages of nonpolar (a), polar uncharged (b), and charged (c) amino acids in their MT PCGs. The significance levels between compared groups were calculated by a t-test, with p b 0.05 as the significant threshold level (denoted by *) and p b 0.01 as the very significant threshold level (denoted by **).

significantly lower content of charged amino acids (10.01 ± 0.16%) than do their shallower counterparts (10.46 ± 0.26%) (Fig. 4c), but their MT PCGs display no significant differences in terms of polar uncharged and nonpolar amino acid constitution (Fig. 4a, b). Notably, the percentages of nonpolar amino acids in the MT PCGs of deep sea amphipods exhibit an increasing trend with depth. 3.5. rRNAs The lengths of rrnS and rrnL are 536 bp and 1076 bp, respectively, and they are located between trnL1 and CR on the heavy strand. Unlike the rrnS and rrnL in other amphipods, the rrnS and rrnL in A. gigantea are adjacent to each other without any tRNAs (such as trnV or trnL1) between them (Fig. 3). 3.6. Control regions A. gigantea contains two CRs in its MT genome (Fig. 2), both of which show high AT contents (76.70%, 77.37%). Hairpin structures and the nearby conserved elements of typical CR structure [24,43,59] were also identified (Fig. S2). The conserved structures near the hairpin structures of CR1 and CR2 are “TAAT” and “TATA” motifs and a poly T stretch. In CR1, the poly T stretch (position: 8793 nt-8860 nt) and the nearby poly A stretch form the stem part of the hairpin secondary structure. In CR2, the poly T stretch (position: 14,842 nt-14,856 nt) is located

upstream of the hairpin structure, which is not involved in hairpin structure formation.

3.7. Phylogenetic analysis The topologies of the highly resolved trees resulting from the Bayesian and maximum likelihood methods based on the 13 concatenated MT PCG amino acid alignments were consistent (Fig. 5). Crangonyctidae is divided into two separate clades, and the resulting 11 clades in the phylograms correspond to 10 identified superfamilies in Amphipoda. Consistent with the findings of our previous research [27], Dexaminoidea, represented by the hadal species Halice sp. MT-2017, is placed on an independent branch and does not cluster with Lysianassoidea, a superfamily mainly composed of deep sea species in this study. The above result suggested that the deep sea amphipods were non-monophyletic, and they were represented by at least two separate lineages (shaded in yellow and blue in Fig. 5). Regarding the other three deep sea amphipods evaluated in this study, the relationship (abyssal Alicella gigantea + (abyssal Eurythenes maldoror + (hadal Hirondellea gigas + Arctic pack ice-shallow water Onisimus nanseni))) was constructed with high nodal support (bootstrap values N90 and posterior possibilities = 1), which indicated that A. gigantea was placed on the basal branch of the lysianassoid assemblage (H. gigas, E. maldoror and O. nanseni) (Fig. 5).

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Fig. 5. Phylogenetic trees of ten superfamilies in Amphipoda based on Bayesian and maximum likelihood methods. SH-aLRT, ultrafast bootstrap values and posterior possibilities are shown near the node of each branch. Only bootstrap values larger than 50% and posterior possibilities above 0.8 are indicated. The two deep sea lineages are shaded in yellow and blue.

4. Discussion 4.1. PCG amino acid composition As the composition and property of amino acids are related to the function of proteins [60,61], a comparison of the amino acid contents of different categories was performed in our previous research, which indicated a significant difference in nonpolar amino acid compositions between hadal amphipod mitochondrial PCGs and those from the shallow water [27]. Nevertheless, taxa distributed at other depth ranges of the deep sea environment have not been investigated. By investigating the deep sea amphipods A. gigantea, E. maldoror and additional shallow water species in this study, a clear pattern was revealed that the inclination of nonpolar amino acids encoded by MT PCGs increased with depth. Similar studies on the amino acid composition have also been performed in other deep sea organisms [62–65], however, their results revealed only a few amino acid substitutions of certain nucleus-encoded proteins and did not show a tendency of change at global level. The reason was probably the lack of suitable study subjects that only differed in optimal growth pressure [66]. On the other hand, a global propensity for noncharged polar amino acids and an avoidance of hydrophobic amino acids have been considered to be associated with cold adaptation. A case showing this pattern can be exemplified in Methanogenium frigidum and Methanococcoides burtonii, both of which are psychrophilic Archaea discovered in Antarctica [67]. However, the analysis in the above study did not take into account the optimal growth pressures of the compared Archaea. In our study, although the deep sea samples compared were from the abyssal and the hadal depth respectively, the ambient temperatures in these two habitats would not have large differences, because when the depth reached over 2000 m in the deep sea, the temperature would fluctuate within a small range from 2 °C to 4 °C [68]. Therefore, the pressure appeared to be the key driver that contributed to the preference for nonpolar amino acids in the composition of deep sea amphipod MT PCGs.

To be rigorous, however, there is a caveat related to the above analysis that should be addressed, because the close phylogenetic relationship of the individuals within the compared groups could also result in the convergence of amino acid composition pattern in MT PCGs, independent of depth or pressure. In the hadal group, the samples of H. gigas and Halice MT-2017 were from distantly related superfamilies (Lysianassoidea and Dexaminoidea) [27] (Fig. 5), so their consistent tendency for MT amino acid composition bias was certainly not caused by phylogenetic relatedness. However, in the abyssal/cosmopolitan group, although A. gigantea was regarded as a separate superfamily from Lysianassoidea, which the other abyssal species E. maldoror belonged to, their close relationship was still obvious in the phylogenetic tree (Fig. 5). Therefore, to avoid the influence of phylogenetic relatedness, a more detailed examination focusing on the clade composed of Lysianassoidea and Alicellidea was performed, which also revealed a bias toward uncharged amino acids in the MT PCGs of the deep sea species (Fig. S3). In summary, both the large-scale analysis including different phylogenetic groups and the small dataset consisting of only closely related species indicated that the MT genomes of deep sea amphipods had a preference for uncharged amino acids, of course, if MT genomes were available for more species from the two deep sea clades, our conclusion would be more robust. Overall, the study suggests the importance of the amino acid composition of MT PCGs to the survival of amphipods in the extreme deep sea. The protein products of 13 MT PCGs are embedded in the hydrophobic lipid chains of membranes [69], and a decrease in the polarity of PCGs would be conducive to compact interaction between membrane proteins and lipid chains. At a depth of 11,000 m, the strategy adopted by abyssal individuals of decreasing the contents of charged amino acids in PCGs might not be sufficient to mitigate the potential damage caused by the high pressure (up to 110 MPa) at hadal depths. Consequently, in addition to charged amino acids, polar uncharged and nonpolar amino acid contents were found to be altered in hadal amphipods. Considering that mitochondria are centers of energy metabolism in the cells of nearly all eukaryotes [70], an intact and sound condition of the MT

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membrane structure may be a determinant factor influencing the pressure tolerance of deep sea amphipods. 4.2. Phylogeny of deep sea amphipods Gene arrangements may provide valuable phylogenetic information [39], and the general similarity between the gene orders of A. gigantea and E. maldoror revealed that these two deep sea species might share close phylogenetic positions. Unfortunately, another deep sea amphipod, H. gigas, was not included in the comparison. The large number of tandem repeats consisting of both A and T in the H. gigas MT genome gave rise to two unconnected contigs [26], making it impossible to perform a reliable comparison with regard to gene order. In view of the phylogenetic inference based on the 13 concatenated PCGs, the results of this study are congruent with the current taxonomic opinion that A. gigantea belongs to the superfamily Alicelloidea and that E. maldoror shows a close relationship to H. gigas, as they are both from Lysianassoidea [16]. It is believed that the abyssal region constitutes a habitat for deep sea species colonization, as such taxa might be preadapted to the high hydrostatic pressure at hadal depths [71]. Our topology shows that the hadal endemic species H. gigas clusters with the abyssal species E. maldoror and A. gigantea, indicating that a transition from the abyssal to the hadal zone occurred during the evolution of H. gigas. However, Ritchie et al. reported that H. gigas and E. maldoror are located basally among these three deep sea species based on cox116S-rDNA-18S-rDNA concatenated sequences and the 16S marker, respectively [17]. The inconsistency between their results and ours may result from differences in the evolutionary rates of MT PCGs and rRNAs as well as the different phylogenetic signals that mitochondria and nuclei carry [72,73]. Although the nodal support values of the topology among the deep sea species A. gigantea, H. gigas and E. maldoror in this study are more credible than those in Ritchie's work, analyses of more MT genomes from unreported families in Alicelloidea (such as Valettidae and Parargissidae) and Lysianassoidea (such as Uristidae and Tryphosidae) are needed to unveil more comprehensive relationships at the family or superfamily level for deep sea groups. Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2019.09.050. Funding This study was supported by the National Key Research and Development Program of China [grant no. 2016YFC0302500]; the National Key Research and Development Program of China [grant no. 2016YFC0304905]; the National Key Research and Development Program of China [grant no. 2018YFC0309804]; and the “Strategic Priority Research Program” of the Chinese Academy of Sciences [grant no. XDB06010103]. Author contributions Jun-yuan Li performed the experiments, analyzed the data, wrote the paper, and prepared the figures and tables. Zeng-lei Song performed the experiments. Guo-yong Yan collected the samples. Li-sheng He conceived and designed the experiments, contributed reagents/materials/ analysis tools, supervised the work, and reviewed drafts of the paper. Declaration of competing interest The authors declare no competing interests. References [1] R. Väinölä, J. Witt, M. Grabowski, J.H. Bradbury, K. Jazdzewski, B. Sket, Global diversity of amphipods (Amphipoda; Crustacea) in freshwater, Hydrobiologia 595 (2008) 241–255.

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