A deeper insight into the deepest vertebrate of the sea

doi:10.21203/rs.3.rs-1828415/v1

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A deeper insight into the deepest vertebrate of the sea

https://doi.org/10.21203/rs.3.rs-1828415/v1

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As the deepest vertebrate in the ocean, the hadal snailfish, which lives at a depth of about 7,000m, is a representative case for studying adaptation to extreme environments. Three years after the publication of its first genome, we re-investigated this species using more resequencing individuals and more refined comparative genomic analyses. We found that the hadal snailfish have the ability to migrate across different trenches in the Pacific Ocean after fully adapting to the hadal zone. The loss and retention of sensory genes suggests that the auditory and mechanosensory perception of the hadal snailfish may have undergone compensatory enhancement in complete darkness, while the visual system gradually disappeared. The special environment of the hadal zone has also affected this species in ways that are reflected in genetic changes related to circadian rhythms, behavior, bone mineralization, and high-hydrostatic pressure adaptation. We report here further advances in our understanding of the origin, specific characteristics and adaptive mechanisms of hadal snailfish.

Since its discovery in 2008 the hadal snailfish (Pseudoliparis swirei) has captured the attention of biologists and public as the deepest known vertebrate on Earth^1,2. The hadal zone, 6 kilometers below the sea surface where this species lives, and after which the species is named, is characterized by high hydrostatic pressure, complete darkness, barrenness, etc.^3,4. This special organism that survives and thrives in this “forbidden zone for life” provides us with an unusual case of adaptation to an extreme environment. After several years of anatomical and genomic research^5–7, it’s now known that the hadal snailfish has degraded vision and a lack of melanin in its skin⁷. Previous studies have also revealed that having more unsaturated fatty acids in the cell membrane^8,9 and high levels of trimethylamine N-oxide (TMAO) in the body¹⁰ may have played important roles in resistance to high hydrostatic pressure. However, much remains to be discovered about this species; our lack of knowledge can be categorized into three principal areas.

The first concerns the origin of hadal snailfish. They have been observed in several Trenches in the Northwest Pacific Ocean, including the Mariana⁷, Yap¹, Kuril–Kamchatka⁵ and Japan¹ Trenches. The question arises: Do they have the ability to migrate across trenches, or do they enter the hadal zone independently? Furthermore, it has been shown that the divergence time between hadal snailfish and Tanaka’s snailfish (a closely related species distributed in shallow areas) is about 20 million years ago (Mya)⁷; but when did the hadal snailfish enter the hadal zone and how long did they take to complete their adaptation to this ecological niche?

The second aspect concerns its morphological and physiological characteristics. For example, in such a dark environment, what do hadal snailfish rely on to sense the world: is it smell, taste or something else? Do they still have circadian rhythms in the absence of sunlight? Does darkness and high hydrostatic pressure have any effect on their behavior?

The last area concerns the mechanisms by which they tolerate high hydrostatic pressure. While unsaturated fatty acids and TMAO are widespread in marine fishes, why is it that only hadal snailfish have been observed to reach such depths? Some studies suggest that certain genetic alterations may confer tolerance to high hydrostatic pressure⁷, but if so, how do these alterations help hadal snailfish to adapt to this environment, and which alterations are most critical?

Unfortunately, these questions have not been well resolved because it is difficult for us to make long-term observations of deep-sea organisms in situ. During 2018 to 2019, we collected multiple samples of hadal snailfish from the Mariana Trench and Tanaka’s snailfish from the Yellow Sea (Supplementary Table 1). In addition, we further applied an improved method to identify gene loss and duplication, which is effective in reducing false positives and false negatives caused by errors in genome assembly and annotation, as well as multi-species sequence alignments. Based on more data and the new approaches, we have been able to trace the genomic signals left by adaptive evolution in an attempt to more fully understand the evolutionary processes and key changes in this special organism.

Improved genome assembly for Mariana hadal snailfish

A total of four hadal snailfish and four Tanaka’s snailfish individuals were collected for this study (Supplementary Table 1). Using a combination of ONT long reads, BGI short reads, and Hi-C sequencing technologies, we generated a chromosome-level genome assembly for hadal snailfish (Pseudoliparis swirei) (Supplementary Table 2-4 and Supplementary Fig. 2). The genome assembly comprised 1,173 contigs (total length = 626.44 Mb, contig N50 = 4.22Mb), organized into 24 chromosomes with an anchoring rate of 98.24%. The new assembly filled 1.26 Mb of gaps that were present in our previous assembly and have a much higher level of genome continuity and completeness (with complete BUSCOs of 96.0 %) than the two previous assemblies (Supplementary Table 4 and Supplementary Fig. 4)^7,11. Moreover, the genome redundancy caused by mis-assembly is also largely reduced in the new assembly (Supplementary Fig. 4), which ensures the reliability of the subsequent analysis. Meanwhile, we generated a high-quality chromosomal level genome assembly for Tanaka’s snailfish (Liparis tanakae) for a comparative evolutionary study (Supplementary Table 3 and Supplementary Fig. 2). We noticed that there is no major chromosomal rearrangement between hadal snailfish and Tanaka’s snailfish, while the stickleback, their closet relative, had undergone several independent chromosomal fusion events (Supplementary Fig. 5).

Based on the new genome assemblies, we re-examined the genetic changes that occurred in the common ancestor of hadal snailfish in combination with the new resequencing and transcriptome data. After a thorough scan and careful inspection, we identified 51 absent genes, 19 unitary pseudogenes, 21 lineage-specific expanded genes, 33 genes with insertions and deletions (with a length ≥ 3 amino acids) in coding regions, and 33 de novo originated new genes (Supplementary Table 9-11 and Supplementary Data 2-3). Most of them have not been previously reported and we discuss them in the following sections.

Cross-trench migration and high level of genetic diversity

Combining the eight new sequenced individuals (four hadal snailfish and four Tanaka’s snailfish) with five previously reported individuals (four hadal snailfish and one Tanaka’s snailfish), we have been able to form an initial perspective of the hadal snailfish at the population level. The principal component analysis (PCA), neighbor-joining tree and genetic clustering analysis shows that the eight hadal snailfish individuals can be divided into two populations, the first with seven individuals and the second with one individual (Fig. 1A-C). Interestingly, the first population includes samples from both the Mariana and Yap trenches. Using the mitochondrial data, we found that the divergence time of these individuals from different trenches appears to be only about 44,000 years (Supplementary Fig. 6). Combined with additional publicly available mitochondrial data, we noticed that the sample from the Kermadec Trench⁵, about 6,400 kilometers away from the Mariana Trench, is also clustered with individuals from the first population, and the divergence time was estimated to be 1.0 Mya (Fig. 1D and Supplementary Fig. 7). These results indicate that hadal snailfish are quite active and are able to complete long distance migrations, although it may take some time.

Genetic diversity of hadal snailfish is about 3.48 times higher than Tanaka’s snailfish. The F_ST between the two species is close to 0.91, indicating a large genetic divergence (Supplementary Fig. 8). That most polymorphisms are unique to each species, and that they share only 6% of their SNPs, is consistent with this observation. In addition, we also estimated the demographic history of hadal snailfish at the population level and observed a significant expansion in the last 60,000 years (Supplementary Fig. 9).

Step-by-step into the hadal zone

Based on the mitochondrial data, the closest known species related to the hadal snailfish were found to be from the genera Careproctus, Crystallias, Rhodichthys and Paraliparis, which contain many species living at approximately 1000 m depth (Fig. 1D and Supplementary Table 12). The divergence time between hadal snailfish and these species was estimated to be about 9.9 Mya, close to the formation time (8 Mya) of the deepest trough of the Mariana Trench¹². We might therefore speculate that the ancestor of hadal snailfish adapted to the deep-sea environment around 1000 m at about 9.9 Mya, and subsequently gradually adapted to greater depths in the formed or forming trench. Interestingly, the vision-related genes also exhibit a progressive evolutionary pattern.

Fish that inhabit different depths of the sea rely on different vision-related genes¹³. Since light with longer wavelengths is absorbed more quickly than those with shorter wavelengths, (except for the shortest UV wavelengths) high energy light with shorter wavelengths, such as blue, is able to penetrate to greater depths (Fig. 2A). The genes responsible for absorbing these shorter wavelengths (sws2 and rh2) are therefore much more important to deep-sea species in the photic zone (above 200m) than those that absorb longer wavelengths (lws). Similarly, the genes providing monochromatic vision in very dim light (rh1 and gnat1) have been proven to be important for deep-sea species of the disphotic zone (from 200m to 1000m)¹³. Along with the expression data, we noticed that these genes exhibit a different level of relaxation of natural selection in hadal snailfish (Fig. 2B and Supplementary Fig. 10). The lws gene (long wavelength) has been completely lost in both hadal snailfish and Tanaka’s snailfish; rh2 (central wavelength) has been specifically lost in hadal snailfish (Fig. 2C); sws2 (short wavelength) is still present in hadal snailfish but is hardly expressed; while rh1 and gnat1 (perception of very dim light) is both still present and normally expressed in hadal snailfish (Fig. 2D). A previous study has also proven the existence of rhodopsin protein in the eyes of hadal snailfish using proteome data¹⁴. These genetic alterations reflect, on the one hand, the gradual entry of hadal snailfish into the deep sea, and on the other hand, suggest again that genes would be very likely to be lost in the absence of natural selection pressure.

Highly expressed auditory genes

Do hadal snailfish compensate for the lack of vision when perceiving the external environment? The genes associated with the olfactory and auditory systems were investigated using both comparative genomic and transcriptomic methods. While the number of olfactory receptors was largely reduced (Supplementary Fig. 11), we found that the majority of the auditory genes were well preserved in hadal snailfish. Many of the auditory genes also tended to be significantly more upregulated in the brain of hadal snailfish than in Tanaka’s snailfish (Fig. 3A and Supplementary Data 4). The upregulated genes involve many aspects of the auditory system, including the development and tethering of otoliths^15,16, the development^17-20, maturation and maintenance of inner ear hair cells, the development and mechanosensitivity of stereocilia^21,22, and other factors^23,24. (Fig. 3A). Of these, the most significant upregulated gene is tmc1, which plays an important role in auditory perception as a component for mechanical-transduction in hair cells²⁵ and some mutations in this gene have been found to be associated with hearing loss^26,27. Coincidentally, tmc1 is also found to be the gene with the longest deletion specific to hadal snailfish (11 amino acids) in the regions that are generally highly conserved across vertebrate’s genomes (Supplementary Fig. 12 and Supplementary Table 11). This specific deletion is also verified by the short reads of all sequenced hadal snailfish individuals, including both genomic and transcriptomic reads. Our structural modeling also suggests that this deletion is very likely to lead to changes in the three-dimensional structure of its encoded protein (Fig 3B).

Moreover, the gene involved in lifelong otolith mineralization, cldnj, has three copies in hadal snailfish, but only one copy in other teleost species (Fig. 3C and Supplementary Table 10)²⁸. This may be important for hadal snailfish because calcium carbonate, the inorganic component in otoliths, is thought not to accumulate efficiently below the carbonate compensation depth (CCD; > 4,000 m - 5,000 m)⁴. It should be noted that the hadal snailfish survive at depths far beyond the limits of CCD, but their otoliths still maintain densities similar to those of sea-surface species²⁹. Although we do not know how the hadal snailfish breaks the depth limitation on calcium carbonate deposition, it is likely that the expansion of cldnj contributes to otolith mineralization. This may be the reason why it is able to become one of the few species of teleost in hadal zone.

Specific circadian rhythm and behavior in the dark

There is growing evidence that persistent darkness challenges the physiology and behavior of animals, leading to disrupted circadian rhythms, neurological damage and depressive-behavioral phenotypes³⁰. Consistent with previous research in cavefish³¹, we noticed that many of the circadian rhythm genes (per2a, cry1a, cry3, cry5 and gpr19) are lost or have undergone pseudogenization in the hadal snailfish (Fig. 4A and Supplementary Fig. 13). Despite that, we noticed that the essential clock control genes are present with normal expression level in the hadal snailfish, indicating the rhythm cycle is retained, although it is likely to have been largely uncoupled from sunlight. Moreover, gpr19 deficiency has been reported to prolong the cycle of circadian locomotor activity rhythms³², so hadal snailfish may have an extended rhythm cycle like cavefish^32,33.

In addition, in fish closely related to hadal snailfish, there are usually two copies of grpr encoding the gastrin-releasing peptide receptor; we noticed that in hadal snailfish one of them is absent and the other is barely expressed (Fig. 4B). It has been reported that grpr deficient mice show significantly increased locomotor activity in dark conditions³⁴. We might therefore speculate that the absence of that gene might in some way benefit the activity of hadal snailfish under complete darkness.

It should be noted that the above-mentioned missing genes are not sufficient to exhibit the full range of changes that occur in the nervous system of hadal snailfish. Previous studies suggest that high hydrostatic pressure suppressed the compound action potential in nerve trunks of fishes from shallow areas but not from deep areas, and can perturb the function of G protein-coupled receptors³⁵. From our transcriptome data, we also observed that the brain is one of the most divergent organs regarding expression levels between hadal snailfish and Tanaka’s snailfish (Supplementary Fig. 14). In line with this observation, one of our previous studies showed that zebrafish brains have the highest number of differentially expressed genes than the other investigated organs when exposed to high hydrostatic pressure³⁶. We also identified 15 de novo new genes in hadal snailfish that are highly expressed in the brain (Supplementary Fig. 14). The adaptation of the nervous system to high hydrostatic pressure deserves more in-depth study in the future.

Possibility of short-term starvation

In our previous study, we noticed that the individual hadal snailfish we investigated retained a large amount of intact food in its stomach and had larger eggs than might otherwise be expected⁷. It appears that the hadal snailfish have a survival strategy of storing energy, which is often found in species that need to cope with occasional starvation. Here we find another clue that hints at the existence of this possibility: the pseudogenization of the gene gpr27 in hadal snailfish (Fig. 4C and Supplementary Fig. 15). It has been reported that the knockout of gpr27 increases the expression of key enzymes in the carnitine shuttle complex³⁷, especially cpt1, which is essential for the beta-oxidation of lipid metabolism. The transcriptome data further confirm that the gene cpt1 is significantly upregulated in the liver of hadal snailfish. As lipid mobilization is thought to be a common metabolic response to short-term starvation in fish³⁸, the inactivation of gpr27 could help hadal snailfish to better survive periods of food deficiency. Although previous surveys have shown that various types of organisms live in the hadal zone, and that the hadal snailfish can survive by eating amphipods and occasionally polychaetes and decapod shrimp¹⁴, short-term starvation is still possible because although energy sources are limited by complete darkness, but organisms usually tend to “over-reproduce”.

Reduced bone mineralization

Vitamin D synthesis is dependent on UV light, with phytoplankton being the origin of vitamin D in food³⁹. Whether and how vitamin D reaches the hadal zone through various pathways, for instance as particulate organic matter, is still unknown. By investigating the genes associated with vitamin D metabolic pathways, we found that these genes are well conserved in the genome of hadal snailfish and are similarly expressed in both hadal snailfish and Tanaka’s snailfish (Supplementary Fig. 16), suggesting that vitamin D may not be a limiting factor for hadal zone vertebrates.

Nonetheless, micro-CT scans have revealed shorter bones and reduced bone density in hadal snailfish, from which it has been inferred that this species has reduced bone mineralization²⁹; this may be adaptive or it may be the result of high hydrostatic pressure. The gene bglap, which regulates bone mineralization, had been found to be a pseudogene in hadal fish⁷, which may contribute to this phenotype. Here, we found two more lost genes specific to hadal snailfish, tmem251 and tmem263, that contribute to reduced bone mineralization (Fig. 4D and Supplementary Fig. 17). These two genes encode transmembrane proteins, and loss-of-function mutations have now been found that may affect bone mineral deposition and thus bone development and body growth^40,41. Furthermore, many genes that determine chondrocyte differentiation and bone mineralization were found to be differentially expressed in the bones of hadal snailfish and Tanaka’s snailfish. However, it should be noted that this result derives from a single bone sample of a hadal snailfish and needs further verification.

High-hydrostatic pressure adaptation at molecular and cellular levels

A high level of trimethylamine N-oxide (TMAO) is thought to be the most important factor protecting the native structure of proteins in response to high-hydrostatic pressure⁴². However, we found no significant difference in gene expression levels of fmo3, a gene family encoding TMAO-generating enzyme flavin monooxygenase 3, between hadal snailfish and Tanaka’s snailfish (Fig. 5A and Supplementary Table 13). The large amount of TMAO in hadal snailfish may be due to the higher activity of that enzyme; we also identified two species-specific amino acid substitutions in fmo3a, and one in fmo3d, (Supplementary Fig. 18). Another possibility is the existence of an unknown mechanism in hadal snailfish that inhibits the degradation of TMAO in vivo.

Furthermore, in addition to the previously reported expansion of the acaa1 gene, an important gene for synthesizing unsaturated fatty acids, many lineage-specific genetic changes in hadal snailfish that have not been reported before may also have contributed to the high-hydrostatic pressure adaptation at molecular and cellular levels. For example, the genes vbp1 (Fig. 5B)⁴³, encoding Prefoldin subunit 3 that promotes protein folding, pebp1 (Supplementary Table 10), encoding phosphatidylethanolamine-binding protein 1 that coordinates the actin cytoskeleton together with the cell shape⁴⁴, and fthl27, encoding ferritin that influences reactive oxygen species metabolism, are specifically expanded in the hadal snailfish (Supplementary Fig. 19). The genes hace1, encoding a ubiquitin ligase that correctly organizes and renews cytoskeletal proteins⁴⁵, and cdc7, encoding cell division cycle 7-related protein kinase that has roles in DNA repair⁴⁶, both have lineage-specific deletions or insertions in otherwise highly conserved regions across teleosts (Supplementary Fig. 19).

Finally, we identified 34 genes that are significantly more highly expressed in all organs of hadal snailfish in comparison to Tanaka’s snailfish and zebrafish, while only nine genes were found to be significantly more highly expressed in Tanaka’s snailfish using the same criterion (Fig. 5C and Supplementary Table 14 and Supplementary Data 5). The 34 genes are enriched in only one GO category, GO:0000077: DNA damage checkpoint (Supplementary Table 15). Moreover, five of the 34 genes are associated with DNA repair (Supplementary Table 17). Previous studies have shown that high-hydrostatic pressure can induce DNA chain breaks and damage^47-49, and the widespread upregulation of the above genes may help hadal snailfish overcome these challenges.

It is generally accepted that most hadal snailfish are endemic to a single trench⁵⁰, which may support the model that this species has descended into different trenches independently. However, similar morphological types of hadal snailfish also exist in different trenches: whether this is the result of convergent evolution or their ability to migrate across the trench is not known. Here, by resequencing analyses and estimating divergence times from mitochondrial data, we have revealed that snailfish from the Marianas Trench are divided into two groups, with hadal snailfish from the Yap Trench and Kermadec Trench clustered with one of the groups from the Marianas Trench. Furthermore, our estimated divergence times support the notion that hadal snailfish do have the ability to cross trenches, which is also consistent with recent studies on similarities in the composition of gut microbial taxa of two hadal snailfish species from the Kermadec and Mariana Trenches⁵¹. As a complement, the specific loss of genes in hadal snailfish also suggests a possible ability to tolerate short-term starvation and achieve neutral buoyancy through lower skeletal density, all of which may further contribute to their ability to cross trenches^{1,29,37,40,41}. In addition, a previous study suggested that hadal snailfish may develop in shallow waters during juvenile life and then descend to greater depths in adulthood⁵². Although we have no additional supporting evidence, this conjecture does explain why they can cross the trench. The divergence time between the hadal snailfish and Tanaka’s snailfish has been estimated to be about 20 million years ago⁷, but there is no clear evidence or results as to when the hadal snailfish entered the hadal zone. It has been suggested in previous studies that hadal snailfish evolved rapidly compared to other fishes, possibly evolving rapidly into the deep-sea trench habitats, allowing liparids to fill that ecological niche before other taxa invaded the abyss. By contrast, we found in our results that hadal snailfish display a step-by-step signal of their gradual entry into the hadal zone. Firstly, vision-related genes showed a different level of relaxation of natural selection, suggesting a possible pattern of gradual descent into the hadal zone. At the same time, phylogenetic relationships of published mitochondrial data for fishes of the family Liparidae suggest that the ancestor of the hadal snailfish adapted to a deeper environment of about 1,000 m at about 9.9 million years after diverging from Tanaka’s snailfish, and then entered deeper regions in subsequent time, which further supports that this species entered the hadal zone step-by-step.

The hadal zone is a completely dark environment with no sunlight and no evidence of bioluminescence¹. Previous studies have shown that hadal snailfish exhibit a substantial loss of vision-related genes and a significant reduction in the number of olfactory receptors, showing varying degrees of degradation compared to shallower-living relatives. So how does this species perceive the external environment? In situ observations suggest that snailfish tend to rely more on mechanosensation¹, and here we found many of auditory-related genes upregulated in the brain, suggesting enhanced hearing and a possible shift from a visual to an auditory-dependent sensory system. It is worth mentioning that the expansion of cldnj, a gene essential for otolith formation in hadal snailfish²⁸, may be the reason why their otoliths still maintain densities similar to those of sea-surface species despite being far beyond the limits of CCD. Moreover, since the dissolution rate of calcium carbonate increases with higher pressure, keeping the otoliths stable may be one of the reasons limiting the inability of hadal snailfish to dive to even deeper regions. Complete darkness also further affects the circadian rhythms of organisms. Some cavefish, for example, lose their circadian rhythms in darkness⁵³. Consistent with those observations, we also found multiple circadian rhythms to have been lost in hadal snailfish, although other rhythm-related genes were still present and normally expressed, suggesting that this species may still have a light-independent rhythm and a prolonged rhythm cycle due to pseudogenization of gpr19³². Whether this rhythm cycle is food-induced, as in cavefish, is unknown and requires further studies to resolve. There is also evidence that prolonged darkness can trigger neurological damage and depressive phenotypes. We found that grpr gene copies were lost and down-regulated in hadal snailfish, which in turn increased their activity in the dark, allowing them to survive better in a dark and food-poor environment³⁴. Complementary to this, our findings suggest that the brain exhibits the greatest degree of differential expression of genes between hadal snailfish and Tanaka's snailfish, which may imply that the brain of this species is specifically altered to cope with the specific environment of the hadal zone, but its specific adaptive changes still need further study in the future.

High hydrostatic pressure is the most challenging factor to which inhabitants of the hadal zone need to adapt. It has been reported that high hydrostatic pressure can cause DNA strand damage and protein misfolding, which in turn affects the survival of organisms^47,48,54. We identified some genetic alterations in hadal snailfish associated with adaptation to high hydrostatic pressure, including the specific expansion of vbp1 and pebp1, that may help promote proper protein folding and maintain cytoskeletal stability^43,44. Meanwhile, comparative transcriptomic analysis identified multiple genes associated with DNA repair that were significantly more highly expressed in all tissues of hadal snailfish than in other fish, revealing a molecular mechanism allowing this species to cope with high hydrostatic pressure. Nevertheless, many of these results may also be neutral, especially regarding the possible mechanisms of adaptation to high pressure in hadal snailfish. For example, TMAO, which is able to maintain protein function under high pressure, and is thought to be a limiting factor in determining the depth at which fish can survive¹⁰, is found at the highest recorded concentrations in hadal snailfish. Results from our previous analysis suggested that positive selection on fmo3 may be important in promoting TMAO synthesis; but here, after an updated genome and rigorous FDR correction, we found that these genes had not undergone any particular or significant alteration in the constituent amino acids they expressed, although this does not exclude the possibility of a small number of amino acids having an effect on enzyme activity. However, since the expression of this gene is very similar among the various tissues of Tanaka’s snailfish and hadal snailfish, it seems more likely that the mechanism associated with TMAO degradation is altered in hadal snailfish, although we do not yet have any additional evidence to support this because the genes associated with TMAO degradation are still unclear. In addition, there are some gene expansions and upregulations that we cannot yet understand in terms of their putative roles in changing the ability of hadal snailfish to adapt to high hydrostatic pressure in the hadal zone. The specific effects of these changes need to be verified in further future experiments.

In summary, we provide chromosome-level genomes of hadal snailfish and Tanaka snailfish, as well as extensive transcriptome and resequencing data to analyze and determine the adaptive changes of hadal snailfish to the high-hydrostatic pressure, and dark environment of the hadal zone. Our resequencing data provide evidence for cross-trench migration of hadal snailfish, while mitochondrial data and vision-related genes suggest a progressive pattern of gradual descent into the hadal zone. More refined comparative genomic analysis reveals an auditory-dominant sensory system, a dramatically altered rhythm and nervous system, enhanced protein stability, and enhanced DNA repairability. The extensive genetic data provided by our results and the identification of genetic variants unique to hadal snailfish will provide a basis for a deeper understanding of deep-sea high-pressure adaptation mechanisms and future bionomics applications.

Genome sequencing and assembly

Genomic DNA was extracted from the muscle of four hadal snailfish collected from the Mariana Trench, and four Tanaka's snailfish collected from the southern Yellow Sea. We generated a total of 47.8 gigabases (gb) of Nanopore reads, 148.6 gb of BGI short reads, and 123.3 gb of Hi-C reads for hadal snailfish; and 39.0 gb of Nanopore reads, 130.3 gb of BGI short reads, and 99.5 gb of Hi-C reads for Tanaka’s snailfish.

The genome sizes of hadal snailfish and Tanaka’s snailfish were estimated by k-mer distribution analysis (K = 27) of SOApec v2⁵⁵ to be 633.2 Mb and 539.9 Mb, respectively. We then assembled the hadal snailfish and Tanaka’s snailfish genomes based on the filtered Nanopore sequencing data using wtdbg2 v2.4.1⁵⁶ based on default parameters, followed by two rounds of error correction using NextPolish v1.0⁵⁷ based on the filtered BGI sequencing data, and finally assembled them into chromosomal versions using 3D-DNA v180114⁵⁸ based on Hi-C data. Finally, BUSCO v 4.1.2⁵⁹ was used with the library ‘actinopterygii_odb10’ to analyze and evaluate the completeness of the gene set in our draft genome.

Transcriptome sequencing

A total of 11 transcriptomes from six tissues (eye, stomach, heart, liver, muscle, skin) were extracted from three hadal snailfish, while a total of 26 transcriptomes from 10 tissues (brain, spinal cord, eye, bone, cholecyst, stomach, heart, liver, muscle, skin) were extracted from three Tanaka's snailfish (Supplementary Data 1). RNA was subsequently extracted using TRIzol (Invitrogen) and purified using the RNeasy Mini Kit (Qiagen). Transcriptome reads were obtained from the Illumina HiSeq 2000 sequencing platform. The RNA sequences were filtered by Fastp v0.20⁶⁰ and assembled without reference using SPAdes⁶¹ with default parameters. Subsequently, TransDecoder v5.5.0 (https://github.com/TransDecoder/TransDecoder) was used to identify coding regions of the transcripts.

Genome annotation

Both de novo and homology-based predictions were used to identify repetitive elements in hadal snailfish and Tanaka's snailfish. First, we constructed a de novo transposable element library using RepeatModeler v1.0.11⁶², and then used RepeatMasker v4.0.7⁶³ to detect repeats. For homologous annotations, the genome sequences were compared with data from Repbase using RepeatMasker v4.0.7 and RepeatProteinMask v1.36 to predict transposable elements. For tandem repeat sequences, we used Tandem Repeats Finder v4.07⁶⁴ to make predictions.

The repeat masked genome was used for the gene annotation. We used a combination of ab initio gene predictions, homologous gene predictions, and direct gene models produced by transcriptome assembly to identify protein-coding genes structure on the genome as follows.

Step 1: Augustus v3.2.1⁶⁵ was used to generate ab initio predictions with internal gene models.

Step 2: The protein sequences from seven species, medaka, Atlantic cod, flatfish, stickleback, zebrafish, turbot, and fugu (Supplementary Table 6) and the transcriptome predicted protein sequences were used to align genomic sequences with BLAT v. 35⁶⁶.

Step 3: The psl files obtained in the previous step were integrated and the protein sequences that were aligned to the overlapping region of the genome were scored and sorted based on the alignment results using a custom script to filter out the best aligned protein sequences in this region. Then, GeneWise v2.4.1⁶⁷ was used to predict gene models with the aligned sequences as well as the corresponding query proteins. The custom scripts have deposited in GitHub (https://github.com/wk8910/bio_tools/tree/master/42.prediction).

Step 4: The Evidence Modeler (EVM) v1.1.1⁶⁸ was used to integrate the prediction results with different weights for each.

The integrated gene set was translated into amino acid sequences using InterProScan v5⁶⁹ to annotate motifs and domains in protein sequences by searching publicly available databases (including Pfam, PRINTS, PANTHER, ProDom and SMART) and the genes were further annotated using the KEGG databases.

Variant calling using resequencing data

Short reads of seven Mariana hadal snailfish, one Yap hadal snailfish and five Tanaka's snailfish (Supplementary Table 1) were mapped to the hadal snailfish genome assembled in this study with BWA v0.7.12-r1039⁷⁰; then SAMtools v1.4⁷¹ was used to sort and obtain BAM files. To analyze population genetics, we focused on SNPs and small indels (1–10 bp)⁷². The SNPs were called by FreeBayes v0.9.10-3-g47a713e⁷³ with parameters “--gvcf --min-coverage 5 --limit-coverage 200” and filtered by following three thresholds: (1) SNPs with missing rate £ 30%; (2) The highest sequencing depth of SNP position < 200 ×; (3) The lowest sequencing depth for each allele ≥ 5. Subsequently, we calculated the distribution of heterozygosity in genome-wide regions with 500 kb non-overlapping sliding windows.

Inference of phylogeny history

SNP tree, PCA and diversity statistics. PLINK v1.90b6.6⁷⁴ was used to perform PCA and other population divergency statistics, including nucleotide diversity and genetic differentiation (FST). A neighbor-joining tree was constructed with PHYLIP v3.697⁷⁵ for paired genetic distance matrices.

Admixture analysis. Different K values (from 1 to 5) were tested using Admixture v1.3.0⁷⁶ to infer ancestral populations in all hadal snailfish and Tanaka’s snailfish individuals accessions.

Demographic analysis. The demographic history of hadal snailfish and Tanaka’s snailfish was inferred with pairwise sequential Markovian coalescent (PSMC)⁷⁷ analysis, based on a substitution rate of 1.9174e-09 per generation for hadal snailfish and 5.6790e-09 per generation for Tanaka’s snailfish. The analysis was performed using the following parameters: −N25 −t15 −r5 −p ‘4+25 × 2+4+6’.

Mitochondrial genome phylogenetic reconstruction and divergence time estimation. The mitochondria of eight hadal snailfish and five Tanaka's snailfish were assembled by NOVOPlasty v4.3.1⁷⁸ with default parameters and annotated by MITOS (http://mitos2.bioinf.uni-leipzig.de/index.py). Subsequently, mitochondrial data from currently published species of the Liparidae were combined (Supplementary Table 12), nucleic acid sequences of 13 coding genes on mitochondria were aligned by MUSCLE v3.8.425⁷⁹ using default parameters, and alignments of the coding sequences were generated with pal2nal v14 using default parameters. The maximum likelihood (ML) tree was constructed by RAxML-8.2.12⁸⁰ using the following parameters: -f a -m GTRGAMMA -p 15256 -x 271828 -N 100. Finally, divergence times were estimated by MCMCtree v4.9j⁸¹ with 1 soft-bound calibration time-point (snailfish-stickleback: ~32-73 Ma). For Notoliparis kermadecensis, we combined all the above mitochondrial data and performed the same above analysis based on co1 and cytb gene sequences to obtain the ML tree and divergence times.

Gene loss and duplication

Here, we applied an improved read mapping-based method to identify gene loss and duplication, which is effective in reducing false positives and false negatives caused by genome assembly and annotation errors as well as multi-species sequence alignments. The custom scripts have deposited in GitHub (https://github.com/wenjie-xu-nwpu/hadal_snailfish). Although this method may have limitations for identifying gene loss and duplication in species with long divergence times, the divergence times of hadal snailfish and Tanaka's snailfish are about 20 million years⁷, and at least 88% of the reads in all hadal snailfish individuals can be well compared to Tanaka's snailfish genome, indicating that this method is applicable to this study.

For gene loss, the following methods were used for identification. (1) Short reads of eight hadal snailfish and five Tanaka's snailfish (~30X) were compared to the stickleback and Tanaka’s snailfish genome using BWA v0.7.12-r1039⁷⁰ and subsequently sorted by Samtools v1.4⁷¹ to obtain the BAM files. (2) We obtained the reads depth for each sites in the gene coding region based on the annotation information of the reference genome, and subsequently classified the depths we had on individual loci into 3 types (“HIGH” for greater than half of the average coverage, “LOW” for less than 3, and “MID” for the rest). We defined sites with “HIGH” for Tanaka's snailfish and “LOW” for hadal snailfish as hadal snailfish-specific lost sites (SLS). Then, the genes with SLSs accounting for at least 40% of the coding sequence length were selected as the candidate specific loss genes. (3)The protein sequences of the genes selected in the previous step were used as a reference to search through the genome of hadal snailfish by BLAT v. 35⁶⁶ and predict the gene structure using GeneWise v2.4.1⁶⁷ to determine the genes that were completely lost or partially lost in this species. (4) The synteny alignment between the hadal snailfish, Tanaka’s snailfish and stickleback were plotted for partial or fully lost of the gene.

For gene duplication, the following methods were used for identification. (1) Short reads of eight hadal snailfish and five Tanaka's snailfish were compared to the stickleback and Tanaka’s snailfish genome using BWA v0.7.12-r1039⁷⁰ and subsequently sorted by Samtools v1.4⁷¹ to obtain the BAM files. (2) The homologous sites whose average value of reads depth of all hadal snailfish individuals were greater than 1.5 the average value of the Tanaka’s snailfish individuals were retained and defined as hadal snailfish specific high-copy sites (HCS). Then, the genes with HCSs accounting for at least 50% of the coding sequence length were selected as the candidate high-copy genes. (3) We searched for the location of this gene on the hadal snailfish genome by BLAT v. 35⁶⁶ and predicted the gene structure by GeneWise v2.4.1⁶⁷ to determine its copy number. (4) Finally, the expansion of this gene was determined by constructing a gene tree of the protein sequences of this gene family from nine species, hadal snailfish, Tanaka’s snailfish, medaka, Atlantic cod, flatfish, stickleback, zebrafish, turbot, and fugu (Supplementary Table 6).

Identification of unitary pseudogenes

Unitary pseudogenes are non-functional genes that decay at their original location⁸², and we suggest that some missing homologs will exist in hadal snailfish genome as unitary pseudogenes during their adaptation to the special environment of the hadal zone.

We obtained pseudogenes in hadal snailfish by following five steps. (1) Using the stickleback genome sequence as a reference, we performed synteny alignment for three species (hadal snailfish, Tanaka's snailfish and stickleback) with Last v956⁸³ using the parameters ‘-E0.05', generating a total of 382 Mb (of which 290 Mb was informative for all species) of one-to-one alignment sequences with Multiz v1⁸⁴ using the default parameters. (2) Genes with at least 70% of the coding sequences of stickleback or Tanaka’s snailfish present in the MAF and not present in the corresponding regions of hadal snailfish were selected as alternative unitary pseudogene datasets. (3) We used BLAST v2.9.0⁸⁵ to determine if this gene was present in other regions of the hadal snailfish genome. (4) The hadal snailfish corresponding region was extended left and right by 10 KB, and the genes of stickleback and Tanaka's snailfish were used as references for predict the gene structure using GeneWise v2.4.1⁶⁷. (5) Screening for pseudogenes that were consistent in all hadal snailfish individuals.

De novo originated new genes

First, the short reads of eight hadal snailfish and five Tanaka's snailfish were compared to the hadal snailfish genome using BWA v0.7.12-r1039⁷⁰ and subsequently sorted by Samtools v1.4⁷¹ to obtain the BAM files. In the second step, we defined a single sequenced sample with reads depths < 10 at a single locus as a deletion locus. Based on the annotation file of hadal snailfish, we screened all Tanaka's snailfish individuals for genes with deletions >50%. Next, For the genes specifically present in hadal snailfish selected in the previous step, we used BLAST v2.9.0⁸⁵ to align them with the genomes of eight other fishes (Tanaka’s snailfish, medaka, Atlantic cod, flatfish, stickleback, zebrafish, turbot, and fugu) and screened for genes with a matching region < 0.4. Genes with TPM maxima less than 1 in each tissue of hadal snailfish were filtered out. The fully annotated genes (presence of start and stop codons) in the results were defined as novel genes of hadal snailfish.

Lineage-specific changes in amino acid sequences

For 17 species, Tanaka’s snailfish, stickleback, pacific bluefin tuna medaka, platy fish, Atlantic cod, flatfish, zebrafish, turbot, fugu, spotted gar, coelacanth, chicken, mouse, human, brownbanded bamboo shark and elephant shark (Supplementary Table 6), we identified one-to-one orthologues for each species and hadal snailfish by the RBH method, and subsequently selected genes present in 15 species, including hadal snailfish, as conserved gene sets. Next, the protein sequences of the selected genes were aligned using MAFFT v7.471⁸⁶, and a custom script was used to select regions that were consistent in other species and had contiguous specificity at sites greater than 3 bp in hadal snailfish, and that had at least 90% sequence identity for each 5 bp region before and after this variant region⁸⁷. Finally, genes with consistent variants in all hadal snailfish individuals were selected.

We performed protein structure simulation using AlphaFold2⁸⁸ for the amino acid sequences of target genes in hadal snailfish and Tanaka's snailfish. Finally, the highest scoring prediction was selected as the best structure and visualized by UCSF Chimera⁸⁹.

Comparative transcriptome analysis

For the RNA sequences of hadal snailfish and Tanaka's snailfish, we used Fastp v0.20.0⁶⁰ to filter out low quality and contaminated reads, and then used Hisat2 v 2.1.0⁹⁰ to align them to the respective reference genomes. StringTie v1.3.6⁹¹ was then used to count the number of reads paired for each gene with the help of gene annotation information of the species, and then TPM values were calculated for each gene in both species. Next, we identified 17281 one-to-one orthologues of hadal snailfish and Tanaka's snailfish using the RBH method. Subsequently, we identified the genes that were differentially expressed (DEGs) between the same tissues of two species using the R package DESeq2 with |log₂ (foldchange)| ≥ 1 and corrected P < 0.05. For genes that are up-regulated or down-regulated in multiple tissues, we first found by stochastic simulation that a gene is differentially expressed between two species in one organ does not affect the probability that this gene is differentially expressed in any other organ. Subsequently, we counted the genes that were up- regulated or down-regulated in each tissue to obtain a list of genes that were co-altered in multiple tissues.

Data availability

The sequence data and genome assembly files of the hadal snailfish have been deposited in the NCBI BioProject database with accession numbers PRJNA852951 (genome data) and PRJNA855356 (transcriptome data).

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Acknowledgments

The project was supported by the National Natural Science Foundation of China (32122021 to K.W. and 41876179 to S.H.); the 1000 Talent Project of Shaanxi Province to Q.Q., K.W. and S.H.; Fundamental Research Funds of Northwestern Polytechnic University; Strategic Priority Research Program of Chinese Academy of Sciences (Grant No. XDB42000000). The authors thank Dr. Yang Zhou, Dr. Yuan Yuan, and Dr. Tao Qin for their advice and discussions during this project.

Author Contributions

K.W. and S.H. designed and managed the project. B.W., H.X., X.G., J.B. and L.H. collected the samples. M.H. and C.Z. performed genome assembly and Hi-C chromosomal assembly. W.X. performed the repeat annotation and gene annotation. W.X., C.Z., B.W., M.H., J.Z. and C.F. performed the evolutionary analyses. W.X., K.W., S.H., C.F., W.W. and Q.Q. wrote and revised the manuscripts.

Competing Interests statement

The authors declare no competing interests

There is NO Competing Interest.

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A deeper insight into the deepest vertebrate of the sea

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Abstract

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Introduction

Results

Improved genome assembly for Mariana hadal snailfish

Cross-trench migration and high level of genetic diversity

Step-by-step into the hadal zone

Highly expressed auditory genes

Specific circadian rhythm and behavior in the dark

Possibility of short-term starvation

Reduced bone mineralization

High-hydrostatic pressure adaptation at molecular and cellular levels

Discussion

Materials And Methods

References

Declarations

Additional Declarations

Supplementary Files

Status:

Version 1