Adaptation to deep-sea chemosynthetic environments as revealed by mussel genomes

Sun, Jin; Zhang, Yu; Xu, Ting; Zhang, Yang; Mu, Huawei; Zhang, Yanjie; Lan, Yi; Fields,Christopher J.; Hui, Jerome Ho Lam; Zhang, Weipeng; Li, Runsheng; Nong, Wenyan;Cheung, Fiona Ka Man; Qiu, Jian-Wen; Qian, Pei-YuanPublished in:Nature Ecology and Evolution

Published: 01/05/2017

Document Version:Final Published version, also known as Publisher’s PDF, Publisher’s Final version or Version of Record

License:CC BY

Publication record in CityU Scholars:Go to record

Published version (DOI):10.1038/s41559-017-0121

Publication details:Sun, J., Zhang, Y., Xu, T., Zhang, Y., Mu, H., Zhang, Y., Lan, Y., Fields, C. J., Hui, J. H. L., Zhang, W., Li, R.,Nong, W., Cheung, F. K. M., Qiu, J-W., & Qian, P-Y. (2017). Adaptation to deep-sea chemosyntheticenvironments as revealed by mussel genomes. Nature Ecology and Evolution, 1(5), [0121].https://doi.org/10.1038/s41559-017-0121

Citing this paperPlease note that where the full-text provided on CityU Scholars is the Post-print version (also known as Accepted AuthorManuscript, Peer-reviewed or Author Final version), it may differ from the Final Published version. When citing, ensure thatyou check and use the publisher's definitive version for pagination and other details.

General rightsCopyright for the publications made accessible via the CityU Scholars portal is retained by the author(s) and/or othercopyright owners and it is a condition of accessing these publications that users recognise and abide by the legalrequirements associated with these rights. Users may not further distribute the material or use it for any profit-making activityor commercial gain.Publisher permissionPermission for previously published items are in accordance with publisher's copyright policies sourced from the SHERPARoMEO database. Links to full text versions (either Published or Post-print) are only available if corresponding publishersallow open access.

Take down policyContact lbscholars@cityu.edu.hk if you believe that this document breaches copyright and provide us with details. We willremove access to the work immediately and investigate your claim.

Download date: 26/10/2021

nature eCOLOGY & eVOLutIOn 1, 0121 (2017) | DOI: 10.1038/s41559-017-0121 | www.nature.com/natecolevol 1

ArticlesPUBLISHED: 3 APrIL 2017 | VOLUME: 1 | ArTICLE NUMBEr: 0121

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

adaptation to deep-sea chemosynthetic environments as revealed by mussel genomesJin Sun1, 2, Yu Zhang3, ting Xu2, Yang Zhang4, Huawei Mu2, Yanjie Zhang2, Yi Lan1, Christopher J. Fields5, Jerome Ho Lam Hui6, Weipeng Zhang1, runsheng Li2, Wenyan nong6, Fiona Ka Man Cheung6, Jian-Wen Qiu2* and Pei-Yuan Qian1, 7*

Hydrothermal vents and methane seeps are extreme deep-sea ecosystems that support dense populations of specialized macro benthos such as mussels. But the lack of genome information hinders the understanding of the adaptation of these ani-mals to such inhospitable environments. Here we report the genomes of a deep-sea vent/seep mussel (Bathymodiolus plati-frons) and a shallow-water mussel (Modiolus philippinarum). Phylogenetic analysis shows that these mussel species diverged approximately 110.4 million years ago. Many gene families, especially those for stabilizing protein structures and removing toxic substances from cells, are highly expanded in B. platifrons, indicating adaptation to extreme environmental conditions. The innate immune system of B. platifrons is considerably more complex than that of other lophotrochozoan species, including M. philippinarum, with substantial expansion and high expression levels of gene families that are related to immune recognition, endocytosis and caspase-mediated apoptosis in the gill, revealing presumed genetic adaptation of the deep-sea mussel to the presence of its chemoautotrophic endosymbionts. A follow-up metaproteomic analysis of the gill of B. platifrons shows metha-notrophy, assimilatory sulfate reduction and ammonia metabolic pathways in the symbionts, providing energy and nutrients, which allow the host to thrive. Our study of the genomic composition allowing symbiosis in extremophile molluscs gives wider insights into the mechanisms of symbiosis in other organisms such as deep-sea tubeworms and giant clams.

The environment of deep-sea hydrothermal vents and methane seeps is characterized by darkness, lack of photosynthesis-derived nutrients, high hydrostatic pressure, variable tem-

peratures and high concentrations of heavy metals and other toxic substances1,2. Despite this hostile environment, these ecosystems support dense populations of macrobenthos which, with the help of chemoautotrophic endosymbionts, are fuelled by simple reduced molecules such as methane and hydrogen sulfide1–3. Studying vent and seep organisms may answer fundamental questions about their origin and adaptation to the extreme environments1,4. Deep-sea mussels (Mytilidae, Bathymodiolinae) often dominate at hydro-thermal vents and cold seeps around the world. Owing to their ecological importance and remarkable biological characteristics, including their ability to survive an extended period under atmo-spheric pressure5, they are a recognized model for studies of sym-biosis6, immunity7, adaptation to abiotic stress8, ecotoxicology9 and biogeography10.

results and discussionWe sequenced the genomes of both B. platifrons Hashimoto and Okutani, 1994 (a deep-sea mussel, Fig.  1a) and M. philippinarum (Hanley, 1843) (a shallow-water mussel, Fig.  1b) in the family Mytilidae using a whole-genome shotgun approach and compared their features (Supplementary Note 1). Both assembled genomes were highly repetitive (Supplementary Fig. 2). The genome of B. platifrons

(1.64  Gb) was smaller than that of M. philippinarum (2.38  Gb), mainly because it has fewer repeats, particularly the Helitron-transposable elements and unclassified repeats that together contrib-uted a 707 Mb difference between the two genomes (Supplementary Note 9). The N50 value (the shortest sequence length at 50% of the genome size) of the B. platifrons scaffolds and contigs was 343.4 Kb and 13.2 Kb, respectively; and the N50 value of the M. philippinarum scaffolds and contigs was 100.2 Kb and 19.7 Kb, respectively. The deep-sea and shallow-water mussel genome contained 96.3% and 93.7% complete and partial universal single-copy metazoan ortholo-gous genes, respectively, indicating the completeness of the assembly and gene models (Supplementary Note 6). As with other bilateri-ans, the two mussel genomes contained all the expected Hox and ParaHox genes, as well as similar numbers of microRNAs, again sug-gesting that the assemblies encapsulate a nearly complete representa-tion of genic information (Supplementary Note 13 and 14). Although the gills of B. platifrons contain endosymbiotic methane-oxidizing bacteria (MOB)11, the host scaffolds did not contain bacterial nucle-otide sequences, showing a lack of horizontal gene transfer from the symbiont. B. platifrons has a lower rate of heterozygosity compared to the other three bivalves with a sequenced genome (Supplementary Fig. 3a), potentially owing to recurrent population bottlenecks as a result of population extinction and re-colonization12.

The B. platifrons genome had 33,584 gene models, 86.2% of which had transcriptome support, based on transcriptome data from three

1Division of Life Science, Hong Kong University of Science and Technology, Hong Kong, China. 2Department of Biology, Hong Kong Baptist University, Hong Kong, China. 3Shenzhen Key Laboratory of Marine Bioresource and Eco-Environmental Science, College of Life Science, Shenzhen University, Shenzhen, China. 4Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China. 5High Performance Computing in Biology, Roy J. Carver Biotechnology Centre, University of Illinois at Urbana–Champaign, Urbana, Illinois 61801, USA. 6Simon F. S. Li Marine Science Laboratory, School of Life Sciences, Centre for Soybean Research, Partner State Key Laboratory of Agrobiotechnology, the Chinese University of Hong Kong, Hong Kong, China. 7HKUST-CAS Joint Laboratory, Sanya Institute of Deep Sea Science and Engineering, Sanya 572000, China. *e-mail: qiujw@hkbu.edu.hk; boqianpy@ust.hk

2 nature eCOLOGY & eVOLutIOn 1, 0121 (2017) | DOI: 10.1038/s41559-017-0121 | www.nature.com/natecolevol

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Articles NATure ecOlOgy & evOluTiON

tissues in a previous study13, and our new transcriptome data from three additional tissues; whereas the M. philippinarum genome had 36,549  gene models, 86.0% of which had transcriptome sup-port, based on our new transcriptome data from five tissues (Supplementary Note 5). A total of 5,527 B. platifrons gene models were validated by shotgun proteomics using gill samples (false discovery rate (FDR)  <   0.01), and the gene-expression level correlated well (P  <   1  ×   10−5) with their corresponding protein abundance (Supplementary Note 20). Comparisons among five molluscan genomes revealed 6,883 shared protein domains and 474 protein domains unique to B. platifrons (Fig. 2a and Supplementary Table 14).

A phylogenetic tree constructed using 375 single-copy ortho-logues from 11 lophotrochozoan species (Fig. 1c) and another tree constructed using transcriptome sequences of representative mem-bers of Mytilidae (Fig.  1d) showed that, within the genera used, Modiolus is the closest shallow-water relative of Bathymodiolus. Using the lophotrochozoan tree as a reference, the time of diver-gence between B. platifrons and M. philippinarum was estimated to be around 110.4 million years ago (Ma), with a 95% confidence interval of 52.4–209.7 Ma (Fig. 2b), which is close to the upper age limit of deep-sea symbiotic mussels (102 Ma) previously estimated using five genes14. This result supports the hypothesis that the ances-tors of modern deep-sea mussels might have experienced an extinc-tion event during the global anoxia period that has been associated with the Palaeocene–Eocene thermal maximum at around 57 Ma15.

Gene-family analysis among the 11 lophotrochozoans revealed the expansion of 111 protein domains and contraction of 39 domains in B. platifrons (Fig.  2c and Supplementary Table 10). The num-ber of contracted domains in the deep-sea mussel is the smallest among all the species examined. This indicated that adaptation of B. platifrons to the deep-sea chemosynthetic environment was mainly mediated by gene family/domain expansion, and the deep-sea mussel has retained most genes of its shallow-water ancestors.

Among the most numerously expanded gene families in B. platifrons are the HSP70 family (179 proteins) and the ABC transporters (393 proteins). HSP70 is important for protein folding, and thus high expression of these genes in the gill (Supplementary Fig. 15) could indicate their active response to stresses such as changing temperature, pH and hypoxia. However, because it took approximately three hours from the sample collection to preserva-tion, the high expression of some of the stress-related genes could also be caused by the stress experienced during the sampling and transportation. Nevertheless, the expression level of HSP70 has been reported to positively correlate with the level of DNA-strand breakage in a deep-sea mussel16. Therefore, expansion of this gene family may provide additional genetic resources allowing deep-sea mussels to cope with abiotic stressors. ABC transporters are known to move toxic chemicals outside mussel gill epithelial cells17, form-ing the first line of defence against toxic chemicals in the vent/seep fluid. Many of the ABC transporters were highly expressed in the gill (Supplementary Fig. 16), indicating their active role in protect-ing the deep-sea mussel through detoxification.

Deep-sea mussel Bathymodiolus spp. obtain their symbionts from the environment, and house them in the vacuoles of gill epithe-lial cells18. Therefore, they must recognize the bacteria in the water column, incorporate them into the host cell, and control their population growth (Fig.  3). The target microbes carry signature molecules on their surface, which are recognized by the transmem-brane pattern-recognition receptors (PRRs) of the host19. Among the PRRs in B. platifrons, there was a significant expansion of genes with an immunoglobin (Ig) domain and genes with an fibrinogen domain, both of which are critical in allowing the sea anemone Aiptasia to recognize its dinoflagellate symbiont20. In addition, peptidoglycan-recognition proteins (PGRPs) that can bind with peptidoglycan21 on the bacterial cell wall were also expanded in the genome of B. platifrons. Many of these PRR genes were also highly

a b

Helobdella robustaCapitella teleta

Lingula anatina

Octopus bimaculoidesLottia gigantea

Biomphalaria glabrataAplysia californica

Modiolus philippinarumBathymodiolus platifrons

Crassostrea gigasPinctada fucata

0.05

Cephalopoda

Gastropoda

Bivalvia

c

100

100100

100

100100

100

100

Pinctada fucata

Crassostrea gigas

Modiolus philippinarum

Bathymodiolus platifrons

Bathymodiolus azoricus

Perna viridis

Limnoperna fortunei

Mytilus coruscus

Mytilus edulis

Mytilus galloprovincialis

Mytilidae

0.01

d

94

98

100100

100

100

100

Figure 1 | Shell morphology and phylogenetic positions of B. platifrons and M. philippinarum. a,b, Shell of the deep-sea mussel B. platifrons (a, 10.7 cm) and shallow-water mussel M. philippinarum (b, 6.5 cm). c, Phylogenetic tree of 11 lophotrochozoan species based on genome data; the value of bootstrap support is shown at each node. The scale bar indicates 0.05 expected substitutions per site. d, Phylogenetic tree of Mytilidae based on transcriptome data from representative genera/species. The scale bar indicates 0.01 changes per site.

nature eCOLOGY & eVOLutIOn 1, 0121 (2017) | DOI: 10.1038/s41559-017-0121 | www.nature.com/natecolevol 3

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

ArticlesNATure ecOlOgy & evOluTiON

expressed in the gill (Supplementary Note 18), consistent with the gill containing most of the MOB in the deep-sea mussels.

Endocytosis of exogenous bacteria is a critical step in establish-ing the host–symbiont relationship. Several observations showed molecular mechanisms of endocytosis of MOB by B. platifrons. (1) The Toll-like receptor 13 (TLR13) was found to be expanded in the genome of B. platifrons, and these receptors were highly expressed in the gill (Supplementary Fig. 20). TLR13 functions as an endosomal receptor in various groups of animals, which can sense microbes only after their internalization by phagocytosis22. (2) Two adhesion gene families (syndecan and protocadherin) were expanded in the genome of B. platifrons, and these families have been reported to mediate endocytosis in other species23,24 (Supplementary Figs 21 and 22). (3) Positive selection of six genes in the endocytosis pathway was shown by a branch-site evolution-ary analysis (Supplementary Fig. 14 and Supplementary Note 17). Among the positively selected genes, a Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) functions as a nucleation-promoting factor (NPF) to stimulate actin polymerization on endo-somal membranes and consequently regulate vesicular trafficking25.

Apoptosis plays multiple functions in host–symbiont interactions such as post-phagocytic winnowing in coral–dinoflagellate sym-biosis26 and symbiont recycling in insect–bacteria symbiosis27. In B. platifrons, several families of the classic apoptosis system includ-ing TNF, DEATH, CARD and caspases were remarkably expanded and were highly expressed in the gill (Table 1 and Supplementary Note 18). As the central controllers of programmed cell death, cas-pases initiate the transduction of apoptotic signals by activation of members of the TNF superfamily and their DEATH receptor28,

which can eventually result in the release of lysozymes to digest the symbionts29. A TUNEL assay showed the presence of numer-ous apoptotic cells in deep-sea mussels30 and in late-juvenile stages of vent tubeworms when they have acquired symbionts31, further substantiating the evolutionarily conserved role of apoptosis in establishing the host–symbiont relationship in various groups of invertebrates. In addition, as required to maintain homeostasis in symbiosis, B. platifrons has a complicated anti-apoptosis system with 130 inhibitors of apoptosis proteins (IAPs), which exceeds the number of IAPs in any other lophotrochozoan species available for comparison (Fig. 4). The high expression of many IAP genes is consistent with their role in maintaining the symbiont population in the gill of B. platifrons (Fig. 4b). Therefore, the expansion of the apoptosis- and anti-apoptosis-related gene families could contrib-ute to the regulation of the symbiont population in Bathymodiolus.

To better understand the relationship between the host and its symbionts, 220  MOB proteins were identified through metapro-teomics of the B. platifrons gill (Supplementary Note 20), leading to the discovery of three key metabolic pathways in the symbi-onts (Fig.  3). The methane-oxidation pathway, indicated by high expression of two signature enzymes of methanotrophy (that is, methane monooxygenase and methanol dehydrogenase)11, fuels the host–symbiont relationship (Supplementary Fig. 28). The assim-ilatory sulfate reduction pathway, identified by several adenylyl- sulfate kinases, produces sulfur-containing amino acids32. The ammonia-assimilation pathway, identified by glutamate-ammonia ligase and glutamate synthase, detoxifies ammonia and provides the symbionts and host with the much needed organic nitrogen in the deep sea11.

474445

273248

197

357480

624

2249294694 999527

6,883134

312158

2032 3

464

314

311

235

288

261

231

177

288

145

161

128

126 125 181

Bathymodiolus 1

2,033

Modiolus 12,049

Octopus 11,004

Crassostrea 11,070

Aplysia 10,325

Bp Mp Pf Cg Lg Bg Ac Ob La Ct Hr AnnotationImmunoglobulin domainAAA domainAnkyrin repeatC1q domainABC transporterRas familyProtein-tyrosine phosphataseFibronectin type III domainEGF-like domainTIR domainFibrogen CHsp70 proteinDual specificity phosphatase, catalytic domainDeath domainSyndecan domainMitochondria-eating proteinCaspase-recruitment domainInhibitor of apoptosis domainNTPaseGalactosyltransferaseInositol hexakisphosphateTyrosine phosphatase familyTHAP domainZinc knuckleProtocadherinNatural killer cell receptor 2B4Homeodomain-like domainMethyltransferase FkbM domainWinged helix-turn helixMacro domainCaspase domainTissue factorMyb/SANT-like DNA-binding domainTranscriptional coactivator p15 (PC4)Alcohol dehydrogenase transcription factor Myb/SANT-likeCENP-B N-terminal DNA-binding domainTNF (tumour necrosis factor) familyInterferon-inducible GTPase (IIGP)'Paired box' domainLecithin retinol acyltransferaseCell-cycle sustaining, positive selectionREJ domainPPR repeatPancreatic hormone peptideIntegrin-α cytoplasmic regionProtein QIL1VASP tetramerization domainDimerization domain of male-specific-lethal 1Iron-sulfur cluster-binding domain

–1 –2 0 1 2 3Row Z scoreZ

0100200300400500600

Capitella teletaHelobdella robustaLingula anatinaOctopus bimaculoidesLottia giganteaBiomphalaria glabrataAplysia californicaModiolus philippinarumBathymodiolus platifronsCrassostrea gigasPinctada fucata

Ma

629.79

496.64

611.69543.61

+55/–93

+321/–381

+73/–68

+41/–78

+37/–400

+69/–43

+67/-68

477.81

51517.69

225.71

381.32

110.43

282.56

++99/–69

+93/–63

+48/–39+65/–81

+61/–45

+127/–63

+111/–39

b

a c

+101/–115

+80/–95

Figure 2 | Gene family representation analysis and divergence time of 11 lophotrochozoan species. a, Venn diagram showing the number of shared and unique Pfam functional domains in five molluscan species. b, Divergence times of 11 lophotrochozoan species with error bars (purple lines) indicating the 95% confidence interval, and the event of gene family expansions (red) and contractions (blue) analysed by counting Pfam domains. c, Heat map of major annotated Pfam domains that are expanded in B. platifrons, with multiple domains in a given gene being counted as one. Ac, Aplysia californica; Bp, Bathymodiolus platifrons; Bg, Biomphalaria glabrata; Cg, Crassostrea gigas; Ct, Capitella teleta; Hr, Hellobdella robusta; La, Lingula anatina; Lg, Lottia gigantea; Mp, Modiolus philippinarum; Ob, Octopus bimaculoides; Pf, Pinctada fucata.

4 nature eCOLOGY & eVOLutIOn 1, 0121 (2017) | DOI: 10.1038/s41559-017-0121 | www.nature.com/natecolevol

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Articles NATure ecOlOgy & evOluTiON

Our study has provided genomic resources for understanding how the deep-sea mussel has adapted to extreme abiotic stresses and absence of photosynthesis-derived energy and nutrients in the chemosynthetic ecosystems. The general mechanisms of symbiosis revealed in our study are of relevance to other symbi-otic organisms such as deep-sea tubeworms and clams, as well as shallow-water corals.

MethodsMussel collection and DNA extraction. Individuals of B. platifrons were collected using the manned submersible Jiaolong from a methane seep (22° 06.921′  N, 119° 17.131′  E, 1,122 m deep; Supplementary Fig. 1) in the South China Sea in July 201313. The mussels were kept in an enclosed sample chamber placed in the sample basket of the submersible, and it took approximately three hours from sampling to sample preservation. Once the samples were brought to the upper deck of the mothership, the adductor muscle of an individual was dissected, cut into small pieces and immediately fixed in RNAlater. The sample was then transported to Hong Kong Baptist University (HKBU) on dry ice and stored

at − 80 °C until use. DNA was extracted using the CTAB method, and its quality and quantity was checked with standard agarose gel electrophoresis and a Qubit Fluorometer, respectively.

The shallow-water horse mussels M. philippinarum were collected from a sandy shore at Ting Kok, Hong Kong (22° 46.989′  N, 114° 21.389′  E; Supplementary Fig. 1) during low tide in April 2014. The mussels were transported to HKBU and maintained in a seawater aquarium until use. The species identity was confirmed based on a morphological description33. DNA from the adductor muscle was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany).

Genome sequencing and reads cleaning. For B. platifrons, nine libraries with insert sizes ranging from 180 bp to 16 kb were constructed using different preparation methods (see Supplementary Table 1a for details) and sequenced by Illumina Hiseq2000 and Hiseq2500 platforms. For M. philippinarum, six libraries with insert sizes ranging from 180 bp to 5 kb were constructed and sequenced by Illumina Hiseq2000 and Hiseq2500 platforms. Low-quality reads and sequencing-adaptor-contaminated reads were trimmed and error corrected. Reads of mate-pair libraries were de-duplicated and sorted.

Transcriptome sequencing. For B. platifrons, the mantle, foot and gill transcriptomes were generated from a single B. platifrons as previously reported13. The adductor muscle, visceral mass and ovary tissues from another B. platifrons collected from a hydrothermal vent at the Noho site in November 2015 were dissected on board and fixed in RNAlater, and their RNA was extracted using TRIzol and sent to BGI (Shenzhen) for sequencing using a eukaryotic mRNA enrichment and library preparation protocol. For M. philippinarum, RNA was extracted from the mantle, foot, gill, adductor muscle and visceral mass and sequenced using the same method as for B. platifrons. The RNA-sequencing (RNA-seq) data are summarized in Supplementary Table 5. Raw reads were trimmed and assembled using Trinity version 2.0.634.

Genome assembly. The genomes were assembled using Platanus version 1.2135. Our genome-assembly pipeline involved assembling of DNA sequences using different parameters, merging results from different assemblies, removing redundant scaffolds, further scaffolding using transcripts, filling the gaps and correcting the errors (Supplementary Fig. 4).

Identification of repeats and transposable elements. Repeats and transposable elements were screened using the RepeatModeler pipeline version 1.0.8. RMblast was used to search and construct a species-specific repeats library of the genome, and RepeatMasker36 was used to identify, classify and mask the repeats with the species-specific repeats library and repeats in the RepBase with the default settings.

Genome annotation. Trinity was used to generate another version of transcripts using the genome-based mode. The transcriptome data from the de novo and genome-assisted approaches were further assembled using PASA pipeline version 2.0.237 with BLAT as the aligner. The two mussel genomes were annotated using MAKER version 3.038. MAKER was run twice, in order to train the gene models and increase the gene-predication accuracy. The gene models were further filtered by running OrthoMCL39 with the protein models from six species of mollusks (Crassostrea gigas, Pinctada fucata, Lottia gigantea, Aplysia californica, Biomphalaria glabrata, and Octopus bimaculoides) and sequences that could be clustered with these molluscs were retained. Transcriptome reads were then mapped to the remaining ‘orphan’ genes, and only those supported by at least 10 parts per million (ppm) reads were retained. BLASTp was applied to search the predicted mussel protein models against the whole non-redundant database with an E value of 1 ×  10−5. The.xml format files were uploaded to BLAST2GO to obtain the GO and InterprotScan annotation. Pfam annotation was performed by searching the 16,295 Pfam entries (version 29.0) using a hidden Markov model (HMM)40. The clean RNA-seq reads were quantified using kallisto41. Gene-expression levels were quantified based on transcripts per million (TPM).

Phylogenomics. Protein sequences of the nine published or public lophotrochozoan genomes (Supplementary Table 3) were downloaded and analysed together with data from the two mussels used in the phylogenomic analysis. OrthoMCL39 was applied to determine and cluster gene families, and the BBH approach in BLASTp was used with a threshold value of 1 ×  10−5. This analysis resulted in 35,057 gene families among the 11 species. Only single-copy orthologues in each gene cluster were concatenated without partition information for the phylogenetic analysis using RaxML42 with the GTR +  Γ model and slow bootstrapping.

Divergent time among the mollusks was estimated using MCMCTree, which applied the Bayesian estimation method for species divergence43. The maximum-likelihood tree obtained using the 11 lophotrochozoan species was used to provide a reference tree topology, and the WAG +  Γ protein substitution model was employed on each site. The tree was calibrated with the following time frames to constrain the age of the nodes between the species: minimum =  305.5 Ma and soft maximum =  581 Ma, for C. teleta and H. robusta44; minimum =  168.6 Ma and soft

CH4

C

O

H H

NH4+

NH4+

L-Glutamate

Apoptosis

MOB Ig

Amt

Fibrinogen

Syndecan TLRProtcadherin

PGRP

Glyceraldehyde3-phosphate

Glycolysis

AP-1WASH

VPS

Endocytosis

DEATH

Caspase CARD

TNF

IAPLysozyme

Recognition

Gill epithelial cell

M

G

AMVM and O

F

Figure 3 | a model of symbiosis between B. platifrons and methane oxidizing bacteria. A model of symbiosis between B. platifrons and MOB. Top left, the diagram of the six tissues used for transcriptome sequencing, including the major MOB-containing gill. AM, adductor muscle; F, foot; G, gill; M, mantle; O, gonad; VM, visceral mass. Bottom, the host has mechanisms for MOB recognition, endocytosis and population control through apoptosis. The symbiont has various metabolic pathways (that is, methanotrophy and ammonia metabolism) that are beneficial to the host. Amt, ammonia transporter; AP-1, adaptor protein complex 1; CARD, caspase-recruitment-domain protein; IAP, inhibitor of apoptosis; Ig, immunoglobulin; LRP, low-density lipoprotein-receptor-related protein; PGRP, peptidoglycan recognition protein; TLR, Toll-like receptor; VPS, vacuolar protein-sorting protein; WASH, Wiskott–Aldrich syndrome protein and SCAR homologue.

nature eCOLOGY & eVOLutIOn 1, 0121 (2017) | DOI: 10.1038/s41559-017-0121 | www.nature.com/natecolevol 5

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

ArticlesNATure ecOlOgy & evOluTiON

maximum =  473.4 Ma, for A. californica and B. glabrata44; minimum =  470.2 Ma and soft maximum =  531.5 Ma, for A. californica (or B. glabrata) and L. gigantea44; minimum =  532 Ma and soft maximum =  549 Ma, for the first appearance of mollusks45; and minimum =  550.25 Ma and soft maximum =  636.1 Ma, for the first appearance of Lophotrochozoa45.

To understand the phylogenetic positions of these two mussels in the family Mytilidae better, transcriptome sequences from six additional mytilid species were downloaded from NCBI and re-assembled where appropriate, with details shown in the Supplementary Information. Protein sequences from each species were grouped into gene families using OrthoMCL39 as described above. Only the one-to-one orthologues were aligned, trimmed and concatenated. Maximum-likelihood trees were built using RaxML42 with the GTR +  Γ model and slow bootstrapping.

Gene-family evolution. We applied a sensitive HMM scanning method on known pfam functional protein domains to classify the gene families46. Annotation of Pfam domains were performed on all 11 lophotrochozoan species after excluding protein domains of various transposable elements. A domain with multiple copies in a protein was counted only once. Thereafter, in order to identify gene-family expansion/contraction events in B. platifrons, a two-tailed Fisher’s exact test was conducted. The Pfam domain counts in B. platifrons were compared against the background average domain counts of the 9 non-mussel mollusks (that is, species other than B. platifrons and M. philippinarum). In addition, OrthoMCL39 was applied to cluster gene families and CAFÉ version 3.1 was applied to determine the significance of gene family expansion/contraction47. For each gene family, CAFÉ generated a family-wide P value, with a significant P value indicating a possible gene-family expansion or contraction event. A branch-specific P value was also generated for each branch/node using the Viterbi method. A family-wide P value less than 0.01 and a branch/node Viterbi P value less than 0.001 was considered as a signature of gene family expansion/contraction for a specific gene family and specific species, respectively, as recommended for CAFÉ version 3.1 in ref. 47.

Proteomics characterization. Gill samples from three individuals of B. platifrons fixed separately in RNAlater were used in proteomic analysis48. In brief, the samples were homogenized in a lysis buffer containing 8 M urea and 40 mM HEPES (pH =  7.5), sonicated to break the cells up, centrifuged at 4 °C at 15,000g for 15 min. The supernatant that contained the proteins was collected, purified with a methanol/chloroform protein-precipitation method, and re-constituted in lysis buffer. Three samples were then pooled in equal amounts, reduced using dithiothreitol, alkylated using iodoacetamide, and digested using sequencing-grade Trypsin. The digested sample, containing approximately 1 mg peptides, was fractionated using a strong cation-exchange (SCX) column following the procedure described previously48.

Each SCX fraction was re-dissolved in 0.1% formic acid and analysed on a LTQ-Orbitrap Elite mass spectrometer coupled to an Easy-nLC (Thermo Fisher, Bremen, Germany) equipped with an Acclaim PepMap RSLC C18 column (Thermo Fisher Scientific, Sunnyvale, California, USA).

The MS/MS data were uploaded into Mascot version 2.3.2 (Matrix Sciences, London, UK) to search against the deduced B. platifrons protein sequences, with their reversed sequences used to calculate the FDR. The searching parameters were as follows: peptide charge: + 2, + 3 and + 4; fixed modification: carbamidomethyl (C); variable modification: oxidation (M) and deamidated (NQ); maximum missed

cleavage: 2; peptide mass tolerance: 4 ppm; and fragment mass tolerance: 0.05 Da. The FDR was dynamically controlled at 0.01.

Because the gills of B. platifrons contain MOB11,49 that belong to the family Methylococcaceae13, we also identified and quantified the bacterial proteins in the gill samples to gain insights into the symbiotic relationship between the bacteria and their host. Protein sequences from 24 published bacterial genomes in Methylococcaceae were downloaded and appended with the 1,402 bacterial that were identified in our previous meta-transcriptome analysis13. This created a database with 86,023 bacterial proteins. The database search criteria were identical to those described previously for the mussel proteins. The identified/matched protein sequences were further annotated by BLASTp against the KEGG database using an E value of 1 × 10−7.

Determination of positively selected genes. Single-copy orthologues were selected among B. platifrons, M. philippinarum, C. gigas and P. fucata after running the OrthoMCL pipeline39 as mentioned above. For each single-copy orthologue group, they were aligned, trimmed and concatenated. The phylogenetic tree was constructed using RaxML42 with the GTR +  Γ model and rapid bootstrapping.

Signatures of positively selected genes along a specific branch can be detected by branch-site models implemented in the codeml module of the phylogenetic analysis by maximum likelihood (PAML) package version 4.850. The branch-site model requires a lineage designated as ‘foreground’ phylogeny (that is, B. platifrons in the former phylogenetic tree) and the rest of the lineages as ‘background’ phylogeny (that is, M. philippinarum, C. gigas, and P. fucata in the former phylogenetic tree). Alternative model (model = 2, NSsite = 2, fix_omega = 0) and null hypothesis (model = 2, NSsite = 2, fix_omega = 1 and omega = 1) likelihood values were tested using χ2 analysis. Only genes that had Bayesian Empirical Bayes (BEB) sites  >   90 % and a corrected P value less than 0.1 were considered to have undergone positive selection. A KEGG-pathway enrichment was performed with a χ2 test (if any of the components are over 5) or the Fisher’s exact test (all other analyses).

Data availability. Raw reads, assembled genome sequences and annotation are accessible from NCBI under BioProject numbers PRJNA328542 and PRJNA328544, Sequence Read Archive accession numbers SRP078287 and SRP078294, and Whole Genome Shotgun project numbers MJUT00000000 and MJUU00000000. The genome assemblies and annotations are also available from the Dryad Digital Repository at http://dx.doi.org/10.5061/dryad.h9942.

received 3 August 2016; accepted 16 February 2017; published 3 April 2017

references1. Van Dover, C. L. The Ecology of Deep-Sea Hydrothermal Vents

(Princeton Univ. Press, 2000).2. Levin, L. A. Ecology of cold seep sediments: interactions of fauna with flow,

chemistry and microbes. Oceanogr. Mar. Biol. 43, 1–46 (2005).3. Sibuet, M. & Olu, K. Biogeography, biodiversity and fluid dependence

of deep-sea cold-seep communities at active and passive margins. Deep Sea Res. II 45, 517–567 (1998).

Table 1 | Distribution of protein Pfam domains associated with apoptosis among 11 lophotrochozoans.

Pfam Domain Bp Mp Pf Cg Lg Bg Ac Ob La Ct Hr

PF00656 Caspase 75* 42 45 38 18 17 21 38 71 42 15

PF00653 IAP repeat 130* 95 68 49 26 43 52 16 37 26 6

PF00452 Bcl-2 12 10 12 8 11 9 8 14 16 15 12

PF00020 TNFR_c6 37 40 27 28 31 5 18 12 49 28 12

PF00229 TNF 50* 33 25 29 11 4 9 8 24 22 5

PF05729 NACHT 245 204 352* 127 105 53 170 102 194 246 84

PF00931 NB-ARC 78 69 95 52 104* 27 55 47 88 77 37

PF00531 DEATH 155* 98 125 55 40 8 53 186* 126 37 23

PF01335 DED 26 18 63* 22 10 2 7 6 47 8 2

PF00619 CARD 133* 94 66 57 32 6 36 29 212* 197* 16

total 941 703 878 465 388 174 429 458 864 698 212Each number shows the number of proteins containing the specific domain. Protein family expansion is indicated by an asterisk, with a significant corrected P value (P <  0.05). In B. platifrons, five gene families are significantly expanded when compared to other species. In any of the other 10 species, two gene families are significantly expanded at the most. Ac, Aplysia californica; Bg, Biomphalaria glabrata; Bp, B. platifrons; Cg, Crassostrea gigas; Ct, Capitella teleta; Hr, Hellobdella robusta; La, Lingula anatine; Lg, Lottia gigantean; Mp, Modiolus philippinarum; Ob, Octopus bimaculoides; Pf, Pinctada fucata. Domain abbreviations: IAP, inhibitor of apoptosis; Bcl-2, apoptosis regulator proteins, Bcl-2 family; TNFR, tumour necrosis factor receptor; TNF, tumour necrosis factor; NACHT, a domain that can be found in NAIP, CIITA, HET-E and TEP1 proteins; NB-ARC, a nucleotide-binding adaptor shared by APAF-1, certain R gene products and CED-4; DED, death effector domain; and CARD, caspase recruitment domain.

6 nature eCOLOGY & eVOLutIOn 1, 0121 (2017) | DOI: 10.1038/s41559-017-0121 | www.nature.com/natecolevol

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Articles NATure ecOlOgy & evOluTiON

4. Little, C. T. S. & Vrijenhoek, R. C. Are hydrothermal vent animals living fossils? Trends Ecol. Evol. 18, 582–588 (2003).

5. Colaço, A. et al. LabHorta: a controlled aquarium system for monitoring physiological characteristics of the hydrothermal vent mussel Bathymodiolus azoricus. ICES J. Mar. Sci. 68, 349–356 (2011).

6. Dubilier, N., Bergin, C. & Lott, C. Symbiotic diversity in marine animals: the art of harnessing chemosynthesis. Nat. Rev. Microbiol. 6, 725–740 (2008).

7. Bettencourt, R. et al. High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus. BMC Genomics 11, 559 (2010).

8. Boutet, I., Jollivet, D., Shillito, B., Moraga, D. & Tanguy, A. Molecular identification of differentially regulated genes in the hydrothermal-vent species Bathymodiolus thermophilus and Paralvinella pandorae in response to temperature. BMC Genomics 10, 222 (2009).

9. Bougerol, M., Boutet, I., LeGuen, D., Jollivet, D. & Tanguy, A. Transcriptomic response of the hydrothermal mussel Bathymodiolus azoricus in experimental exposure to heavy metals is modulated by the Pgm genotype and symbiont content. Mar. Genomics 21, 63–73 (2015).

10. Johnson, S., Won, Y.-J., Harvey, J. & Vrijenhoek, R. A hybrid zone between Bathymodiolus mussel lineages from eastern Pacific hydrothermal vents. BMC Evol. Biol. 13, 21 (2013).

11. DeChaine, E. G. & Cavanaugh, C. M. in Molecular Basis of Symbiosis: Symbioses of Methanotrophs and Deep-Sea Mussels (Mytilidae: Bathymodiolinae) (ed. Overmann, J.) 227–249 (Springer Berlin Heidelberg, 2006).

12. Faure, B., Schaeffer, S. W. & Fisher, C. R. Species distribution and population connectivity of deep-sea mussels at hydrocarbon seeps in the Gulf of Mexico. PLoS ONE 10, e0118460 (2015).

13. Wong, Y. H. et al. High-throughput transcriptome sequencing of the cold seep mussel Bathymodiolus platifrons. Sci. Rep. 5, 16597 (2015).

14. Lorion, J. et al. Adaptive radiation of chemosymbiotic deep-sea mussels. Proc. Biol. Sci. 280, 20131243 (2013).

15. Olu-Le Roy, K., von Cosel, R., Hourdez, S., Carney, S. L. & Jollivet, D. Amphi-Atlantic cold-seep Bathymodiolus species complexes across the equatorial belt. Deep Sea Res. I 54, 1890–1911 (2007).

16. Pruski, A. M. & Dixon, D. R. Heat shock protein expression pattern (HSP70) in the hydrothermal vent mussel Bathymodiolus azoricus. Mar. Environ. Res. 64, 209–224 (2007).

17. Luckenbach, T. & Epel, D. ABCB- and ABCC-type transporters confer multixenobiotic resistance and form an environment-tissue barrier in bivalve gills. Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R1919–R1929 (2008).

18. Won, Y.-J. et al. Environmental acquisition of thiotrophic endosymbionts by deep-sea mussels of the genus Bathymodiolus. Appl. Environ. Microbiol. 69, 6785–6792 (2003).

19. Hoffmann, J. A., Kafatos, F. C., Janeway, C. A. & Ezekowitz, R. Phylogenetic perspectives in innate immunity. Science 284, 1313–1318 (1999).

20. Baumgarten, S. et al. The genome of Aiptasia, a sea anemone model for coral symbiosis. Proc. Natl Acad. Sci. USA 112, 11893–11898 (2015).

Bpl s

caf 1

061

2-0

.51

Bpl s

caf 6

3703

-4.15

Mph

sca

f 223

07-1

.7M

phsc

af 2

8741

-0.2

Mph

sca

f 287

41-0

.6M

ph s

caf 4

8689

-0.4

9998

6485

56

Bpl s

caf 5

7222

-0.10

Bpl s

caf 5

0748

-0.18

Bpl s

caf 2

8331

-0.6

Bpl s

caf 5

0748

-0.2

0

087

81

Bpl s

caf 9

962-

0.2

Bpl s

caf 1

7866

-1.8

Bpl s

caf 5

3657

-2.13

Bpl s

caf 1

7125

-0.2

Bpl s

caf 4

8590

-3.12

90

0

18

Bpl s

caf 2

1361

-1.3

1Bp

l sca

f 524

03-0

.4Bp

l sca

f 325

41-0

.0

70

Bpl s

caf 5

8950

-1.2

Bpl s

caf 1

373-

8.16

73

Bpl s

caf 3

6057

-0.0

Bpl s

caf 4

9105

-0.10

Bpl s

caf 3

1475

-0.17

89

96

78

99

Bpl s

caf 4

9378

-4.8

Bpl s

caf 1

6617

-0.7

Bpl s

caf 5

8975

-3.17

89

Bpl s

caf 9

450-

5.0

Bpl s

caf 5

2311-

0.1

Bpl s

caf 3

4540

-1.11

Bpl s

caf 9

507-

1.6

99

59

0

0

4796

Mph

scaf

489

28-0

.15

Bpl s

caf 4

3082

-1.16

Bpl s

caf 2

9314

-0.19

Mph

scaf

1669

2-0.19

0

Bpl s

caf 3

9147-

0.5

Mph

scaf

22932

-0.1

Mph

scaf

12896-0

.4

Mph s

caf 7

1468-0

.7

Mph sc

af 8362-0

.0

Mph sc

af 55521-0

.2

96

80

44

36

76

84

7395

Bpl sca

f 21361-1

.32

Bpl sca

f 63127-0

.0

69

Mph scaf 2

8535-0.10

Bpl scaf 5

5632-0.5

Bpl scaf 4

4217-3.12

9060

Mph scaf 2

5992-0.20

Mph scaf 2

859-0.6

Mp h scaf 4241 8

-0.20

Mph scaf 60939-0.23

Mph scaf 44777-0.2

Mph scaf 72144-0.2

82049

7999

92

Bpl scaf 62587-3.12

Mph scaf 29020-0.21

Mph scaf 2487-0.8

10095

Bpl scaf 20345-0.3

Bpl scaf 16935-0.14

Bpl scaf 16935-0.10

4794 Bpl scaf 60678-0.2

Bpl scaf 58975-3.16

Bpl scaf 3669-2.10

548

93Mph scaf 43719-0.10

Mph scaf 4112-0.5

Mph scaf 60911-0.4

Mph scaf 13346-0.34

Mph scaf 71142-0.27

72

10Mph scaf 41985-0.31

Mph scaf 5289-0.2

Mph scaf 23881-0.2

957379

7299

35

30

80

98

70

96

780

100

78

Cgi 405978024

Cgi 40597802590

Cgi 405978023

Cgi 405978020

100

93

Pfu 217.1 23677.t1

Bpl scaf 40569-10.6

Mph scaf 9227-0.0

Cgi 405978032

2471

100

Pfu 174.1 13739.t1

Pfu 174.1 13738.t199

Cgi 405954175

Cgi 405970397Cgi 405972132Cgi 405965687

8669

99

Cgi 405964327Cgi 405961627

94

Pfu 369.1 30682.t1Pfu 225.1 17144.t1Pfu 953.1 11324.t1Pfu 1360.1 01575.t1Pfu 12316.1 23022.t1

8086

58

98

Cgi 405965161Cgi 405970790Cgi 40595998280

96

84

24

97

96

68

94

Mph scaf 17036-0.0Cgi 405973931

84

Bpl scaf 13258-0.4Pfu 862.1 07964.t1Cgi 405971607

94

99

85

Pfu 656.1 21026.t1Pfu 656.1 21025.t1

Pfu 4402.1 09320.t1

90

Pfu 31 35.1 19035.t1Pfu 656.1 21023.t1

50

100

89

Pfu 1269.1 31575.t1

Mph scaf 71942-0.14

Bpl scaf 5295-2.5

90

Bpl scaf 62500-9.1 3

Bpl scaf 1831 1-0.11

Mph scaf 3607 4-0.8

Mph scaf 9742-0.6

83

77

98

97

86

81

36

77

88

Bpl scaf 54651-5.15

Mph scaf 26204-0.3

99

Cgi 405961841

Pfu 2126.1 21946.t1

Pfu 9131.1 06381.t1

Cgi 405971608

41

99

97

99

Pfu 819.1 31 187.t1

Pfu 3027.1 25614.t1

Pfu 1559.1 31760.t1

99

Pfu 1866.1 21829.t1

Bpl scaf 22825-0.10

Mph scaf 60456-0.2

99

Pfu 112.1 06954.t1

Pfu 3032.1 09046.t1

Pfu 1 12.1 06953.t1

Pfu 21322.1 09976.t1

12

24

30

86

98

90

Cgi 405950313

Cgi 405959887

100Pfu 2310.1 02085.t1

Pfu 12377.1 26430.t1

54Pfu 2299.1 32136.t1

Pfu 5650.1 02768.t1

10094

Pfu 143.1 10339.t1

Pfu 5801.1 06165.t1

92Pfu 819.1 31188.t1

Mph scaf 71627-0.17

Cgi 405957896

Cgi 405950727

89Cgi 405976607

Pfu 542.1 07658.t1

884

7790

46

87M

ph scaf 31601-0.5

Mph scaf 43894-0.1 7

Mph scaf 10419-0.3

10093

Bpl scaf 38273-0.6

Mph scaf 34625-0.2

Mph scaf 13673-0.0

99Bpl scaf 64881-2.27

Bpl scaf 16610-0.3

Bpl scaf 16610-1.1 8

85Bpl scaf 64881-1 .10

Bpl scaf 38141-1.10

96Bpl scaf 38141-2.35

Bpl scaf 38141-0.1 4

5562

95100

3072

99

Cgi 405969007

Cgi 40596976799

Cgi 405971683

Cgi 40596109699

Cgi 405953039

Cgi 405953040

10065

95

Cgi 405969765

Cgi 405969762

Bpl scaf 6172-0.376

94

Bpl scaf 64029-1.12

Bpl scaf 12477-0.199

Pfu 739.1 31113.t1Pfu 22681.1 06646.t1

99

Pfu 3553.1 12489.t1Pfu 2875.1 18953.t1C

gi 405959453

9682

71

32

69

Cgi 405969764

Bpl scaf 54651-4.5M

ph scaf 37795-0.3M

ph scaf 58020-0.5

9698

28

Cgi 405946675

Pfu 352.1 07397.t1M

ph scaf 72046-0.31Bpl scaf 14491-1.29Bpl scaf 59306-0.8

Bpl scaf 64881-1.21Bpl scaf 59306-0.14

Bpl scaf 20380-0.11Bpl scaf 64881-2.16

Bpl scaf 38141-0.15Bpl scaf 64881-2.17

Bpl scaf 20380-0.18

8667

64

100

9985

9998

7781

60

68

70

72

91

56

5

63

60

44

71

Mph

scaf

474

18-0

.6

Mph

scaf

669

47-0

.2

Cgi 4

0596

7461

0

Bpl s

caf 5

0304

-3.11

Bpl s

caf 6

4029

-1.2

0

98

Bpl s

caf 4

4653

-3.2

7

Mph

scaf

406

44-0

.7

Mph

scaf

416

21-0

.4

100 49

Mph

scaf

374

70-0

.3

Bpl s

caf 6

0224

-2.8

Mph

scaf

684

95-0

.10

Mph

scaf

465

1-0.5

100

Mph

scaf

465

1-2.7

Mph

scaf

684

95-0

.11

Bpl s

caf 6

4881-2

.10

29

80

52

66

36

87

94 95

Pfu 7

6.1 20

178.t

1

Mph

scaf

69270-0

.5

Bpl s

caf1

4002-1.8

Bpl sc

af 44217

-0.9

Mph sc

af 69270-1.

14

99

94 86

87

46

Bpl sca

f 13236-1.

16

Mph sc

af 312

14-1.

10

99

Mph sc

af 7887-1.

18

Bpl scaf 14

002-1.10

Mph scaf 11

822-0.40

Mph scaf 11

822-0.0

Mph scaf 3

5545-0.6

96 66 92

73

Bpl scaf 4

8726-1.21

Mph scaf 7

887-1.9

Bpl scaf 64635-0.3

Bpl scaf 53797-3.1

100 61 8493

Cgi 405952425

Cgi 405958687

Mph scaf 60977-0.4

Bpl scaf 16982-0.22

84 98

87

Cgi 405967807

Bpl scaf 48726-1.19

Bpl scaf 63743-1.17

Mph scaf 42407-2.1

Pfu 575.1 17589.t1

Pfu 1277.1 24833.t1

43 92 98 79 8880

89

4

25

20

92

Mph scaf 89-0.4

Pfu 2888.1 28975.t128

Pfu 4545.1 19322.t1

Bpl scaf 32704-0.12Bpl scaf 8738-0.11

97

84

Bpl scaf 63743-4.11Mph scaf 48369-1.6Bpl scaf 63743-3.7

Bpl scaf 63743-3.6

98 38 9818

77Mph scaf 43212-0.17

Bpl scaf 32300-1.11Bpl scaf 32300-1.15Mph scaf 51546-0.615 91

18Pfu 3964.1 15902.t1Pfu 1222.1 08254.t1 99Cgi 405976682Cgi 405961110Cgi 405976683Cgi 405965335

8653

10040

72

91

82

Pfu 3730.1 02480.t1Pfu 2691.1 05625.t1

99

Pfu 3337.1 25718.t1Pfu 6665.1 19533.t1

100

Pfu 34.1 13439.t1Bpl scaf 61711-1.12

Mph scaf 52145-0.7Mph scaf 56141-0.11 99

4553

28

82

9

Cgi 405974052

Cgi 405978031

91

Pfu 1562.1 08448.t1

Pfu 53.1 10176.t1

Pfu 53.1 10177.t1 10027

66

8

Mph scaf 61372-0.1

Mph scaf 4651-1.3

94

Pfu 2834.1 15640.t2

Bpl scaf 62500-6.18

41

Bpl scaf 52241-1.18

Bpl scaf 44653-3.2499

62

99

83

Pfu 1412.1 08368.t1

Bpl scaf 28262-0.4

Mph scaf 8340-0.496

100

Pfu 2234.1 15452.t1

Pfu 1338.1 28214.t1

Cgi 405951267

82

Mph scaf 1654-0.14

Bpl scaf 61248-0.0

97

100

80

74

Mph scaf 59187-1.0

Mph scaf 4651-0.7

Mph scaf 9266-0.1

80

Bpl scaf 9642-0.1

Bpl scaf 52241-1.16

29

95

Mph scaf 72752-0.2

Bpl scaf 34030-2.5

Bpl scaf 44519-2.42

Bpl scaf 64136-8.7

27

Bpl scaf 64136-8.11

Bpl scaf 15982-1.0

Bpl scaf 61110-0.0

Bpl scaf 64136-8.1

85

80

100

98

99

99

69

Bpl scaf 14490-4.2

Bpl scaf 13698-7.17

Mph scaf 41468-1.21

79

Mph scaf 5553-0.3

Bpl scaf 13698-7.15

Bpl scaf 290-0.8

89

95

92

Mph scaf 50990-0.8

Pfu 2896.1 22226.t1

Pfu 3135.1 19034.t1

959

Pfu 908.1 27897.t1

Mph scaf 57138-0.25

Bpl scaf 23799-1.5

9987

49

Bpl scaf 16610-2.14

Bpl scaf 57815-0.10

Bpl scaf 64881-2.15

7799

Bpl scaf 16610-2.15

Bpl scaf 14491-1.30

Bpl scaf 28185-0.8

6268

84

Bpl scaf 55011-0.4

Pfu 6508.1 29520.t180

Bpl scaf 16610-1.17

Bpl scaf 11809-0.1

Bpl scaf 38141-2.34

99

98

Mph scaf 74210-0.7

Mph scaf 53612-0.5

Mph scaf 74210-0.18

Mph scaf 6618-0.0

9182

91

Bpl scaf 38141-2.33Bpl scaf 16610-1.16

Bpl scaf 64881-0.19

92100

93

91

82

95

8

87

80

89

75

Bpl_scaf_64881

25 kb

Bpl_scaf_38141

Bpl_scaf_16610

AM F O G M VM

Row Z score–2 –1 0 1 2

a

c

b

Figure 4 | expansion of the IaP family in B. platifrons. a, Unrooted genealogy of IAP genes in Bathymodiolus platifrons (Bpl, green), Crassostrea gigas (Cgi, red), Modiolus philippinarum (Mph, yellow) and Pinctada fucata (Pfu, blue). b, Heat map of expression of IAP genes in six tissues. AM, adductor muscle; F, foot; G, gill; O, ovary; M, mantle; VM, visceral mass. c, Genomic arrangement of three IAP clusters. IPA genes are shown in red, whereas other genes are labelled in yellow. Scaffolds Bpl_scaf_16610 and Bpl_scaf_38141 each contain 6 IAP genes that are transcribed in the same direction, whereas scaffold Bpl_scaf_64881 contains 8 IAP genes that are transcribed in opposite directions.

nature eCOLOGY & eVOLutIOn 1, 0121 (2017) | DOI: 10.1038/s41559-017-0121 | www.nature.com/natecolevol 7

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

ArticlesNATure ecOlOgy & evOluTiON

21. Dziarski, R. & Gupta, D. The peptidoglycan recognition proteins (PGRPs). Genome Biol. 7, 1–13 (2006).

22. Biondo, C. et al. The role of endosomal Toll-like receptors in bacterial recognition. Eur. Rev. Med. Pharmacol. Sci. 16, 1506–1512 (2012).

23. Chen, K. & Williams, K. J. Molecular mediators for raft-dependent endocytosis of syndecan-1, a highly conserved, multifunctional receptor. J. Biol. Chem. 288, 13988–13999 (2013).

24. de Beco, S., Gueudry, C., Amblard, F. & Coscoy, S. Endocytosis is required for E-cadherin redistribution at mature adherens junctions. Proc. Natl Acad. Sci. USA 106, 7010–7015 (2009).

25. Derivery, E. et al. The Arp2/3 activator WASH controls the fission of endosomes through a large multiprotein complex. Dev. Cell 17, 712–723 (2009).

26. Dunn, S. R. & Weis, V. M. Apoptosis as a post-phagocytic winnowing mechanism in a coral–dinoflagellate mutualism. Environ. Microbiol. 11, 268–276 (2009).

27. Vigneron, A. et al. Insects recycle endosymbionts when the benefit is over. Curr. Biol. 24, 2267–2273 (2014).

28. Romero, A., Novoa, B. & Figueras, A. The complexity of apoptotic cell death in mollusks: an update. Fish Shellfish Immunol. 46, 79–87 (2015).

29. Detree, C. et al. Multiple I-type lysozymes in the hydrothermal vent mussel Bathymodiolus azoricus and their role in symbiotic plasticity. PLoS ONE 11, e0148988 (2016).

30. Guezi, H., et al. The potential implication of apoptosis in the control of chemosynthetic symbionts in Bathymodiolus thermophilus. Fish Shellfish Immunol. 34, 1709 (2013).

31. Nussbaumer, A. D., Fisher, C. R. & Bright, M. Horizontal endosymbiont transmission in hydrothermal vent tubeworms. Nature 441, 345–348 (2006).

32. Canfield, D. & Des Marais, D. Aerobic sulfate reduction in microbial mats. Science 251, 1471–1473 (1991).

33. Lee, S. & Morton, B. in The Malacofauna of Hong Kong and Southern China II: The Hong Kong Mytilidae (eds Morton, B. & Dudgeon, D.) 49–76 (Hong Kong Univ. Press, 1985).

34. Grabherr, M. G. et al. Full-length transcriptome assembly from RNA-seq data without a reference genome. Nat. Biotechnol. 29, 644–652 (2011).

35. Kajitani, R. et al. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Res. 24, 1384–1395 (2014).

36. Smit, A., Hubley, R. & Green, P. RepeatMasker Open-3.0. (1996–2010).37. Haas, B. J. et al. Improving the Arabidopsis genome annotation

using maximal transcript alignment assemblies. Nucleic Acids Res. 31, 5654–5666 (2003).

38. Cantarel, B. L. et al. MAKER: an easy-to-use annotation pipeline designed for emerging model organism genomes. Genome Res. 18, 188–196 (2008).

39. Li, L., Stoeckert, C. J. & Roos, D. S. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 13, 2178–2189 (2003).

40. Finn, R. D. et al. Pfam: the protein families database. Nucleic Acids Res. 42, D222–D230 (2014).

41. Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol. 34, 525–527 (2016).

42. Stamatakis, A., Ludwig, T. & Meier, H. RAxML-III: a fast program for maximum likelihood-based inference of large phylogenetic trees. Bioinformatics 21, 456–463 (2005).

43. dos Reis, M. & Yang, Z. Approximate likelihood calculation on a phylogeny for Bayesian estimation of divergence times. Mol. Biol. Evol. 28, 2161–2172 (2011).

44. Benton, M. J., Donoghue, P. C. J. & Asher, R. J. in The Timetree of Life: Calibrating and Constraining Molecular Clocks (eds Hedges, S. B. & Kumar, S.) 35–86 (Oxford Univ. Press, 2009).

45. Benton, M. J. et al. Constraints on the timescale of animal evolutionary history. Palaeontol. Electron. 18, 18.1.1FC (2015).

46. Albertin, C. B. et al. The octopus genome and the evolution of cephalopod neural and morphological novelties. Nature 524, 220–224 (2015).

47. Han, M. V., Thomas, G. W. C., Lugo-Martinez, J. & Hahn, M. W. Estimating gene gain and loss rates in the presence of error in genome assembly and annotation using CAFE 3. Molecul. Biol. Evol. 30, 1987–1997 (2013).

48. Sun J. et al. First proteome of the egg perivitelline fluid of a freshwater gastropod with aerial oviposition. J. Proteome Res. 11, 4240–4248 (2012).

49. Barry, J. P. et al. Methane-based symbiosis in a mussel, Bathymodiolus platifrons, from cold seeps in Sagami Bay, Japan. Invertebr. Biol. 121, 47–54 (2002).

50. Yang, Z. PAML 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24, 1586–1591 (2007).

acknowledgementsWe thank the crew of research vessel (R/V) Xiangyanghong 9 and operation team of the Jiaolong, and the crew of R/V Kairei, operation team of remotely operated vehicle (ROV) Kaiko Mk-IV, and on-board scientists during the cruise of KR15-17 for facilitating sampling of B. platifrons. We also thank M. Sun for her help with the figures and her continuous encouragement to the first author. This study was supported by the Strategic Priority Research Program of Chinese Academy of Sciences (grant XDB06010102 to P.-Y.Q.), Hong Kong Baptist University (grant FRG2/14-15/002 to J.-W.Q.), and Scientific and Technical Innovation Council of Shenzhen and Guangdong Natural Science Foundation (grant 827000012, JCYJ20150625102622556, 2014A030310230 to Yu Z.). Genome sequencing was conducted by BGI (Shenzhen), Macrogen (Seoul), and the Roy J. Carver Biotechnology Center of University of Illinois Urbana–Champaign (UIUC). The mass spectrometry analysis was performed at the Instrumental Analysis & Research Center, Sun Yat-Sen University.

author contributionsJ.S., J.-W.Q. and P.-Y.Q. conceived and led the study. J.W.Q. and J.S. collected the samples. T.X. extracted the DNA and RNA, constructed the phylogenetic tree, and analysed the apoptosis-related gene families. J.S. and C.J.F. assembled the genome. J.S., C.J.F. and R.L. annotated the genomes. J.S. and Yang Z. analysed the immune-related gene families. Yanj Z. analysed the HSP70 gene family. J.H.L.H., W.N., and F.K.M.C. analysed microRNA and hox genes. Y.L. analysed the positively selected genes. H.M., Yu Z., and W.Z. analysed the proteome. J.S. performed the other bioinformatic analysis. All of the authors read, wrote and approved the manuscript.

additional informationSupplementary information is available for this paper.

Reprints and permissions information is available at www.nature.com/reprints.

Correspondence and requests for materials should be addressed to J.-W.Q. and P.-Y.Q.

How to cite this article: Sun, J. et al. Adaptation to deep-sea chemosynthetic environments as revealed by mussel genomes. Nat. Ecol. Evol. 1, 0121 (2017).

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Competing interestsThe authors declare no competing financial interests.

This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as

you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/