bioRxiv preprint doi: https://doi.org/10.1101/2021.03.18.435778; this version posted March 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Comparative genomics reveals evolutionary drivers of sessile life and 2 left-right shell asymmetry in bivalves 3 4 Yang Zhang 1, 2 # , Fan Mao 1, 2 # , Shu Xiao 1, 2 # , Haiyan Yu 3 # , Zhiming Xiang 1, 2 # , Fei Xu 4, Jun 5 Li 1, 2, Lili Wang 3, Yuanyan Xiong 5, Mengqiu Chen 5, Yongbo Bao 6, Yuewen Deng 7, Quan Huo 8, 6 Lvping Zhang 1, 2, Wenguang Liu 1, 2, Xuming Li 3, Haitao Ma 1, 2, Yuehuan Zhang 1, 2, Xiyu Mu 3, 7 Min Liu 3, Hongkun Zheng 3 * , Nai-Kei Wong 1* , Ziniu Yu 1, 2 * 8 9 1 CAS Key Laboratory of Tropical Marine Bio-resources and Ecology and Guangdong Provincial 10 Key Laboratory of Applied Marine Biology, Innovation Academy of South China Sea Ecology and 11 Environmental Engineering, South China Sea Institute of Oceanology, Chinese Academy of 12 Sciences, Guangzhou 510301, China; 13 2 Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 14 511458, China; 15 3 Biomarker Technologies Corporation, Beijing 101301, China; 16 4 Key Laboratory of Experimental Marine Biology, Center for Mega-Science, Institute of 17 Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; 18 5 State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-sen University, 19 Guangzhou 510275, China; 20 6 Zhejiang Key Laboratory of Aquatic Germplasm Resources, College of Biological and 21 Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China; 22 7 College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China; 23 8 Hebei Key Laboratory of Applied Chemistry, College of Environmental and Chemical 24 Engineering, Yanshan University, Qinhuangdao 066044, China. 25 26 # These authors contributed equally to this work. 27 * Corresponding authors 28 E-mail: [email protected] (Yu Z), [email protected] (Wong N), 29 [email protected] (Zheng H) The updated email and affiliation of Nai-Kei Wong: [email protected], Department of Pharmacology, Shantou University Medical College, Shantou 515041, China bioRxiv preprint doi: https://doi.org/10.1101/2021.03.18.435778; this version posted March 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 30 31 32 Running title: Yang Z et al. / Genomic drivers of bivalve sessility and shell asymmetry. 33 34 Total word counts (from “Introduction” to “Conclusions” or “Materials and methods”): 5770 35 Total figures: 4 36 Total tables: 0 37 Total references: 120 38 References from 2014: 31 39 Total supplementary figures: 13 40 Total supplementary tables: 15 41 Total supplementary files: 2 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.18.435778; this version posted March 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 60 Abstract 61 Bivalves are species-rich mollusks with prominent protective roles in coastal ecosystems. 62 Across these ancient lineages, colony-founding larvae anchor themselves either by byssus 63 production or by cemented attachment. The latter mode of sessile life is strongly molded by 64 left-right shell asymmetry during larval development of Ostreoida oysters such as 65 Crassostrea hongkongensis. Here, we sequenced the genome of C. hongkongensis in high 66 resolution and compared it to reference bivalve genomes to unveil genomic determinants 67 driving cemented attachment and shell asymmetry. Importantly, loss of the homeobox gene 68 antennapedia (Antp) and broad expansion of lineage-specific extracellular gene families are 69 implicated in a shift from byssal to cemented attachment in bivalves. Evidence from 70 comparative transcriptomics shows that the left-right asymmetrical C. hongkongensis 71 plausibly diverged from the symmetrical Pinctada fucata in expression profiles marked by 72 elevated activities of orthologous transcription factors and lineage-specific shell-related gene 73 families including tyrosinases, which may cooperatively govern asymmetrical shell formation 74 in Ostreoida oysters. 75 76 77 KEYWORDS: Comparative genomic, Ostreoida oysters, attachment, shell asymmetry, 78 bivalves 79 80 81 82 83 84 85 86 87 88 89 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.18.435778; this version posted March 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 90 Introduction 91 Bivalves belong to the ancient lineages of Mollusca comprising nearly 9,600 species that 92 thrive in aquatic environments, with notable economic and ecological importance [1]. As 93 bilaterian organisms, they rely nutritionally on filtering phytoplankton, and primarily follow a 94 life cycle that transitions from free-swimming larvae to attached juveniles, culminating in 95 sessile life [2, 3]. Among filter-feeding bivalves, oysters of the superfamily Ostreoidea serve 96 as crucial guardians of marine ecosystems by forming oyster reefs that clean up water and 97 sustain biodiversity [4,5]. Due to climate change and coastal degradation, however, bivalves 98 face profound challenges from warming waters and ocean acidification, which destabilize 99 habitats, raise infection risks and dampen the bivalve capacity of acquiring carbonate for shell 100 formation [6-8]. 101 To cope with diverse ecosystems, a variety of sessile strategies has emerged in bivalves 102 during evolution, among which two modes of sessile life prevail. Characteristically, majority 103 of the bivalves, including Mytilidae (mussel), Pectinidae (scallop), and Pteriidae (pearl oyster) 104 secret adhesive byssal threads to stabilize themselves against marine turbulences [9-13]. In 105 contrast, Ostreoida oysters have evolved a highly sophisticated machinery of cemented 106 attachment through producing organic-inorganic hybrid adhesive substances in place of 107 byssus, which allows them to permanently fuse the left shell with rock surfaces or shells of 108 other individuals in intertidal zones [14]. Compared with byssus, cemented attachment 109 exhibits superiority in physical adhesion and mechanical tension, enabling oysters to 110 efficiently create and thrive in large reef communities [2]. Developmentally, as a salient 111 feature of their exoskeleton, shell formation processes in bivalves are strongly molded by 112 their preferences for sessile life [15]. Quite distinctively, byssally attached bivalve species 113 tend to possess a bilaterally symmetrical shell, whereas cement-attached oysters present a 114 high degree of phenotypic variability and morphological asymmetry characteristic of their 115 radically distinct left-right (L/R) shells [15]. Nevertheless, the molecular mechanisms driving 116 these extraordinary innovations in bivalve evolution remain enigmatic, particularly in 117 genomic contexts. 118 The Hong Kong oyster (Crassostrea hongkongensis, first described as Crassostrea rivularis 119 by Gould, 1861) is an economically valuable aquacultural species endemic to the South China bioRxiv preprint doi: https://doi.org/10.1101/2021.03.18.435778; this version posted March 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 120 coastline [16]. As an ideal model for studying shell asymmetry, C. hongkongensis larvae 121 follows a typical developmental cycle of cemented attachment and asymmetrical 122 differentiation of the L/R shells. In order to elucidate the genetic basis underpinning the 123 evolution of bivalve sessile life and asymmetry of shell formation, we sequenced and 124 analyzed the complete genome of C. hongkongensis and performed comparative genomic 125 analysis along with several other bivalve species, including two congeneric Ostreoida oysters, 126 Crassostrea gigas, and Crassostrea virginica [12,17-21]. In addition, we monitored 127 transcriptomic changes of C. hongkongensis embryos during the critical window of larval 128 attachment, and compared any asymmetry-related gene expression patterns in the L/R mantles 129 of adult C. hongkongensis and byssus-producing pearl oyster (Pinctada fucata). Our 130 comparative genomic data and associated functional assays reveal extensive molecular 131 adaptations across the oyster genome that support the evolutionary switch from byssal to 132 cemented attachment and divergence from symmetrical shell in Ostreoida oysters. 133 134 Results 135 Genome sequencing, annotation and Hi-C, phylogenomics and evolutionary rate 136 Efforts on genome sequencing and assembly are inherently challenging for many marine 137 invertebrates such as mollusks, annelids, and platyhelminths due to their remarkable genetic 138 heterozygosity (or polymorphisms) [17,18,21,22]. Based on k-mer analysis, the genome size 139 of a single wild-stock Hong Kong oyster (C. hongkongensis) individual was estimated to be 140 695 Mb with 1.2% of heterozygosity (Figure S1), which is broadly comparable to that of the 141 Pacific oyster (1.3%) [17]. To circumvent limitations of short-read next-generation 142 sequencing in assembling highly polymorphic genomes,