Tracing the evolution of tissue inhibitor of metalloproteinases in Metazoa with the Pteria penguin genome

Summary Tissue inhibitors of metalloproteinase (TIMPs) play a pivotal role in regulating extracellular matrix (ECM) dynamics and have been extensively studied in vertebrates. However, understanding their evolution across invertebrate phyla is limited. Utilizing the high-quality Pteria penguin genome, we conducted phylogenomic orthology analyses across metazoans, revealing the emergence and distribution of the TIMP gene family. Our findings show that TIMP repertoires originated during eumetazoan radiation, experiencing independent duplication events in different clades, resulting in varied family sizes. Particularly, Pteriomorphia bivalves within Mollusca exhibited the most significant expansion and displayed the most diverse TIMP repertoires among metazoans. These expansions were attributed to multiple gene duplication events, potentially driven by the demands for functional diversification related to multiple adaptive traits, contributing to the adaptation of Pteriomorphia bivalves as stationary filter feeders. In this context, Pteriomorphia bivalves offer a promising model for studying invertebrate TIMP evolution.


Highlights
The TIMP gene family originated in the common ancestor of eumetazoans TIMP gene family sizes vary dramatically across metazoan clades Pteriomorphia bivalves exhibit the largest and most diversified TIMP repertoires

INTRODUCTION
The evolutionary dynamics of key gene families have long been a topic of great interest in evolutionary biology. 1,2This includes studying the variations in gene family size and functional divergence during the evolutionary process.Analyzing the patterns of these events, while often difficult to achieve, can provide insights into the forces driving gene family evolution and their impact on the adaptation and evolutionary success of certain species. 3,4he tissue inhibitor of metalloproteinase (TIMP) family is an ancient gene family that is widely distributed in the Metazoa kingdom. 5,6TIMPs primarily function as endogenous inhibitors of metalloproteinases, particularly matrix metalloproteinases, which are enzymes responsible for extracellular matrix (ECM) degradation. 7This inhibitory function is mainly mediated by the netrin-like (NTR) domain, which is conserved and corresponds to the N-terminal domain in TIMPs.The NTR domain can independently interact with the active site of MMPs, forming a tight 1:1 complex and inhibiting MMP activity. 8,9By regulating MMP activity, TIMPs play a crucial role in maintaining the balance between ECM synthesis and degradation, ensuring proper tissue remodeling and repair processes. 10,11As a key gene family instrumental in ECM modification, investigating the evolutionary trajectory of TIMPs across metazoans can provide valuable insights into the mechanisms that govern ECM dynamics in organismal development and survival.
Promising research has been conducted on the evolution and functional diversification of the TIMP gene family within specific clades in vertebrates. 6,10A comprehensive study revealed four TIMP members, commonly found in jawed vertebrates, originated from a common ancestor through three successive duplications during early vertebrate radiation. 12Throughout their evolutionary divergence, different members of the vertebrate TIMP family have exhibited varying rates of evolutionary change and have undergone gain and loss of functions. 12For instance, some members have lost inhibitory activity toward specific metalloproteinases while developing new interactions with specific targets such as integrins and receptors, while retaining their roles in regulating ECM structure. 6,13These findings highlight the complex evolutionary history of the TIMP gene family.
The TIMPs in invertebrates share a similar domain architecture but exhibit substantial sequence divergence compared to vertebrate TIMPs, indicating distinct evolutionary paths. 6,10A notable example is the study conducted on the evolutionary trajectory of TIMPs in echinoderms, 14 which belong to the Deuterostomia clade along with vertebrates.Phylogenetic analysis has revealed that the TIMP gene family underwent diversification in the ancestral deuterostome.Unlike chordates, which retained only a single copy in the ancestral stage and subsequently diversified into four members in vertebrates, echinoderms retained numerous early-lineage TIMPs and continued to diversify after their divergence from chordates.Despite significant sequence differences compared to vertebrates, invertebrate TIMPs retain inhibitory activities against metalloproteinases. 10,12TIMPs from cnidarians exhibit a canonical TIMP fold structure, like mammalian TIMPs, with only minor structural changes, suggesting the evolutionary conservation of a common structure across metazoans. 15However, comprehensive studies focusing on the evolutionary events of invertebrate TIMPs, particularly in protostomes, are still limited despite the vast diversity within this group.A more comprehensive understanding of TIMP repertoires in invertebrates, which are currently underrepresented in evolutionary studies centered around the TIMP gene family, is essential for gaining insights into the evolution of the TIMP gene family across the entire animal kingdom and facilitating further functional studies within an appropriate evolutionary context.
Despite Mollusca being the second largest phylum of invertebrates, our understanding of the evolution of TIMP genes in this clade is still lacking.Present studies in molluscs have primarily centered around the bivalves from the Pteriomorphia clade, including Tegillarca granosa (Arcida), 16 Mytilus spp.(Mytilida), 17,18 Magallana spp.and Pinctada spp.9][30][31] These studies have revealed that TIMPs found in Pteriomorphia bivalves play important roles in the immune system 16,19 and formation of the bivalved shell and the hinge ligament, 22,23,25 as well as the development of the bivalve byssus. 18,25,26,31These findings suggest that the TIMP gene family is crucial for the survival and adaptation of Pteriomorphia bivalves and may have undergone expansion in family size during their evolution.Moreover, Pteriomorphia bivalves can serve as valuable model organisms for studying the evolutionary patterns and diversification of the TIMP gene family in invertebrates.In addition to the Pteriomorphia bivalves, Pteria penguin, commonly known as the penguin's wing oyster, belongs to the family Pteriidae, within the order Ostreida.Native to the western and central Indo-Pacific region, this species is widely cultivated for pearl production. 32While genomic resources and TIMP information for P. penguin are currently unavailable, it is reasonable to speculate that this species may possess a rich repertoire of TIMPs based on its phylogenetic relationship with other bivalves, as well as notable features such as a well-developed nacre layer and byssus in the adult stage.Conducting a comprehensive phylogenomic analysis with a specific focus on molluscs would significantly contribute to our understanding of the underappreciated TIMP resources in Pteriomorphia bivalves and provide insights into the broader distribution of TIMPs across metazoans.
In this study, we aim to address the gaps in our understanding of the evolution of the TIMP gene family in Mollusca and investigate the origin and diversification of canonical TIMPs across the phylogeny of Metazoa.While the size of the TIMP gene family in various molluscan clades remains inconclusive, previous studies have provided insights into the diverse functions of TIMPs in Pteriomorphia bivalves. 19,22,28ence, we hypothesize that the TIMP gene family undergoes multiple duplication events and exhibits an extensive repertoire in Pteriomorphia bivalves.To address this hypothesis, we initiated the study by generating a high-quality genome assembly for P. penguin and conducted comprehensive phylogenomic and orthology inference analyses incorporating other metazoan genomes.Our findings offer a comprehensive overview of the distribution patterns of the TIMP gene family across the animal kingdom, suggesting the TIMP repertoires in metazoans originated from ancestral genes that emerged during the early radiation of eumetazoans.The most significant revelation from our research is that Ostreida bivalves belonging to the Pteriomorphia infraclass possess the most extensive and diverse repertoire of TIMPs in the animal kingdom.We propose that the abundance of TIMP repertoires in these species results from multiple independent duplication events driven by the various functional demands of different ECM protein-related tissues.These insights of this study could pave the way for further research into the role of TIMPs in a wide range of biological processes across metazoans by providing a crucial evolutionary context.

RESULTS
A high-quality P. penguin genome Genomic DNA was extracted from a single individual of P. penguin (Figure 1A) and sequenced using Illumina short-read and PacBio long-read sequencing platforms (Table S1).The de novo assembly process, followed by heterozygosity removal and polishing, resulted in a final genome assembly of 838.71 Mb.The assembly had a contig N50 of 5.03 Mb and a scaffold N50 of 60.66 Mb, comparable to the estimated size by k-mer analysis (812.32 Mb) (Figure S1).Additionally, leveraging 46 Gb Hi-C sequencing data, approximately 99.83% of the obtained contigs were successfully anchored into 14 pseudo chromosome linkage groups (Table S2).To assess the completeness of the genome assembly, Benchmarking Universal Single-copy Ortholog (BUSCO) analysis was performed, revealing 95.8% metazoan BUSCOs present (Table S3).These results confirm the high quality and completeness of the assembled P. penguin genome.
For genome annotation, repeat elements constituted 43.89% (368.14Mb) of the assembled genome (Table S4).A total of 36,733 proteincoding genes were identified within the P. penguin genome using ab initio methods and transcripts assembly constructed from Illumina and PacBio sequencing data (Table S5).Among these genes, 99.85% (36,677) were anchored to the 14 pseudo chromosome linkage groups.BUSCO analysis further demonstrated the completeness of the protein-coding gene set in the P. penguin assembly, with a metazoan BUSCO score of 97.8%.Additionally, a total of 16,884 noncoding RNA genes were annotated across the P. penguin genome (Table S6).
To investigate the phylogenetic relationship of P. penguin within the molluscs, a set of 273 single-copy genes was identified among seventeen molluscan species and one brachiopod Lingula anatina (for detailed information, see Table S7).These genes were used to construct a phylogenomic tree, which placed P. penguin together with the Pacific oyster Magallana gigas in the Ostreida order (Figure 1B).Both species, along with the mussel Mytilus californianus and the scallop Mizuhopecten yessoensis, clustered within the Pteriomorphia infraclass.This infraclass represents the sister clade to Heteroconchia, which includes the freshwater mussel Dreissena polymorpha, the soft-shell clam Mya arenaria, the hard clam Mercenaria mercenaria, and the freshwater pearl mussel Margaritifera margaritifera.

Phylogenetic orthology analysis reveals the distribution of the TIMP gene family across Mollusca
Using Orthofinder software, a total of 38,849 orthogroups (OGs) were identified among eighteen species, representing 88.7% of all assigned genes.For each OG, a rooted gene tree was constructed by introducing the outgroup species L. anatina.Subsequently, 40,746 orthogroups at each hierarchical level (HOGs) were inferred from the OGs by analyzing the rooted gene trees.The gene copy numbers in each HOG were counted to estimate gene family expansion and contraction events across the molluscan phylogenomic tree.Among the 17,411 HOGs owned by the Ostreida clade (P.penguin or M. gigas), 82 gene families experienced significant expansion, while 13 gene families showed significant contraction compared to their parent clade (p value <0.05).Gene Ontology (GO) enrichment analysis of the aforementioned expanded HOGs in P. penguin genome revealed 276 highly significantly enriched GO categories (adjusted p value <0.01; listed in Table S8), of which fifteen were related to the TIMP gene family and TIMP activity (Figure 2A), indicating a massive expansion of the TIMP family in the species belong to the Ostreida order.
To quantify the copy numbers of TIMP genes in the eighteen species, their genomes were scanned, resulting in the identification of 144 putative TIMP sequences.These putative TIMPs had protein signature matches with ''Tissue inhibitor of metalloproteinase'' in the Pfam database, ''METALLOPROTEASE INHIBITOR'' in the PANTHER database, and ''NTR domain profile'' in the PROSITE database, all simultaneously.These 114 TIMP sequences were further classified into two groups based on whether they matched the conserved NTR domain in the SMART database.One group included 89 sequences, which were named ''canonical TIMPs'' because they had matches to SMART NTR domain.The other group consisted of 55 sequences, which were referred to as ''pseudo TIMPs'' since they did not have matches to the domain.Analysis of the canonical TIMP gene distribution revealed significant divergence in copy numbers among molluscan species.Cephalopods, gastropods, and some bivalves from the infraclass Heteroconchia showed either one copy or no canonical TIMP gene, except for M. margaritifera, which possessed three copies.In contrast, Pteriomorphia bivalves exhibited a dramatic expansion of canonical TIMP copies, ranging from eight in M. yessoensis to twenty-eight in M. gigas and P. penguin (Figure 2B); notably, species from the order Ostreida possessed the highest number of canonical TIMP genes in their genomes.The distribution pattern of pseudo TIMPs differed from canonical TIMPs and was mainly found in seven bivalves and one gastropod (Pomacea canaliculata).Notably, two Heteroconchia bivalves, D. polymorpha and M. mercenaria, possessed the highest number of pseudo TIMPs compared to other species.
The HOGs containing putative TIMP sequences were further analyzed.One HOG named HOG0001471, containing the majority of canonical TIMPs (53 out of 89 sequences) from ten out of eighteen surveyed species that encompassed canonical TIMP genes, was considered as the ''core Mollusca-Brachiopoda TIMP HOG'' that possessed ''core Mollusca-Brachiopoda TIMPs.''Additionally, eighteen HOGs containing other canonical TIMPs were considered ''branch TIMP HOGs'' with ''branch TIMPs'', while ten HOGs containing no canonical TIMPs but pseudo TIMPs were named ''pseudo TIMP HOGs'' (Table S9).Through inferring the gene duplication events in each HOG, the core Mollusca-Brachiopoda TIMP HOG was found to have experienced multiple duplication events across different species (Figure S2), representing the mainstream evolutionary trajectory of TIMPs from their potential common ancestor.Two major duplication events were identified in the evolution of core Mollusca-Brachiopoda TIMPs within the molluscan phylogeny (marked as ''Non-Terminal'' in Table S10).These events   were shared by more than one species and linked to the speciation events.The first event occurred during the separation of cephalopods from gastropods and bivalves.At the same time, the second event happened within the Pteriomorphia clade, specifically during the divergence of the Pectinida order from Mytilida and Ostreida.In contrast to the core Mollusca-Brachiopoda TIMP HOG, the branch TIMP HOG underwent species-specific or lineage-specific duplication events, giving rise to several TIMP subgroups specific to particular species.Similar to the previous two groups, the pseudo TIMP HOGs were derived from multiple duplications.Leveraging our chromosome-level P. penguin assembly as well as the publicly available M. gigas genome, the genomic location of putative TIMPs in these two species was retrieved, and the tandem duplications contributing to the TIMP expansion were detected and shown (Figure S3).Phylogenetic trees were constructed using the aforementioned 144 putative TIMPs and 53 core Mollusca-Brachiopoda TIMPs, with two L. anatina sequences chosen as outgroups (Figures S4 and S5).Both trees divided molluscan sequences into two groups: a minor group consisting of single-copy canonical TIMPs from the four Heteroconchia bivalves and a major group comprising other sequences, primarily from Pteriomorphia bivalves.In the tree constructed from putative TIMPs, the pseudo or branch canonical TIMPs of D. polymorpha (eighteen pseudo TIMPs), M. mercenaria (eleven pseudo TIMPs), and M. margaritifera (three pseudo TIMPs and two branch TIMPs) were not clustered with the canonical TIMPs of these species but instead grouped within the major group.In the tree constructed from core Mollusca-Brachiopoda TIMPs, the TIMP sequence from the octopus Octopus sinensis clustered with the Pteriomorphia TIMPs rather than serving as an outgroup for both Heteroconchia and Pteriomorphia.Ten sequence motifs were discovered in the 144 putative TIMPs and used to compare the sequence composition of canonical and pseudo TIMPs from different phylogeny clades (Figure S4).Five motifs (motif1 to motif5) were present across the majority of putative TIMPs, although absent in several sequences.Compared to other molluscan putative TIMPs, motif7 was found only in some pseudo TIMPs of D. polymorpha, and motif8 was barely present in canonical TIMPs from the two Ostreida bivalves, P. penguin and M. gigas, as well as their most closely related species, M. californianus.The motifs motif9 and motif10 were completely absent in the four sequences from P. canaliculata, and motif10 was also absent in all sequences from Heteroconchia.Additionally, the arrangement order of motifs in core Mollusca-Brachiopoda TIMPs showed no reshuffling of order, but the position of motif10 in molluscan TIMPs differed from that in L. anatina (Figure S5).The distribution and position of signal peptides and NTR domain in core Mollusca-Brachiopoda TIMPs were also demonstrated (Figure S5).Finally, the exon numbers in each putative TIMP gene were examined and found to range from four to six exons, with 132 out of 142 molluscan sequences encompassing five exons, similar to the exon number found in vertebrate TIMP genes. 12This suggests that five exons are common in molluscs.

Phylogenetic orthology analysis reveals the distribution of the TIMP gene family across Metazoa
The Orthofinder software was employed to identify OGs among forty-one metazoans (Table S11).A total of 59,702 OGs were detected, encompassing 93.3% of all assigned genes.For each OG, a rooted gene tree was constructed using the sponge Amphimedon queenslandica as the outgroup.Subsequently, a total of 61,700 phylogenetic HOGs were inferred from the OGs by analyzing the rooted gene trees.negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of endopeptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity negative regulation of peptidase activity  (B) The number of putative TIMP copies in each species and node labels were marked on the node of the phylogeny tree.With deeper color denoting the higher values, the BUSCO numbers indicating the completeness of protein sets are marked in green boxes, the number of sequences having matches in different databases as well as the finally identified canonical TIMPs are marked in red boxes, TIMP numbers in core Mollusca-Brachiopoda TIMP HOG (HOG0001471) are marked in blue boxes, and the numbers of pseudo TIMPs are marked in purple boxes.The cladogram tree is inherited from the results of this research.

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Through the examination of a total of forty-one metazoan genomes, we identified 165 putative TIMPs, including 125 canonical TIMPs and 40 pseudo TIMPs (Table S12).The distribution of canonical TIMPs in the surveyed species was consistent with previous findings, showing the widespread presence of orthologs across Metazoa. 10 However, the copy numbers varied among different metazoan clades (Figure 3).In the  The distribution of TIMP copy numbers in each species and node labels were marked on the node of the phylogeny tree.][35][36][37][38][39][40] poriferan (Amphimedon queenslandica), TIMP gene was absent, while in the clade containing placozoans (Trichoplax adhaerens and T. sp.) and cnidarians, the copy numbers ranged from one to multiple copies (Cnidaria, ranging from two to five).In Ambulacraria within Deuterostomia, Echinodermata species exhibited multiple TIMPs in their genomes, ranging from seven (Anneissia japonica, Asterias rubens) to eleven (Lytechinus variegatus).In Chordata of Deuterostomia clade, basal species such as cephalochordates (Branchiostoma belcheri) and tunicates (Styela clava, Ciona intestinalis) possessed one canonical TIMP, while the vertebrates included in this study, along with the jawless fish Petromyzon marinus, were found to have four canonical TIMPs.In Ecdysozoa of Protostome, only one or no copy of canonical TIMPs was detected.In Spiralia of Protostome, canonical TIMP copy numbers varied significantly.For instance, in Platyzoa, Macrostomum lignano had two copies, while Rotaria socialis had none.Among Trochozoa, which includes Annelida, Brachiopoda, and Mollusca, the TIMP copy numbers varied not only between sister clades but also within a single clade.Annelida exhibited a range from no copies in the leech Helobdella robusta to up to fourteen copies in the spindle worm Owenia fusiformis.And, as previously mentioned, the range of TIMP repertoire sizes in molluscs varied from zero copy to a remarkable twenty-eight copies, which were found in P. penguin, making it the largest repertoire of TIMP copies among all animals employed in this study.The distribution of pseudo TIMPs was also investigated and found to be present in species from Cnidaria (Rhopilema esculentum), Echinodermata (Holothuria leucospilota, L. variegatus), Chordate (B.belcheri), Annelida (O.fusiformis), and Mollusca (D. polymorpha, P. canaliculata, and P. penguin).Notably, the bivalves D. polymorpha had the highest number of pseudo TIMPs compared to other species.Among the HOGs derived from the forty-one surveyed metazoans, the ''core Eumetazoa TIMP HOG'', named HOG0001662, comprised the majority of canonical TIMPs (114 out of 125 sequences) from twenty-nine out of forty-one surveyed animals that encompassed canonical TIMP genes and all belong to Eumetazoa clade, the sister group of the Porifera (sponges).The number of core Eumetazoa TIMPs from each species followed the pattern observed in canonical TIMPs, with P. penguin possessing the highest number.Additionally, eight branch and pseudo TIMP HOGs were identified.Multiple gene duplication events were detected within each HOG containing putative TIMPs (Figure S6).Two major duplication events were identified in the evolution of core Eumetazoa TIMPs within the metazoan phylogeny; one occurred during the divergence of bilaterians from cnidarians and placozoans, while the other took place during the separation of protostomes from deuterostomes (Table S13).
Phylogenetic trees were constructed using the aforementioned 165 putative TIMPs and 114 core Eumetazoa TIMPs, with the two sequences of Placozoa species chosen as the outgroups (Figures S7 and S8).Among the ten sequence motifs discovered in the 165 putative TIMPs, motif1 and motif3 were present in the majority of putative TIMPs, with only a few sequences lacking them (Figure S7).The motif2 and motif4 were commonly found in canonical TIMPs, but one or both were absent in the pseudo TIMPs.Consistent with the previous findings, motif10 was exclusively observed in some of the pseudo TIMPs from D. polymorpha.Four motifs (motif1 to motif4) and seven motifs (motif1 to motif5, motif7, motif8) were concurrently present in certain canonical TIMPs from eleven phyla representing the four major metazoan clades.These clades included Placozoa and Cnidaria (which were sister groups), Echinodermata, Chordata (both belonging to Deuterostomia), Arthropoda, Onychophora, Priapulida (belonging to Ecdysozoa of Protostomia), Annelida, Brachiopoda, Mollusca, and Platyhelminthes (belonging to Spiralia of Protostomia).No reshuffling of motif order was observed in the 114 core Eumetazoa TIMPs (Figure S8).The distribution and position of signal peptides and NTR domain in core Eumetazoa TIMPs were also demonstrated (Figure S8).Lastly, the exon number in each gene of the canonical and pseudo TIMPs was calculated.Out of the 165 sequences, 127 encompassed four to six exons, with 60 sequences having five exons.In Echinodermata, 25 out of 41 sequences had four exons, while in Placozoa and Cnidaria 10 out of 13 sequences encompassed two exons, indicating the common exon numbers in these two clades.

DISCUSSION
The primary objective of this study is to investigate the evolutionary dynamics and distribution of the TIMP gene family within the animal kingdom, with a particular focus on molluscs.To achieve this, we performed comprehensive analyses of the TIMP gene family across the metazoan phylogeny.By assembling a high-quality genome assembly of P. penguin and comparing it with other animals, we identified TIMP orthologs and examined their distribution in various animal clades.While the canonical TIMP gene is absent in the genome of the poriferan A. queenslandica, its orthologs are widely distributed among the eumetazoans, spanning from simple placozoans to bilaterians with complex tissue structures.It is worth mentioning that the phylogeny orthology analyses reveal a shared TIMP orthology group among the analyzed animals with canonical TIMPs, indicating a common ancestral origin in the evolution of the TIMP gene family.These findings also suggest the TIMP gene family likely emerged in the common ancestral lineage of eumetazoans after diverging from poriferans but before the split between the Placozoa + Cnidaria clade and Bilateria.Within the TIMP repertoires of various animal clades, a remarkable expansion was observed in the bivalves of the infraclass Pteriomorphia.From the common ancestor of eumetazoans to Pteriomorphia bivalves, four major duplication events of TIMP family during speciation events have occurred, along with multiple lineage-or species-specific duplication events after speciation events, potentially driven by adaptive demands.Accompanying the TIMP duplication events are the secondary loss events of the TIMP members in some lineage or species along the metazoan phylogeny.The duplication and loss events shape the distribution pattern of TIMPs in extant animals (Figure 4).
Our study provides comprehensive genomic analyses of TIMP gene copy number distribution across the Metazoa kingdom.Regarding the protostomes, ecdysozoans either completely lose TIMPs or retain a single-copy TIMP within their genomes.In contrast, the Spiralia displays two distinct expansions.One occurs in Annelida, where we identified fourteen canonical TIMPs in O. fusiformis from the Polychaeta class.Conversely, no TIMP gene was found in H. robusta from the Clitellata class.The variation in the size of the TIMP gene family within Annelida necessitates further elucidation through future studies incorporating additional genomic resources.
Another noteworthy expansion was observed in the Mollusca phylum, which exhibited considerable variation in the size of the TIMP gene family across its different clades.Some cephalopods have one TIMP gene, while gastropods have lost the canonical TIMP family during evolution.This observation highlights the need for further studies to investigate the evolutionary mechanisms behind the loss of TIMP members in gastropods.Compared with other molluscs, the Pteriomorphia bivalves exhibit a general expansion of the TIMP gene family.Within this clade, the copy number of the TIMP family has a linear relationship along the evolution phylogeny and reaches its peak in the Ostreidae bivalves with the most recent origins. 41The Ostreidae bivalves employed in this study, P. penguin and M. gigas, possess the highest number of canonical TIMP genes within their genomes, surpassing all other animals included in our research.The distribution of TIMP genes in the genomes of P. penguin and M. gigas provides compelling evidence supporting frequent gene tandem duplication as the primary mechanism underlying the notable expansion of the TIMP gene family.This mechanism is also implicated in the expansion of other gene families to unparalleled sizes in bivalves. 42,43Shifting the focus to the non-protostomes, our findings generally align with previous reports on TIMP copy numbers in cnidarians, 15 echinoderms, 14 and vertebrates; 12 with a few exceptions.In the jawless fish Petromyzon marinus, we observed four TIMP genes, which deviates from the previously reported number of three. 12The four TIMPs in jawed vertebrates most likely originated from three successive duplications before the split of Chondrichthyes (cartilaginous fishes) and Euteleostomes (ray-fins and lobe-fins).In contrast, similar duplications are considered to occur only once or twice in Agnatha (jawless fishes).These suggest an additional Agnatha-specific duplication for the TIMP gene after their separation from the vertebrate stem.Another exception pertains to echinoderms, where the size of the TIMP repertoire identified in our study is relatively smaller than that previously reported. 14This discrepancy may arise from alternative splicing events, wherein a single TIMP gene can generate different transcript isoforms. 44,45Previous transcriptome-based methods may have consequently overestimated the distribution of TIMPs in echinoderms to some extent.In brief, our phylogenomic analysis offers a comprehensive investigation of the size variations in the TIMP gene family across the Metazoa kingdom.It highlights an unparalleled expansion of the TIMP gene family in the Pteriomorphia bivalves, emphasizing a unique evolutionary process.
High diversity is also a prominent feature of the TIMP repertoires of Pteriomorphia bivalves.In contrast to the echinoderms and O. fusiformis, which also exhibit an expansion of the TIMP gene family in the genome, all their TIMP orthologs are grouped within the Eumetazoa orthology group shared by other species containing TIMP genes, namely the core Eumetazoa HOG identified in our results.This indicates the limited diversity of the TIMP family in echinoderms and O. fusiformis, suggesting a relatively recent origin from a single expansion event. 42The canonical TIMPs from P. penguin display a high diversity and are clustered into five different orthology groups, including the core Eumetazoa orthology group and the other four restricted to P. penguin.Indeed, 25% of the canonical TIMPs (seven sequences) from P. penguin were not included in the core Eumetazoa orthology group but dispersed in other branch orthology groups, suggesting the evolution novelty of these sequences. 42In our molluscan orthology analysis, the diversity of the TIMP gene family in Pteriomorphia bivalves is particularly pronounced.Along with the core Mollusca-Brachiopoda orthology group, the canonical TIMPs of Pteriomorphia bivalves disperse into twelve different orthology groups.Seven of these groups are shared among different species, while the remaining five orthologs are specific to a single species.This suggests that the large-scale TIMP expansion in Pteriomorphia bivalve results from multiple, independent duplication events occurring at distinct stages along the evolutionary phylogeny.The initial expansion likely occurred in the latest common ancestor of Pteriomorphia bivalves, contributing to their larger repertoire compared with their sister clade, the Heteroconchia.Subsequent independent expansions occurred during the radiation of Pteriomorphia bivalves, with some leading to recent species-specific evolutionary innovations, resulting in observed phylogenetic TIMP orthology branches exclusive to Pteriomorphia bivalves.The phylogeny tree represents relationships among major metazoan clades employed in this study.Branch lengths are not indicative for evolutionary distances.Solid branches signify clades retaining TIMP gene copies, while dashed branches denote complete TIMP gene loss within a clade.Red branches highlight three remarkable TIMP expansions across metazoans: in echinoderms, the Polychaeta annelid O. fusiformis, and Pteriomorphia bivalves.Circles on tree nodes indicate four major duplication events of TIMP family along metazoan phylogeny, including the divergence of bilaterians from cnidarians and placozoans, protostomes from deuterostomes, bivalves and gastropods from cephalopods, and the separation of Mytilida and Ostreida order from Pectinida order.Plus signs (+) along the branches indicate lineage-or species-specific duplication events of TIMP genes within these clades, while minus signs (À) indicate complete TIMP gene loss in at least one species.
The remarkable expansion and diversity of the TIMP family in Pteriomorphia bivalves can be attributed to their adaptation to habitats.This adaptation-driven expansion of the TIMP family has been indicated in echinoderms, as these animals have high demands for tissue remodeling as adaptive mechanisms. 14For Pteriomorphia bivalves, these marine bivalves primarily inhabit intertidal zones or shallow waters as stationary filter feeders, which expose them to constant threats, including aquatic pathogens and pervasive predators, as well as the fluctuating physicochemical conditions of the surroundings. 46Faced with these multiple stresses, Pteriomorphia bivalves require robust adaptive mechanisms, which can be facilitated by the TIMP family and may further drive the subsequent duplication and neofunctionalization events of this family in Pteriomorphia bivalves.Firstly, these bivalves have developed an effective innate immunity system involving multiple adaptive evolutionary processes. 47A crucial aspect of this adaptation is the expansion of specific immune-related gene families, such as the Toll-like receptor (TLR) and inhibitors of apoptosis (IAPs) families. 42,43These genetic changes are closely associated with the immune needs of bivalves in response to the stress from their living environments.In Pteriomorphia bivalves, certain TIMP members specifically expressed in hemocytes have been confirmed to play a pivotal role in immune mechanisms, including wound healing and the defense against microbial pathogens. 16,19,21The demand for immune processes via TIMP members may drive the adaptive duplication of a subset of TIMP repertoires related to the immune system.Secondly, the survival of Pteriomorphia bivalves relies on their bivalved shells, mainly serving as a defensive barrier against external predators.In the case of true oysters like those in the Ostreidae family, the shell firmly cements these oysters to substrates.The components within the shell of Pteriomorphia bivalves contain various matrix proteins contributing to shell formation and growth. 48,49Among these shell matrix proteins of Pteriomorphia bivalves, the TIMP family has consistently been identified as one component, further substantiating its role in facilitating shell formation and growth.Some TIMP members in the Iwagaki oyster M. nippona are specifically expressed in the mantle, 27 the tissue responsible for shell development.Moreover, studies conducted through RNA interference experiments in the pearl oyster Pinctada fucata have provided evidence of TIMP proteins participating in the formation of shell and ligament structure. 23,25Additionally, some TIMP proteins or TIMP-related domains have been identified as integral components of the shell structure, 17,22,24,27,49 possibly serving to protect the shell protein components from degradation by external proteinases.These studies indicate the involvement of the TIMP family in shell development can be a key driver for its general expansion and diversity in Pteriomorphia bivalves.This expansion aligns with the observed duplication events in other genes encoding shell matrix proteins, regarded as fundamental mechanisms in the evolution of these families. 27,50Thirdly, byssus, an adhesion structure made of protein filaments and secreted by the foot, serves as an adaptive trait in Pteriomorphia bivalves.Byssus is crucial for the larvae's settlement and metamorphosis.In adults, many Pteriomorphia bivalves, such as mussels in the Mytiloidea family and pearl oysters in the Pteriidae family, retain byssus for attachment to hard substrates. 51Specific TIMP members in Pteriomorphia bivalves not only play roles in byssus fabrication, as indicted by multiple studies based on foot transcriptomes, 18,28,29,31 but also have been shown to be integrated into the protein components of byssus. 18,26,28Some of these structural TIMPs even have evolved beyond their inhibitory function, transforming into cross-linkers that may facilitate the self-assembly of byssus. 30,31These byssus-related TIMPs undergo duplication and subsequent neofunctionalization events as adaptive mechanisms in Pteriomorphia bivalves to meet their dependence on byssus.Lastly, some other factors contributing to the expansion of the TIMP family in Pteriomorphia bivalves may be attributed to neutral or non-adaptive processes, such as neutral gene duplication events, 52,53 and contribute to the initial duplication event in the ancestor of Pteriomorphia bivalves.Nevertheless, the adaptive force is still recognized as the main driving force behind the multiple duplication events of this family, as supported by the close relationship between the functional diversity exhibited by multiple TIMP members and the adaptive traits in Pteriomorphia bivalves.In summary, the expansion of the TIMP family can be driven by the need for multiple adaptive traits in Pteriomorphia bivalves and plays a crucial role in the survival and adaptation of these bivalves in living environments.Further investigation is needed to explore more precise reasons behind the expansion of this family.
5][56][57] This process contributes to a better understanding of the lineage-specific expansion of the TIMP family in Pteriomorphia bivalves.Beyond Pteriomorphia bivalves, this study also investigated the distribution of TIMP families in other animals such as stationary filter-feeding animals in aquatic environments, but none of them exhibited the remarkable TIMP expansion seen in Pteriomorphia bivalves.Among these animals, Heteroconchia bivalves, which are the sister group to Pteriomorphia bivalves and share similar adaptive traits, 58 including effective innate immunity, bivalved shells, and adhesive byssus, have much smaller TIMP repertoires in comparison to Pteriomorphia bivalves.Considering the more than 500 million years of divergence between these two groups, despite the deep conservation in fundamental biological processes, specific divergences remain in the genetic mechanisms underlying some similar traits.In the case of gene families regulating innate immunity in bivalves, although some families have ancestrally conserved duplications across bivalve lineages, the expansion of specific families is identified as lineage or species specific. 46And regarding the shell and byssus of bivalves, despite the conservation in the morphology, the molecular components of these structures have diverged across bivalve lineages, indicating independent evolution in multiple gene families related to shell formation and byssus fabrication. 51,59,60Therefore, despite their limited TIMP family, it is speculated that Heteroconchia bivalves may have evolved specific alternative genes to achieve functions related to adaptive traits similar to those carried out by Pteriomorphia TIMPs.For other stationary filter-feeding animals beyond bivalves, the reasons for not experiencing TIMP expansion may follow a similar pattern.One example comes from the barnacles, which are sessile arthropods and have undergone convergent evolution with bivalves.A comparative genomics analysis in barnacles and Pteriomorphia bivalves revealed genome-wide substantial convergent molecular evolution between these two groups.However, there are still multiple divergences in the genetic mechanisms related to convergent adaptive traits, including shell formation and substrate settlement. 57The results of this study indicate that barnacles have much less TIMP members than Pteriomorphia bivalves.These suggest that, in the convergent evolution of barnacles and Pteriomorphia bivalves, the TIMP families in these two groups have divergent evolutionary processes.In brief, diverse functions of large TIMP families contribute to some adaptive traits in Pteriomorphia bivalves, while other animals may achieve similar functions through distinct genetic mechanisms.This divergence is hypothesized to be caused by the initial duplication of the TIMP family in Pteriomorphia bivalves, specific to the latest common ancestor of this bivalve clade.The resulting several TIMP gene copies in the ancestral Pteriomorphia bivalves may have undergone neofunctionalization 61,62 and further contribute to the multiple adaptive duplication events of TIMP families in their descendants.More studies are required to compare further the evolution dynamics of the TIMP family in Pteriomorphia bivalves and other animals.
Moreover, our findings reveal the widespread presence of pseudo TIMPs in metazoans, which lack conserved NTR domains compared to canonical TIMPs.These pseudo TIMPs exhibit the most extensive repertoire and diversity in bivalves, reaching unprecedented sizes in two Heteroconchia bivalves (18 copies in D. polymorpha, 11 copies in M. mercenaria).Among the pseudo TIMPs in molluscs, the majority of pseudo TIMPs in Pteriomorphia bivalves were found grouped within the same orthology groups as canonical TIMPs, suggesting a coeval origin with the multiple independent duplications that contribute to the extensive TIMP repertoire in these bivalves, as discussed earlier.In these cases, the pseudo TIMPs may have emerged through gene duplication events of TIMP, resulting in excess copies, which subsequently underwent N-terminal structural changes to balance the overall functionality of TIMPs within the organism.Conversely, most pseudo TIMPs in Heteroconchia bivalves and the four pseudo TIMPs found in the gastropod P. canaliculata belong to the orthology groups that do not contain any canonical TIMP, indicating the existence of specific and independent duplications.The expansion of these pseudo TIMPs may have played specific functional roles within the organism, conferring advantages for the survival of certain species and leading to positive natural selection.One hypothesis is that these multiple pseudo TIMPs partially compensate for the functional deficiencies and losses of canonical TIMPs in certain species, such as Heteroconchia bivalves and the gastropod P. canaliculate.However, the precise roles of these pseudo TIMPs in these molluscs require further investigation in future studies to be fully understood.
In conclusion, our study utilized a high-quality genome of P. penguin to investigate the evolutionary dynamics of the TIMP gene family within the Metazoa kingdom.Through comparative genomic analyses, we investigated the distribution of the TIMP family across the animal phylogeny and suggested this family originated in the common ancestor of eumetazoans.Our investigation revealed variations in TIMP gene copy numbers across animal lineages, with a massive expansion occurring in the Pteriomorphia infraclass and peaking in the Ostreoida order.These bivalves also exhibit highly diverse TIMP repertoires, which contribute to their multiple adaptive traits, including an effective immune system, and the formation of bivalved shell and byssus.Moreover, our study unveils the presence of abundant pseudo TIMPs, primarily concentrated in Heteroconchia bivalves, highlighting potential future research directions to uncover the exact functions of these genes.Overall, our study provides valuable insights into the evolution of the TIMP gene family in molluscs, bridging the gap in our understanding of the TIMP gene family evolution across the entire Metazoa kingdom.Further investigations into the diverse functions and evolutionary trajectory within the TIMP gene family in Pteriomorphia bivalves are warranted to deepen our understanding.

Limitations of the study
This study investigates the distribution of TIMP numbers across various metazoan clades using comparative genomic analyses.The reliability of our findings relies on the quality of the investigated genomes, their annotations, and the quantity of genomes studied in each metazoan clade.We gathered metazoan genomes from sources like NCBI (https://www.ncbi.nlm.nih.gov/) and relevant literature and then assessed genome annotation completeness using the BUSCO score as an index.Despite these efforts, a scarcity of publicly available genomes with highly comprehensive annotations, especially in some clades like the Heteroconchia infraclass within bivalves, potentially limited our research scope.A more thorough exploration of TIMP family distribution across animals awaits the release of future high-quality genomes.Additionally, previous research has established the involvement of TIMP family members in various organismal processes within Pteriomorphia bivalves, like immune responses and shell formation.This study refrains from conducting extra systematic experiments to further validate the functions of multiple and diverse TIMP orthologs in P. penguin or other Pteriomorphia bivalves.Finally, the sex of P. penguin individuals utilized for genome and transcriptome sequencing was undetermined, as species within the Pteriidae family are usually protandrous hermaphrodites.The gender does not have an impact on the quality of our genome assembly.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:  85 which conducted the inference of orthogroups and orthologs, constructed the complete set of gene trees for all orthogroups and the rooted maximum-likelihood species tree.Based on the phylogeny relationship among the species analyzed, the Orthofinder rooted the gene tree for each orthogroup identified, and further conducted a hybrid duplication-loss-coalescence (DLC) algorithm of the rooted gene trees to identify gene duplication events.The hybrid method integrates the advantages of with DLCpar (full) method the highest accuracy, 95 and the species-overlap method with high efficiency. 96MCMCtree, 97 part of Phylogenetic Analysis by Maximum Likelihood (PAML) version 4.9, 86 was used to yield the time-calibrated tree, with nodes constrained by fossil records and geographic events.The time frames were used to constrain the nodes in the MCMC tree: minimum = 168.6Ma and soft maximum = 473.4Ma for Panpulmonata and Tectipleura; 98 hard minimum bound = 390 Ma for Caenogastropoda and Heterobranchia; 99 minimum = 470.2Ma and soft maximum = 531.5 Ma for A. californica and L. gigantea; 98 hard minimum bound = 419.2Ma for the first appearance of Mytilidae; 100,101 hard minimum = 465.0Ma for the first appearance of Pteriomorpha; 102 hard minimum bound = 520 Ma for the first appearance of bivalves; 103 minimum = 532 Ma and soft maximum = 549 Ma for the first appearance of molluscs; 104 and minimum = 550.25 Ma and soft maximum = 636.1 Ma for the first appearance of Lophotrochozoan. 104The rooted tree from Orthofinder results was used to provide a reference tree topology, and the burn-in, sample frequency, and number of samples were set as 1000000, 10, and 500000, respectively.

Orthologous gene family size change analysis in Mollusca
The Orthofinder inferred HOGs, orthogroups at each hierarchical level by analyzing the rooted gene trees for each orthogroups.The dataset containing the species gene number in each HOG was used for the gene family expansion and contraction analysis by Computational Analysis of gene Family Evolution (CAFE) pipeline version 5.1, 87 and the rooted ultrametric tree from the MCMCtree results were used to provide timecalibrated evolution information.In the calculating process with CAFE 5, the root equilibrium frequency was assumed to be not a uniform distribution and the -p flag is given without a parameter.Gamma model was used to run calculations as if each gene family could belong to a different evolutionary rate category, and K = 3-4 gamma rate categories were tested in two independent analyses. 87The highest likelihood was found using K = 3, with predicted l = 0.00226 and a = 0.781194, because of its maximum Model Gamma Final Likelihood (-lnL) (254497).The cutoff of the p-value was set to 0.05 to determine which families underwent a significant expansion/contraction.
The Gene Ontology (GO) Enrichment Analysis of gene sets in P. penguin significantly expanded gene families was conducted on R software version 4.2.2. 88The R package AnnotationForge 89 was first used for building a comprehensive annotation data package (org.Ppenguin.eg.db) by incorporating the gene pathway annotation results from InterProScan and eggNOG-mapper website; then the R package clusterProfiler 90 was used for enrichment analysis.

Phylogenetic orthology inference in Metazoa
Full protein sets of forty-one publicly available metazoan genomes (from sixteen Phylum, thirty-nine Class) public for using were downloaded, evaluated using BUSCOs version 5.3.2analyzed by searching metazoan gene sets (metazoa_odb10) with protein mode, and used for HOGs inference together with P. penguin by Orthofinder version 2.5.4.The longest protein sequence was selected as the representative that was used when a gene in each set possessed multiple isoforms.[35][36][37][38][39][40]

TIMP gene family analyses with Mollusca and Metazoa
In two independent analyses, the TIMP gene family was surveyed across the Molluscan and then Metazoan to determine the genomic distribution and calculate the copy numbers.This gene family was first searched from the full protein sets of eighteen molluscs as well as forty-one metazoans mentioned above first using hmmsearch program within HMMER software version 3.2.1 (http://hmmer.org/) with an E-value threshold of 1e -5 against a TIMP hmm file (PF00965) from Pfam-A database; 105 in this procedure, the identical sequences were removed and isoforms coded by one gene (like two sequences in M. gigas: NP_001292268.1 and XP_011421049.2,coded by the same gene with Gene symbol as LOC105323677) were checked to only keep one.The TIMP sequences were then identified from the sequences obtained using InterProScan; 106 which searches for protein signature matches, including ''METALLOPROTEASE INHIBITOR'' in the PANTHER version 17.0, ''NTR domain profile'' in PROSITE version 2022_05, 107 and ''NTR_2 domain'' in SMART version 9.0 108 databases.Sequences failing to have matches in both the former two protein structure databases were excluded, the remaining were named ''putative TIMP'' and divided into two groups according to whether matching the conserved NTR domain in the SMART database 109,110 : one having matches were considered as canonical TIMP, while the other failing to have matches were considered as ''pseudo TIMPs''.All of the putative TIMPs were used as inputs for MEME website (https://meme-suite.org/meme/tools/meme) for protein motif discovery; 111 and in each run, ten motifs were set and expected to be found.According to the accession ID of each TIMP protein, the gene structure information was derived from the genome annotation files in Gff3 format; the whole length and number of exons in each TIMP gene were calculated.
For the phylogenetic analyses of molluscan and metazoan TIMP gene family, the putative TIMP sequences (144 sequences in molluscan TIMP gene family and 114 sequences in the metazoan) and the canonical TIMP sequences from core Mollusca-Brachiopoda TIMP HOG (53 sequences) and core Eumetazoa TIMP HOG (114 sequences) were aligned in Mafft version 7.505 112 using strategy ''L-INS-i'', both the original Mafft alignment of putative sequences and sequences from the two core HOGs were put into IQ-Tree version 2.2.2.6 91 for Maximum Likelihood (ML) tree construction.In each run, branch supports were evaluated using both 1000 SH-aLRT bootstrap replicates 113 and 10000 ultrafast Tracing the evolution of tissue inhibitor of metalloproteinases in Metazoa with the Pteria penguin genome

Figure 1 .
Figure 1.The penguin's wing oyster Pteria penguin and its phylogenetic positions (A) The photograph of P. penguin.Circos plot showing the 14 pseudomolecules.The outer ring (red peaks) indicates gene density in each pseudo chromosome, and the inner ring shows GC content; the sliding window size is 1 MB, megabases.(B) Time-calibrated phylogenomic tree showing the position of the P. penguin among seventeen molluscan species and one brachiopod species Lingula anatina.The color labeling scheme of taxa: Bivalvia: red; Gastropoda: blue; Cephalopoda: orange; other two Lophotrochozoan species: gray.The blue horizontal bars indicate the 95% confidence intervals of the divergence times.Red stars indicate the calibrated nodes based on fossil records and geographic events.The node labels are marked on the tree nodes.
cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion negative regulation of cell−cell adhesion

Figure 2 .
Figure 2. The TIMP family has dramatic expansion in P. penguin and other relative bivalves (A) The GO pathways related to the TIMP gene family and TIMP activity, which were clustered from the expanded gene families within the order Ostreida.The molecular function (MF) GO pathways are shown in red, the biological process (BP) GO pathways are shown in blue, and the cellular component pathways are shown in green.(B)The number of putative TIMP copies in each species and node labels were marked on the node of the phylogeny tree.With deeper color denoting the higher values, the BUSCO numbers indicating the completeness of protein sets are marked in green boxes, the number of sequences having matches in different databases as well as the finally identified canonical TIMPs are marked in red boxes, TIMP numbers in core Mollusca-Brachiopoda TIMP HOG (HOG0001471) are marked in blue boxes, and the numbers of pseudo TIMPs are marked in purple boxes.The cladogram tree is inherited from the results of this research.

BFigure 3 .
Figure 3.The distribution and phylogenetic relationship of the TIMP family from 41 metazoansThe distribution of TIMP copy numbers in each species and node labels were marked on the node of the phylogeny tree.The detailed interpretation can be referred to Figure2B; with some differences are the core Eumetazoa TIMP HOG named HOG0001662, and the cladogram tree is constructed by referring to existing studies.[33][34][35][36][37][38][39][40]

PFigure 4 .
Figure 4. Schematic of features in the evolution of TIMP gene family in Metazoa kingdomThe phylogeny tree represents relationships among major metazoan clades employed in this study.Branch lengths are not indicative for evolutionary distances.Solid branches signify clades retaining TIMP gene copies, while dashed branches denote complete TIMP gene loss within a clade.Red branches highlight three remarkable TIMP expansions across metazoans: in echinoderms, the Polychaeta annelid O. fusiformis, and Pteriomorphia bivalves.Circles on tree nodes indicate four major duplication events of TIMP family along metazoan phylogeny, including the divergence of bilaterians from cnidarians and placozoans, protostomes from deuterostomes, bivalves and gastropods from cephalopods, and the separation of Mytilida and Ostreida order from Pectinida order.Plus signs (+) along the branches indicate lineage-or species-specific duplication events of TIMP genes within these clades, while minus signs (À) indicate complete TIMP gene loss in at least one species.

TABLE
Proteomes from all eighteen species were used as the input to Orthofinder version 2.5.4, d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability d EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS B The penguin's wing oyster d METHOD DETAILS used.