Evolutionary convergence and trophic diversity in hot vent and cold seep shrimps showcase a continuum of symbiosis

Evolutionary convergence and trophic diversity in hot vent and cold seep shrimps showcase a continuum of symbiosis | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Evolutionary convergence and trophic diversity in hot vent and cold seep shrimps showcase a continuum of symbiosis View ORCID Profile Pierre Methou , View ORCID Profile Margaux Mathieu-Resuge , View ORCID Profile Loïc N. Michel , View ORCID Profile Valérie Cueff-Gauchard , View ORCID Profile Hiromi Kayama Watanabe , View ORCID Profile Emily J. Cowell , View ORCID Profile Jonathan T. Copley , View ORCID Profile Roxanne Beinart , View ORCID Profile Magali Zbinden , View ORCID Profile Florence Pradillon , View ORCID Profile Marie-Anne Cambon , View ORCID Profile Chong Chen doi: https://doi.org/10.1101/2025.08.27.672534 Pierre Methou 1 Univ Brest, Ifremer, BEEP , F-29280 Plouzané, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pierre Methou For correspondence: pierre.methou{at}ifremer.fr Margaux Mathieu-Resuge 1 Univ Brest, Ifremer, BEEP , F-29280 Plouzané, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Margaux Mathieu-Resuge Loïc N. Michel 2 Animal Systematics and Diversity, Freshwater, and Oceanic Sciences Unit of reSearch (FOCUS), University of Liège , Liège, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Loïc N. Michel Valérie Cueff-Gauchard 1 Univ Brest, Ifremer, BEEP , F-29280 Plouzané, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Valérie Cueff-Gauchard Hiromi Kayama Watanabe 3 X-STAR, Japan Agency for Marine-Earth Science and Technology (JAMSTEC) , 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hiromi Kayama Watanabe Emily J. Cowell 4 Department of Biology, Temple University , Philadelphia, PA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Emily J. Cowell Jonathan T. Copley 5 Ocean and Earth Science, University of Southampton , Southampton, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jonathan T. Copley Roxanne Beinart 6 Graduate School of Oceanography, University of Rhode Island , Narragansett, RI, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Roxanne Beinart Magali Zbinden 7 Laboratoire de Biologie des Organismes et Ecosystèmes Aquatiques (BOREA), MNHN, CNRS-8067, IRD-207, Sorbonne Université, UCN, UA , France, 75005 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Magali Zbinden Florence Pradillon 1 Univ Brest, Ifremer, BEEP , F-29280 Plouzané, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Florence Pradillon Marie-Anne Cambon 1 Univ Brest, Ifremer, BEEP , F-29280 Plouzané, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Marie-Anne Cambon Chong Chen 3 X-STAR, Japan Agency for Marine-Earth Science and Technology (JAMSTEC) , 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chong Chen Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Convergent evolution offers a powerful lens through which to examine the selective forces shaping life in extreme environments. In deep-sea hot vents and cold seeps, invertebrates have independently evolved symbioses with chemosynthetic bacteria, but repeated origins of such associations within a family remain rare. Here, we investigate the evolutionary emergence of chemosymbiosis in the shrimp family Alvinocarididae across 22 species collected globally. Electron microscopy identified a gradient of epibiotic bacterial colonization within the cephalothoracic cavity, ranging from absent to dense filamentous mats, suggesting distinct trophic strategies. Isotope and lipid trophic markers confirmed difference in reliance on chemosynthetic production among sympatric species with different bacterial colonization from a single vent. Phylogenetic analysis reveals at least two independent origins of chemosymbiosis, suggesting evolutionary convergence. Microhabitat association data further show that symbiotic phenotypes are most common in shrimps occupying the hottest, most geofluid-enriched microhabitats, though exceptions suggest contributions from additional ecological or physiological constraints. Our findings reveal many alvinocaridids as gradually evolving towards symbiosis, highlighting the importance of intermediate cases to understand the pathways to chemosymbiosis. This study contributes to a broader understanding of the predictability of evolutionary outcomes in distinct habitats such as vents, with broader implications for resilience of deep-sea ecosystems. Introduction Repeated evolution of similar phenotypic traits within distinct lineages is often considered as the most compelling evidence for the action of natural selection [ 1 , 2 ]. The recurrence of similar adaptive solutions in response to comparable environmental contexts is unlikely to arise by chance alone [ 3 , 4 ]. This can result from parallel evolution towards a new phenotype if ancestral phenotypes are similar, or from convergent evolution when the ancestral phenotypes are distinct [ 4 ]. While evolutionary constraints can limit phenotypic variation, the repeated emergence of analogous traits act like replicated experiments of natural selection and shed light on the underlying factors and mechanisms that govern the adaptation of species [ 3 ]. At hydrothermal vents and other deep-sea chemosynthetic ecosystems such as hydrocarbon seeps, many invertebrate species have independently evolved a symbiotic partnership with chemosynthetic bacteria as an adaption to the particular conditions of these environments [ 5 ]. In the absence of sunlight, these associations result in ‘holobiont’ organisms where the host is dependent on the primary production of their microbial partners, which make use of chemical energy generated by geochemical gradients in the mixing zone where seawater collides with geofluids [ 6 ]. However, not all species there are symbiotrophic – several other feeding strategies such as bacterial grazing, deposit feeding, suspension feeding, scavenging and/or predation have been reported in other species endemic to chemosynthetic environments [ 7 – 9 ]. For most families with symbiotrophic members, the appearance of symbiosis is a unique event either at the common ancestor of the family such as siboglinid tubeworms, mussels in the subfamily Bathymodiolinae, paskentanid snails in the sister genera Alviniconcha and Ifremeria and yeti crabs in the family Kiwaidae [ 5 , 10 – 12 ], or in a single member of a family such as the squat lobster Shinkaia crosnieri within Munidopsidae [ 13 ]. A notable exception are peltospirid snails, in which chemosynthetic symbioses seem to have appeared at least twice, in two distantly related genera Chrysomallon and Gigantopelta [ 14 ], and potentially even a third case in Peltospira gargantua [ 15 ]. In the evolutionary history of these families, the acquisition of a chemosynthetic symbiosis is not necessarily concomitant with colonization of a chemosynthetic-based habitat, questioning the respective role of selective pressures from the environment, evolutionary constraints of these hosts, and historical contingencies in the making of such symbiotic relationships. As with peltospirid snails, alvinocaridid shrimps likely acquired chemosymbiosis several times independently, once in the sister species pair Rimicaris exoculata / R. kairei and another in the species complex R. chacei / R. hybisae [ 16 ]. These shrimps host dense communities of filamentous bacteria in their cephalothoracic cavities and on the surface of their mouthparts that harbour a great metabolic versatility, adapted to different geochemical contexts depending on the hydrothermal fields they occupy [ 17 – 19 ]. Three of these shrimp species ( R. exoculata, R. hybisae and R. kairei ) display a hypertrophy of their symbiont-hosting surfaces, with an inflated cephalothoracic chamber and enlarged mouthparts [ 16 ], and are considered to have a nutrition based mainly on their symbionts [ 20 , 21 ] with a direct nutritional transfer demonstrated for R. exoculata [ 22 ]. These symbiont-hosting shrimps gather in dense swarms of thousands of individuals near the vent fluid orifices, often constituting the most dominant fauna there [ 23 , 24 ]. Conversely, several other species in the family or even genus such as Nautilocaris saintlaurentae, Rimicaris acuminata , and Rimicaris variabilis show limited colonisation by chemosymbiotic bacteria [ 25 ]. These species tend to feed on other food sources available such as bacterial mats and/or detritus [ 25 – 28 ]. In R. chacei , the presence of a dense bacterial colonization is not accompanied with the enlargement of cephalothorax, but results from isotopic and anatomical analyses indicate it partially depends on symbionts for nutrition in a mixed diet, while using other food sources [ 21 , 26 , 29 ]. This suggests that chemosymbiosis might be more widespread in alvinocaridid shrimps than previously identified simply by external morphology. Yet, the observation of bacterial colonization patterns remained limited to a few species and we still lack an overview of chemosymbiotic associations and diets in this family. Our study aims to reconstruct the evolutionary history of chemosymbiosis in alvinocaridid shrimps and link it to the habitat they occupy. We address the following questions: 1) What are the patterns of bacterial colonization in different alvinocaridid species and how many time have dense bacterial colonization appeared? 2) Does alvinocaridid species with distinct colonization degrees exhibit different trophic strategies? 3) Is dense colonization by epibiotic filamentous bacteria linked to specific microhabitat occupancy along the environmental gradient of geofluid emissions in the local environment? Materials and Methods Sample collection A total of 22 alvinocaridid shrimp species were gathered from samples originating from 32 different vent and seep localities around the globe (Table S1), collected during 31 different oceanographic expeditions between 2004 and 2023. They were mostly collected using suction samplers on either remotely operated vehicles (ROVs) or human-occupied vehicles (HOVs) and supplemented by samples from baited traps or epibenthic sledges (see Table S2 and S3 for detailed information). For scanning electron microscopy (SEM) observations, shrimp specimens were preserved either in 80% ethanol, frozen whole at −80°C, or fixed for 16h in 2.5% glutaraldehyde / filtered seawater solution, rinsed, and then preserved in filtered seawater solution at 4°C on-board of the ship. For specimens frozen whole on-board, post-fixation in 2.5% glutaraldehyde was done in the laboratory before SEM analyses. DNA extractions were carried out using individuals preserved in 80% ethanol or frozen at −80°C. Additional specimens collected at the Puy des Folles vent field on the Mid-Atlantic Ridge (MAR) during the BICOSE 3 expedition were stored frozen at −80°C for stable isotopes and lipid analyses (Table S4 and S5). Stable isotope and lipid analysis The Puy des Folles hydrothermal site was selected to investigate stable isotope and fatty acid profiles of alvinocaridids because it offered the widest range of species showing distinct bacterial colonization patterns available to us, with four different species within a single hydrothermal vent locality including Rimicaris exoculata, R. chacei, Mirocaris fortunata , and Alvinocaris markensis . For stable isotopes analyses, pieces of abdominal muscle and mouthparts (scaphognathites – i.e., exopodite of the second maxilla – and exopodites of the first maxillipeds, hereafter called just exopodites)from three specimens per species were isolated, freeze dried and homogenized with a manual potter. Homogenized pieces of abdominal muscles from the same individuals were also used for fatty acid analyses. Measurements of stable isotope ratio were performed by continuous flow–elemental analysis isotope ratio mass spectrometry (CF-EA-IRMS) at University of Liège (Belgium), using a vario MICRO cube C-N-S elemental analyser (Elementar Analysensysteme GMBH, Hanau, Germany) coupled to an IsoPrime100 isotope ratio mass spectrometer (Isoprime, Cheadle, United Kingdom). Isotopic ratios were expressed in ‰ using the widespread δ notation [ 30 ] relative to the international references: Vienna Pee Dee Belemnite (for carbon), atmospheric air (for nitrogen) and Vienna Canyon Diablo Troilite (for sulphur). IAEA (International Atomic Energy Agency, Vienna, Austria) certified reference materials sucrose (IAEA-C-6; δ 13 C = −10.8 ± 0.5‰; mean ± SD), ammonium sulphate (IAEA-N-2; δ 15 N = 20.41 ± 0.12‰; mean ± SD), and barium sulphate (IAEA-SO5, δ 34 S = 0.5 ± 0.2‰) were used as primary analytical standards. Sulfanilic acid (Sigma-Aldrich; δ 13 C = −25.6 ± 0.4‰; δ 15 N = −0.13 ± 0.4‰; δ 34 S = 5.9 ± 0.5‰; means ± SD) was used as a secondary analytical standard. Standard deviations on multi-batch replicate measurements of secondary and internal lab standards (seabass muscle) interspersed with samples (one replicate of each standard every 15 analyses) were 0.2‰ for δ 13 C, 0.1‰ for δ15N and 0.3‰ for δ 34 S. Δ 13 C was defined as the δ 13 C isotopic ratio of abdominal muscle tissue (the “consumer”) from which was subtracted the δ 13 C isotopic ratio of mouthparts more or less colonized by symbiotic bacteria (the “nutrition source”) depending on the shrimp species. Following the same principle, Δ 15 N and Δ 34 S were calculated for each individual as follows: Δ 15 N = δ 15 N muscle - δ 15 N mouthpart and Δ 34 S = δ 34 S muscle - δ 34 S mouthpart . A minimum of 2 mg of abdominal tissue (n = 3 per species) powder were placed in pre-combusted glass vials and 6 ml of solvent mixture (CHCl3: MeOH, 2:1, v:v) were added to extract lipids. Lipid extracts were then flushed under nitrogen, sonicated, vortexed, and rested at −20°C for 24h. As described in a previous study [ 31 ], an aliquot of total lipid extract was used for transesterification. Briefly, after adding C23:0 as internal standard (free fatty acid form), lipids were first transesterified with 1ml KOH-MeOH and then with 1.6ml of H 2 SO 4 -MeOH, to form fatty acid methyl ester (FAME) that were then rinsed with distilled water saturated in hexane, and recovered within 0.4ml of hexane. FAME were analyzed on a TRACE 1300 gas chromatograph (Thermo Scientific) programmed in temperature and equipped with a splitless injector, a ZB-WAX column (30m×0.25mm IDx0.2μm) and a flame-ionisation detector, using hydrogen as vector gas. Obtained chromatograms were processed with Chromelon 7.2 (Thermo Scientific). Sixty-four FAME were identified by comparing their retention time with references from commercial mixtures (37 components FAME, PUFA1 and PUFA3, Sigma) and from house-made standards GC-MS certified; as well as by checking certain samples using mass spectrometry (GC-MS TRACE 1300 coupled to an ISQ 7000 mass spectrometer, equipped with the same apolar column and using the same program as GC-FID). Quantification of FAME in µg was based on the internal standard recovery, and then expressed in mg g −1 of dry weight. Fatty acids relative proportions were expressed as mass percentages (%) of the total identified fatty acids. All statistical analyses with Kruskal-Wallis and Wilcoxon tests were performed in a R v. 4.4.1 statistical environment [ 32 ]. Scanning electron microscopy SEM observations were carried out on 17 alvinocaridid species sampled to investigate the bacterial colonization on the cephalothoracic cavity. 48 dissected branchiostegites (inner side of the cephalothoracic cavity) and mouthparts (scaphognathites and exopodites) of individuals fixed in 2.5% glutaraldehyde were dehydrated with an ethanol series from 50% to 100% ethanol by 10% increments or directly into 100% ethanol for individuals preserved in 80% ethanol. Samples were then dehydrated for 5 h in a critical point dryer CPD 020 (Balzers Union, Balzers, Liechtenstein) and gold-coated with a SCD 040 sputter-coater (Balzers Union). Observations and imaging were performed using a Quanta 200 SEM (FEI-Thermo Fisher, Hillsboro, OR, USA). We defined a colonization score for consistency when comparing our multiple SEM observations. Branchiostegites (Br) were divided in three equivalent areas (anterior, center and posterior) and mouthparts (Mp) in three features: the central surface, the marginal setae on the edge of the mouthparts, and the additional bacteriophore setae covering the surface, present only in some species (Figure S1). For each area/feature, a score from 0 to 4 was attributed according to the following criteria, 0: no visible bacterial colonization; 1: a single layer of rod-shaped bacterial mats; 2: colonization by rod-shaped bacteria and sparsely distributed filamentous bacteria; 3: localized spots colonized by filamentous bacteria; 4: extremely dense colonization by filamentous bacteria, the surface of shrimp cuticle barely visible. The final colonization score is obtained by the addition of each areas/features colonization scores for a total score of 0 to 24. We reanalysed the SEM dataset from a previous study [ 25 ] to define the colonization scores of species analyzed by this study ( R. variabilis, R. acuminata and N. saintlaurentae ). We based our colonization scores of R. exoculata and R. kairei from descriptions of their cephalothoracic symbioses in the literature [ 17 , 19 ]. DNA extraction and sequencing We performed DNA extractions on individuals from the same 22 alvinocaridid species sampled using pieces of abdominal tissues or the 4th/5th pleopods with the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Partial fragments of the nuclear 18S rRNA (905 bp), and the mitochondrial 16S rRNA (515 bp) and the cytochrome oxidase c subunit I (COI) gene (743 bp) were amplified respectively with the 18S-1f & 18S-5r, the Cari16S-1F and Cari16S-1R [ 33 ], and the CariCOI-1F and CariCOI-1R primers [ 21 ]. We used ExTaq polymerase (Takara) or GoTaq polymerase (Promega) in 25μl reactions for PCR amplifications with the following cycling conditions: 40 cycles at 51°C as the annealing temperature for 18S, 35 cycles at 55°C for 16S, and 35 cycles at 50°C for COI. Bi-directional Sanger sequencing was conducted by Eurofins (Köln, Germany) or by the FASMAC Corporation (Kanagawa, Japan) for the different PCR products. We used Geneious Prime® 2023.1.2 ( https://www.geneious.com ) to edit and align sequence chromatograms. We carried out phylogenetic reconstruction using the Mr Bayes plugin v3.2.6 [ 34 ] in Geneious. Bayesian inference was run with the GTR + G + I substitution model and 1 million MCM steps, discarding the first 10 000 steps and with a subsampling frequency of 4000 steps. Best fitting model was selected with Modeltest [ 35 ] within Geneious. Ancestral state reconstruction was conducted with the ace function of the phytools v2.4-4 package [ 36 ] in RStudio v4.4.1. Results Stable isotope and lipid analysis Within a single hydrothermal vent field - Puy des Folles on the Mid Atlantic Ridge - each of the four alvinocaridid species displayed clearly distinct fatty acid and stable isotope composition ( Figure 1 ). The δ 15 N of most alvinocaridid mouthparts was significantly lower than the δ 15 N of abdominal muscles from the same species (Wilcoxon test; W= 118, p-value < 0.01), except for A. markensis that showed the opposite trend with equivalent or higher δ 15 N values of mouthparts compared to abdominal muscles. Inter-specific differences in nitrogen trophic shift (Δ 15 N) were present (Kruskal–Wallis; χ 2 = 9.46, p-value < 0.05), with higher values for the two Rimicaris species ( R. exoculata : Δ 15 N = 3.17 ± 0.17; R. chacei : Δ 15 N = 3.34 ± 0.95) compared to A. markensis (Δ 15 N = −0.05 ± 1.53). The δ 13 C did not vary between muscles and mouthparts (Wilcoxon test; W= 90, p-value > 0.05), but varied among species (Kruskal–Wallis; χ 2 = 17.79, p-value < 0.001) with a significant 13 C-enrichment for R. exoculata and M. fortunata compared to R. chacei and A. markensis . Carbon trophic shift (Δ 13 C) was high in R. chacei compared to other alvinocaridids, which exhibited low Δ 13 C values in R. exoculata and M. fortunata or even negative values for A. markensis . As for carbon, δ 34 S did not vary between tissues (Wilcoxon test; W= 57, p-value > 0.05) but varied among species (Kruskal–Wallis; χ 2 = 15.9, p-value < 0.001) with a significant 34 S-depletion for M. fortunata and A. markensis compared to the two Rimicaris species (Figure S2). Download figure Open in new tab Figure 1. Trophic strategies of alvinocaridid species displaying different degree of bacterial colonization at the Puy des Folles vent field. Colors indicates shrimp species and shape indicates organ analyzed. A. Principal Component Analyses (PCA) of fatty acid composition (expressed in mass %) of the abdominal muscles from the distinct alvinocaridid species B . Carbon and nitrogen isotopic ratios of abdominal muscles and mouthparts from alvinocaridid shrimps. Δ 13 C and Δ 15 N display variations of carbon and nitrogen ratios between muscles and mouthpart tissues of each individual. Overall, species differed in their total fatty acid content (Kruskal–Wallis test, H25 = 9.67, p-value < 0.05; Table S5). Rimicaris species had the higher significant total contents with 207.0 ± 34.0 and 181.0 ± 27.0 mg g −1 for R. exoculata and R. chacei , respectively. M. fortunata (24.7 ± 2.4 mg g −1 ) and A. markensis (20.8 ± 0.9 mg g −1 ) contained fatty acid contents 10 and 8.3 times lower than R. exoculata , respectively. The fatty acid compositions of shrimp abdominal muscles varied significantly among species (PERMANOVA, df = 3, F = 5.34, r 2 = 0.59, p-value < 0.001), as highlighted by the PCA on which the two first axis explained 48% of the total inertia (26.76 % on the first axis and 22.17 % on the second, Figure 1A ). The fatty acids profiles of the two Rimicaris species were positively related to axis 2, while that the two other species were negatively related to this axis. Individuals of R. exoculata and two of R. chacei were characterized by high proportions of short chain fatty acids mainly composed of 14:0, 14:1n-7, 16:1n-5, 16:2n-4, 18:2n-4, 18:3n-4, 18:2n-7 and by the 20:3nmi (non-methylene interrupted) ( Figure 1A ). Individuals of A. markensis and one of R. chacei were discriminated positively on the first principal axis and characterized by higher proportions of longer chain fatty acids, such as 20:4n-6, 20:5n-3, 22:5n-3, 22:6n-3 and the 18:1n-9DMA (dimethylacetal) ( Figure 1A ). Individuals of M. fortunata were negatively discriminated on the first and second principal axis, and characterized by higher proportions of monounsaturated fatty acids, such as 16:1n-7, 18:1n-7, 18:1n-9, 22:1n-7 and the 20:2nmi ( Figure 1A ). SEM observations For most of the 22 alvinocaridid species observed, some general trends of bacterial colonization on branchiostegites and on mouthparts emerge ( Figure 2 and Table S2). Overall, branchiostegites were generally devoid of colonization with only a few spots of rod-shaped bacteria in a single layer or a few scattered filamentous bacteria, most often on the anterior one-third ( Figure 2 ; exemplified by R. leurokolos and M. fortunata ). Mouthparts – scaphognathites and exopodites – show similar colonization patterns on most of their surfaces but contrast by a denser colonization on the plumose setae structures aligned along their margins ( Figure 2 ; exemplified by R. leurokolos and M. fortunata ). This results in total colonization scores between 3 and 10 score for most alvinocaridid species. Download figure Open in new tab Figure 2. Evolutionary history of the chemosynthetic symbiosis in alvinocaridid shrimps (left) Phylogenetic tree of Alvinocarididae based on Bayesian inference (GTR + G + I model) generated from a 2177 bp concatenated alignment comprising partial fragments of the 18S, 16S, and COI genes. Coloured numbers associated to each species indicate the range of colonization scores defined from SEM observations and colours correspond to the different categories of chemosymbiotic phenotypes (see legend). Numbers on each node indicate Bayesian Posterior Probabilities (BPP) of the phylogeny and pie charts represent ancestral phenotypes displaying posterior probabilities of each node being in each category of phenotypes. (right) Representative examples of SEM observations for the cephalothoracic cavities and mouthparts linked to the alvinocaridid species observed. Scale bars from top to bottom for cephalothoracic cavities: A. muricola overview (1 mm); A. costaricensis (2 mm); R. hybisae (2 mm); R. leurokolos (1 mm); R. vandoverae (500 μm); M. fortunata (2.5 mm). Scale bars from top to bottom for mouthparts: A. muricola overview (1 mm) and close up (100 μm); A. costaricensis (250 μm); R. hybisae (1 mm); R. leurokolos (1 mm); R. vandoverae (250 μm); M. fortunata (50 μm). Additional SEM observations for all studied species are provided in Figure S4 and S5. Apart from these general trends, some species show more specific colonization patterns ( Figure 2 and Table S1). Among them, Rimicaris hybisae and R. chacei differ drastically from the other species by a very dense colonization of filamentous bacteria on all structures except on the posterior part of the branchiostegite. These two species were mainly distinguished by a more advanced colonization of dense filamentous bacteria covering the anterior two-thirds of the branchiostegite for R. hybisae ( Figure 2 ) and the anterior one-third only for R. chacei . These two species also show additional bacteriophore setae structures (less numerous in R. chacei ) on the entire mouthpart surfaces offering additional colonisation areas compared to other alvinocaridids, further enhancing their colonization score ( R. hybisae : 22; R. chacei : 18 to 20). Based on the literature, a similarly dense colonization of filamentous bacteria on all structures except on the posterior third of the branchiostegite with a colonization score of 22 can be attributed to R. kairei and R. exoculata [ 17 , 19 ]. Two other Rimicaris species – R. vandoverae and R. cambonae – also departs from the general pattern with a dense colonization by filamentous bacteria on the anterior one-third of their branchiostegite ( Figure 2 ), and to some extent, on their mouthpart surfaces as well, leading to total colonization scores of 12 to 13 for R. vandoverae and of 11 to 13 for R. cambonae . In addition, two Alvinocaris species – A. lusca and A. costaricensis – consistently exhibit dense spots of filamentous bacteria, not only on the marginal setae but also on the surfaces of their mouthparts in all individuals observed ( Figure 2 ), as well as on the anterior one-third of the branchiostegites in some individuals ( A. lusca scores: 11 to 12; A. costaricensis scores: 9 to 12). Finally, in contrast to all other species, A. markensis shows a nearly complete absence of bacterial colonization on all of these structures ( Figure 2 ), even on marginal setae of their mouthparts which were typically colonized in all other species ( Figure 2 ), leading to a score of 0 or 1. Phylogenetic and Ancestral state reconstruction Our phylogenetic reconstruction of Alvinocarididae based on multi-gene alignment ( COI, 16S and 18S ) using Bayesian inference ( Figure 2 ) recovered a strongly supported monophyly of the family (Bayesian Posterior Probability (BPP) = 1) as well as the monophyly of the genera Rimicaris (BPP = 0.99) and Alvinocaris (BPP = 0.89). The clade formed by Mirocaris fortunata + Nautilocaris saintlaurentae was the early-diverging lineage, sister to all the other alvinocaridid shrimps. All nodes were well-supported (BBP > 0.8), except one node within Rimicaris (BPP = 0.77). Ancestral state reconstruction supported two distinct origins for the appearance of a symbiotic phenotype: once at the common ancestor of R. exoculata and R. kairei and once in the R. chacei / R. hybisae species complex ( Figure 2 ). It also supported multiple origins for the appearance of mixed or potentially mixed phenotypes ( Figure 2 ). Ancestral state reconstruction of habitats revealed that a vent origin was most likely for the most recent common ancestor of Alvinocarididae with a secondary colonisation of hydrocarbon seeps in A. costaricensis and in the common ancestor of one of the two major clades within genus Alvinocaris clade (Figure S3). Habitat and colonization score The relationship between the colonization score of an individual and its type of habitat was not entirely straightforward ( Figure 3 ). All species with high colonization scores – i.e., score > 18 – such as the R. exoculata, R. kairei, R. hybisae/R. chacei complex – were always found in close proximity to vent fluid emissions. However, not all alvinocaridid species occupying similar habitat, such as R. leurokolos or some individuals of R. variabilis , necessarily showed such high colonization scores ( Figure 3 ). Indeed, R. leurokolos shrimps showed more similar colonization scores to A. longirostris or A. dissimilis individuals from the same site but inhabiting cooler habitats within mussel beds or near Shinkaia aggregates. In addition, species with mid-range colonization score – i.e., score >10 – like R. vandoverae, R. cambonae , or A. lusca were found to inhabits cooler habitats than chimney surfaces such as Alviniconcha snail colonies or even cooler ones such as mussel beds. Alvinocaris markensis which displayed lowest colonization score were always found in peripheral areas away from the venting fluid. Download figure Open in new tab Figure 3. Individual colonization scores of each alvinocaridid species as a function of their habitat along the geochemical gradient. The habitats are ordered according to their distance to the venting or seepage emissions from close distance on the bottom and further away on top of the plot. Sampling of Alvinocaridids shrimps off New Zealand with an epibenthic sledge did not allow to associate a habitat type with their individual colonization scores. *: Species collected from methane seeps. **: R. cambonae and R. loihi were collected within a mussel bed in close vicinity to a hot venting habitat. These shrimps were found in both habitats [ 46 ] with individuals likely moving from one to another. Habitats types of alvinocaridid shrimps are illustrated by (from top to bottom): a peripheral rock habitat at Puy des Folles (Mid Atlantic Ridge); a mussel bed habitat at Minami-Ensei (Okinawa Trough); a Ifremeria snail colony at Susu Knolls (Manus Basin); an aggregation of Alviniconcha snails at Snail site (Mariana back-arc basin); a shrimp assemblage near hot venting fluids at PACMANUS (Manus Basin). Discussion This study shows the presence of distinct trophic strategies among species of alvinocaridid shrimps that display different degrees of bacterial colonization within the same locality, as supported by our stable isotope and fatty acid analyses ( Figure 1 & S1). As indicated by previous studies, the nutrition of Rimicaris exoculata is mainly based on its bacterial ectosymbionts [ 17 , 21 , 22 ]. Here, we recorded important values for Δ 15 N between the host organs (i.e., mouthparts) and muscle tissues of this species. This is generally expected between a consumer and its food items, which consistent with ectosymbionts being a major food source for this species. This is further supported by a fatty acid composition enriched in 16:2n-4 and 18:2n-4 sulfuric oxidative filamentous bacterial markers [ 37 ]. On the other end of the spectrum, Alvinocaris markensis showed a near absence of colonization, which is consistent with a non-symbiotic nutrition. The isotopic composition of its mouthparts and muscles were similar (absence of marked trophic shift), which is expected for two tissues synthesized independently by the same animal and does not suggest any trophic relationship. Their fatty acid compositions were also enriched in omega-3 and -6 long chain polyunsaturated fatty acids, which can be either associated to the ingestion of phytodetritus [ 37 ] and/or biosynthesised by bacteria [ 38 , 39 ]. In the same way, the limited bacterial colonization observed in M. fortunata is in line with limited inputs from its symbionts, demonstrated by a lower trophic shift and a distinct monounsaturated fatty acid composition often associated to thio-oxidizing bacteria [ 40 ]. Our trophic marker data on R. chacei individuals are also consistent with previous works suggesting a mixotrophic diet for this species [ 21 , 29 ]. Taken together, our results illustrate niche theory predicting that sympatric species are partitioned in their feeding habits and resource use to avoid competitive exclusion among the four studied species [ 41 ]. Through their symbiotic associations, host species such as Rimicaris are likely to access novel niches that are expanded by the addition of symbiont’s physiological and metabolic capacities [ 5 ]. A partnership with symbionts likely guarantees access to larger and more consistent quantities of food, as highlighted by the higher total fatty acid contents found in these two species. In chemosynthesis-based ecosystems, niche partitioning in relation to symbioses have been demonstrated in Alviniconcha snails where different hosts harbour variable symbiont phylotypes within the same region [ 42 ]. In alvinocaridid shrimps, the partitioning among sympatric species seems to be driven by various degrees of reliance on the production of their filamentous bacterial symbionts, from none to full dependence. By expanding our assessment of bacterial colonization patterns to a global dataset of 22 alvinocaridid species, we revealed the presence of a gradient of colonization phenotypes within the family, with lineages associated with symbiotrophic, mixotrophic (or potentially mixotrophic) diets. This suggests an evolutionary convergence with multiple emergences of symbiotic phenotypes along the evolutionary history of Alvinocarididae ( Figure 2 ), implying that this group has high flexibility in the development of distinct trophic strategies. Although uncommon in other chemosymbiotic holobionts, nutritional flexibility with limited contribution from heterotrophy has also been shown in several bathymodioline mussels which retain some ability to filter-feed [ 43 , 44 ], or Alviniconcha snails which can still digest some food with their reduced gut [ 45 ]. In alvinocaridid shrimps, this trophic flexibility can be further extended when considering the case of the R. chacei / R. hybisae species complex that display a developmental plasticity during their metamorphosis in the colonization surfaces of their symbiont-hosting organs, allowing individuals to perform either mixotrophic or symbiotrophic feeding depending on the environment [ 16 ]. This developmental variability could be due to competitive interactions for niche occupancy with R. exoculata at juvenile stages promoting a shift toward mixotrophy on MAR vents [ 24 ], while R. hybisae in the Mid-Cayman Spreading Centre has no local competitor and can fully develop its symbiotrophic phenotypes [ 23 ]. We suggest that other cases of niche partitioning are driving feeding strategies and symbiont colonization in alvinocaridids such as between A. marimonte, R. cambonae , and R. loihi living in sympatry on the Mariana Arc [ 27 , 46 ]. Our analyses also support habitat specialization as a key factor shaping symbiotic relationships in alvinocaridid shrimps ( Figure 3 ). At an ecosystem-scale, the acquisition of sulfur-oxidizing or methanotrophic symbionts in bathymodioline mussels and vesicomyid clams coincides with their repeated colonisations in different types of chemosynthetic habitats along their evolutionary histories [ 11 , 47 ]. Similarly, the origin of chemosymbiosis in the abyssochrysoidean snails coincide with the Aptian oceanic anoxic event which allowed them to colonize hydrothermal vents, though the other major clade that split at the same time, containing genera such as Provanna and Desbruyeresia , did not evolve symbiosis [ 48 ]. Symbiosis in ‘vestimentiferan’ worms in the family Siboglinidae also evolved only once at the start of their association with reducing habitats, but its origin traces back widespread reducing, anoxic sediments instead of sparse and ephemeral vents or seeps [ 49 ]. These scenarios contrast with the emergence of chemosynthetic symbiosis in alvinocaridid shrimps, which is not directly linked to their colonization of reducing habitats, as our ancient habitat reconstruction indicated that association with hydrothermal vents is ancestral to the family. Instead, the symbiosis appeared secondarily, more similar to the case of some peltospirid snails [ 14 , 15 ]. Yet, at the microhabitat scale, the habitat preference of alvinocaridid species along the geochemical gradient appears to be important, as all species with high colonization scores occupy the hottest parts of the vent chimney with the greatest exposure to high concentrations of geofluids. This emphasizes the strong influence of the geochemical gradient in structuring the distribution and zonation of chemosynthetic holobiont species [ 5 , 6 ]. However, this link is not always clear-cut, with some alvinocaridid species of potentially mixed phenotypes inhabiting coolers habitats (e.g., A. lusca and R. cambonae ) and species displaying limited epibiont colonization inhabiting hot venting areas (e.g., R. leurokolos ), suggesting that other factors, including the co-occurrence with other abundant holobionts, are also at play for establishment of their realized niches. The limited number of known chemosynthetic symbioses in crustaceans compared to annelids and molluscs [ 5 , 50 ] may be linked to evolutionary constraints of the phylum such as their moulting cycle. In symbiotic insects, moulting only during the early phases of their life cycles poses a challenge to the transmission and maintenance of symbiotic partnerships [ 51 ], which is further enhanced in crustaceans that moult throughout their adult life. In R. exoculata , the moulting does not hamper the establishment of a chemosynthetic symbiosis but the very rapid moulting frequency, estimated at every ten days, imposes a rapid renewal rate of the symbiont community [ 52 ], probably resulting in a significant metabolic cost. The high mobility of decapod crustaceans could also be another barrier to the emergence of symbiotic relationship in this group. Indeed, global functional trait analyses of hydrothermal vent ecosystems have highlighted that most species with a symbiotic feeding mode were sessile or only slightly mobile [ 53 ]. A high motility allows species to significantly expand accessible food resources instead of relying on a single trophic strategy, which could explain the gradual variations of symbiont colonization phenotypes we observe among alvinocaridid species. These two factors – i.e., moulting and mobility – should also be mentioned as potential sources of bias in our observations which could contribute partly to the observed inter-individual variability of bacterial colonization observed. For our observations, we excluded individuals just after moulting – characterised by soft carapaces and a lack of mineral deposition – that have reset their epibiont colonization [ 52 ]. However, we cannot rule out that different individuals observed could be at slightly different moult – and thus bacterial colonization – stages. Similarly, with alvinocaridid shrimps being mobile, we cannot be sure if the sampling habitat differs or not from the habitat of that individual in the past few hours/days, especially for species known to occupy a wide range of habitat types such as A. longirostris, R. variabilis or R. chacei [ 24 , 28 ] (see also Figure 3 ). However, these potential biases are unlikely to affect the general trends we highlight, with several species, such as R. leurokolos , being restricted to a single type of habitat [ 28 , 54 ]. Overall, rather than a division in two categories of symbiotic and non-symbiotic species, our results indicate that chemosymbiosis gradually emerged in alvinocaridid shrimps, under the influence of the geochemical gradient but not only that. Our observations highlight a more complex and ongoing dynamic in the emergence of symbioses in vent and seep endemic species. As R. chacei and other shrimps with mixotrophic diet, several species in other phyla could be partially symbiotic. To some extent, mixotrophy has already been demonstrated in bathymodioline mussels. Many other examples inferred from stable isotopic compositions and/or observations of bacterial ectosymbionts could represent cases of partially chemosymbiotic species such as the amphipod Exitomelita sigynae [ 55 ] or the small vent snails Peltospira smaragdina and Cyathermia naticoides [ 8 , 56 ]. We argue that studying these gradual cases of chemosymbiosis is as essential as studies of symbioses from emblematic foundational species to fully understand the evolutionary dynamics of how symbiotic relationships are established. Our results also have significant implications for the conservation of hydrothermal vent ecosystems. A recent study [ 53 ] highlighted the high proportion of functionally unique species with little redundancy in hydrothermal vents resulting in rapid collapse of these ecosystems using simulated species extinction – but the feeding modes of all alvinocaridids were categorized as bacterivores. Our work suggests there is more nuance with a continuum in terms of feeding modes within this family. Rather than diminishing the conclusions of that study [ 53 ], we further enhance the view of high functional uniqueness in vent endemic species which points again to the vulnerability of these environments in the face of mining threats. Conclusion By investigating bacterial colonisation and trophic markers in alvinocaridid shrimps in an unprecedented global-scale study of 22 species, we underscore that the evolution of chemosynthetic symbiosis in alvinocaridid shrimps is a dynamic and recurrent process shaped by complex factors including ecological context, developmental plasticity, and phylogenetic constraints. The independent emergence of densely colonized symbiotic phenotypes, coupled with a spectrum of intermediate forms, points to evolutionary convergence driven by strong environmental selection. Yet, the incomplete correspondence between symbiosis and habitat, and the persistence of mixed trophic strategies, highlight the role of other factors like the physiological and developmental constraints due to moulting and mobility in modulating evolutionary trajectories. Our study exemplifies how complex traits such as symbiosis at vents and seeps can, and are being, evolved incrementally, through flexible and sometimes facultative intermediates, rather than through abrupt transitions as is seen in other groups like abyssochrysoid snails and vesicomyid clams. This continuum challenges simplistic categorizations of broader taxonomic groups inhabiting vents and seeps under one or a couple of feeding modes, since Alvinocarididae clearly includes a whole spectrum of feeding modes. Our work contributes to a more nuanced understanding of the repeatability — and limitations — of evolution in ‘extreme’ environments. Funding PM was supported by ISblue project, Interdisciplinary graduate school for the blue planet (ANR-17-EURE-0015) and co-funded by a grant from the French government under the program “Investissements d’Avenir” embedded in France 2030. Analyses conducted during this study also benefited from French State aid managed by the National Research Agency under France 2030 LIFEDEEPER: ANR-22-POCE-0007. This study was supported by the Cooperative Research Program of Atmosphere and Ocean Research Institute, The University of Tokyo (R/V Yokosuka , cruise YK23-16S). Sample collection on TN401 by EC and RAB was supported by the US National Science Foundation award OCE-1736932 to RAB. This study was also supported by Council for Science, Technology, and Innovation (CSTI), Japan as the Cross Ministerial Strategic Innovation Promotion Program (SIP), Next-generation Technology for Ocean Resource Exploration. RRS James Cook cruise JC82 was funded by the UK Natural Environment Research Council grant NE/F017774/1 to JTC. The FKt230303 expedition was funded by the Schmidt Ocean Institute, who also provided funding for making this paper Open Access. The AT42-03 expedition off Costa Rica was funded by the NSF grant NSF OCE 1635219. Acknowledgments We thank the captain, crew, and scientists on-board R/V Pourquoi Pas? during research expeditions BICOSE2014 ( https://doi.org/10.17600/14000100 ), BICOSE2 ( https://doi.org/10.17600/18000004 ), BICOSE3 ( https://doi.org/10.17600/18002399 ), BIOBAZ ( https://doi.org/10.17600/13030030 ), HERMINE2 ( https://doi.org/10.17600/18001851 ), and WACS ( https://doi.org/10.17600/11030010 ), R/V Atalante during expeditions CHUBACARC ( https://doi.org/10.17600/18001111 ), FUTUNA3 ( https://doi.org/10.17600/12010040 ), and MOMARSAT21 ( https://doi.org/10.17600/18001296 ), R/V Shinsei Maru during expeditions KS-21-20 and KS-22-2, R/V Natsushima during expedition NT15-02, R/V Kaimei during expedition KM23-E05, R/V Kaiyo during expedition KY14-01, R/V Yokosuka during expeditions YK10-11, YK19-10, YK16-E02, YK22-05, YK23-06, and YK23-16, R/V Haiyang 6 during expedition HYDZ6-2020-05, R/V Tangaroa during expeditions TAN0411, TAN1206, and TAN2102, R/V Ka’imikai-o-Kanaloa during expeditions KOK0506 and KOK0507, R/V James Cook during expedition JC82, R/V Falkor (too) during expedition FKt231024, R/V Atlantis during expedition AT42-03 and R/V Thomas G. Thompson during expedition TN401. We extend the same to the pilots and technical teams of ROVs Victor 6000, Hyper-Dolphin, KM-ROV, Haima2, Isis, Ropos, SuBastian , and Jason II as well as the HOVs Nautile, Shinkai 6500, Pisces V , and Alvin during those expeditions. We gratefully acknowledge the cruise PIs for leading the expeditions: François Lallier, Sorbonne Université (BIOBAZ); Didier Jollivet and Stéphane Hourdez, CNRS (CHUBACARC); Yves Fouquet (FUTUNA3); Cécile Cathalot and Ewan Pelleter, Ifremer (HERMINE2); Marjolaine Matabos and Jozée Sarrazin, Ifremer (MOMARSAT21); Karine Olu, Ifremer (WACS); Ken Takai, JAMSTEC (KY14-01, YK19-10, YK16-E02, YK22-05, KM23-E05); Masahiro Yamamoto, JAMSTEC (NT15-02, YK23-06); Shigeaki Kojima, AORI, the University of Tokyo (YK10-11); Takafumi Kasaya, JAMSTEC (NT15-02), Tatsuo Nozaki, Waseda University (KS-21-20); Hironori Komatsu, National Museum of Nature and Science (KS-22-2); Kenichiro Tani, National Museum of Nature and Science, Japan (YK23-16S); Jun Tao, Guangzhou Marine Geological Survey (HYDZ6-2020-05); Gary Massoth and Bob Embley, NOAA (KOK0505, KOK0506, KOK0507); Malcolm Clark, NIWA (TAN1206); Laura Wallace, New Zealand Institute of Geological and Nuclear Sciences (TAN2102); John W. Jamieson, Memorial University of Newfoundland (FKt231024) and Erik Cordes, Temple University (AT42-03). Biological sampling within the Commonwealth of the Northern Mariana Islands was carried out under the Marine Scientific Research permit number MSR U2022-047 from the United States government. This is a GACHINKO cruise Episode I (YK19-10) and Episode III (YK22-05) output. We are indebted to the Kingdom of Tonga and the government of Papua New Guinea for permitting access to their national waters and resources. We obtained the agreement to sample in Wallis et Futuna waters from the Haut Commissariat à la République in New Caledonia and the Préfecture in Wallis and Futuna. We thank Kareen Schnabel (NIWA) for providing samples from the Kermadec Arc and Hikurangi Margin, Aotearoa/New Zealand, Jianwen Qiu for providing samples from methane seep of the South China Sea and Hidetaka Nomaki (JAMSTEC) for providing facilities and discussions that helped in drafting this manuscript. We also thank Nicolas Gayet (UMR BEEP; Ifremer) and Katsuyuki Uematsu (Marine Work, Japan) for their work in preparing samples for scanning electron microscopy. Funder Information Declared ISblue, https://ror.org/01pczmy17 , ANR-17-EURE-0015 ANR LIFEDEEPER , ANR-22-POCE-0007 Cooperative Research Program of Atmosphere and Ocean Research Institute, The University of Tokyo US National Science Foundation , OCE-1736932 , OCE-1635219 Natural Environment Research Council, https://ror.org/02b5d8509 , NE/F017774/1 Schmidt Ocean Institute, https://ror.org/02bkxyz57 Footnotes Correct author family name Margaux Mathieu-Resuge References 1. ↵ Arendt J , Reznick D. 2008 Convergence and parallelism reconsidered: what have we learned about the genetics of adaptation? Trends in Ecology & Evolution 23 , 26 – 32 . ( doi: 10.1016/j.tree.2007.09.011 ) OpenUrl CrossRef PubMed Web of Science 2. ↵ Losos JB . 2011 Convergence, adaptation, and constraint . Evolution 65 , 1827 – 1840 . ( doi: 10.1111/j.1558-5646.2011.01289.x ) OpenUrl CrossRef PubMed Web of Science 3. ↵ Sackton TB , Clark N. 2019 Convergent evolution in the genomics era: new insights and directions . Phil. Trans. R. Soc. B 374 , 20190102 . ( doi: 10.1098/rstb.2019.0102 ) OpenUrl CrossRef PubMed 4. ↵ Cerca J. 2023 Understanding natural selection and similarity: Convergent, parallel and repeated evolution . Molecular Ecology 32 , 5451 – 5462 . ( doi: 10.1111/mec.17132 ) OpenUrl CrossRef 5. ↵ Sogin EM , Kleiner M , Borowski C , Gruber-Vodicka HR , Dubilier N. 2021 Life in the Dark: Phylogenetic and Physiological Diversity of Chemosynthetic Symbioses . Annual Review of Microbiology 75 , 695 – 718 . ( doi: 10.1146/annurev-micro-051021-123130 ) OpenUrl CrossRef PubMed 6. ↵ Sogin EM , Leisch N , Dubilier N. 2020 Chemosynthetic symbioses . Current Biology 30 , R1137 – R1142 . ( doi: 10.1016/j.cub.2020.07.050 ) OpenUrl CrossRef PubMed 7. ↵ Govenar B. 2012 Energy Transfer Through Food Webs at Hydrothermal Vents: Linking the Lithosphere to the Biosphere . Oceanography 25 , 246 – 255 . ( doi: 10.5670/oceanog.2012.23 ) OpenUrl CrossRef 8. ↵ Portail M , Brandily C , Cathalot C , Colaço A , Gélinas Y , Husson B , Sarradin PM , Sarrazin J. 2018 Food-web complexity across hydrothermal vents on the Azores triple junction . Deep-Sea Research Part I: Oceanographic Research Papers 131 , 101 – 120 . ( doi: 10.1016/j.dsr.2017.11.010 ) OpenUrl CrossRef 9. ↵ Van Audenhaege L , Fariñas-Bermejo A , Schultz T , Lee Van Dover C. 2019 An environmental baseline for food webs at deep-sea hydrothermal vents in Manus Basin (Papua New Guinea) . Deep-Sea Research Part I: Oceanographic Research Papers 148 , 88 – 99 . ( doi: 10.1016/j.dsr.2019.04.018 ) OpenUrl CrossRef 10. ↵ Roterman CN , Lee W , Liu X , Lin R , Li X , Won Y. 2018 A new yeti crab phylogeny: Vent origins with indications of regional extinction in the East Pacific . PLOS ONE 13 , e0194696 . ( doi: 10.1371/journal.pone.0194696.s011 ) OpenUrl CrossRef PubMed 11. ↵ Lorion J , Kiel S , Faure B , Kawato M , Ho SYW , Marshall B , Tsuchida S , Miyazaki JI , Fujiwara Y. 2013 Adaptive radiation of chemosymbiotic deep-sea mussels . Proceedings of the Royal Society B: Biological Sciences 280 , 20131243 . ( doi: 10.1098/rspb.2013.1243 ) OpenUrl CrossRef PubMed 12. ↵ Breusing C , Genetti M , Russell SL , Corbett-Detig RB , Beinart RA . 2022 Horizontal transmission enables flexible associations with locally adapted symbiont strains in deep-sea hydrothermal vent symbioses . Proceedings of the National Academy of Sciences 119 , 1 – 11 . ( doi: 10.1073/pnas.2115608119 ) OpenUrl CrossRef PubMed 13. ↵ Watsuji T , Yamamoto A , Motoki K , Ueda K , Hada E , Takaki Y , Kawagucci S , Takai K. 2015 Molecular evidence of digestion and absorption of epibiotic bacterial community by deep-sea crab Shinkaia crosnieri . The ISME Journal 9 , 821 – 831 . ( doi: 10.1038/ismej.2014.178 ) OpenUrl CrossRef PubMed 14. ↵ Chen C , Uematsu K , Linse K , Sigwart JD . 2017 By more ways than one: Rapid convergence at hydrothermal vents shown by 3D anatomical reconstruction of Gigantopelta (Mollusca: Neomphalina) . BMC Evolutionary Biology 17 , 62 . ( doi: 10.1186/s12862-017-0917-z ) OpenUrl CrossRef PubMed 15. ↵ Chen C , Pradillon F , Diaz-Recio Lorenzo C , Alfaro-Lucas JM . 2025 Integrative taxonomy of two new peltospirid gastropods from Mid-Atlantic Ridge hot vents, including a potentially symbiotic species . Zoological Journal of the Linnean Society 204 , zlaf055 . ( doi: 10.1093/zoolinnean/zlaf055 ) OpenUrl CrossRef 16. ↵ Methou P , Guéganton M , Copley JT , Kayama Watanabe H , Pradillon F , Cambon-Bonavita M-A , Chen C. 2024 Distinct development trajectories and symbiosis modes in vent shrimps . Evolution 78 , 413 – 422 . ( doi: 10.1093/evolut/qpad217 ) OpenUrl CrossRef PubMed 17. ↵ Zbinden M , Cambon-Bonavita M. 2020 Biology and ecology of Rimicaris exoculata, a symbiotic shrimp from deep-sea hydrothermal vents . Marine Ecology Progress Series 652 , 187 – 222 . ( doi: 10.3354/meps13467 ) OpenUrl CrossRef 18. Cambon-Bonavita M-A , Aubé J , Cueff-Gauchard V , Reveillaud J. 2021 Niche partitioning in the Rimicaris exoculata holobiont: the case of the first symbiotic Zetaproteobacteria . Microbiome 9 , 1 – 16 . ( doi: 10.1186/s40168-021-01045-6 ) OpenUrl CrossRef PubMed 19. ↵ Methou P , Hikosaka M , Chen C , Watanabe HK , Miyamoto N , Makita H , Takahashi Y , Jenkins RG . 2022 Symbiont Community Composition in Rimicaris kairei Shrimps from Indian Ocean Vents with Notes on Mineralogy . Applied and Environmental Microbiology 88 , 1 – 16 . ( doi: 10.1128/aem.00185-22 ) OpenUrl CrossRef 20. ↵ Streit K , Bennett SA , Van Dover CL , Coleman M. 2015 Sources of organic carbon for Rimicaris hybisae: Tracing individual fatty acids at two hydrothermal vent fields in the Mid-Cayman rise . Deep-Sea Research Part I: Oceanographic Research Papers 100 , 13 – 20 . ( doi: 10.1016/j.dsr.2015.02.003 ) OpenUrl CrossRef 21. ↵ Methou P , Michel LN , Segonzac M , Cambon-Bonavita M-A , Pradillon F. 2020 Integrative taxonomy revisits the ontogeny and trophic niches of Rimicaris vent shrimps . Royal Society Open Science 7 , 200837 . ( doi: 10.1098/rsos.200837 ) OpenUrl CrossRef 22. ↵ Ponsard J et al. 2013 Inorganic carbon fixation by chemosynthetic ectosymbionts and nutritional transfers to the hydrothermal vent host-shrimp Rimicaris exoculata . ISME journal 7 , 96 – 109 . ( doi: 10.1038/ismej.2012.87 ) OpenUrl CrossRef PubMed 23. ↵ Nye V , Copley JTP , Tyler PA . 2013 Spatial Variation in the Population Structure and Reproductive Biology of Rimicaris hybisae (Caridea: Alvinocarididae) at Hydrothermal Vents on the Mid-Cayman Spreading Centre . PLoS ONE 8 . ( doi: 10.1371/journal.pone.0060319 ) OpenUrl CrossRef 24. ↵ Methou P , Hernández-Ávila I , Cathalot C , Cambon-Bonavita M-A , Pradillon F. 2022 Population structure and environmental niches of Rimicaris shrimps from the Mid-Atlantic Ridge . Marine Ecology Progress Series 684 , 1 – 20 . ( doi: 10.3354/meps13986 ) OpenUrl CrossRef 25. ↵ Methou P , Cueff-Gauchard V , Michel LN , Gayet N , Pradillon F , Cambon-Bonavita M. 2023 Symbioses of alvinocaridid shrimps from the South West Pacific: No chemosymbiotic diets but conserved gut microbiomes . Environmental Microbiology Reports 15 , 616 – 630 . ( doi: 10.1111/1758-2229.13201 ) OpenUrl CrossRef 26. ↵ Gebruk AV , Southward EC , Kennedy H , Southward AJ . 2000 Food sources, behaviour, and distribution of hydrothermal vent shrimps at the Mid-Atlantic Ridge . Journal of the Marine Biological Association of the United Kingdom 80 , 485 – 499 . ( doi: 10.1017/S0025315400002186 ) OpenUrl CrossRef 27. ↵ Stevens CJ , Limén H , Pond DW , Gélinas Y , Juniper SK . 2008 Ontogenetic shifts in the trophic ecology of two alvinocaridid shrimp species at hydrothermal vents on the Mariana Arc, western Pacific Ocean . Marine Ecology Progress Series 356 , 225 – 237 . ( doi: 10.3354/meps07270 ) OpenUrl CrossRef 28. ↵ Methou P , Nye V , Copley JT , Watanabe HK , Nagai Y , Chen C. 2023 Life⍰history traits of alvinocaridid shrimps inhabiting chemosynthetic ecosystems around Japan . Marine Biology 170 , 1 – 17 . ( doi: 10.1007/s00227-023-04221-4 ) OpenUrl CrossRef 29. ↵ Apremont V , Cambon-Bonavita M-A , Cueff-Gauchard V , François D , Pradillon F , Corbari L , Zbinden M. 2018 Gill chamber and gut microbial communities of the hydrothermal shrimp Rimicaris chacei Williams and Rona 1986: A possible symbiosis . PLoS ONE 13 , e0206084 . ( doi: 10.1371/journal.pone.0206084 ) OpenUrl CrossRef PubMed 30. ↵ Coplen TB . 2011 Guidelines and recommended terms for expression of stable-isotope-ratio and gas-ratio measurement results . Rapid Communications in Mass Spectrometry 25 , 2538 – 2560 . ( doi: 10.1002/rcm.5129 ) OpenUrl CrossRef PubMed Web of Science 31. ↵ Mathieu-Resuge M , Le Grand F , Brosset P , Lebigre C , Soudant P , Vagner M , Pecquerie L , Sardenne F. 2023 Red muscle of small pelagic fishes’ fillets are high-quality sources of essential fatty acids . Journal of Food Composition and Analysis 120 , 105304 . ( doi: 10.1016/j.jfca.2023.105304 ) OpenUrl CrossRef 32. ↵ R Core Team . 2021 R: A language and environment for statistical computing . See https://www.r-project.org . 33. ↵ Aznar-Cormano L , Brisset J , Chan TY , Corbari L , Puillandre N , Utge J , Zbinden M , Zuccon D , Samadi S. 2015 An improved taxonomic sampling is a necessary but not sufficient condition for resolving inter-families relationships in Caridean decapods . Genetica 143 , 195 – 205 . ( doi: 10.1007/s10709-014-9807-0 ) OpenUrl CrossRef PubMed 34. ↵ Ronquist F , Teslenko M , Van Der Mark P , Ayres DL , Darling A , Höhna S , Liu L , Suchard MA , Huelsenbeck JP . 2012 MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice Across a Large Model Space . Systematic Biology 61 , 539 – 542 . ( doi: 10.1093/sysbio/sys029 ) OpenUrl CrossRef PubMed 35. ↵ Darriba D , Posada D , Kozlov AM , Stamatakis A , Morel B , Flouri T. 2020 ModelTest-NG: A New and Scalable Tool for the Selection of DNA and Protein Evolutionary Models . Molecular Biology and Evolution 37 , 291 – 294 . ( doi: 10.1093/molbev/msz189 ) OpenUrl CrossRef 36. ↵ Revell LJ . 2011 phytools: an R package for phylogenetic comparative biology (and other things) . Methods in Ecology and Evolution 37. ↵ Pond DW , Dixon DR , Bell MV , Fallick AE , Sargent JR , David W , Sargentl JR . 1997 Occurrence of 16:2(n-4) and 18:2(n-4) fatty acids in the lipids of the hydrothermal vent shrimps Rimicaris exoculata and Alvinocaris markensis: nutritional nutritional and trophic implications . Marine Ecology Progress Series 156 , 167 – 174 . ( doi: 10.3354/meps156167 ) OpenUrl CrossRef Web of Science 38. ↵ Russell NJ , Nichols DS . 1999 Polyunsaturated fatty acids in marine bacteria — a dogma rewritten . Microbiology 145 , 767 – 779 . ( doi: 10.1099/13500872-145-4-767 ) OpenUrl CrossRef PubMed Web of Science 39. ↵ Nichols DS . 2003 Prokaryotes and the input of polyunsaturated fatty acids to the marine food web . FEMS Microbiology Letters 219 , 1 – 7 . ( doi: 10.1016/S0378-1097(02)01200-4 ) OpenUrl CrossRef PubMed 40. ↵ McCaffrey MA , Farrington JW , Repeta DJ . 1989 Geochemical implications of the lipid composition of Thioploca spp. from the Peru upwelling region—15°S . Organic Geochemistry 14 , 61 – 68 . ( doi: 10.1016/0146-6380(89)90019-3 ) OpenUrl CrossRef GeoRef Web of Science 41. ↵ Schoener TW . 1974 Resouce partitioning in ecological communities: Research on how similar species divide resources helps . Science 185 , 27 – 39 . OpenUrl Abstract / FREE Full Text 42. ↵ Beinart RA et al. 2012 Evidence for the role of endosymbionts in regional-scale habitat partitioning by hydrothermal vent symbioses . Proceedings of the National Academy of Sciences , 241 – 250 . ( doi: 10.1073/pnas.1202690109 ) OpenUrl Abstract / FREE Full Text 43. ↵ Riou V et al. 2010 Mixotrophy in the deep sea: A dual endosymbiotic hydrothermal mytilid assimilates dissolved and particulate organic matter . Marine Ecology Progress Series 405 , 187 – 201 . ( doi: 10.3354/meps08515 ) OpenUrl CrossRef Web of Science 44. ↵ Demopoulos AWJ , McClain-Counts JP , Bourque JR , Prouty NG , Smith BJ , Brooke S , Ross SW , Ruppel CD . 2019 Examination of Bathymodiolus childressi nutritional sources, isotopic niches, and food-web linkages at two seeps in the US Atlantic margin using stable isotope analysis and mixing models . Deep Sea Research Part I: Oceanographic Research Papers 148 , 53 – 66 . ( doi: 10.1016/j.dsr.2019.04.002 ) OpenUrl CrossRef 45. ↵ Yang Y , Sun J , Chen C , Zhou Y , Van Dover CL , Wang C , Qiu J-W , Qian P-Y. 2022 Metagenomic and metatranscriptomic analyses reveal minor-yet-crucial roles of gut microbiome in deep-sea hydrothermal vent snail . Animal Microbiome 4 . ( doi: 10.1186/s42523-021-00150-z ) OpenUrl CrossRef 46. ↵ Methou P , Johnson SB , Sherrin J , Shank TM , Chen C , Tunnicliffe V. 2024 A tale of two shrimps – Speciation and demography of two sympatric shrimp species from hydrothermal vents ., 2024.12.18.629080. ( doi: 10.1101/2024.12.18.629080 ) OpenUrl Abstract / FREE Full Text 47. ↵ Gao K et al. 2025 Phylogenomic analyses of Pliocardiinae (Bivalvia: Vesicomyidae) update genus-level taxonomy and shed light on trait evolution . Cladistics , cla.70001. ( doi: 10.1111/cla.70001 ) OpenUrl CrossRef 48. ↵ Breusing C , Johnson SB , Tunnicliffe V , Clague DA , Vrijenhoek RC , Beinart RA . 2020 Allopatric and sympatric drivers of speciation in alviniconcha hydrothermal vent snails . Molecular Biology and Evolution 37 , 3469 – 3484 . ( doi: 10.1093/molbev/msaa177 ) OpenUrl CrossRef PubMed 49. ↵ Li Y , Kocot KM , Whelan NV , Santos SR , Waits DS , Thornhill DJ , Halanych KM . 2017 Phylogenomics of tubeworms (Siboglinidae, Annelida) and comparative performance of different reconstruction methods . Zoologica Scripta 46 , 200 – 213 . ( doi: 10.1111/zsc.12201 ) OpenUrl CrossRef 50. ↵ Joey Pakes M , Mejía-Ortiz L , Weis AK . 2014 Arthropods host intracellular chemosynthetic symbionts, too: cave study reveals an unusual form of symbiosis . Journal of Crustacean Biology 34 , 334 – 341 . ( doi: 10.1163/1937240X-00002238 ) OpenUrl CrossRef 51. ↵ Hammer TJ , Moran NA . 2019 Links between metamorphosis and symbiosis in holometabolous insects . Philosophical Transactions of the Royal Society B: Biological Sciences 374 , 20190068 . ( doi: 10.1098/rstb.2019.0068 ) OpenUrl CrossRef PubMed 52. ↵ Corbari L , Zbinden M , Cambon-Bonavita MA , Gaill F , Compère P. 2008 Bacterial symbionts and mineral deposits in the branchial chamber of the hydrothermal vent shrimp rimicaris exoculata: Relationship to moult cycle . Aquatic Biology 1 , 225 – 238 . ( doi: 10.3354/ab00024 ) OpenUrl CrossRef Web of Science 53. ↵ Alfaro-Lucas JM , Chapman ASA , Tunnicliffe V , Bates AE . 2024 High functional vulnerability across the world’s deep-sea hydrothermal vent communities . Proc. Natl. Acad. Sci. U.S.A . 121 , e2403899121 . ( doi: 10.1073/pnas.2403899121 ) OpenUrl CrossRef PubMed 54. ↵ Zhang H , Yahagi T , Miyamoto N , Chen C , Jiang Q , Qian P-Y , Sun J. 2025 Circatidal control of gene expression in the deep-sea hot vent shrimp Rimicaris leurokolos . Proc. R. Soc. B . 292 , 20242970 . ( doi: 10.1098/rspb.2024.2970 ) OpenUrl CrossRef PubMed 55. ↵ Tandberg AH , Rapp HT , Schander C , Vader W , Sweetman AK , Berge J. 2012 Exitomelita sigynae gen. et sp. nov.: A new amphipod from the Arctic Loki Castle vent field with potential gill ectosymbionts . Polar Biology 35 , 705 – 716 . ( doi: 10.1007/s00300-011-1115-x ) OpenUrl CrossRef 56. ↵ Zbinden M , Marqué L , Gaudron SM , Ravaux J , Léger N , Duperron S. 2015 Epsilonproteobacteria as gill epibionts of the hydrothermal vent gastropod Cyathermia naticoides (North East-Pacific Rise) . Marine Biology 162 , 435 – 448 . ( doi: 10.1007/s00227-014-2591-7 ) OpenUrl CrossRef View the discussion thread. Back to top Previous Next Posted September 04, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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Copley , Roxanne Beinart , Magali Zbinden , Florence Pradillon , Marie-Anne Cambon , Chong Chen bioRxiv 2025.08.27.672534; doi: https://doi.org/10.1101/2025.08.27.672534 Share This Article: Copy Citation Tools Evolutionary convergence and trophic diversity in hot vent and cold seep shrimps showcase a continuum of symbiosis Pierre Methou , Margaux Mathieu-Resuge , Loïc N. Michel , Valérie Cueff-Gauchard , Hiromi Kayama Watanabe , Emily J. Cowell , Jonathan T. Copley , Roxanne Beinart , Magali Zbinden , Florence Pradillon , Marie-Anne Cambon , Chong Chen bioRxiv 2025.08.27.672534; doi: https://doi.org/10.1101/2025.08.27.672534 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Evolutionary Biology Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41911) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13371) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15599) Genomics (22482) Immunology (17728) Microbiology (40363) Molecular Biology (17163) Neuroscience (88536) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15129) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)