Accepted Article Woods Hole, MA 02543 Woods Hole, MA 02543 MA Woods Hole, 02543 Hole Rd Woods 266 Institution Woods Oceanographic Hole MAWoods Hole, 02543 Rd Hole Woods 266 Institution Woods Oceanographic Hole Biology Dr. Department Authors: *Corresponding Massachusetts Timothy Institute M. Keywords of Shank Technology Japan Kanagawa, Santiago Herrera USA MA 02543, [email protected] [email protected] [email protected] [email protected] 3 2 1 Santiago Herrera hydrothermal vents pa andbiogeographicalEvolutionary Article type : Original Article Accepted Date:20-Dec-2014 Revised Date :14-Dec-2014 Received Date 26-Sep-2014 : This article This protectedis by Allcopyright. rights reserved. 10.1111/mec.13054doi: lead to differences betweenversionthis and the Versionof Record. Please cite this article as through thebeen typesetting,copyediting, pa This article hasbeen accepted for publicationand undergone fullpeer review but has not Biology Department, Woods Hole Oceanographic Institution, 266 Woods Hole Road, Woods Hole, Road, Woods 266 Hole Woods Institution, Oceanographic Hole Biology Department, Institute of Biogeoscie of 77Mass Technology, Institute Massachusetts : Biogeography, , Hydrothermal Vents, Deep-Sea, RAD-seq, Dispersal Dispersal RAD-seq, Deep-Sea, Vents, Hydrothermal Barnacles, Biogeography, :

1,2* nces, nces, Japan forMarine-EarthAgency Science Yokosuka, andTechnology, , HiromiWatanabe, gination and process, which proofreading may achusetts Avenue, Cambridge, MA 02139, USA MA 02139, Avenue,Cambridge, achusetts 3 , Timothy M. Shank M. , Timothy tterns of barnacles from deep-sea 2* Accepted Article Japan 02543, USA 3 2 1 Santiago Herrera Vents Hydrothermal Evolutionary and BiogeographicalPatterns Barnaclesof from Deep-Sea Title Running Tel: 508-289-3392 Tel: 508-289-3906 This article This protectedis by Allcopyright. rights reserved. have barnacles vent that testsindicate hypothesis andtopology inferences Phylogenetic each species. from individuals on DNA sequencing associated performed restriction-site genera.described Wealso Our approach was totarget a ofmultiple loci suite in samples representing seven of out the eight and diversification. time infer their to vent barnacles of diversity genetic global the characterize we Here fauna. vent modern of diversity dispersal, and the origin, regarding vent model for hydrothermal hypotheses an biogeographic testing excellent barnacles represent deep-sea distribution, discontinuous but widespread their Due to biodiversity. driving factors identify biogeograp evolutionary and of The characterization Abstract Biology Department, Woods Hole Oceanographic Institution, 266 Woods Hole Road, Woods Hole, MA Woods Hole, Road, Hole Institution,266Woods Woods Hole Biology Oceanographic Department, Instituteof Biogeosciences, Japan for Mari Agency Massachusetts Instituteof Technology,77Massachusetts Avenue, Cambridge,MA USA 02139, : Phylogeography of Deep-Sea Vent Barnacles Barnacles Vent Phylogeography Deep-Sea : of 1,2 , Hiromi, Watanabe 3 , Timothy M.Shank , Timothy ne-Earth Science and Technology, Yokosuka, Kanagawa, Kanagawa, Yokosuka, Technology,ne-Earth Science and of radiation, place of radiation, of to isoffundamentalimportance patterns hical 2

origin, mode of dispersal, modeofdispersal, origin, Accepted Article The characterization of evolutionary and biogeo evolutionary and of Thecharacterization Introduction which effectsassess to with ofanth potential Th diversity. faunal deep-sea roleshaping important an have played and taxa marinemany of other patterns distribution current explain canhere discussed mechanisms dispersal and processes geological the that wesuggest Thus, extinctions. mass and occurrencethe andarecorrelated with non-vent geological ofmajor species, taxa, events including deep-sea other for made recentinferences with consistent are and dispersal radiation origin, mode of vent of taxa. antiquity the and The of timing hypotheses classic rejects time radiation of The inferred Neogene. the during hemisphere eastward the southern through colonization followed by circumglobal vent lineage, of major the origin of place was the Pacific the western that suggest analyses aOur of ecosystems we radiation likely find barnaclesCenozoic. the during vent estimates, in at tw least vents hydrothermal colonized deep-sea This article This protectedis by Allcopyright. rights reserved. Due totheir (Van Dover2010). potentially lead tosignificant widespread losses ofbiodiversity havedi could effectsadditive mining tonatural ofeachgeophysicalthe nature particular syst at frequencyand the intensity ephemeral habitats, these to inherent disturbances natural tocope at ventsliving with adapted hydrothermal deep-sea have Van resources mineral 2010; Dover of exploiting Dover (Van these el fauna the with found connection and these areas its targetsindustrial forexploitation (Boschen massiveat deep-sea hydr deposits seafloor sulphide in (Thurber deep-sea the extraction resource incommercial interest increasing the isocean of patterns in This relevantinthe & the particularly (McClain light Mincks 2010). to identify the factors shapethat the taxa, ranges deep-sea of and ultimatelythat drive biodiversity et al. et ropogenic disturbances on deep-sea ecosystems. ecosystems. deep-sea on disturbances ropogenic em (Baker & German 2004). Thus, em Thus, from disturbance (Baker &German 2004). sturbances at unprecedented scales, which could could which scales, atunprecedented sturbances 2013). Understanding the biodiversity contained in contained in the 2013). biodiversity Understanding ice in history. Consistent preliminarywith Consistent inhistory. ice which these depending vary which occur can greatly on othermal ventfields has oneofthemain become ese results also results ese constitute critical data baseline sewhere is critical to assess the potential impacts potential the assess to iscritical sewhere graphical patterns is of fundamental importance importance isoffundamental graphical patterns

et al. et 2012). Although organisms organisms Although 2012). et al. et 2014). Mining of of Mining 2014). Accepted Article ribosomal genes and the histone and the genes ribosomal (Newman description awaiting several newspecies ventwith species, approximately barnacle 13 described (Newman barnacles) asacorn known (commonly 1818 Lamarck, are Hydrothermal ventbarnacles intofour grouped presently families belonging to the orders Lamarck, asstal 1818(commonly Pedunculata known Cubelio levels & trophic Tunnicliffe VanDover & Southward (Southward Newman 1998; 2002; 2004; energyof thro flow the and funnelling engineers Southward 2004;Marsh Southward & (Tunnicliffe per meters square high atoverindividuals highdensities 1500 biomass reach with areas oflo ventbarnacles inhabit Hydrothermal and arelike 2000), (Buckeridge KermadecArc 1998), Southward 2004), 2004), FijiNorth BasinSouthward (Desbruyeres et al. Mariana (Ohta1990), Okinawa Through &LonsdaleTrough (Hessler TalaudSangihe (Herrera 1991), (Ohno Izu-Ogasawara Arc 2005), (Southward Ridge Pacific-Antarctic 1993), Jones 1979; al. (Van Dover Ridge Indian Central the including (Fig. 1), arcs worldwide andisland systems of ridge most themajor spreading in fields These ventworldwide. ecosystems sea hydrothermal BarnaclesBurmeister, are (Cirripedia 1834) s ecosystems. deep-sea on disturbances anthropogenic of effects potential assess to which the hold barnacles therefore, fauna; chemosynthetic disp oforigin, patterns biogeographic historical an vent (Fig. distribution modelfor represent excellent barnacles 1), hypothesestesting regarding the This article This protectedis by Allcopyright. rights reserved. 2011), 2011), East Scotia Ridge (Rogers 2010;Shank et al. et etal. 2007; Rogers 2007; 2006). A molecular phylogenetic study of the Cirripedia, employing nuclear employing Cirripedia, nuclear the study of 2006).Amolecular phylogenetic et al. et 2010), Manus Basin (Tufar 1990), Edison Seamount (Tunnicliffe & Seamount (Tunnicliffe Edison 1990), (Tufar Manus Basin 2010), et al. et etal. 2012), 2012), playing vent key in communities asroles micro-habitat H3 2012; Reid 2012; etal. gene, indicates that these morphologically-based taxonomic morphologically-based these gene, indicates that et al. et 2001;Nakamura 2012), northern and southern East Pacific Rise (Newman (Newman Rise EastPacific and southern northern 2012), etal. ersal, and current diversity of modern deep-seamodern of diversity current ersal, and w-temperature diffuse fluid flow. Populations can Populations flow. fluid diffuse w-temperature ugh ecosystems from ughprimar ecosystems et al. et 2013). 2013). ome of the most conspicuous organisms in deep- in organisms conspicuous ome most ofthe promise ofproviding critical baseline data with sessile can be found in active canin found vent be crustaceans sessile ly to be present in other unexplored areas. areas. unexplored other in be present ly to ked or gooseneck barnacles) and Sessilia barnacles) gooseneck or ked 1994), Lau Basin (Southward & Newman (Southward Lau 1994), Basin et al. et 2012), Southwest Indian Ridge (Tao Ridge Indian Southwest 2012),

et al. et 2006). There are are There 2006). y producers tohigher et al. et 1996), 1996), et et

Accepted Article ( I subunit gene c the cytochrome region, oxidase one mitochondrial We markers. genetic targeted significantl studies by phylogenetic previous and diversity, evolutionary genetic study,global weaimtocharacterize In the this on build to was approach vents. Our hydrothermal deep-sea barnacles from of history biogeographic 2011). (Moalic Ocean Indian the and Rise Pacific East the Pacific, Northeast the with paths) exchange edgeconnect exclusive vents,hydrothermal having of the formodern Pacific western fauna with position associated ancestral a possible hypothesizes 2011; Herrera 2011; and marine ofterrestrial groups in organisms, a biodiversity considered Pacific isalso western and centre place theis considered possible (Newman oftheir distribution oforigin which Pacific, western isthe richness) species as (measured species barnacle chemosynthetic putative of diversity highest of the region Byfar, speciation. mechanism of an important as discontinuity (Watanabe scales these to appear species andsignifican vent barnacles arc neighbouring and back-arc putative basins, Many variability of markers examined to date. among morphos relationships due thepaucity study. the Furthermore, remains anopenquestion oftaxonomic to in that sampling origin a single of possibility the however, the Cretaceous; in originated likely that clade monophyletic These results, together with those from(Linse incongru and thus polyphyletic are groupings This article This protectedis by Allcopyright. rights reserved. from 18vent worldwide. fields We also performed ofthe described seven eight out genera, representing for 94individuals, completeobtaining sequences coxI ), ), 28S, and two nuclearand the histone gene the large sub-unit regions, ribosomal etal. 2012). In a similar way, a recent biogeographic analyses using network theory network 2012). using analyses way,aIn recent asimilar biogeographic et al. et 2005). Together these observations suggest a suggest Together of role 2005). habitat observations these pecies of vent barnacles also remain unresolved due to the low the remaindue low ofventto unresolved also pecies barnacles y andnumberof sampling expanding taxonomic the ent with evolutionary history with ent (Pérez-Losada cluding deep-sea taxa (Cairns 2007; deep-sea cluding Carpenter2007; taxa (Cairns et al. et hotspot and potential place of origin of many modern manyof modern origin of place potential and hotspot ions (indicating faunal similarity possible faunal ions possible (indicating similarity restriction-site associated DNA sequencing (RAD- DNA sequencing associated restriction-site 2013), also suggest that vent barnacles forma 2013), suggest also that t population structure has also been found at at been has found also structure population t be restricted to particular ridge systems and and systems ridge particular to be restricted

et al. et H3 2006). The etal. gene, et al. et 2008). 2008). etal.

Accepted Article nuclear markers (histone nuclear markers (histone the and Arc, Trough. Okinawa (Bonin) Izu-Ogasawara Manus Basin, Tonga Arc, LauMariana Basin, Arc, Ridge, Kermadec EastScotia Trough, the Ridge, Indian Southwest Rise, Individuals the were collectedCentral from Indian Ridge, East Pacific Rise, southern East Pacific as reference. species descriptions and stereo-microscopy using vents deep-sea hydrothermal Methods & Materials hypotheses. them tocurrentmorphospecies ofvent and andcompare boundaries barnacleand colonization (4) worldwide; species identify taxa dispersal of patterns historical (3)infer time; geologic in vent barnacles radiation of and origin of time evolutionary of barnaclessingle origin) vents; hydrothermal (2)infer fromdeep-sea the place and from species. each we:(1)testthe hypothesisseq) onindividuals Here (i.e., ofmonophyly identified a This article This protectedis by Allcopyright. rights reserved. pH 8.0), (3) precipitating nucleic acids with 100% ethanol (1:1), and (4) washing the precipitate twice twice washing and(4) precipitate the ethanol (1:1), precipitating100% with acids nucleic pH 8.0), (3) chloroform: alcohol chloroform: (24:1) isoamyl Trisand phenol: isoamyl alcohol (25:24:1, buffered at and A/T1 for with proteinase with acids K RNAse CTAB buffer (2) 1 hour, nucleic separating laboratoryMolecular methods Sanger-basedmarkers. traditional of analyses species-tree from obtained topologies tocompare to a tree,and infer be used could phylogenetic backbonerobust barnacle ofthevent to a S1) eachfrom genome-wide obtain to markers (Table selected individuals morphospecies that of set (http://www.ncbi.nlm.nih.gov/genbank/ GenBank in and ) theincluded analysesS2). (Table Supero the from sequences Additional individual. Partial DNA sequences of one mitochondrial (cytochrome c oxidase subunit I)andtwo c subunit oxidase (cytochrome mitochondrial one of DNA sequences Partial S1)from on94barnacle (Table performed were specimens identifications Morphological Total genomic DNA was extracted from tissue samples by: (1) digesting the tissue in 2 X 2 in (1)digesting by: tissue the samples extracted tissue from Total genomic DNA was Associated Restriction-site H3 gene 28S) and were large sub-unit the generated ribosomal for each DNA sequencing (RAD-seq) (Baird (RAD-seq) DNA sequencing rder Darwin, 1854 were retrieved from from were 1854 retrieved rder Darwin, Thoracica

et al. et 2008) was performed on on wasperformed 2008) Accepted Article sequencer. anHi-Seq 2000 onaIllumina multiplex of single lane (http://ww.genomesize.com) for barnaclesgenome size were from the obtained Genome Size Database in the model, genome ofthe composition observed frequency ofthe recognitionSbfI seque (Tabl barnacle athoracican genome of the in times fidelity SbfI restriction enzyme, is predic which ahigh- with digested DNA from produced were libraries Individual sequencing. RAD and preparation were assembled and edited using GENEIOUS v6.1.6 (Drummond v6.1.6 using GENEIOUS edited and assembled were chromatograms Laboratory.Complementary Biological Marine the of Center Paul Bay Josephine the analyzer using DNA a3730xl was completed sequencing was done Subsequentpurification protocols. manufacturer CarlsBad, following CA) v3.1 BigDye Terminator kit (Life Corp., technologies Cycle-sequen protocols. manufacturer’s following min at72 cycles (35 for stepof The denaturation an with reactions carried out were 5 initial contamination. 94 at min (Folmer (Colgan & H3R H3F for 28S, (Whiting 2002) 28S_B & this study) in (designed CGTGAAGCTGCCAVTATGG-3’ 5’- 28SF_330 were: amplifications MgCl mM 2.0 mM(Promega), 1X Buffer XBSA, mM dNTPs1.0 Flexi each), (0.25 enzymes: 2.5 GoTaq volumefinal of25 a to were prepared markers Sanger-based traditional of Polymerase 70% reactions ethanol. chain with This article This protectedis by Allcopyright. rights reserved. 2 , 1U , Taq (GoTaq,polymerase Promega), and 0.3 etal. Concentration-normalized genomic DNA was submitted to Floragenex Inc. for library library Inc. for Floragenex to genomicDNA submitted was Concentration-normalized ° C. PCR products were cleaned using the Mi the using cleaned were products PCR C. 1994) for coxI ) of 60s at 94 μ l (1 coxI μ l of template)resulting in the followin . . Negative controls were included in every experiment to test for testfor to inevery were experiment included . controls Negative Barcode tags were 10-base pairs long. Libraries were sequenced by 96- weresequenced Libraries Barcode pairs long. tags were 10-base ° C, 90s C, at 48 through isopropanol precipitation. Automated precipitation. isopropanol through ° ted to cut approximately between 5,000 and 15,000 5,000 ted tocutapproximately between C, and 90 s at 72 and 90sat 72 C, nce, and its probability using a trinucleotide a using probability trinucleotide andits nce, cing reactions reactions were cing theperformed using ABI e S3). This predicted range was obtained the Thispredictedusing range obtained was e S3). Daphnia pulex Daphnia et al. et nElute PCR Purification Kit (Qiagen)Purification nElute PCR μ (Life technologies Corp., CarlsBad, CA) at CA) CarlsBad, Corp., technologies (Life M of each primer. Primer pairs used for used for pairs Primer each M of primer. 1998) for

g final concentrations ofreagentsand ° et al. et C, and a final elongation step of 10 10 step of elongation final and a C, (Herrera H3 2011). 2011). , and , & LCO1490 HC02198 etal. 2014). Ranges of Ranges of 2014). ° C, 32 C, Accepted Article variation among partitions. Nucleotide frequencies, substitution rates, site shape and invariant amonggamma frequencies, substitution Nucleotide partitions. variation vary account undera distribution to frequencies wereallowed for the to prior Dirichlet flat rate State evolution. modelsof selected their with along independently concatenated region, each generations (burn-in = 25%). Combined analyses explicit were performed with character partitions for Monte (4chains) (MCMC) wereCarlo generations Criterion (Table S4). Information Markov of200million Four Bayesian independent analyses Chain (http://www.phylo.org), for a first-pass phylogenetic inference using the maximum likelihood likelihood for using the maximum phylogenetic inference a first-pass (http://www.phylo.org), v3.3 Gateway Science the CIPRES in asimplemented 2006), (Stamatakis v8.0 HPC2 Phylogenetic Inferences region. eachcoding for evaluated wasalso partitions codon in Saturation transversions. and transitions of number the against model) (K80 distances genetic by plotting and 2013), (Xia v5.3.48 test(Xia Xia the implementing by evaluated was sequences (Lopez unlikely was nuclearpseudogenes of amplification indicating wereidentified, substitutions suspicious framesor (Drummond v6.1.6 GENEIOUS acids in of alignments all mistakes, offset value= for 0.07) protein and possible Tocorrect coding ribosomal regions, respectively. (Katoh selection model and analysis saturation Alignments, This article This protectedis by Allcopyright. rights reserved. (Darriba v2.0 JMODELTEST region using gene each non-saturated for wereselected substitution nucleotide of CIPRESv3.3. Gateway the Science Models in implemented (Ronquist v3.2.2 MRBAYES in datasets these with was performed inference Bayesian through estimation Phylogenetic dataset. concatenated the in markers included were the three prog inthis analysed also was dataset concatenated Branch was by 500bootstrap assessed optimality criterion. support replicates. A Thoracica-wide et al. et Non-saturated datasets from individual Sanger-based markers were analysed in RAxML- Sanger-based analysed markersin datasets were fromindividual Non-saturated Eachset sequencesof forSanger-based ma 2002), andQ-INS- the employing 2002), G-INS-i et al. et 1994; Bensasson 1994; protein coding sequences were visually inspected and translated to amino toamino and visually translated coding inspected were protein sequences etal. et al. 2011). No unusual stop codons, misplaced reading stop 2011). misplaced reading codons, No unusual 2001). Possible substitution saturation in the DNA the in saturation substitution Possible 2001). run with a runwith sampling of 20 frequency thousand ram. Only outgroups with data for at least two of at least two for data with outgroups Only ram. rkers was aligned independently usingMAFFT aligned independently rkers was i algorithms (gap opening penalty= 1.53, 1.53, penalty= opening (gap algorithms i et al. et

as2003), inDAMBE implemented et al. et 2012), following the the following 2012), etal. 2012), as 2012), Accepted Article sizes (EES) (>200), in addition to high correlations be to addition sizes (>200), in (EES) scale reduction potential frequencies (<0.01), partition by runs was supported among independent Convergence each concatenated region, along with their independently selected models of evolution. Three fossil Three fossil evolution. modelsof selected their with independently along concatenated region, each as tree 2009) weretested (Stadler model sampling taxonomic rates with incomplete andextinction speciation and constant 2008), Birth-Death the (Gernhard model rates and extinction (Yule constant speciation the 1925), extinction no Birth-Death uncorrelated relaxed lognormalmolecular clock model. an assuming by allowed was branches among rates in mutation Variation dataset. markers based Sanger- concatenated Thoracica-wide the using v3.3, Gateway Science CIPRES the in implemented of out through the carried was a Bayesian-MCMC Time-calibration hypothesis phylogenetic estimation of joint phylogeny inBEAST (Drummond and v1.7.5 times divergence Estimations Time Divergence (<0.01). frequencies of partition deviation standard bythe diagnosed was runs among generations, foreachmillion ot model.topology All mill 100 of runs independent two in performed was (Fan method stepping-stone the through estimated was model each topology for likelihood marginal The dataset. concatenated non-mono of hypothesis andthe alternative hypothesis vents Totest thatbarnacles the fromdeep-sea a form hydrothermal hypothesis monophyletic comparison ( we performed group, factor a Bayes Testing Hypothesis Topological eachparameter. of traces distribution posterior (Rambaut & Drummond 2007) 2007) and AWTY(Rambaut & Drummond (http assumed forall other parameters. MCMC runs were analysed with the programs TRACER v1.5 part across unlinked parameters were proportion This article This protectedis by Allcopyright. rights reserved.

et al. et 2011; Xie et al. et itions. Default prior distribution settings Default priordistribution were itions. ://ceb.csit.fsu.edu/awty) (Nylander ://ceb.csit.fsu.edu/awty) Kass & Raftery 1995) between this topological topological & between Raftery Kass 1995) this 2011) in MRBAYES using 50 steps. The estimation estimation The steps. MRBAYES 50 usingin 2011) priors. partitions character Unlinked were setfor ion generations, with ion a diagnostic with frequency generations, 1 of her parameters were set to default. herConvergence parameters default. setto were tween runs and the flat shapes of the stationary shapesof flat and the runs tween phyly ofthegroup using the Thoracica-wide factors (PSRF) (ca. effective and sample1.00), The Yule constant speciation rate model rate and model speciation The Yuleconstant observed values of standard deviation of deviation ofstandard values observed

et al. et etal. 2012), 2012), as 2008). Accepted Article (see Results section) were carried out using data from the Sanger-based markers in the program the program in markers the Sanger-based from data using out carried were section) estimation Results species-trees of (see analyses Bayesian Species Estimation Tree processes. coalescent and intraspecific processes speciation–extinction interspecific between points bran in increases identifying by boundaries species fromhttp://r-forge.r-proj SPLITSR-package (available Monaghan we section), Results A inClade (see barnacles vent method for (Pons likelihood (GMYC) boundaries Yule-coalescent mixed identifyemployed generalized species To Species Delimitation above. as described tree wereinference th to weremapped locations sampling geographical (Lemey nodes ancestral for location geographic states of discrete character of reconstruction a Bayesian in performed dispersal Toinferhistoricalof patterns Biogeography Historical (Drummond median with tree credibility maximum clade Mostabove. probable tree TRACER as mentioned diagnostics frequencyConvergence of20thousand. MCMC parameters. Three analyses were independent other all settings withmean values were assumed prior for my.25 Default of distribution distributions exponential prior assuming Fossil ages constraints aslowerboundary were used hypothesis. Linse Pérez-Losada by studies the from wereselected C7) and C2, (C1, points calibration This article This protectedis by Allcopyright. rights reserved. et al. et etal. (2013) based on well-supported topological congruencies with our phylogenetic phylogenetic our with congruencies topological well-supported based on (2013) et al. et et al. et 2009; Fujisawa & Barraclough 2013), with a single threshold, as implemented in the the in asimplemented a single with threshold, 2013), 2009;FujisawaBarraclough & 2012). 2009) using BEAST v1.7.5 (Heled 2010). & (Heled In v1.7.5 BEASTusing the 2009) this framework, Drummond node heights using TREEANNOTATOR v1.7.1 v1.7.1 using TREEANNOTATOR node heights deep-sea hydrothermal vent barnacle lineages, we barnaclelineages, vent deep-seahydrothermal s, after 25% after25% a were s, burn-in, into summarized ching rates that are characteristic of transition transition of characteristic are that rates ching etime-scaled phylogenetic tree. Parametersfor were examined were forthe combined runs in for vent barnacle species identified A Clade in identified vent species for barnacle run for 200 million generations with a generations 200million with sampling run for ect.org/ projects/splits/). Thisect.org/ method estimates projects/splits/).

et al. et et al. 2006; (2008) and (2008) Accepted Article individuals to identify to loci individuals performed theusing programwas wasRAD-tagsand enabled betweenAdditionaland filtering, clustering (-r). the within algorithm of 15% window of sliding the read were (-w (-r). The0.15) discarded length rescue and barcodes a within 10) 10(-s score below an quality average with Reads (-c). discarded were bases ambiguous 90bp 90) from(-t Readsto adapters wereexcluded truncated each readandwas with length and quality control loci clustering RAD-seq data above. as described were inference size population constanttree model. parameters root Other for and linear aWeassumed piecewise evolution. of models selected independently their with along marker, for each wereset tree partitions and clock, character, Unlinked analyses. delimitation species the after defined Species were 2008). on the hypothesis & evolutionary concatenation barnacles Edwards ofthese Edwards(Brito 2008; evaluate effects and the to ofgene- andgene heterogeneity, processes tree coalescent evolutionary 2010).Thisappr *BEAST &Drummond (Heled v1.7.5 This article This protectedis by Allcopyright. rights reserved. a with model substitution DNA time-reversible ageneralized We v8.0. assumed RAxML-HPC2 taxon andminimum pa coverage threshold clustering RAD Phylogenetics matrices. sites a set to3 (p 3)polymorphic locusin was shared of The maximumnumber 10). and 8, 6, 4, (m explored were locus given a in coverage taxon and 0.90). 0.85 0.80, were thresholds (c explored coverageabuild required to cluster was As 5(d5). Hipp in below score aquality bases with 33 more than from the package STACKS v1.19 (Catchen v1.19 STACKS package the from Phylogenetic inferences of RAD-seq matrices, built with built matrices, Phylogenetic inferences ofRAD-seq and quality de-multiplexed were Sequence reads et al. . . combinedRAD-seq Loci wereconcatenated into 20 were also discarded. The 20 were of also discarded. depth minimum 2011; Catchen Similarly, fourdifferent values for the minimum rameters (asabove), outlined were carried in out oach was employed to take into account account into take to employed oach was filtered with filtered program with the process_radtags et al. py

RAD v2.01 (Eaton 2014). Reads with with v2.01Reads 2014). (Eaton RAD etal. py (2014), (2014), clustering three different RAD under each combination of RAD undereach combination 2013). Barcodes and Illumina and Illumina Barcodes 2013). Accepted Article number of loci number of loci per individual asth was higher the As X 13X). expected, (SD approximately 54 was locus coverage per of depth The average predictedfor and 30,000, between 10,000 RAD-tags, This per number individual. 18,000, expected 4 Xwas iscongruent the approximately with number of thenumberofR with high-integrity DNA, individuals In steps. filtering quality retained after wereall reads, of roughly 78% 546,846), (SD individual reads per 712,306 of average An electrophoresis. gel agarose by as S6), determined DNA (Table degraded notably had samples some as integrity DNA of degrees different of the product largely was yield in sequencing variability NCBI.great of The Archive (SRA) Read attheSequence stored are species representing barnacle vent werein from13individuals the obtained RAD-seq datasets study S1). averageread An (Table this of843,541 published phylogenetic hypotheses forthe Thoracica hypotheses (Pérez-Losada published phylogenetic topologies The material). electronic supplementary a (Fig. generated and resolved phylogeny 2, overall better-supported dataset concatenated electroni 80)(supplementary < support bootstrap < 80, probability branches posterior (i.e. supported poorly showed all yet degree resolution the of in phylogenetic inferences markerproduced congruent thatvaried mostly trees eachfrom Sanger-based (Table Little saturationS5). was all other found in partitions. H3 foreach sequence lengths Approximate markerwere bpfor 700 28S, 657 bp for numbers). accession for S1 see Table (NCBI, Information Biotechnology for Center National of specimens Results replicates. by wasbootstrap 500 assessed support Branch across rate sites. variation gamma-distributed This article This protectedis by Allcopyright. rights reserved. . Xia tests indicated substantial saturation at the Thoracica-wide level third codon positions of positions of codon third Thoracica-widesaturationat the level substantial Xia tests indicated . Complete Sanger-based marker datasetswere obtained for all 94 individuals, exceptfor 2 osheai. osheai. Vulcanolepas Sequences are stored at the GenBank database of the U.S. U.S. the of the are at database stored GenBank Sequences e clustering threshold was (Table S7). e larger threshold clustering c material). Analyses of the Thoracica-wide Thoracica-wide the Analyses of c material). s (SD 589,377) were obtained per individual. Reads perindividual. wereobtained (SD 589,377) s of theseof trees were with congruent previously aSbfI (Table barnacle, using enzyme the S3). AD-tag loci with depth of coveragedepth greaterthan AD-tagwith loci

Maximum likelihood and Bayesian and Bayesian Maximum likelihood

et al. et 2008; Linse 2008; coxI, coxI, et al. et and bpforand 327 2013). 2013). coxI

Accepted Article respect to a well-supported and well-resolved clade made up by up made clade well-resolved and well-supported a to respect history with in earlier appearhave to diverged tolineages that belong Arc, Mariana and Basin, Lau Vulcanolepas Vulcanolepas of aparatype sub-clade includes latter The Arc. and theKermadec as(identified from the Izu-Og individuals onegrouping clades, Ashinkailepas The rest of support. the genus Family genera A inClade . The the to belong orderlineages of inCladetheir A;however, dive placement. and the within lack ofsupport resolution phylogenetic later group prevents anunambiguous CladeB 2013). Eochionelasmus Neolepas ) Yamaguchi taxon to the predominantly cl thepredominantly taxon deep-sea to Brachylepadomorpha) genera the contains Clade A 2): (Fig. are (posterior two well-supported pr divided into Phylogenetic Inferences data. based Sanger- with produced trees the with congruent and werelargely resampling, bootstrap by indicated fromthe RAD-seqdatase obtained trees Phylogenetic This article This protectedis by Allcopyright. rights reserved. Neobrachylepas Neobrachylepas Analyses of Sanger-based markers revealed that barnacles from deep-sea hydrothermal vents deep-sea hydrothermal from barnacles that revealed markers Sanger-based of Analyses Newman, 1979 Scalpellomorpha); an 1979 (Suborder Newman, el al. el al. Ashinkailepas seepiophila Ashinkailepas nor belongs to the earliest divergent lineage in family the in (Fig. 3),andcontains lineage theearliest divergent belongs sub- two to Eochionelasmus Yamaguchi, 1990 (Suborder Balanomorpha). Cl Balanomorpha). 1990 (Suborder Yamaguchi, 2004 (Suborder Scalpellomorpha) (Suborder 2004 , Vulcanolepas , Neolepas Neolepas , Neoverruca , and is monophyletic. The is monophyletic. Neoverruca Neoverruca is associated with the paraphyletic Balanomorpha group, however the however the group, the Balanomorpha paraphyletic with associated is Southward & Jones, 2003 (Suborder (Suborder Scalpellomorpha) & 2003 Jones, Southward Newman, 1989 (Suborder (Suborder Verrucomorpha) 1989 Newman, Neobrachylepas Neobrachylepas ade oftheScalpellidae (Pérez-Losada ), and the second grouping individuals from the Lau Basin, Lau Basin, from the individuals grouping second and the ), appear as the extant divergent extant representatives as ofthe earliest appear the obability = bootstrap 1, support > 99)main clades rgence is unclear due to lack of strong branch strong of lack due to unclear is rgence asawara (Bonin) Arc and the Trough Okinawa asawara (Bonin) Vulcanolepas/Leucolepas Vulcanolepas/Leucolepas , Leucolepas , ts were completely resolved, highly as werecompletely supported ts resolved, Newman & Yamaguchi, 1995 (Suborder (Suborder 1995 & Yamaguchi, Newman d Clade B was restricted to the genus genus the to restricted was B d Clade

N. zevinae/rapanuii N. zevinae/rapanuii Southward & Jones, 2003 (Suborder & (Suborder Jones, Southward 2003 ade A is well supported as the sister sister asthe supported well A is ade A. kermadecensis. A. from the Kermadec Arc, Arc, Kermadec the from et al. et , Ashinkailepas , Ashinkailepas 2008; Linse 2008; from the East from the Neither genus Neither , and etal.

Accepted Article Pacific Rise and its sister sub-clade of of sub-clade sister its and Rise Pacific This article This protectedis by Allcopyright. rights reserved. occurred 1.7 Myr BP (95% HPD: 0.4-3.8) under BD and 2.3 (95% HPD: 0.5-4.9) under Yule. The underYule. 0.5-4.9) HPD: (95% 2.3 and under BD 0.4-3.8) HPD: (95% Myr BP occurred 1.7 between the Ridgeand theIndian Scotia The Ocean lineages East split models, Yule respectively. a 2.3-8.5) HPD: (95% BP Myr4.8 have occurred andnon-Pacific Pacific Divergence between and 13.8 HPD: 15.4-53.7) Myr HPD: 12.1-43.3) and Myr(95% underBD, HPD:10.5 and 31.2BP Myr 5.4-17.3) (95% BP (95% of Eolepadidae and the Myr HPD:79.3 underBP 47.1-121.5) (95% the Yule modelofconstantrate. The speciation TMRCAs and models(BD) the under Birth-Death 38.2-105.9) [HPD]: Interval Density Highest Posterior (95% estimate Awas Clade of (TMRCA) common ancestor recent themost to The time 1). < (log-likelihood models among encountered were differences score; undertheBirth-Death significant obtained however, no likelihood model hadthebest Linse from estimates with consistent are estimates divergence These material). models(Fig. electronic 2and supplementary prior theBirth-Death under estimates obtained identical estimates divergence produced dataset Sanger-based combined the of Treetime calibrations whencompared the with nearly of constant speciation, the Yule under treeolder prior slightly History andBiogeographic Divergence Estimates as barnacles ofvent against monophyly the difference 5)(Kass (> log-likelihoods & in Raft and The -16928.21 (marginal -16908.62 respectively). non-monophyly large log-likelihoods thanthe hypothesis of less probable wassignificantly monophyly of hypothesis the test showedthat Thetopological vents. hydrothermal deep-sea from barnacles of themonophyly support dataset based Testing Hypothesis Topological Indian from the and Ridge. Southwest Central None ofthephylogenetic hypotheses inferred from Sanger- concatenated the Thoracica-wide Neolepas-Vulcanolepas-Leucolepas Neolepas-Vulcanolepas-Leucolepas BP (95% HPD: under 7.5-23.1) model, theHPD: Yule BP (95% respectively.

V. scotiaensis scotiaensis V. originally suggested by originally suggested Pérez-Losada Neolepas-Vulcanolepas ery 1995) constitutes strong contradictory evidence evidence contradictory strong constitutes ery 1995) nd 6.4 Myr BP (95% HPD: 3.0-11.2) under nd 6.4BD under Myrand HPD:3.0-11.2) BP(95% from the East Scotia Ridge and and Ridge Scotia East the from d years million d at 68.0 (Myr before present BP) sub-clade were estimated at 25.1 Myr BP BP Myr 25.1 at estimated were sub-clade

eolepadids was estimated to to estimated was eolepadids et al. et al. et (2013). The (2013). tree Neolepas Neolepas (2008). sp. 1 1 sp. Accepted Article GMYC model showed a significant ( a showed significant GMYC model The (Fig. modeltree the 3). prior Birth-Death with estimated phylogeny combined Sanger-based speciation– interspecific between point atransition identified A Clade of analyses GMYC time-scaled the for BP Myr 0.6 at processes coalescent intraspecific and processes extinction Species Delimitation of and Ocean Ocean Indian thethe south Southern 3). (Fig. Atlantic, in westernthe and Pacific, thelate during Miocenecolonized the –Pliocene Eastern Pacific, the hydrothermal that with ventbarnacles fromClade high-probability A originated suggest biogeography historical of Yule. analysis under The 1.3-8.8) HPD: Myr and 4.2 BP(95% model Birth-Death B Clade of TMRCA This article This protectedis by Allcopyright. rights reserved. of Mariana andbasally the positions Trough, among of lineages relationships values (Fig. equal 4). branch support aremostly of topology the with congruent fully is tree species Sanger-based inferred the dataset andthe markers of withtheconcatenated Sanger-based obtained Thetopology thehypothesis phylogenetic Species Estimation Tree ranged between 0and 0.9%. distance was 0.9%).Similarly inClade B ranged between (except 2andthe 17.8% for two (Table Genetic S8). ranged between 0and0.9% di ( distances Genetic new be to species. alr tospecies largelywhich corresponded identified, 25.9 = ratio (likelihood rates branching coalescent

Eochionelasmus

Vulcanolepas/Leucolepas Vulcanolepas/Leucolepas α was estimated at 3.2 3.2 Myr estimated at HPD: BP(95% was underthe1.1-6.7) coxI coxI 0.05)better= fit to the model ofuniform datathanthenull Eochionelasmus uncorrected distances) among individuals within clusters clusters individuals among distances) within uncorrected Neoverruca Neoverruca Poorly-resolved regions of the tree include the the include tree the of regions Poorly-resolved Neolepas zevinae/rapanuii Neolepas zevinae/rapanuii , p < , p< There 0.0001). were stances among individuals from different clusters clusters different from among individuals stances eady described or populations that were that presumed eady orpopulations described and and the genetic distances among individuals among individuals distances the genetic from the Kermadec Arc, Laufrom the Kermadec Arc, Basin, Neobrachylepas Neobrachylepas

12 distinct clusters clusters distinct 12 clusters whose maximum within Clade A. within Accepted Article

This article This protectedis by Allcopyright. rights reserved. al. ventstwicehydrothermal at least in history. Th s thus 2), (Fig. B) Clade and A (Clade clades main there are two that dataset indicate aexpanded of significantly analyses Our barnacles. vent and Arc, of andtheKermadec Basin of clades the supporting branches cases, the these In m10. of parameter coverage taxon a minimum with generated matrices from theones for except branches, all for we topologies trees RAD-based Ridge. Indian Central Rise, Pacific by madeup the clade to sister as trees RAD-based the in appearing tree Sanger-basedobtained with markers (Fig. 4),only differing in the of position material). Theseelectronic topologies from RAD-seq (supplementary each wereto other eachmatrix identical of inferences from phylogenetic obtained and valuesvalularger ofclustering thresholds - %and2.26- 13.18 4. matrices rangedbetween 6.81 The ofvari S9fordetails). see Table percentages and and 52 600 %, loci, %, 44 3,800, 33 and 21 form % 6, missing8,% 4, and data, 10respectively; parameter value forthe used taxon minimum coverage numbers similar contained oflociandsi 0.90) Pérez-Losada the with consistent not is barnacles as vent propos hydrothermal ancestry) (single of monophyly history of evolutionary hypothesis Theinferred Are vent monophyletic? barnacles Discussion respectively. and 71, 94 than greater values support have bootstrap RAD Phylogenetics Phylogenetics RAD (2014) provide support to this inference (2014) to Clade provide support this B placing by ( RAD-seq matrices resulting from resulting thematrices from RAD-seq three explor V. scotiaensis scotiaensis V. et al. et (2008) and Linse (2008) and from the East Scotia Ridge and Ridge Scotia theEast from etal. Leucolepas-N. zevinae/rapanuii Leucolepas-N. (2013), which (2013), of included only two the four families of uggesting that barnacles have colonized deep-sea barnaclescolonized haveuggesting that milar percentages of missing data per clustering per milar percentagesclustering ofmissing data es of minimum taxon coverage. The tree topologies tree topologies The taxon coverage. es ofminimum e results from a concurrente study byPerez-Losada results able sites and parsimony informative sites across across sites informative parsimony and sites able ed by the smaller taxon-sampling studies of studies taxon-sampling the smaller ed by 22 %respectively, smaller being higher with re highly supported with bootstrap bootstrap values of100 highly with re supported matrices were also mostly similar to the species thespecies to similar mostly also were matrices in a given locus (approximately 15,500, 9,600, 9,600, 15,500, (approximately locus agiven in ed clustering thresholds (c 0.80, 0.85ed and (c thresholds clustering 0.80, Neolepas Neolepas

Eochionelasmus Eochionelasmus N. zevinae/rapanuii zevinae/rapanuii N. - sp. 1fromthe Southwest sp. and V. scotiaensis- Neolepas Neolepas scotiaensis- V. Vulcanolepas ohtai) nestedwithin ohtai) Leucolepas from theLau from the East , sp. 1 1 sp. et Accepted Article the Balanomorpha, the phylogenetic position of position the phylogenetic the Balanomorpha, ohtai Perez-Losada contains only genus the 1995). Clade B & barnacles (Newman ‘primitive’ Yamaguchi sessilian of thelineage most considered of placement example incongruence is thephylogenetic noteworthy ofthis taxonomic and phylogenetic (Pérez-Losada hypotheses phylogenetic molecular multilocus of in Cirripedia light in need tobe define to taxonomic groups revised the higher characters used observati state.This isaderived Neobrachylepadidae Losada of TheClade sisterrelationship A and the 1989)). early during & Hessler have ontogenesis (Newman a stage barnacles neoverrucid that pedunculated (note (Fig. 4) forms Neobrachylepadidae) and (Neoverrucidae sessile and (Eolepadidae) pedunculate (Neoverruci asymmetric including morphologies, thebarnaclevent inthis seven genera Deep-sea A is themore containing diverse included hydrothermal ofthetwo, Clade six of (Yang decapods (Lorion mussels e.g. taxa, other asof aswell barnacles, of lineages by multiple (Shock history earth’s geologic throughout environments chemosynthetic vent of persistence Thewidespread 2007). allows the presence of organisms with specialized adaptations (Tunnicliffe adaptations specialized with organisms of presence the allows thatfilter only act and organisms environmental challenges as an and ecological to physiological (VanDove supply and food oxygen chemicals, extremepatchyand ephemeral with habitats spatia areasexplicitly tested ventenvironments inthat study. characterized hydrothermal Deep-sea being not was barnacles vent of monophyly of hypothesis the although barnacles, balanomorph the This article This protectedis by Allcopyright. rights reserved. N. relica relica N. within the balanomorphs. This instability is likely caused by to the long branch supporting this supporting branch the long to by caused likely is instability This balanomorphs. the within et al. et nested in Clade A Clade in nested 2008; Linse 2008; et al. et et al. et etal. (2014) forfound low support the branchesresolving the position 1995) has likely been an important factor enabling the independent colonization 1995) colonization enablingfactor an theindependent has been important likely 2013). Eochionelasmus et al. . N. relica. N. 2013) suggests that the sessile state of the Neoverrucidae and Neoverrucidae the state of sessile the that suggests 2013) study. This clade also contains a remarkable diversity of study.Thiscladealso contains a of remarkable diversity . Despite its morphological and phylogenetic affinities with affinities with morphological andphylogenetic its . Despite is the nowwas sole species andbrachilepadoform living until r 2000). These conditions present significant present significant r These 2000). conditions Eochionelasmus dae) and symmetric (Neobrachylepadidae), dae) andsymmetric (Neobrachylepadidae), deep-sea pedunculate (Fig. 2)(Pérez- Scalpellidae pedunculate deep-sea l and temporal gradients of temperature, gradients l andtemporal reduced of on is consistent the mountingwith evidence that et al. et

is this study is unstable. Similarly, 2008; Linse 2008; et al. et etal. et al. et 2003; Fisher 2003; 2013) 2013) and Eochionelasmus Eochionelasmus 2013). 2013). A etal.

Accepted Article also the region where the oldest lineages and the highest diversity are found. The heterogeneity of of heterogeneity The arefound. diversity highest the and lineages oldest the where region the also by andhighly Pacific,supported asindicated western Harnik Rogers 2000; (Jacobs & Lindberg 1998; epochboundary Paleocene-Eocene the at and period Cretaceous the during occurred also changes and temperature acidification eventsocean to Events, Anoxic Oceanic regional extinction linked Horne 1982; 1999;Harnik boundary (Raup& Sepkoski period the Cretaceous-Paleogene event during extinction deep-sea a mass the inferencewith of major &Smith deep-sea taxa (Little 2003; StrugnellVrijenhoek Stockley 2005; Arecent (2013). and Vrijenhoek of (2003) and radiation modern vent origin most taxa and other many Little and by BP;alsosee Vrijenhoek at ca. reviews Myr 30 yeti starting of kiwaid crabs radiation (Chevaldonne systematics for use character of review (Fig.electronic material). supplementary Furthe 2, evolutionaryundersampling rate, taxonomic divergence, old a rapid or clade, whichmay indicate This article This protectedis by Allcopyright. rights reserved. bresiliid shrimp 6.7-11.7 Myr (Shank6.7-11.7 BP shrimp bresiliid of and radiation in origin of radiation e.g., other estimates taxa, to the comparable chemosynthetic byLinsedescribed as phylogenetic inthe because placement uncertainty of points calibration fossil of exclusion Pérez-Losada from the results with The Newmandiscrepancy 1985). 1979; ba hydrothermal-vent that proposed taxa, which vent byPérez-Losada proposed origin Cretaceous the Linse byinferred timing with isconsistent study this in inferred in lived barnacles common ancestor the to from Clade recent A likely time most The hydrothermal-vent Cenozoic. of early or ancestor lateMesozoic the The recent most common Biogeography Historical The most probable place of origin of modern vent barnacle lineage from CladeA the vent of modern from lineage is barnacle origin of Theplace mostprobable et al. et etal. 2002); radiation of chemosynthetic mussels at ca. 45 Myr BP (Lorion Myr BP 45 ca. at mussels chemosyntheticof radiation 2002); (2013). (2013). The timing of radiation ofClade A during the Cenozoic is should helpresolv should et al. et et al. 1999); origin of siboglinid ca.60Myr of 1999); BP siboglinid tubeworms origin et al. et (2008) and with classic hypotheses of antiquity of of and antiquity (2008) classicof with hypotheses r taxonomic ofrelated sampling genera andcareful et al. et rnacles were mid-Mesozoic relict taxa (Newman taxa relict (Newman mid-Mesozoic were rnacles 2012). 2012). Bayesian ancestral state reconstruction. This is This state reconstruction. ancestral Bayesian et al. et (2013), withthelower contrasts but e its systematics. systematics. e its 2012) (see Fig. 3). Several smaller-scale 2012) Several smaller-scale (see Fig. 3).

et al. et et al. et (2008) (2008) is duetothe 2008) is consistent consistent is 2008) et al. et 2013); 2013); Accepted Article portion of this largelymajor mid-oceansouthern remains unexplored. ridge at Mid At been have found barnacle species vent western the South RidgeIndian and Central Indian Ridge duringthePliocene/Pleistocene epochs. No reaching the late epoch, during Miocene Arc Sandwich and South Ridge to Scotia followed East the hemisphere likely southern the through A the Juan spreading along Ridge. Pacific de Fuca vents whichcanexplain BP (Fig.1989), 3)(Atwater Ridgeby Pacific-Farallon of the disruption Oligocene the to posterior is that point acoalescence Rise have Pacific East the from Neolepadids The along the Pacific-Antarcti vent hydrothermal habitats following of the was Pacific western a epoch, theMiocenepossibly during eastward migration (Moalic diversification the re-colonization driving factors ofvent as andsubsequent important environments been suggested and seeps ascold such ecosystems chemosynthetic wes the systemsin vent hydrothermal in depths This article This protectedis by Allcopyright. rights reserved. hypothesis proposed by Moalic by proposed hypothesis membranacea dogfish spiny octocoral the as such taxa hemisphere epoch ha Miocene the during southern thethroughout andcolonization eastward bynorthwestern Pacific followed the western or migration (see Roterman Ridge West andthe South Indian Ridge Scotia tothe East Pacific the from eastern dispersal historical of pattern anidentical almost share ) Anomura: ventyeti (Decapoda: crabs Hydrothermal mid Mioceneof formation the Scotia East Rise, (ACC), Current Circumpolar the eastward-flowing Antarctic epoch, Eocene the mid during Passage The proposed history ofdispersal history The congruenproposed is Squalus acanthias Squalus (Schwaninger 2008). These observations provide support 2008). These for support provide (Schwaninger biogeographic observations the et al. al. et (2013) for an detailed hypothesis of vicari of hypothesis detailed an (2013) for etal. Paragorgia arborea Paragorgia 2011). Our the analyses 2011).Our suggest that et al. et (Verissimo (2011) that the western Pacific was a centre of origin of modern modern of origin of centre wasa Pacific the western that (2011) ca. 41 41 Myr the lateEoceneBP of establishment (Scher 2006), subduction under theNorth subduction American Plate, et al. et (Herrera why barnacles are absent from the north-eastern thenorth-eastern from absent are why barnacles tern Pacific,tern and the close proximitytoother ca. lantic Ridge hydrothermal vents, although the lantic vents, although Ridge hydrothermal 2010) and the bryozoan s also been inferred for other non-vent deep-sea deep-sea non-vent for other beeninferred also s organic enrichments, both shallow and deep, shallow have both enrichments, organic 15 Myr BP (Livermore 2003) (see Fig. 3). 3). Fig. (see 2003) (Livermore Myr BP 15 c Ridge, and colonization of the eastern Pacific. Pacific. oftheeastern colonization and Ridge, c t with thet with ofopening ofthetiming Drake etal. ca. ca. 2012), and other 2012), marine taxasuch asthe

ance in this ance this group). Alikely origin in in 34 Myr BP (Scher 2006), andthe Myr 2006), 34 BP (Scher most probable path of dispersal out out pathofdispersal probable most Membranipora Membranipora ca. ca. 28.5 Myr28.5 Accepted Article ca. 2 %are clusters of species among distance minimum and 0.9 %, speciesthe clusters of within distance genetic maximum The the species clusters. for in distances genetic inferred isa There overlap lack of byBuckeridge defined asgenera andNeolepas Leucolepas inclusive proposed ofthe by Mariana arc, (Bachraty boundary the with biogeographic largely consistent Inferences of species boundaries in Clade A, based on the generalized mixed Yule-coalescent mixedYule-coalescent onthegeneralized based Clade A, in boundaries ofspecies Inferences Relationships and Delimitation Species of the evolution provideof Clade greater resolution B. paquensis other from data group. Additional this in undersampling (Fig. 2).However thisinferred antiquity is likely tobe anartefact caused by taxonomic presented here hypothesis phylogenetic The constrained. well not is B Clade the lineage era Balanomorpha occurred the Mesozoic of in divergence ofthis the that within suggests Thehistory diversity faunal deep-sea role shaping manyan of patterns marine taxaandhaveextant other played important distribution current explainthe can here discussed mechanisms and dispersal processes geological the that suggest We of ACC. the the direction hemisphere inthe following dominant stone modeofdispersal southern rather a but Ocean, the Indian stepping- and Pacific communities between links western direct the idea of the support not datado our However, globally. taxa most dispersed from which fauna vent This article This protectedis by Allcopyright. rights reserved. in among congeners Divergences 4). and 3 (Figs. arc basins island andneighbouring system spreading ridge by geographically are mid-ocean well-constrained clusters of descriptions with arecongruent largelymethod, 2 % to define species boundaries through DNA barcoding in Crustacea (Hebert Crustacea DNAbarcoding in through 2 %to species boundaries define from the eastern Pacific, as well as aswell Pacific, from the from eastern species clusters remain contentious due to the non-monophylyduethe to three ofall remainclusters contentious species et al. et (2013) and thus require substantial revision. revision. substantial require thus and (2013)

putative morphospecies. The identified species The identified putative morphospecies. between the northwest and southwest Pacific, andsouthwest between northwest the confamilial speciesconfamilial andrelated groups would et al. et consistent with the proposed threshold value of threshold with proposed the consistent Echionelasmus Echionelasmus 2009). Relationships among coxI

barcode marker within barcodeand among markerwithin Ashinkailepas Ashinkailepas populations, e.g., e.g., populations, and and et al. et Neoverruca Vulcanolepas, Vulcanolepas, 2003; 2003; E. are Accepted Article difficulties inherent to the use inherent to in manydifficulties approaches taxa. oftraditional Jones 2013; (Wagner organisms non-model in phylogenetics for available and accessible increasingly resources made genomic have as RAD-seq, such and platforms methodologies, sequencing next-generation developmentsin and methodological Recent technological 2007). and identifying thousands of variable regions across the genome variable (Rokas genome across regions the of thousands and identifying individuals various from sequences DNA genomic large of numbers comparing by model organisms for in accounted and have problems been recognized Such comparative datasets. locus genome-wide oftheseare The effects others. among pseudogenes, poor genomic repr sampling, biased loci variability, sequence traditional few the of the use with associated been have uncertainty of low and ribosomal including genes), sources Several organisms (e.g., mitochondrial available fornon-model markers RAD phylogenetics available. regions become geographical explored from newly as specimens assignments enable species rapid will framework developed delimitation forCladeproposed A. The species to the species further delimitations support distance data provides evolutionary history of vent barnacles inferred with traditional Sanger-based markers, and allow the allow Sanger-basedand markers, traditional with vent barnacles inferred of history evolutionary and in crustaceans in general (Costa general in and incrustaceans ohtai Lefebure This article This protectedis by Allcopyright. rights reserved. consistently found in other barnacle taxa (Tsang taxa otherbarnacle in found consistently has been amongand The barcoding species gap within differentiation. indicative ofspecies-level not of division the that suggesting Rise, and southern East thus Rise Pacific se geographic or this split for support phylogenetic S8). There is0.9 (Table % is clusters no between where distance the maximum pair, cluster is 0.9%. The only exception to this pattern is found in the found in is pattern this to exception only The 0.9%. is In this study, we demonstrated that RAD-seq RAD-seq that study, we demonstrated In this et al. et et al. et 2006). Similarly, the maximum genetic distance among individuals of Similarly, individuals among distance genetic themaximum2006). 2013; Reitzel 2013; et al. et et al. et 2013), 2013), offering overcome athus greatto opportunity the 2007; Matzen Matzen 2007; da Silva et al. et gregation between specimens from the East Pacific EastPacific the from between specimens gregation esentation, low statistical power, and inclusion of inclusion and power, statistical low esentation, often often hard to duetotheidentify paucity multi- of 2008; Tsang 2008; data strong support for provide the overall

Neolepas zevinae/rapanuii Neolepas zevinae/rapanuii etal. et al. et 2011), 2011), thus our etal. Neolepas zevinae/rapanuii Neolepas 2009; Yoshida 2009; et al. et 2003;Clark 2012; Eaton&Ree 2012; Echionelasmus Echionelasmus coxI et al. et et al. et species species genetic genetic 2011)

is Accepted Article for Exploringfor Planetsprogram oftheNational based on generalized mixed Yule-coalescent methods and DNA barcoding are largely congruent with congruent with largely are DNA barcoding and methods Yule-coalescent mixed based generalized on the hemisphere during thesouthern along eastward ofof origin the major hydrothermal colonization byvent barnacle lineage, circumglobal followed the place was Pacific western the that suggest analyses Our events. extinction major of occurrence the timing the with consistent Cenozoic, late the during The likelygroup. ventbarnaclemonophyletic hydrothermal in of timing ecosystems radiation was Conclusions in known poorly taxa such as invertebrates. deep-sea inference validation phylogenetic ofcomparative theutility demonstrates Sanger-bas clarify be ofthe the validity currently needed genera.revisionto would described This study does a have sampling Further taxonomic implications. and morphological follow-up taxonomic regardinghistor of the conclusions biogeographical the anyalter not trees does RAD-seq andthe markers Sanger-based with tree species inferred between the topology in difference small The tree. phylogenetic supported and resolved afully of inference This article This protectedis by Allcopyright. rights reserved. Science of the National (PLR- Foundation (OCE-1 Foundation Science National the of Sciences of Ocean TS); the to Division (NA09OAR4320129 Administration and OceanicAtmospheric National the of Research and Ocean Exploration of Office bythe supported was research This Acknowledgements ondeep-seaecosystems. effects disturbances anthropogenic of potential consti also results These tree. phylogenetic barnacle ventthe of backbone resolved a fully and Sanger-based markers from inferred history evolutionary of puta descriptions morphological Phylogenetic

inferences and topologyand indicateinferences tests that tive species. RAD-seq data provide strong support for for support overall the strong data provide RAD-seq species. tive 0739675 Aeronautics and Space Administration (NNX09AB76G (NNX09AB76G and SpaceAeronautics Administration ed and RAD sequencing as a means of comparative ofcomparative as a means RAD and sequencing ed Neogene period. Inferences of species boundaries ofspecies Inferences boundaries Neogene period. of other specific deep-sea taxa, ofand correlatedto specific deep-sea other taxa, toTS); the Astrobiology and TechnologyScience tute critical baseline da 131620 to to TS);131620 the Division of PolarPrograms y or species delimitation of vent barnacles, but

hydrothermal vent barnacles are not a arenot vent barnacles hydrothermal ta with which to assess to which with ta Accepted Article Bensasson D, Zhang D, Hartl D, Hewitt G (2001) Mitochondrial pseudogenes: evolution's misplaced misplaced evolution's pseudogenes: Mitochondrial G (2001) Hewitt D, Hartl Zhang D, D, Bensasson On the hydrothermal Baker ET, Germanof distribution vent CR global (2004) fields. Baird P,N, Etter Atwood T hydrothermal among relationships Biogeographic D (2009) Desbruyeres P, Legrende C, Bachraty 2005 NOAA Ring of Fire Expedition, part of NIWA’s Seamount Program (FRST CO1X0508). Program (FRST Seamount NIWA’s of part Expedition, Fire of NOAA Ring 2005 fu communities (CO1X0906), vulnerable deep-sea on use resource of Impact NewZealand; by LandInformation funded 20/20 Survey Ocean WHOI; and Science (GNS), and Geological Nuclear of Institute University, Auckland (MBIE), Employment ofBusiness, by& programs:Minerals, Zealand Ministry theInnovation New funded Kermadec Arc research under collected were (NIWA) Research Water of Atmospheric and Institute by and the National provided Specimens TN234,YK06-13, TN236, TAN1206, YK09-13. TAN1104, TAN1007, RR1211, NT99-09, NT97-14, NT97-10, KOK0506, KOK0505, KM0912, KM0417, JC067, JC042, AT07-06, AT03-28, agencies expeditions of and funding personnel, scientific crew, themasters, Lilley (UW), M. (UNH), K.VonDamm (Harvard), Langmuir C. (UH), P. Fryer R. Embl (NOAA-PMEL), Resing J. (NIWA), S. Mills (NIWA), K.Schnabel (NIWA), Rowden A. (NIWA), M. Clark (Oxford), A. Rogers (NOCS), Copley J. (NOCS), Tyler P. Nakagawa (JAMSTEC), S. Tokyo), Suzuki (U. Y. Tokyo), (U. K.Iizasa we thank key specimens to access For enabling TMSandSH). grant to (internal Institution support TMS) (Fellowship to and the Life Woodsthe Oceanographic Institute Hole Ocean of Institute Office (Ocean Academic Programs the and TS); to This article This protectedis by Allcopyright. rights reserved. America. In: North Pacific andwestern of northeast the history tectonic Plate T (1989) Atwater References this manuscript. improved and helpful Tarrant,comments A.Reitzel, thank for providing A. three that anonymous reviewers, witnesses. witnesses. Oceans between and the Lithosphere Ridges:Interactions Hydrothermal RAD markers. 1371-1378. scale. faunas onaglobal vent Colorado. Boulder, America, Society of Geological OceanHawaii and Pacific Eastern

Trends in Ecology & inEcology Evolution Trends Plos One Plos

, et al.

3 (2008) Rapid SNP discovery and genetic mapping using sequenced sequenced SNP and genetic(2008) usingRapid mapping discovery , 3376. 3376. , ey (NOAA-PMEL), A. Reysenbach (PSU), M.K. Tivey (WHOI), (WHOI), Tivey M.K. (PSU), A. Reysenbach ey (NOAA-PMEL), Deep-Sea Research Part I-Oceanographic Research Papers Research I-Oceanographic Part Research Deep-Sea (eds.Winterer EL, Hussong DM, Decker RW), pp. 21-72.

nded by MBIE; andtheJointNewZealand-USA Ventures Fund to Fund theOcean SH), to Exploration Ventures 16 , 314-321.

148 , 245-266. Mid-Ocean Mid-Ocean

We We The

56 , Accepted Article barnacle (Eolepadidae: Neolepadinae) from hydrothermal vents in the Scotia Sea, Antarctica. fromhydrothermal inthe vents Sea, Scotia Antarctica. Neolepadinae) barnacle (Eolepadidae: Cubelio SS, Tsuchida S, Watanabe S (2007) Vent associated associated Vent S (2007) Watanabe S, Tsuchida SS, Cubelio Costa FO, deWaard JR, Boutillier J Buckeridge JS, Linse K, Jackson JA (2013) (2000) J Buckeridge Colgan DJ, McLauchlan A, Wilson GDF Wilson A, McLauchlan DJ, Colgan DR EisenSmith MB, AG, Clark Lutz P, Desbruyeres Jollivet RA D,Chevaldonne D, W, P,Cresko JM,Amores A, Hohenlohe Catchen an corals: Deep-water overview with S (2007) Cairns Fan Y, Wu R, Chen MH, Kuo L, Lewis PO (2011) Choosing among partition Wumodels L,Bayesian in among Choosing Fan MH, Lewis partition Y, (2011) PO Chen Kuo R, Eaton RH(2013) DAR, InferringRee phylogenyand analyses. phylogenetic for loci novo RADseq de of assembly (2014) PyRAD: DA Eaton Brito Edwards SV(2008) PH, Multilocus phyl Brito Catchen J, Hohenlohe PA, Bassham S, Amores A, Cr A, Amores S, Bassham PA, Hohenlohe J, Catchen Carpenter KE, Barber PH,Crandall ED Fisher CR, Takai K, Le Bris N (2007) Hydrothermal vent ecosystems. vent Hydrothermal ecosystems. LeN Takai(2007) Bris K, CR, Fisher IsEdwards aSV (2008) new and general D, MA, Xie Suchard AJ, Drummond Boschen RE, Rowden AA, Clark MR, Gardner JPA JPA Gardner MR, Clark AA, Rowden RE, Boschen This article This protectedis by Allcopyright. rights reserved. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of amplification DNA primers for R(1994) W, Lutz R, Vrijenhoek M, Hoeh O,Black Folmer Desbruyeres D, Alayse-Danet A-M, Ohta S, the S, A-M, Ohta Alayse-Danet D, Desbruyeres PosadaR, D(2012) Doallo GL, D, Taboada Darriba Drummond BA,S A, Drummond B the case of the Crustacea. Crustacea. the of case the and molecular evolution. molecular evolution. and arthropod Loci genotyping de novofrom short-read sequences. scalpelliform Research Freshwater Ocean. Pacific south-west Caldera, the Brothers from phylogeny. phylogeny. COI clock calibration. mitochondrial worms (Ampharetidae vent hydrothermal Pacific genomics.population corals. scleractinian deep-water 3745 phylogenetics. phylogenetics. 19. (Pedicularis: from flowering plants Orobanchaceae). Bioinformatics BEAST 1.7. Triangle and implications Triangle for marine management. andimplications markers. 67. regulatory frameworks and management strategies. mining, from impacts benthic communities, their deposits, the reviewA of sulfides: http://www.geneious.com/ newspecies. one Seamoun from Brothers Galatheidae) Molecular Marine Biology andBiotechnology Marine Molecular I cytochrome from diverse mitochondrial coxidase invertebrates. subunit metazoan Basins): Composition, microdistribution and foodweb. and microdistribution Composition, Basins): Lau and Fiji North (the back-arc basins Southwestern Pacific in communities hydrothermal computing. parallel , 551-568. 551-568. , Genetica Nature Neolepas osheai Neolepas osheai Molecular Biology and Evolution and Molecular Biology . Molecular Biology and Evolution and Biology Molecular , et al. , et Journal of Biology Journal Crustacean

135 450 Nat Methods Nat

34 Molecular Ecology Molecular (2011) Geneious v5.4. Available from from (2011)Available Geneious v5.4. , 439-455. , 203-218. 203-218. , , et al. , et , 409-418. 409-418. , Canadian Journal of Fisheries and Aquatic Sciences andAquatic Fisheries of Journal Canadian , etal. Rambaut A (2012) Bayesian phylogenetics with BEAUti and the with A (2012)Bayesian Rambaut phylogenetics sp. nov., a new deep-sea vent barnacle (Cirripedia: Pedunculata) Pedunculata) (Cirripedia: barnacle vent deep-sea a new nov., sp. (2007) Evolution of (2007) the Evolution genes and genomes on of , etal. Bulletin of Marine Science Marine of Bulletin . , et , et al.

theory of molecular systematics emerging? emerging? systematics molecular of theory 9 (2007) Biological identifications through DNA barcodes: through DNA barcodes: identifications Biological (2007) , 772. , Australian Journal of Zoology of Journal Australian Vulcanolepas scotiaensis Vulcanolepas t, Kermadec Arc, Southwest Pacific, with description of of description with Pacific, Southwest Arc, Kermadec t, Cahiers de Biologie Marine deCahiers Biologie (2011) Comparative phylogeography of the Coral Coral the of phylogeography Comparative (2011) ogeography and phylogenetics using sequence-based sequence-based using andphylogenetics ogeography (1998) Histone H3 and U2 snRNA DNA sequences DNA (1998) H3andU2snRNA Histone sequences Scientific Parties ofbi Scientific

Postlethwait JH (2011) Stacks: building and building Stacks: JH (2011) Postlethwait 22 , Vrijenhoek RC (2002) Sister-species of eastern Sister-species of (2002) RC Vrijenhoek , jModelTest 2: more models, new heuristics and new more models, 2: heuristics jModelTest (2013) Mining ofdeep , 3124-3140. 3124-3140. , special reference to diversity and distribution of referencespecial todiversity and distribution esko WA set Stacks: ananalysis tool esko for (2013) introgression using data:AnexampleRADseq introgression

3 29 , 294-299. 294-299. ,

, Alvinellidae, Vestimentifera) provide Vestimentifera) new Alvinellidae, , 27 28 , 1969-1973. 1969-1973. , Journal of Marine Biology Marine of Journal , 513-519. , 513-519. Ocean and Coastal Management Coastal and Ocean , 523-532. 523-532. , Munidopsis Systematic Biology Systematic G3 New Zealand Journal of of Marine and Journal New Zealand

Marine Geology Marine

1 , 171-182. , 171-182.

81 sp. nov., a new deep-sea deep-sea a new sp. nov., , 311-322. , 311-322. olauand s (1994) Deep-seaolauand s (1994) Oceanography (Decapoda : Anomura : :Anomura (Decapoda

43

46 -sea seafloor massive seafloor -sea , 367-370. , , 419-437. 419-437. ,

. 116 , 227-242. , Evolution

20

Drosophila 2011

64 , 14-23. , 14-23. , 272-295. , , 1-14. , 1-14.

84 Zootaxa

63 , 54-

, 1-

Accepted Article Linse K, Jackson JA, Fitzcharles E, Sands CJ, CJ, E, Sands Fitzcharles JA, Jackson Linse K, Lopez JV, Yuhki N, Masuda R, Modi W, O'Brien N, Masuda Modi R, Yuhki Lopez JV, Are RC(2003) hydr CTS,Vrijenhoek Little SuchardMA DrummondAJ, A, Lemey Rambaut ( P, (2006) between Relationship morphological J taxonomy M, Gouy Lefebure CJ, Douady T, Gilbert Livermore R (2003) Back-arc spreading and ma and Back-arcspreading Livermore (2003) R (2002) T K,Miyata Kuma K,MisawaK, Katoh Lorion J, KielS,Faure J, B Lorion Herrera Spatial a S,ShankTM, Sanchez (2012) JA ShankTMGen (2014) PH, Herrera S,Reyes-Herrera Herrera S, Munro C, Nganro N Bayesian infere DrummondAJ (2010) Heled J, JR SL, Ball deWaard A, PDN,Cywinska Hebert LotzeHarnik PG, HK, Anderson SC Gernhard T reconstructed(2008) The process.conditioned single-locus theGeneralized Fujisawa species Delimiting using data T, Barraclough and (2013) TG This article This protectedis by Allcopyright. rights reserved. Hessler RR, Lonsdale PF (1991) Biogeography of Mariana Trough hydrothermal vent communities. Biogeography PF Lonsdale communities. ofMariana (1991) vent Trough Hessler hydrothermal RR, Hipp AL, Eaton DAR, Cavender-Bares AL,J Cavender-Bares Hipp DAR, Eaton Jacobs DK, Lindberg DR (1998) Oxygen DR(1998) and evolut Lindberg DK, Jacobs the for deep- of antiquity implications modes phanerozoic: the of Oceancirculation DJ (1999) Horne Jones JC, Fan S, Franchini P, Schartl Fan P, M, MeyerJones Franchini Schartl JC, S, A(2013) The evolutionary ofXiphophorus history Thor Anew Jones (1993) DS Neolepas (Cirripedia: Kass RE, Raftery AE AE Raftery RE, Kass Oceanographic Research Papers Research Oceanographic Antarctic Scalpelliformes (Crustacea:Thoracica). Cirripedia: Biology PLoS Computational London Special London Publications. processes andmagmatic tectonic systems: subduction Evolution &amp; delimitation. species help to threshold amolecular of Proposal Crustacea: within divergence molecular and Fourier alignment basedonfast transform. 795. Molecular Evolution Molecular of DNAto mitochondrial thedomestic genome nuclear cat. the of amplification Proceedings of the Royal Society B: Biological Sciences SocietyB: Biological Royal the of Proceedings widespread antitropical deep-sea coral coral deep-sea antitropical widespread life. of tree eukaryotic the across Abstracts Meeting AGU Fall expedition. 2010 theINDEX-SATAL from Observations Indonesia: region, Sangihe-Talaud Biology andEvolution barcodes. Ecology & Evolution 778. Biology Systematic onsimulateddata A and evaluation revisedsets. method approach: Mixed Coalescent Yule Deep-Sea Research Part a-Oceanographic Research Papers Research a-Oceanographic Part Research Deep-Sea 6067. clade based on sequenced RAD data. data. RAD sequenced on clade based vent, southeast Pacific. Pacific. southeast vent, America States OfThe Of Sciences United of deep-sea faunas. recruitment and recent trends invertebrates. sea benthonic associated DNA sequencing. sequencing. DNA associated site- restriction using approach sexually a genome-wide sword: and their selected fish Proceedings of Sciences the of B: Royal Society Biological Proceedings Molecular Phylogenetics and Evolution and Phylogenetics Molecular (1995) Bayes factors. Bayes factors. (1995) , et al. , et

62

39 27 , 707-724.

, et al. 27 18 (2013) Adaptive radiation of chemosymbiotic deep-seamussels. ofAdaptive chemosymbiotic (2013) radiation Bulletin of Marine Science Marine of Bulletin , 174-190., , 608-617. , 608-617. , 570-580. , 570-580. , 582-588. , , et , et al. Crustaceana

(2010) Biodiversity of the deep-sea benthic fauna in the the in fauna deep-seabenthic of the Biodiversity (2010)

1 Molecular Ecology Molecular 5 , 1234. , 1234. , e1000520. e1000520. , , etal.

bioRxiv 73 (2012) Extinctions in seas. in ancientandmodern Extinctions (2012) othermal vent living fossils? fossils? living animals vent othermal Journal of the American Statistical Association of theJournal Statistical American , 99-116. Plos One Buckeridge JS (2013) Phylogenetic position of position Phylogenetic Buckeridge JS (2013) (2014) A framework phylogeny of the of American phylogeny Oak A framework (2014) Paragorgia arborea Paragorgia nce of species trees nce ofspecies multilocus from data. ntle flow in the East Scotia Sea. In: the flow In: Sea.East Scotia in ntle MAFFT: a novel method for rapid multiple sequencemultiple forrapid MAFFT: method novel a (2003) Biological identifications through DNA through Biological identifications (2003) , 007781. , 007781.

72 SJ (1994) Numt, Numt, a (1994) recentSJ

nd temporal patterns ofgeneticin variation the 95 Nucleic Acids Research acica: Scalpellidae) an abyssal from acica: hydrothermal Scalpellidae) ionary patterns in the sea: Onshore /offshore /offshore inthe sea:Onshore ionary patterns , 999-1018. , ome-wide of predictability restriction sites 2009) Bayesian phylogeography finds its roots. roots. its finds Bayesian2009) phylogeography , 9396-9401. 9396-9401. ,

9

, e93975. , e93975. 22 Proceedings Of The National Academy Of OfThe National Proceedings

52 Journal of Biology JournalTheoretical of , 2986-3001. 2986-3001. ,

, 937. 937. , 40 , pp. 315-331. 315-331. Geological Society pp. of ,

, 435-447. 435-447. ,

280 . Molecular Ecology

, 20131243-20131243. , 20131243-20131243. 38 Deep-Sea Research Part I- Part Research Deep-Sea , 185-199. , 185-199.

270 transfer and and tandem transfer 30 , 3059-3066. , 3059-3066. , 313-321. 313-321. , Trends in Ecology Ecology in Trends Intra-oceanic

Journal of Journal 21 Molecular Molecular

253 Trends in , 6053- ,

90 , 773- , 769- , Accepted Article Rambaut A, Drummond AJ (2007) Tracer v1.4, Available from Available Tracer AJ (2007) A,Drummond v1.4, Rambaut Barraclough TG,Pons J, J Gomez-Zurita Reid WDK,Reid Wigham BD CJ, Sweeting (1982) in Mass JJJ the marine Raup DM, record. extinctions fossil Sepkoski Marsh L, Copley JT, Huvenne VAI Huvenne L,JT, Marsh Copley This article This protectedis by Allcopyright. rights reserved. Reitzel AM, Herrera S, Layden MJ, Martindale Reitzel AM, Herrera MQ, MJ, Martindale Shank Layden TM GoingS, where (2013) traditional Nakamura K, Watanabe H, Miyazaki J Miyazaki H, Watanabe K, Nakamura Duarte CM D, Y, Desbruyeres Moalic thedeep sea. ranges in The ofbiogeographic Mincks dynamics C, SL (2010) McClain Matzen da Silva J, Creer S, dos Santos A Newman WA (1985) The abyssal hydrothermal vent invertebrate fauna. A glimpse of antiquity? antiquity? of glimpse A fauna. ventinvertebrate hydrothermal abyssal The WA (1985) Newman Newman WA (1979) Monaghan MT, Wild R, Elliot M Newman WA, Yamaguchi T (1995) A new sessile barnacle (Cirripedia, Brachylepadomorpha) from from Brachylepadomorpha) barnacle (Cirripedia, A new T sessile (1995) WA, Yamaguchi Newman Sessilia): (Cirripedia; verrucomorphan A new hydrothermal RR abyssal (1989) WA, Hessler Newman Nylander JA, Wilgenbusch JC, JC, Warren DL,Nylander JA, Wilgenbusch Swofford DL (2008) AWTY a we system yet?): (are there Cirripedia. Crustacea, Arthropoda, Segonzac M AJ, (2006) Southward T, WA, Yamaguchi Newman Perez-Losada M, Hoeg JT, Simon-Blecher N M, JT, Perez-Losada Hoeg Pérez-Losada M, Harp M, Høeg JT Høeg M, M, Harp Pérez-Losada ventnortheastern the on community thehydrothermal of survey submersible S(1990) Deep-sea Ohta The VentCommunity Hydrothermal Y,SegawaS Fiujiwara J, (1996) Hashimoto Fujikura T, K, Ohno DNA taxonomy of undescribed insects. insects. undescribed of taxonomy DNA Phylogenet Evol trophodynamics. of vent webs:hydrothermal influences food http://beast.bio.ed.ac.uk/Tracer the Royal Society B: Biological Sciences Ocean. Southern the Ventsin hydrothermal invertebrate phylogeography and population genomics. population and invertebrate phylogeography gone and promisemarkers utility of have marine in forRADsequencing not before: chemosynthetic fauna on the Central Indian Ridge at 18 degrees -20 degrees S. -20 at18degrees Ridge degrees fauna on theCentralIndian chemosynthetic delineation. species of models coalescent-based communities. vent hydrothermal of history retracing the e19449. Malacostraca). inthe Decapoda mtDNACOI diversity (Crustacea: barcode from Bulletin of the Biological Society of Washington Societythe Biological of of Bulletin spring hydrothermal e32965. écologie animales écologie et A,Zoologie, biologie Section naturelle. d'histoire du Bulletin national Muséum theMiocene. record Tonga; Lau of since first a livingthe Basin, Back-Arc representative History the mostprimitive sessile living barnacle. morphological evolution of Balanomorpha). evolution morphological Sessilia: acorn barnaclesthe (Thoracica: Evolution and Phylogenetics Molecular 24 phylogenetics. Bayesian in convergence MCMCof exploration graphical for Denisia. 356-357. pp. M), Bright In: (JAMSTEC). Research slope ofthe Iheya Okinawa Trough. In: ridge, the (JAMSTEC) /Japan Marine Science Japan, pp. 221-229. Islands, nearSouth Ogasawara at theKaikata (Bonin) Seamount , 581-583. Handbook of Deep-Sea Hydrothermal Deep-Sea Vent Fauna Hydrothermal of Handbook

21 , pp. 145-156. 145-156. (JAMSTEC), pp. /Japan andMarine Center Science Technology , 259-273. , 259-273. A new scalpellid (Cirripedia) : a Mesozoic relic living near an abyssal abyssal an near living relic Mesozoic a : (Cirripedia) A new scalpellid

81 Plos One Plos

17 , 147-158. 147-158. , San Diego Society of Natural Natural of Society San Diego , 221-243. 221-243. , , et al. , et

, et al. 8 , et al. , e65553. e65553. , , et al. , et al. , et , etal. (2009) Accelerated species inventory on madagascar using madagascar on inventory Accelerated (2009) species , et al. , et , et al. , et (2008)The tempo barnacle evolution. andmode of (2012) Microdistribution of at faunal assemblages deep-sea (2011) Biogeography revisited with network theory: theory: network with (2011) Biogeography revisited , et al. (2012) Discovery (2012)Discovery (2013) Spatial (2013) differencesSpatial ridge east scotia in and Technology Center (JAMSTEC). Center and Technology . (2006) Sequence-based species delimitation the for delimitation (2006) species Sequence-based (2011) Systematic and evolutionary insights derived derived andevolutionary insights (2011) Systematic

Systematic Biology Systematic 46 , 10.1098/rspb.2010.1057. , 10.1098/rspb.2010.1057. Transactions of the San Diego Society of Natural DiegoSociety the of Natural of San Transactions , 328-346. (2014) Molecular phylogeny, systematics and (2014) Molecular phylogeny, systematics Plos One chemistry, microbiology and predation on predation and microbiology chemistry,

Systematic Biology Systematic 6 , 231-242. , 231-242. The Sixth Symposium on Symposium Deep-Sea on The Sixth History], San Diego, Calif. Calif. San Diego, History],

(eds. DesbruyeresSegonzac D, M, 7

, e48348. of new hydrothermal activity and activity of new hydrothermal Molecular Ecology Molecular Systematic Biology Systematic

55 , 595-609. , 595-609.

58 Science , 298-311.

22

61 215 Bioinfomatics , 2953-2970. 2953-2970. , Proceedings of of Proceedings , 127-137., Plos One , 1501-1503. , 1501-1503. Plos One Mol

7

, 6

, Accepted Article Schwaninger HR (2008) Global mitochondrial DNA phylogeography and biogeographic history of of the history and biogeographic DNA phylogeography mitochondrial Global HR(2008) Schwaninger Scher TimingScher climatic conseque HD (2006) and Roterman CN, Copley JT, Linse KT, Tyler PA, Rogers AD (2013) The biogeography of the yeti crabs yeti the of Thebiogeography (2013) AD Rogers PA, Tyler KT, Linse JT, Copley CN, Roterman toresolving approaches Genome-scale SB(2003) N,Carroll King BL, Williams A, Rokas DP PA, AD, Rogers Connelly Tyler Shank TM, Black MB, Halanych KM, Lutz RA, Vrijenhoek RC (1999) Miocene radiation of deep-sea Miocene LutzMB, (1999) radiation Shank TM, Vrijenhoek RC KM, Black Halanych RA, E S,Bors Herrera Shank T, Tsang LM, Chan BKK, Shih F-L, Chu KH, Allen Chen C (2009) Host-associated speciation in the in the speciation (2009) Host-associated C Chen Allen KH, F-L, Chu Shih BKK, LM, Chan Tsang T Wu B, Chan L, Tsang BE Sweetman Narayanaswamy AR, AK, Thurber analyseswith maximum likelihood-based phylogenetic Stamatakis ARAxML-VI-HPC: (2006) Shock EL, Mccollom T, Schulte MD (1995) Geochemical Constraints on Chemolithoautotrophic Chemolithoautotrophic on Constraints Geochemical MD(1995) Schulte T, EL, Mccollom Shock F, Teslenko vanderRonquist M, MarkP The roleRogers AD(2000) ofthe oceanic oxygen mini This article This protectedis by Allcopyright. rights reserved. Tao C, LinTao GuoS J, C, RogersJM, Strugnell Prodohl PA, AD, Collins M, Allcock The AL expressway: (2008) thermohaline Tufar W Modern activity, hydrothermal fo (1990) Smith AB, Stockley B (2005) The geological history of deep-sea colonization by echinoids: Stockley roles AB, Smith ofby echinoids: B(2005) The deep-seaof geological colonization history Southward A (2005) A Systematicsand(2005) ecologyofaSouthward Southward AJ, Newman WA (1998) Ectosymbiosis between filamentous sulphur and abacteria sulphur filamentous between Ectosymbiosis WA Newman AJ, (1998) Southward Stadler T (2009) On incomplete sampling under birth-death models and connections to the sampling- to models birth-death andconnections T under Stadler On (2009) sampling incomplete antitropically and longitudinally disjunct marine bryozoan marine bryozoan disjunct and longitudinally antitropically 428-430. Talaud region, Northern Sulawesi, Indonesia. Indonesia. Sulawesi, Northern region, Talaud Sangihe- the in relepresence via discovered communities chemosynthetic Triangle: Coral Evolution and Another cryptic marine species complex? sibling (Cheilostomata): provided by the deep sea. deep by the provided oxidase subunit oxidase I. subunit (Caridea:Bresiliidae vent hydrothermal shrimp Sciences SocietyB: Biological Royal the of Proceedings Anomura). notes onthe with (Kiwaidae) Chirostyloidea the (Decapoda: phylogeny of a andmodelchoice acrossinference large modelspace. phylogenies. molecular incongruence in e1001234. for the oceanbiogeography. andimplications southern in communities II Part Research Deep-Sea coral barnacle Oceans. malayensis Indian Ridge. Southwest oftaxa models. and mixed thousands Reactions in Hydrothermal Systems. Systems. Hydrothermal in Reactions Guinea). Guinea). New Sea,Papua (bismarck basin theManus back-arc in communities vent associated Ecology Molecular octopuses. origin deep-sea of Oceanfor asacentre Southern the Biological Sciences Biological ventilation. deep-water and surfaceproductivity 159. 38° S. Thoracica: Scalpellomorpha: Eolepadidae: Neole Cahiers de Biologie Marine Biologie de Cahiers Neolepadi (Scalpellomorpha, barnacle stalked based coalescent. coalescent. based Marine Biodiversity Marine Marine Ecology Ecology Series Progress Marine Mitteilungen der Oesterreichischen Geologischen Gesellschaft Geologischen derOesterreichischen Mitteilungen : postglacial colonization and recent Indian connectivity across the Pacific and colonization : postglacial , et al. , et

Wanella milleporae milleporae Wanella 49 , etal. , 893-908. 893-908. , Journal of Biology Theoretical of Journal (2011) First First (2011) active ve hydrothermal Molecular Phylogenetics and Evolution and Molecular Phylogenetics

, et al. , et

18 272 (2008) Population differentiation in the barnacle inthe differentiation Population (2008) , 1463-1475. , 1463-1475. , 865-869. 865-869. , Geology (2010) Hydrothermal vents and or and vents (2010)Hydrothermal Biogeosciences

35 47 , et al. , et

39 , 147-156. 147-156. , , 119-148. , 119-148. , 259-262. , 259-262. , et al. , et

(2012)The venthydrothermal discovery ofnew deep-sea 40 (Cirripedia: Pyrgomatidae) inhabiting the Milleporacoral. Pyrgomatidae)Milleporacoral. the inhabiting (Cirripedia: , 47-50. 47-50. , Origins of Life of and Evolution BiosphereLife the of Origins Bioinformatics (2012) MrBayes 3.2: Efficient Bayesian phylogenetic Bayesian (2012)phylogenetic Efficient MrBayes 3.2: nces of the opening of Drake Drake Passage. of opening the of nces Nature

, et , et al. 364 rmation of complex massive sulfide deposits and rmation ofcomplex deposits sulfide massive 11 new species of stalked barnacle (Cirripedia: (Cirripedia: barnacle stalked of species new , 3941-3963. , 3941-3963. , 107-118. 107-118. , AGU Fall Meeting Abstracts Meeting Fall AGU nae) from the Launae) from Tonga. the Back Arc Basin, ma in generating biodiversity in the deep sea. deepsea. the in biodiversity generating main ): evidence from mitochondrial cytochrome mitochondrial evidence from ):

(2014) Ecosystem function and services services and Ecosystem function (2014) 425 Proceedingsthe Society Royal of B:

padini) from thepadini) Pacific-Antarctic Ridge at 261 , 798-804. , 798-804.

22 , 58-66. , 58-66. nts on an ultraslow-spreading center: center: an nts on ultraslow-spreading

, 2688-2690. , 2688-2690. Systematic Biology Systematic

280

13 Membranipora membranacea Membranipora ganic falls in the heart of the of heart the in falls ganic , 20130718-20130718. , 20130718-20130718. , 244-254. 244-254. , Cladistics Molecular Phylogenetics PLoS biology Chthamalus

82

, 183-210. , 24 1 61 , 04. , 853-860. , , 539-542. , 539-542. Science

25

10

312 , 141- , , L. L. , Accepted Article Verissimo A, McDowell JR McDowell A, Verissimo WatanabeH, Tsuchida S, Fujikura K S Wittwer I, Keller CE, Wagner R (2013)On Vrijenhoek the instability and evolutionary age chemosynthetic ofdeep-sea J Ardron CR, Smith Van Dover CL, Van Dover Humphris SE, Fornari CL, D what is at risk? and biodiversity: massiveVan DoverMining sulphides (2010) CL seafloor Xia XH (2013) inmolecularbiology software package DAMBE5: for data analysis Xia XHAcomprehensive (2013) Z, saturation and Salemi X,Xie Xia M, L,its indexAn Chen Y Wang ofsubstitution (2003) and Mecoptera of and phylogeny genes multiple paraphyletic: is Mecoptera MFWhiting (2002) Van Dover CL (2002) Trophic relationships amongVan Dover Trophic CL (2002) i relationships Xie WG, Lewis PO, Fan Y, Kuo L,WG,Lewis FanY,Kuo Improving Xie PO, for Chen MH(2011) estimation likelihood marginal Yang JS,Lu B, Chen DF Van Dover C (2000) (2000) Dover C Van SouthwardAJ V, Tunnicliffe floor. deep-sea the environments of Reducing M(2003) SK, Sibuet Juniper V, Tunnicliffe This article This protectedis by Allcopyright. rights reserved. Yoshida R, Osawa M, Hirose M, Hirose E (2011) M, M, A newfenus E (2011) species Osawa Hirose ofPeltogastridae R, Yoshida Hirose newand two Yule GA (1925) A Yule GA(1925) mathematical ofevoluti theory SRP051026). number accession and Table (SRA S1) numbers SequenceArchive in Read (accession GenBank Nation U.S. at the available are Raw DNA sequences - Raw data are Dryad in files, tree alignments,available DOI: are input doi:10.5061/dryad.7kn5k files Nucleotide material electronic Supplementary - Accessibility Data Squalus acanthias Squalus sea. deep the in reserves vent activity inferred from structur genetic inferred activity vent adaptive radiation. of an boundaries resolution species unprecedented communities. 1651-1662. and evolution. and evolution. application. Siphonaptera. Series Ecology-Progress vents. Ocean hydrothermal Science ofMarine Journal Indian (Central Ridge). Bayesian phylogenetic model selection. selection. model phylogenetic Bayesian Princeton, N.J. the western Pacific and Indian Oceans. Oceans. Indian and Pacific western the 121-132. Papua off New seamount Guinea. longa (Ryukyu Archipelago, Japan), and their DNA-barcodes. DNA-barcodes. and their Japan), (Ryukyu Archipelago, OkinawaIsland from Crabs Hermit parasitizing Rhizocephala) Cirripedia: (Crustacea: Philosophical Transactions of the of Royal Transactions Philosophical (Cirripedia: Scalpellomorpha: Eolepadidae: Neolepadinae) inhabiting a volcanic Eolepadidae: Neolepadinae) inhabiting Scalpellomorpha: (Cirripedia:

Molecular Phylogenetics and Evolution Phylogenetics and Molecular The ecology of deep-sea hydrothermal vents hydrothermal deep-sea of ecology The Deep-Sea Research Part II Part Research Deep-Sea Zoologica Scripta Zoologica Molecular Biology and Evolution Biology Molecular and , etal. , a temperate shark with an antitropical distribution. , a an temperate shark with antitropical distribution. Molecular Ecology , Graves JE (2010) Global popula Global (2010) , GravesJE (2004) Growth and breeding of breeding of and (2004) Growth (2013) When invade decapods did Marine Biology , et al.

Marine Policy 288

68 Science , et , et al. , et , et al. , 233-240. , , 341-348. , 341-348. provide (2012) Genome-wide sequence data RAD

, et , et al. 31 (2012) Designating networks of chemosynthetic ecosystem ecosystem chemosynthetic of networks Designating (2012) Journal of the Marine Biological Association of the UK ofthe Association Biological Marine the of Journal (2005) Population history as history Population (2005)

, 93-104. , 93-104. 294 , n/a-n/a. n/a-n/a. , ofIndian and setting ecological (2001) Biogeography

Molecular Biology and Evolution and Biology Molecular , 818-823. , 818-823.

36 141 Systematic Biology Systematic Society ofLondonB Biological Sciences . on, based on the conclusions of Dr. J.C. Willis. Willis. J.C. Dr. on, of based ontheconclusions , 378-381. , 378-381. e ofneoverrucid barnaclesJapan. around , 761-772. , nvertebrates at vent nvertebrates the Kaireihydrothermal field al Center for Biotechnology Information (NCBI)Information Biotechnology for al Center

30 d relationships in the Lake Victoria cichlid cichlid LakeVictoria in the relationships d , 1720-1728. , 1720-1728.

26 tion structure of structure of tion dogfish spiny the a primitive stalked barnacle primitive stalked a

, 1-7. , Zoological Science Zoological Princeton University Princeton Press, hydrothermal vents? from vents? Clues hydrothermal

60 , 150-160. , sociated with hydrothermal with sociated Molecular Ecology

30

, 305-309. , 28 , 853-862. , 853-862.

Leucolepas 213 ICES Marine Marine , 21-87.,

19 84 , , Accepted Article HW. TMS and from comments paper with the SH andreagents. samples wrote collected and contributed TMS and HW data. SH, SHanalyzed research. SH research. performed anddesigned SH conceived Author Contributions This article This protectedis by Allcopyright. rights reserved. study).Red lines indicate active tectonicmargins (solid lines: spreading centers;dotted lines: subduction zones). vent barnaclesde havebeen 1. Figure Global distributionvent Globalof map hydrothermal barnacles.where Ovals indicatehydrothermal regions scribed (yellow: regions sampled not sampled blue: study; in regions inthis sampled this scribed regions (yellow:

Accepted Article

This article This protectedis by Allcopyright. rights reserved. Losada probabilities.Blue circles posterior labelsBranch show intervals. density posterior highest 95% the represent bars Node B. Clade vent hydrothermal indicates square vent Yellow CladeA. indicates hydrothermal square concatenatedthe Thoracica-wideRed forprior, dataset. 2. Figure etal. Maximum clade credibilityultrametric time-scaled 2008; 2008; Linse et al. 2013). * Indicates data generated in this study. study. this in generated data * Indicates 2013). in nodesindicatein calibration fossil points as (Pérez- in tree, generated under theBirth-Death model tree

Accepted Article This article This protectedis by Allcopyright. rights reserved. Figure 3. 3. Figure (Oli), Miocene(Oli), Pliocene (Mio), eras are indicatedhorizontal with bars: Cretaceous upper (U)), Paleocene (Cre Eocene(Pal), (Eoc), Oligocene theFarallon Pacific Ridge of FPR), formation (black, East the ScotiaRise Geologic (black, ESR). periods and establishmentofthe (black, DP), Circumpolar Drake ACC), disruption Current Antarctic of the Passage (black, Paleogene mass extinction (fuchsia, C-P), Paleocene- geologicglobalOAEr regional),events for OceanicCretaceous-forand in OAEg time: (red, Events Anoxic for the speciation-coalescent threshold for species delimitation Vertical (GYMC). dotted lines indicate important labelsshow posterior probabilities. Purple vertical dashed line indicates maximumthe likelihood-inferred time charts show theposterior probabilities of locationstates eachfor ancestral node (totalareapie =1). Branch in blue, eastern Pacific ingreen, Southern of the Ocean south Atlanticyellow andIndian in inorange. Ocean Pie generated theunder Birth-Death model. Clade A combined28S, (Pli), and Pleistocene (Ple). H3 , and , and Branchshow colours the most probable location states: western Pacific coxI maximumclade credibilityultrametric time-scaled tree Eocene Thermal Maximum (brown, P-ETM), opening of of opening P-ETM), (brown, Maximum Thermal Eocene

Accepted Article This article This protectedis by Allcopyright. rights reserved. in this study. supportvalues. Scale bar indicates substitutions per site 26,955 areparsimony This informative. matrixdata. missing 43.54% contains Branch bootstrap labels show ofcoverage Thismatrix6. nucleotide contains828,960 inof loci.sites 9,766 76,353 sitesare the variable and data.matrix Theused for obtainedtree wasthis witha clustering of0.85 threshold minimum and taxon 4. Figure topologiesyellow arecolored or red. with the same topologyas themaximum clade credibilityspecies tree are coloured in blue. Trees with different clade credibility species tree is shown with thicker bran in thespecies treeshigh with topologyagreement. Different colors represent different Thetopologies.maximum Top . of. the ofClaudogram distribution posterior species color densitytrees. isHigh of indicative areas Bottom . Maximum likelihood phylogenetic tree inferred with RAD-seq ches. Branch labels show posteriorprobabilities. Trees . Barnacle species images arefrom individuals included

Accepted Article Table S3. Table S2. Table S1. Information Supporting This article This protectedis by Allcopyright. rights reserved. known barnaclegenome sizesobtained from the AnimalSize Genome Database (http://ww.genomesize.com) the following generated were model offrequency and recognition sequences calculated probability basedon a genometrinucleotide composition obtained the from National CenterU.S. Biotech for Table S9. Table S8. Table S7. Table S6. Table S5. in jModeltest. Table S4. Predictionsofof number RAD-tags in Collection and sequence information for the specimens inused this study. Accession numbers RAD-seq matrices statistics genetic pairwise Uncorrected RAD clustering statistics. statistics. and filtering results sequencing RAD Xia from Results genetic marker. for eachsaturation test Sanger-based Nucleotide substitutionmodels for each Sanger-basedgenetic marker, as selected by the BIC criterion

for sequences from the SuperorderThoracica retrieved from GenBank

methodology described by Herrera coxI distancesamong (%) specimensA from Clade thoraciacan barnaclesusing SbfI. Data for nology Information (NCBI)WGS database. Observed

et al. (Herrera et al. et 2014). Data for Daphnia pulex Daphnia

.