New Results Preprint is of , the 2 cor- revi- spec- e 1901, 1994; Main, ossella ossella, of W and depend- R R formerly ossella common DOI here! Antarctic am R Molecular 1907). , of existence hexactinel- subantarctic suspension- need the München, was years endal, opsent, Ocean genus Janussen that species in including T T (Scolymastra), the Germany and markers. the a Frankfurt the -80333 localities, and within and ossella D flock of nuda phylogeny R shown Dorte ossella over , . Antarctic Antarctic 10, , -60325 (Kirkpatrick, rDNA) Southern R . München, The copyright holder for this preprint (which preprint this for holder copyright The 2 components D of some Str t unclear have supporting the ossella of species 25, A Anoxycalyx levis analysis (28S R (Barthel a . in -80333 are Geobiology agner systematics 2007). Göcke D Molecular Ocean W consulted. and Germany circum-Antarctic and and 10, dramatically ossella important a 1901, . and ossellidae): analyses 2005). R evidence nuclear Str R Gutt, components Richard- 1872 and al., ossellidae, , and , ossella and distributed ossellidae) R phylogenetic abundance München, shows R et authority key agner R Christian Senckenberganlage opsent, changed phylogeny W it , T provide alaeontology 1998; 1 P cbd COI) and Geology Southern are Carter 1910, Hexactinellida, group has -80333 ossella, relationships D phylogenetic and al., and abundant R and genera CC-BY-ND 4.0 International license International 4.0 CC-BY-ND Richard- where molecular 10, the et the . Sciences, a taxonomic genus are , Senckenberg, this version posted January 22, 2016. 2016. 22, January posted version this the McClintock racovitzae Str ; biomass in ossella yssacinosida: sponges, rDNA antarctica R the this of orifera, Dohrmann (L yssacinosida, Kirkpatrick, Ocean, München, P molecular L agner of of alaeontology by , & 1,3,4 size, monophyletic P provide (16S W 2007; § ossella Glass (Arnaud However of R Hexactinellida) sponges) ossella 1872 flock available under a under available we R , flock, Environmental Naturmuseum used as species Martin & content , Richard- Southern endal, Schulze , 1 However und (class (glass T Carter putatively monophyly the Collections Herein, Earth- namely Species a Antarctica, criteria remarkable Wörheide of and to argas species as species mitochondrial the State V fibulata communities. tlantic. communities ossella the determined a A R on sponges particularly The Gert Introduction https://doi.org/10.1101/037440 words reach species, on Correspondence author: [email protected] § orschungsinstitut doi: doi: ey ossella Department F Bavarian GeoBio-CenterLMU (Hexactinellida: and bioRχiv Nuclear and mitochondrial Sergio and Ludwig-Maximilians-Universtität 1 2 Germany 3 Janussen 1 sion. Germany 4 R K can Glass feeder Abstract Hexactinellida genus systematics, lids, distribution. ing regarded North restricted benthic imens based roborate one bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint Rossella bioRχiv Rossella Rossella is an im- Rossella . The copyright holder for this preprint (which preprint this for holder copyright The Rossella antarctica, all re- is characterized by the presence species still require a revised and 2 Rossella Rossella specimens can harbour a diverse community of CC-BY-ND 4.0 International license International 4.0 CC-BY-ND this version posted January 22, 2016. 2016. 22, January posted version this ; Rossella available under a under available based on mitochondrial (16S rDNA, COI) and nuclear (28S Rossella https://doi.org/10.1101/037440 From a morphological perspective, Clarifying the systematic relationships within the genus doi: doi: of calycocomes (Fig. 1)though similar and spicules its also typical2012). occur microhexasters in In (Tabachnick, contrast a 2002), to few al- relationships the other remain relatively genera unclear stable and (see genus-level most Dohrmann systematics, et intra-generic al., rDNA) markers and includingby 5 Barthel and of Tendal the (1994). 8 We aim species tocbd currently test different recognized morphology-based as taxo- valid species dominate the seafloor (Fig.ity 1), (Gutt and and thus increase Starmans,1999; its Dayton 1998; spatial et heterogene- al., Janussen 2013;(Barthel, and Fillinger 1992a,b), et Reiswig, and al. 2009; locally 2013), playGutt Starmans structure a et benthic et major al. communities role 2013). al., in Large invertebrates silicon and cycling juvenile (Gatti, stages of 2002; manytum other for organisms, and various serve taxa asGutt substra- and of Schickan, other 1998; sessile Kunzmann, 1996; invertebrates Kersken (epibionts) et al., (Barthel, 2014). 1997; clear morphological delineation (Barthel and Tendal, 1994,2013). Göcke As and Janussen, a result, thethe number past, of ranging species from recognized two(e.g. for to the Burton, 21 genus 1929; species has Barthel depending varied andet in on Tendal, al. 1994; the 2015). Koltun, taxonomic 1976; The authority great seeextent, variation also of not van the Soest, surprising. number ofmaining recognized species species With lack is, the clear to some morphological1994), sole or apomorphies exception their (cf. diagnostic of Barthel charactersand and are size Tendal, weak of (e.g. dermal external megascleres).species morphology, In shape addition, delimitation the in majority of thespecies characters genus mainly used gradual for are and continuous, subjectfor to making instance, diverse tend differences interpretations. to overlap Calycocome between between sizes, cally species, important as spicules does (Barthel the and sizeExternal Tendal, of 1994; other body Göcke taxonomi- shape and is Janussen, variable 2013). the even lack within of species (Tabachnick, appropriate 2002).evaluation sampling Finally, has, of to the some variabilityferent extent, of species. hampered the the main systematic characters used for distinguishingportant dif- task of potentialspecies benefit are structurally to important othertion, in areas as Antarctica of that (see of Antarctic above), many(Janussen research. and and other their Reiswig, Antarctic distribu- 2009; sponges, Sara isspecies et thought al., play to 1992). in be The structuring circum-antarctic roleall Antarctic that species different communities, are, as indeed,clear a well circumpolar delineation as cohesive of whether unit, those somethe strongly species. or depends genus Here, upon we the provide a phylogenetic analysis of Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint Rossella bioRχiv - Rossella C 30 s, 50 ◦ C were used ◦ . The copyright holder for this preprint (which preprint this for holder copyright The µl reaction volumes of 3 C 3 min, 35 to 40 cycles of 94 ◦ CC-BY-ND 4.0 International license International 4.0 CC-BY-ND this version posted January 22, 2016. 2016. 22, January posted version this ; C 60 s, and a final extension of 5 min at 72 available under a under available ◦ spp. from the Antarctic Weddell Sea with the molecular results C 30 s, 72 Rossella ◦ https://doi.org/10.1101/037440 Three different molecular markers —partial 28S rDNA (ca. 1.2kb), partial doi: doi: C/40 GoTaq (Promega) supplemented with BSA.an Three-step initial PCR protocols, denaturation including step of 94 16S rDNA (ca.1994) of 0.5kb), COI and (ca. the 0.6kb)— standard were barcoding amplified fragment using 12.5 (Folmer et al. obtained here to further clarify the evolution of this important SO taxon. 2 Materials and Methods 2.1 Specimens and laboratorySpecimens procedures (Table 4) were collected by(2006/2007) trawling and during ANT the XXIV/2-SYSTCO German Expedition ANT (2007/2008)Sea XXIII/8 (Atlantic to sector the of Weddell West Antarctica), photographedAll and specimens fixed are in 96% deposited ethanol. inGermany) the and Senckenberg were Naturmuseum catalogued (Frankfurtdetermined a.M., in to the species online2004) level database and using SESAM. pertinent Sponges standard literature. were with procedures DNA the (e.g. was NucleoSpin DNA extractedmanufacturer’s Janussen tissue from protocol. extraction small et kit pieces al., (Macherey-Nagel) of following tissue the cbd nomic arrangements that have beennomic proposed history for and the attempt genus throughout totion its reconcile of taxo- the current morphology-based classifica- ◦ for all markers (seetemperature the for supplementary each materials primer).specific for primers details (see For on Suppl. 28Sorganisms; the rDNA 16S Materials) annealing rDNA and to primers COI avoid werewere we co-amplification as cleaned of in designed by Dohrmann non-target standard etIT ammonium (Affymetrix) al. acetate-ethanol enzymatic 2008. precipitation PCR or clean-up PCRthe and ExoSap- products same sequenced in primers both used directionsplied for using Biosystems). PCR Sequencing and reactions were theethanol precipitated BigDye with and Terminator sodium 3.1 subsequently acetate- chemistrySequencing analyzed (Ap- Service on of an thewere ABI assembled Department in 3700 of CodonCode Genetic Aligner Biology,origin (CodonCode LMU Analyzer of Corporation); all München. at obtained sequences the 2008). Trace was Sequences files verified are using deposited NCBI at BLASTHE80223. EMBL (Johnson under et accession al., numbers HE80191 to Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint Rossella https: bioRχiv . The copyright holder for this preprint (which preprint this for holder copyright The 4 ( incertae sedis). Leucopsacidae CC-BY-ND 4.0 International license International 4.0 CC-BY-ND this version posted January 22, 2016. 2016. 22, January posted version this ; have been shown to be successive sister groups to Rosselli- available under a under available Clathrochone clathroclada analysis https://doi.org/10.1101/037440 C. clathroclada doi: doi: 2.2 Outgroup choice andNew sequence alignment sequences werepublished manually alignments aligned (Dohrmann in ettaxon SeaView set al., 4 to 2012b). (Gouy representativeswell as et of However, the we al., families restricted 2010) Leucopsacidae the to and , as Vargas et al.and Molecular phylogeny of dae (Dohrmann et al.ments were 2012b), and concatenatedgions were into removed. therefore a used The supermatrix as final outgroups. alignment and is ambiguously Align- alignable 1.2 kb re- long and is available at . //bitbucket.org/sevragorgia/rossella 2.3 Phylogenetic analysis Using the concatenated alignment, weand inferred Bayesian both phylogenies Maximum likelihood2.0, with (ML) respectively. RAxML The GTR 7.2.8 modelused (Stamatakis, of for nucleotide 16S 2006) substitution rRNA, and COI (Tavaré, 1986) asAmong-site PHASE well was rate as for variation 28S rRNAgamma was single-stranded distribution regions modelled (loops). with using 4regions a categories (paired discrete sites) (+G; of approximation Yang,quence the 1994, of evolution 28S 1996). (Savill rRNA a et weML For al., used the tree 2001) the for S16 using stem the and 20port ML S7A independent with analysis. models 1000 tree-search bootstrap of We replicates pseudo-replicates se- searched (Felsenstein,bootstrap” and 1985), for assessed algorithm using the the branch described “rapid sup- byanalysis, two Stamatakis independent et Markov Chain al.for Monte 10,000,000 Carlo generations (MCMC) (2008). after chains a wereery burn-in In run of 100 the 250,000 generations, generations. Bayesian samplingsame ev- Model as specifications for the foronly ML the used analysis the Bayesian (i.e. S7A analysis modelto GTR+G for were achieve for the chain the 16SrDNA 28S convergence rRNA and using stem COI); the regions S16 however because model. we it was difficult 2.4 Partition addition bootstrap and alternative lineage attachment To assess the influencethe of ML the topology individual inferred partitionsidence from or ML the combinations tree), concatenated we thereof data performedfor on matrix each ML individual (i.e. bootstrap marker and analyses the for (10007.2.8. all total pseudoreplicates) combinations For ev- of all two these markers analyses, usingidence we RAxML analysis used for the the same corresponding model settings partition as (e.g. in GTR+G the for totalcbd 16S ev- rDNA and bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint R. Rossella and bioRχiv Rossella R. racov- R. nuda R. nuda, . can be translated into The copyright holder for this preprint (which preprint this for holder copyright The Rossella Rossella 5 , which was present in the ML phylogeny CC-BY-ND 4.0 International license International 4.0 CC-BY-ND this version posted January 22, 2016. 2016. 22, January posted version this ; R. racovitzae + spp. which nested deeply within the family Rossellidae. available under a under available specimens formed a highly supported clade in both Bayesian Rossella R. fibulata species using linmove from the phyutility package. In brief, linmove https://doi.org/10.1101/037440 or of these two species were performed in RAxML and the best constrained Our phylogenetic analyses (Fig. 2) recovered a well supported clade com- doi: doi: S16 for 28S rDNAspecific bootstrap stems). support (sensu Afterin Struck each the et analysis, total al., evidence we 2006)and tree Dunn, for determined using 2008). the the consensus branches We partition- Rossella also from present assessed the alternative phyutility branching packagescreens positions (Smith of a different set ofnative phylogenetic placements trees of and a branch reportsalization occur the of in frequency that alternative with set. branching which Thesupport positions alter- analysis values, of facilitates which a the allows lineage visu- have to showing only determine low a whether bootstrap few poorly attachmentat supported several points branches multiple occurring positions with with high low frequency frequency. or branch off 2.5 Testing hypotheses of relationshipsDifferent within taxonomic arrangements proposed for Vargas et al. Molecular phylogeny of specific phylogenetic hypotheses(e.g., and evaluated Goldman with et1997; available al., Whelan statistical et 2000; al., tests Huelsenbeck, 2001).modaira We and 1997; used Hasegawa, the Huelsenbeck 2001; AU testvalues Shimodaira, and implemented 2002) obtained Crandall, in with CONSEL from site-wise (Shi- RAxML log-likelihood 8.2.4 to test the monophyly of and ML analyses. Specimens belonging to other morphologically defined cbd prising all Rossella antarctica with moderate support. Bothconverged independent to MCMC runs the of same theto consensus Bayesian topology. model analysis The choice ML forresulted topology in the was the 28S not same sensitive rDNA phylogeny. stem sites, as analyses using S7A and S16 racovitzae, the twoBriefly, species ML analyses for constrained which toitze more enforce the that monophyly one of ML specimen trees was were compared available. against the best unconstrained phylogeny. 3 Results 3.1 Total evidence phylogenetic analysis We recovered a phylogenetic tree congruentHexactinellida with (Dohrmann published et al., analyses 2008, of 2009, theses 2012a,b). class recovered generally Bayesian similar and ML trees, analy- buta the clade Bayesian phylogeny of did not include bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint R. and Rossella bioRχiv R. nuda clade. Within expanding from . The copyright holder for this preprint (which preprint this for holder copyright The Rossella species were not recovered as Rossella racovitzae -concept(Rossella of Koltun, 1976), Rossella 6 CC-BY-ND 4.0 International license International 4.0 CC-BY-ND clade were generally low (< 50 %), with only R.) racovitzae showed higher log likelihood val- R. racovitzae clade were not monophyletic in any of the boot- or this version posted January 22, 2016. 2016. 22, January posted version this ; and R. nuda available under a under available R. racovitzae clade, support was low when single partitions or combinations R. racovitzae monophyletic). These results were insensitive to model selection, R. antarctica clade was not supported by 16S rDNA sequences alone but received https://doi.org/10.1101/037440 R. racovitzae doi: doi: species formed a large clade hereafter named the Vargas et al. Molecular phylogeny of this clade, other morphologically defined monophyletic, but were polyphyletic invalues both within the ML the and Bayesian tree. Support some branches showing moderate (50ysis. - 80 In %) contrast, bootstrap the support> Bayesian in the 0.95) analysis ML to assigned anal- most high branches posterior within probabilities this (PP clade. 3.2 Partition addition bootstrapBootstrap analysis values and assigned lineage to movement between the different branches partitions of or the combinationsstrap total thereof support evidence (Fig. increased ML when 2). treethere more In was varied data general, conflict were boot- between added partitions to in the some specific analysis. cases. However, For instance, the ues than trees constrained to make all species monophyletic (e.g. antarctica high bootstrap support from the COIbined, partition. bootstrap When the support two was markerssupport only were com- moderate (> (50 80%) - assignedthe 80%) to in this contrast clade to the in high the total evidence analysis. Within strap pseudo-replicates, invariably formingdifferent clades morphospecies. with specimens belonging to 3.3 Hypothesis testing All constrained phylogenetic hypothesesbe explored significantly in worse this (pThe study decay < in were 0.001) the likelihood than found valuesrelated the to of to the unconstrained the constrained ML ML number phylogenies treemonophyletic of was (e.g. (Table constraints. highly 2). Trees constrained to make single species of two partitions were usedevidence for phylogeny. the analysis, Lineage andincluded movement was only in analysis moderate the revealed in that the total morphospecies leading to identical conclusions whenplied to either 28S the rDNA S16 stems. or the S7A model was ap- 4 Discussion The last 50 years of taxonomic history have seen genus R. racovitzae two species, cbd bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint clade clade. Rossella species bioRχiv Rossella R. antarctica R. racovitzae species based can be readily R. racovitzae R. racovitzae R. racovitzae . divides into two main Rossella The copyright holder for this preprint (which preprint this for holder copyright The R. antarctica Rossella clade, in contrast to the well clade in the molecular phylogeny by Koltun (1976). Here, we have clade is not straightforward. Most species: a well supported 7 R. racovitzae Rossella using an independent set of characters not used CC-BY-ND 4.0 International license International 4.0 CC-BY-ND R. racovitzae R. racovitzae this version posted January 22, 2016. 2016. 22, January posted version this ; Rossella racovitzae Rossella clade in the standard barcoding partition. clade revealed that specimens morphologically assigned available under a under available R. racovitzae R. racovitzae R. antarctica, is a species flock. Species flocks are monophyletic, di- https://doi.org/10.1101/037440 The analysis of the alternative branching positions of specimens included doi: doi: flock includes 4Antarctica, out are of morphologically 5 diverse species and here appearcbd sampled, to have these evolved species rapidly are as endemic to verse (morphologically, ecologically, and taxonomically)related assemblages species of closely which evolvedand rapidly ecologically within dominant an (Lecointre area et where al., they 2013). are endemic The presented here could reflect aConsequently, recent or we ongoing propose speciation process thatdefined in the the genus. within the identified and can be clearlyon distinguished morphology from as all well other trast, as no their diagnostic clear molecularwithin diagnostic the characters molecular were characters; found in for con- all the other species species together also rejectedexpected the to monophyly be of the poly-non-monophyly species or within tested. paraphyletic the after Species or are during speciation. Therefore, the to the same nominal speciestotal were evidence not ML monophyletic analysis in anyapplied nor bootstrap to in some tree the phylogenetic of Bayesian hypotheses the tree; constrained to the group AU different tests of monophyly clades corresponding to Koltun’s (Barthel and Tendal, 1994;clade Göcke can and be Janussen, roughly 2013). subdividedcohesiveness into and Yet, to several some the groups extent that correspondthis display to clade morphological the (Göcke described and species Janussen, included in 2013). In contrast, by previous authors. Our results reveal that genus of its species lack clearfor apomorphic species characters delimitation and many inside(subjective) of this interpretation the clade characters (Table used overlap 3).tion or in are In both prone external addition, and to broad spicule authoritative morphological morphology is varia- found in the to eight species-concept (Rossella of Barthelrently and accepted Tendal, 1994) as to valid 19 inAll species the cur- six World species Porifera Database resurrectedbroad (van by and Soest Barthel et ‘highly al. and polymorphic’ sequenced Tendal 2015). two (1994) mitochondrial were markers included andclarify one in the nuclear systematics the of marker in an attempt to clade was recovered as sistersigned to to a various nominal moderately species supported andBoth group here clades of referred have specimens to as- as clear the i.e., diagnostic molecular molecular characters synapomorphies in (Fig.the their species 3). COI included sequences, within The the morphology-based of Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint s.l.). Rossella R. van- bioRχiv species in (cf. Göcke and R. racovitzae Rossella . The copyright holder for this preprint (which preprint this for holder copyright The corroborating its mono- R. fibulata in this region. We consider species (i.e. clade remains to be tested. In . Rossella Rossella spp. in Antarctic waters, hamper from east Antarctica (collected in are more often collected and more Rossella for molecular phylogenetic analyses. Rossella (Dohrmann et al. 2013). This age ac- 8 Rossella R. nuda R. racovitzae and specimens beloging other species are col- Rossella R. racovitzae CC-BY-ND 4.0 International license International 4.0 CC-BY-ND Rossella and was only collected 3-5 times in 4 expeditions to this version posted January 22, 2016. 2016. 22, January posted version this clade without requiring the synonimization of most ; R. levis have not been collected after years of field work in the and a diverse assemblage of species, here considered a R. racovitzae available under a under available R. racovitzae R. racovitzae and members of the R. villosa and is well known for its abundance in Antarctica, however most specimens species in a “highly polymorphic” R. antarctica https://doi.org/10.1101/037440 Finally, we would like to highlight the difficulties in getting access to fresh From a biogeographic perspective, the circum-Antarctic cohesiveness of both doi: doi: 5 Conclusion We have obtained a phylogeny of the genus hoeffeni Weddell Sea. This difficulties,ical somewhat given normal the in reported thethe abundance deep-sea, thorough of testing but of paradox- thehere monophyly the of first most molecular species study in oftant the the genus. phylogenetic genus relationship We in provide within Antarctica thismolecular impor- and evidence pursue given to the reconcilefuture available the work morphological to material further and and clarify to the phylogeny open of new avenues for phyly and showing the existence ofspecies two clades corresponding to the well-defined cbd material is generally available from these species. Two other species Janussen, 2013). lected less frequently. the Antarctica by DJ and something similar occurs to Rossella collected belong to material of all valid species of this study, the only specimenTerra Adelie) of included in theSea. analysis was sister However, to anythe specimens conclusion SO from the derived about Weddell from thegeographic our coverage biogeography current of of our dataset sample. seems premature given the restricted R. antarctica judged by their poly-graphic or history. paraphyletic Molecular status divergenceof (observed time here) estimations 40±20 resulted and Ma in their for a biogeo- crown-group mean age Vargas et al.cords well with thethat opening resulted of the in Drake theand Passage could final ( have isolation 30 caused of Ma), thethis diversification a Antarctica-concept”, “Rossella of geological (Lawver including event and onerently Gahagan, clear best 2003) species reconcile and alland a available to species-flock, evidence provide (i.e. to an cur- tion evolutionary morphological within framework and the to molecular) interpretRossella the high levels of varia- Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint Rossella bioRχiv . The copyright holder for this preprint (which preprint this for holder copyright The species flock. flock. Future sampling and the use of 9 CC-BY-ND 4.0 International license International 4.0 CC-BY-ND this version posted January 22, 2016. 2016. 22, January posted version this R. racovitzae ; R. racovitzae available under a under available ,Rossella the causes of the high morphological diversity and https://doi.org/10.1101/037440 Barthel, D., Tendal, O.S., 1994. Antarctic Hexactinellida. Synopses of the Barthel, D., 1997. Fish eggs and pentacrinoids in Weddell Sea hexactinellids: Barthel, D., 1992b: Antarctic hexactinellids: a taxonomically difficult, but Barthel, D., 1992a: Do hexactinellids structure Antarctic sponge associa- Arnaud, P., Lopez, C., Olaso, I., Ramil, F., Ramos-Espla, A., Ramos, A., 1998. doi: doi: cbd Antarctic Benthos vol. 6.Champaign, Ill., Eds., 154 p. J.W. Wägele & J. Sieg, Koeltx Scientific Books, further examples for the structuringtems. role Polar of Biol. sponges 17, in 91-94. Antarctic benthic ecosys- ecologically important benthic component.271-276. Verhandl. Deutschen Zool. Ges., tions? Ophelia 36, 111-118. Semi-quantitative study of macrobenthic faunaland in Islands the and region the Antarctic of Peninsula. the Polar South Biol. Shet- 19, 160-166. species flock and termed genome-wide molecular markers will certainlystanding contribute of to these expand important our Antarcticlar under- species, distribution in of particular about the circumpo- Acknowledgements We thank Annamarie Gabrenyain and the Astrid Schuster laboratory. for ConstructiveMolecular assistance comments Geo- and of support and pastproject. and Palaeobiology This present Lab, study members was LMU of possibledation München thanks the (DFG), to through greatly funding the of improved SPPto the 1158 the G. German “Antarktisforschung”, Science Wörheide, grants Foun- Wo896/9-1,2 andtively. DJ1063/14-2,2 MD and was DJ1063/17-1 funded toN. through D. Villalobos DFG Janussen, Trigueros, Grants respec- M. DOconstant Vargas 1742/1-1,2. support Villalobos during SV and the is S. course of indebted Vargas the to Villalobos study. for their 6 References the relationships within the Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint Rossella bioRχiv Anoxycalyx joubini. . The copyright holder for this preprint (which preprint this for holder copyright The gen. nov., for North Atlantic” “Rossella 10 CC-BY-ND 4.0 International license International 4.0 CC-BY-ND this version posted January 22, 2016. 2016. 22, January posted version this Nodastrella ; available under a under available https://doi.org/10.1101/037440 Douady, C.J., Delsuc, F., Boucher, Y., Doolittle, W.F., Douzery, E.J.P, 2003. Dohrmann, M., Vargas, S., Janussen, D., Collins, A.G., Wörheide, G., 2013. Dohrmann, M., Göcke, C., Reed, J., Janussen, D. 2012a. Integrative taxon- Dohrmann, M., Haen, K.M., Lavrov, D.V., Wörheide, G., 2012b. Molecu- Dohrmann, M., Collins, A.G., Wörheide, G., 2009. New insights into the phy- Dohrmann, M., Janussen, D., Reitner, J., Collins, A., Wörheide, G., 2008. Dayton, P.K., Kim, S., Jarrell, S.C., Oliver, J.S., Hammerstrom, K., Fisher, Cardenas, P., Xavier, J.R., Reveillaud, J., Schander, C., Rapp, H.T., 2011. Burton, M. (1929) Porifera. Part II. Antarctic sponges. British Antarctic Buckley, T., 2002. Model misspecification and probabilistic tests of topology: doi: doi: Comparison of Bayesian and maximum likelihood bootstrapcbd measures of phylo- Molecular paleobiology of early-branching :elucidates integrating the DNA and evolutionary fossils history95-108. of hexactinellid sponges. Paleobiology 39, omy justifies a new genus, lar phylogeny of glass spongespling (Porifera, and Hexactinellida): inclusion increased of taxondase the subunit sam- mitochondrial I. protein-coding Hydrobiologia, 687, gene, 11-20. cytochrome oxi- logeny of glass spongesand (Porifera, Hexactinellida): Euplectellinae, monophyly and ofgenet. the Lyssacinosida Evol. phylogenetic 52, position 257-262. of Euretidae. Mol. Phylo- species (Porifera: Hexactinellida: Rossellidae). Zootaxa 3383:1-13. Phylogeny and evolution of glass57, sponges 388-405. (Porifera, Hexactinellida). Syst. Biol. PLoS ONE 8, e56939. J.L., O’Connor, K., Barber, J.S., Robilliard,growth G., Barry, and J., et mortality al. of 2013. an Recruitment, Antarctic hexactinellid sponge, Molecular phylogeny of theunexpected Astrophorida high (Porifera, level of Demospongiae) spicule reveals homoplasy. an PLoS ONE 6, e18318. (‘Terra Nova’) Expedition, 1910.seum (Natural Natural History). History Zoology, 6, Report, 393-458, London, pls. British I-V. Mu- evidence from empirical data sets. Syst. Biol. 51, 509-523. Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint Rossella bioRχiv ,Rossella of ANT . The copyright holder for this preprint (which preprint this for holder copyright The 11 CC-BY-ND 4.0 International license International 4.0 CC-BY-ND this version posted January 22, 2016. 2016. 22, January posted version this ; available under a under available https://doi.org/10.1101/037440 Gutt, J., Schickan, T., 1998. Epibiotic relationships in the Antarctic benthos. Gutt, J., 2007. Antarctic macro-zoobenthic communities: a review and an Gouy, M., Guindon, S., Gascuel, O., 2010. SeaView version 4: A multi- Goldman, N., Anderson, J., Rodrigo, A., 2000. Likelihood-based tests of Göcke, C., Janussen, D. 2013 Hexactinellida of the genus Gatti, S., 2002. The role of sponges in high-Antarctic carbon and silicon cy- Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers Fillinger, L., Janussen, D., Lundälv, T. Richter, C. 2013. Rapid glass sponge Felsenstein, J., 1985. Confidence limits on phylogenies —an approach using Erpenbeck, D., Breeuwer, J., Parra-Velandia, F., Van Soest, R., 2006. Spec- Emerson, B., Ibrahim, K., Hewitt, G., 2001. Selection of evolutionary models doi: doi: Antarct. Sci. 10, 398-405. cbd ecological classification. Antarct. Sci. 19, 165-182. platform graphical user interface forbuilding. sequence Mol. alignment Biol. and Evol. phylogenetic 27, tree 221-224. topologies in phylogenetics. Syst. Biol. 49, 652-670. XXIV/2 (SYSTCO I) Expedition —Antarctic(1), Eastern 102-122. Weddell Sea. Zootaxa, 3692 cling —a modelling approach. Berl. Polar Meeresforsch. 434, 1-134. for amplification of mitochondrialmetazoan cytochrome invertebrates. c Mol. oxidase Mar. subunit Biol. I Biotech. from 3, diverse 294-299. expansion after climate-induced Antarctic23, 1-5. ice shelf collapse. Current Biology, the bootstrap. Evolution 39, 783-791. ulation with spiculation?characters Three versus independent morphology gene in demosponge fragmentsgenet. higher and Evol. classification. biochemical 38, Mol. 293-305. Phylo- for phylogenetic hypothesis testing using620-631. parametric methods. J. Evol. Biol. 14, genetic reliability. Mol. Biol. Evol. 20, 248-254. Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint Rossella bioRχiv . The copyright holder for this preprint (which preprint this for holder copyright The 12 CC-BY-ND 4.0 International license International 4.0 CC-BY-ND this version posted January 22, 2016. 2016. 22, January posted version this ; available under a under available https://doi.org/10.1101/037440 Koltun, V.M., 1976. Porifera —Part 1: Antarctic Sponges. Report B.A.N.Z. Kunzmann, K., 1996. Associated fauna of selected sponges (Hexactinellida Kersken, D., Göcke, C., Brandt, A., Lejzerowicz, F., Schwabe, E., Seefeldt, Johnson, M., Zaretskaya, I., Raytselis, Y., Merezhuk, Y., Mcginnis, S., Mad- Janussen, D., Reiswig, H.M., 2009. Hexactinellida (Porifera) from the AN- Janussen, D., Tendal, O.S., 2007. Diversity and distribution of Porifera in the Janussen, D., Tabachnick, K., Tendal, O., 2004. Deep-sea Hexactinellida Huelsenbeck, J., Crandall, K., 1997. Phylogeny estimation and hypothesis Huelsenbeck, J.P., 1997. Phylogenetic methods come of age: Testing hy- Gutt, J, Böhmer, A., Dimmler, W. 2013. Antarctic sponge spicule mats shape Gutt, J., Starmans, A., 1998. Structure and biodiversity of megabenthos in doi: doi: Antarctic Research Expedition 1929-1931 (B, Zoology and Botany) 5, 153-198. cbd and Demospongiae) from the1-93. Weddell Sea, Antarctica. Berl. Polarforsch. 210, M.A., Veit-Köhler, G., Janussen, D. 2014.sponge The species infauna (Hexactinellida of and three Demospongiae) widely fromin distributed the the deep Ekström Weddell Sea, Shelf Antarctica. Deep-Sea Res. Pt. II 108:101-112. den, T.L., 2008.W5-W9. NCBI BLAST: a better web interface. Nucl. Acids Res. 36, DEEP III Expedition to the Weddell Sea, Antarctica. Zootaxa, 2136, 1-20. bathyal and abyssal Weddell1864-1875. Sea and adjacent areas. Deep-Sea Res. Pt. II 54, (Porifera) of the Weddell Sea. Deep-Sea Res. Pt. II 51, 1857-1882. testing using maximum likelihood. Annu. Rev. Ecol. Syst. 28, 437-466. potheses in an evolutionary context. Science 276, 227-232. macrobenthic diversity and act57-71. as a silicon trap. Mar. Ecol. Prog. Ser. 480, the Weddell and Lazarev Seasters (Antarctica): and biological ecological interactions. role Polar of Biol. physical 20, parame- 229-247. Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint Rossella bioRχiv . The copyright holder for this preprint (which preprint this for holder copyright The 13 CC-BY-ND 4.0 International license International 4.0 CC-BY-ND this version posted January 22, 2016. 2016. 22, January posted version this ; available under a under available https://doi.org/10.1101/037440 Starmans, A., Gutt, J., Arntz, W., 1999. Mega-epibenthic communities in Stamatakis, A., Hoover, P., Rougemont, J., 2008. A rapid bootstrap algorithm Stamatakis, A., 2006. RAxML-VI-HPC: Maximum likelihood-based phyloge- Smith, S.A., Dunn, C.W., 2008. Phyutility: a phyloinformatics tool for trees, Shimodaira, H., Hasegawa, M., 2001. CONSEL: for assessing the confidence Shimodaira, H., 2002. An approximately unbiased test of phylogenetic tree Savill, N., Hoyle, D., Higgs, P., 2001. RNA sequence evolution with sec- Sara, M., Balduzzi, A., Barbieri, M., Bavestrello, G., Burlando, B., 1992. Nichols, S.A., 2005. An evaluation of support for order-level monophyly and McClintock, J., Amsler, C., Baker, B., van Soest, R., 2005. Ecology of Antarc- Lecointre G., Améziane N., Boisselier M-C., Bonillo C., Busson F., et al. Lawver, L., Gahagan, L., 2003. Evolution of Cenozoic seaways in the circum- doi: doi: Arctic and Antarctic shelf areas. Mar. Biol.cbd 135, 269-280. for the RAxML web servers. Syst. Biol. 57, 758-771. netic analyses with thousands2688-2690. of taxa and mixed models. Bioinformatics 22, alignments and molecular data. Bioinformatics 24, 715-716. of phylogenetic tree selection. Bioinformatics 17, 1246-1247. selection. Syst. Biol. 51, 492-508. ondary structure constraints: comparison of substitutionlikelihood rate methods. models using Genetics maximum- 157, 399-411. Biogeographic traits and checklist559-585. of Antarctic demosponges. Polar Biol. 12, interrelationships within thelarge class subunit Demospongiae rDNA using and34, partial cytochrome 81-96. oxidase data subunit from I. the Mol. Phylogenet. Evol. tic marine sponges: An overview. Integr. Comp. Biol. 45, 359-368. (2013) Is thePLoS Species ONE Flock 8(8): e68787. Concept Operational? The Antarctic Shelf Case. Antarctic region. Palaeogeography, Palaeoclimatology, Palaeoecolgy 198, 11-37. Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint http: Rossella bioRχiv . The copyright holder for this preprint (which preprint this for holder copyright The 14 CC-BY-ND 4.0 International license International 4.0 CC-BY-ND this version posted January 22, 2016. 2016. 22, January posted version this ; available under a under available https://doi.org/10.1101/037440 Yang, Z., 1996. Among-site rate variation and its impact on phylogenetic Yang, Z., 1994. Maximum likelihood phylogenetic estimation from DNA se- Whelan, S., Lio, P., Goldman, N., 2001. Molecular phylogenetics: state-of- van Soest, R.W.M., Boury-Esnault, N., Hooper, J.N.A., Rützler, K., de Voogd, Tavaré, S., 1986. Some probabilistic and statistical problems in the analysis Tabachnick, K.R., 2002. Family Rossellidae Schulze, 1885. in J.N.A. Hooper Struck, T., Purschke, G., Halanych, K., 2006. Phylogeny of Eunicida (Annel- doi: doi: cbd analyses. Trends Ecol. Evol. 11, 367-372. quences with variable rates over306-314. sites: approximate methods. J. Mol. Evol. 39, the-art methods for looking into the past. Trends Genet. 17, 262-272. //www.marinespecies.org/porifera N.J., Alvarez de Glasby, B.,Janussen, Hajdu, D., E., Tabachnick, Pisera, K.R., A.B.,Dohrmann, Manconi, Klautau, M., R., Díaz, M., Schönberg, C., Picton, C., Cárdenas, B., P., Kelly, 2015. M., Vacelet, World Porifera J., Database. of DNA sequences. Lect. Math. Life Sci. 17, 57-86. and R.W.M. van Soest (eds.),Sponges., Systema Kluewer Porifera: Academic/Plenum A Publishers, Guide New York, to pp. the 1441-1505. Classification of ida) and exploring dataation congruence (PABA) approach. using Syst. a Biol. partition 55, addition 1-20. bootstrap alter- Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint Rossella bioRχiv

. T5AV9G47 (SVR68) STN54AEV393GD4075 sp. Rossella ? ?

The copyright holder for this preprint (which preprint this for holder copyright The

◦ ◦ ANTXIII-8/700-4/SMF11730 nuda Rossella 60’S 60 S; 56.08’ 65 211.1 W 20.28’

◦ ◦ ANTXIII-8/697-1/SMF11731 racovitzae Rossella 53’S 59 S; 15.38’ 63 143.7 W 3.94’

◦ ◦ Systco/48-1/SMF11735 antarctica Rossella 39’S 8 S; 23.94’ 70 602.1 W 19.14’

◦ ◦ Systco/48-1/SMF11734 antarctica Rossella 39’S 8 S; 23.94’ 70 602.1 W 19.14’

◦ ◦ Systco/48-1/SMF11732 fibulata Rossella 39’S 8 S; 23.94’ 70 602.1 W 19.14’

◦ ◦ Systco/48-1/SMF11728 levis Rossella 39’S 8 S; 23.94’ 70 602.1 W 19.14’

◦ ◦ Systco/48-1/SMF11715 nuda Rossella 39’S 8 S; 23.94’ 70 602.1 91’W 19.14’ 15

◦ ◦ Systco/48-1/SMF11736 racovitzae Rossella 39’S 8 S; 23.94’ 70 602.1 W 19.14’

◦ ◦ Systco/48-1/SMF11733 racovitzae Rossella 39’S 8 S; 23.94’ 70 602.1

CC-BY-ND 4.0 International license International 4.0 CC-BY-ND W 19.14’

◦ ◦ R sel racovitzae ossella Systco/48-1/SMF11729 91’W 19.14’ 8 S; 23.94’ 70 602.1

this version posted January 22, 2016. 2016. 22, January posted version this

; Species xeiinSainVuhrnme oriae Lttd;ogtd)Dph(meters) Depth (Latitude;Longitude) Coordinates number Expedition/Station/Voucher

T be1: able olcindtiso eune specimens. sequenced of details Collection available under a under available https://doi.org/10.1101/037440 doi: doi: cbd Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint Rossella bioRχiv 0.001 0.024 0.001 . N.A. < = < The copyright holder for this preprint (which preprint this for holder copyright The p p p -6178.947402 -6229.382186 -6206.034912 -6245.485101 16 CC-BY-ND 4.0 International license International 4.0 CC-BY-ND this version posted January 22, 2016. 2016. 22, January posted version this ; available under a under available Constrained phylogenetic hypotheses tested using the AU-test. MonophylyconstraintNo constraint R. nuda BestR. racovitzae log likelihoodR. racovitzae+R. nuda AU-test AU-test Table 2: https://doi.org/10.1101/037440 doi: doi: cbd Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was

Preprint

µ )241-561 5?<4? <14 ? 25 6-12 10-25 2-4 l) ( size piece center

Calycocome

µ )1-51 -21 ? 15 8-12 16 12-15 l) ( length ray primary 4? 14

Rossella

Calycocome bioRχiv

µ 01014301020>5 0-0 4-8 185-260 240-380 200-400 >250 130-230 164-350 70-100 l) ( diameter

Calycocome

1=present

, 1 1 1 1 0,1 0=absent 1 1 pcl tuft spicule

. Basal

The copyright holder for this preprint (which preprint this for holder copyright The

1=hexactine

, 1 1 0 0,1 1 0=pentactine ? 1

tilspicules Atrial

1=hexactine

, , , ? 0,1 0,1 0,1 0 0 0 0=pentactine emlspicules Dermal

17

1=pentactine

, ()0 ?(1) 0 0 0 0,1 0=diactine 0

CC-BY-ND 4.0 International license International 4.0 CC-BY-ND spicules surface Protruding this version posted January 22, 2016. 2016. 22, January posted version this

;

1=present

0=absent , , 1 0,1 1 0,1 1 , 0,1 0,1 Conules

available under a under available

a.daee c)1 03 010 30 33 70 15 (cm) diameter Max. 616 26

Max. egt(cm) height 08 07 20 75 30 80 30 030 30

antarctica blt ei uarcvta ahefn villosa vanhoeffeni racovitzae nuda levis fibulata Character

R ossella https://doi.org/10.1101/037440 doi: doi:

cbd Vargas et al. Molecular phylogeny of

T be3: able eetdmrhlgclcaatr sdfrtetxnm fteSO the of taxonomy the for used characters morphological Selected fe ate n edl(1994). Tendal and Barthel after Rossella bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint Rossella bioRχiv NL4F dgHCO2189 Nichols (2005) . Folmer et al. (1994) The copyright holder for this preprint (which preprint this for holder copyright The C C ◦ ◦ 50 40 18 CC-BY-ND 4.0 International license International 4.0 CC-BY-ND this version posted January 22, 2016. 2016. 22, January posted version this 5’->3’ Temperature name and source ; available under a under available Primers and annealing temperatures used for amplification of 28S rDNA and Name Sequence’ Annealing Reverse primer Hexa28SInt4 CTCAGCTTTTCARGGGGTC https://doi.org/10.1101/037440 RossellaCOI_F1 ATATCGGYACATTATACC doi: doi: cbd Table 4: COI. Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint Rossella bioRχiv . The copyright holder for this preprint (which preprint this for holder copyright The Rossella; antarctica Photo: C. spp. in the Weddell Sea, Antarctica (lat- 19 Rossella CC-BY-ND 4.0 International license International 4.0 CC-BY-ND this version posted January 22, 2016. 2016. 22, January posted version this ; available under a under available Seafloor dominated by https://doi.org/10.1101/037440 doi: doi: cbd Figure 1: itude: -70.8717,mans, longitude: A. -10.5233; (2004):doi:10.1594/PANGAEA.198695. depth=254m); Sea-bed Inset: Photo: photographsGöcke. calycocome (benthos) of Gutt, along J. ROV and profile Star- PS56/127-1. Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was Preprint is Rossella Rossella bioRχiv L R (T.A.) (T.A.) . The copyright holder for this preprint (which preprint this for holder copyright The in Lyssacinosida. Within Rossellidae, weddelli Rossella racovitzae Rossella nuda Aulosaccus mitsukurii Rhabdocalyptus dawsoni Caulophacus valdiviae Rossella racovitzae Caulophacus arcticus Caulophacella tenuis Leucopsacus sp. Oopsacas minuta Rossella racovitzae Rossella racovitzae Rossella nuda Crateromorpha meyeri Nodastrella asconemaoida Lophocalyx profundum Rossella sp. Rossella fibulata Rossellinae n. gen. Bathydorus spinosus Rossella nuda Rossella racovitzae Rossella antarctica Rossella antarctica Bathydorus laniger Clathrochone clathroclada Rossella racovitzae Rossella levis incertae sedis .Rossella The tree corresponds to the total evidence maximum 20 highlighted in ligth gray. Information about other specimens included in the analysis CC-BY-ND 4.0 International license International 4.0 CC-BY-ND this version posted January 22, 2016. 2016. 22, January posted version this ; 16S rDNA 16S rDNA + COI 16S rDNA BP BP ML COI 50 700 100 ML 0 10095 available under a under available 28S rDNA + 16SrDNA 28S rDNA Rossella racovitzae sensu lato 28S rDNA 28S rDNA + COI 28S rDNA Phyloenetic relationships (cladogram) of Southern Ocean https://doi.org/10.1101/037440 doi: doi: can be found in Dohrmann et al.support values (2008, are 2009). provided in Both the Suppl. topologies, Materials. ML and Bayesian, as well as partitioncbd specific trees with branch-lengths and indicated with a black bar and Figure 2: likelihood topology. The vertices of thepartition stars or above a combination the of branches partitions show (seetotal the inset). evidence bootstrap analysis, value The and centers obtained on of the for the right, aannotate the stars given the posterior show, family branch on probability Rossellidae (R) when obtained the and for using left, Leucopsacidae the (L); the a Clathrochone branch bootstrap single is in currently value the of Bayesian the analysis. maximum Dark likelihood gray bars on the right Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was

Preprint 0.2 Rossella

bioRχiv Clathrochone clathroclada Clathrochone

Oopsacas minuta Oopsacas

sp. Leucopsacus

Caulophacella tenuis Caulophacella

Lophocalyx profundum Lophocalyx Caulophacus arcticus Caulophacus based on the total evi-

. Caulophacus weddelli Caulophacus

The copyright holder for this preprint (which preprint this for holder copyright The Caulophacus valdiviae Caulophacus

Rossella Rhabdocalyptus dawsoni Rhabdocalyptus

Aulossaccus mitsukurii Aulossaccus

Nodastrella asconemaoida Nodastrella

Crateromorpha meyeri Crateromorpha

. laniger Bathydorus

Bathydorus spinosus Bathydorus

Rossellinae n. gen n. Rossellinae

...... 21 Rossella antarctica Rossella

T T C T T A A T G T Rossella antarctica Rossella

C ......

Rossella fibulata Rossella

CC-BY-ND 4.0 International license International 4.0 CC-BY-ND

C ...... Rossella racovitzae Rossella

C ...... Rossella levis Rossella this version posted January 22, 2016. 2016. 22, January posted version this

; C ...... Rossella nuda Rossella

A ...... sp. Rossella

C ...... Rossella racovitzae Rossella

C ...... oslanuda Rossella

available under a under available

C ...... Rossella racovitzae Rossella

C ...... Rossella racovitzae Rossella

C ...... Rossella racovitzae Rossella

C ...... s.s. clades obtained with their corresponding COI diagnostic characters: consensus

Rossella racovitzae Rossella (T.A.)

Maximum likelihood phylogram of Southern Ocean

A G T C A G C C A G

Rossella nuda Rossella

5 7 2 2 3 1 8 0 4 4

0 8 4 6 8 7 6 0 2 6

6 5 5 4 3 3 3 3 2 1 https://doi.org/10.1101/037440 Rossella doi: doi: sequence on top ofScale the bar, expected alignment, number positions of substitutions identical per to site. the consensus representedcbd with dots. Figure 3: dence data matrix. For themain bootstrap values of the nodes see Figure 1. Highlighted are the two Vargas et al. Molecular phylogeny of bioRxiv preprint preprint bioRxiv was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made made It is perpetuity. in preprint the display to license a bioRxiv has granted who author/funder, is the review) peer by certified not was