New Results Preprint , 3 †1,4,5 . , Shirley A. Pomponi 2 The copyright holder for this preprint (which was not was (which preprint this for holder copyright The Divergence times in demosponges (Porifera) , and Gert Wörheide 1,4 cbd , Ingrid S. Knapp 1 this version posted July 9, 2017. 2017. 9, July posted version this CC-BY-NC-ND 4.0 International license International 4.0 CC-BY-NC-ND ; under a under , Dirk Erpenbeck 2 , Sergio Vargas 1 , Richard-Wagner Str. 10, D-80333 München, Germany LMU mitochondrial genomes, molecular clock, next-generation sequencing, De- https://doi.org/10.1101/159806 Correspondence author: [email protected] † Hawaii Institute of Marine Biology,Harbor 46-0007 Branch Lilipuna Oceanographic Road, Institute, 96744 Florida Kaneohe, Atlantic Hawaii, University, USA 5600 U.S.1 North,GeoBio-Center Ft Pierce, FL-34946, SNSB - Bavarian State Collections of Palaeontology and Geology, Richard-Wagner Str. 10, D-80333 Department of Earth- & Environmental Sciences, Palaeontology and Geobiology, doi: doi: 2 3 4 5 Abstract Approximately 80% of all recentdespite sponge their species diversity belong and to importance,most the accurate class demosponge divergence Demospongiae. times clades. Yet, areanswering still fundamental The unknown questions for estimation likecation e.g. of and demosponge the historical origin divergence biogeography. ofon time Demospongiae, Molecular an is their sequence absolute diversifi- data time keysuch scale, alone to as and is fossils. therefore notadvantage, needs Here, informative compared to we to be apply strict "calibrated" the nodelows with fossilized dating for additional with birth-death the data the model inclusion oldestuse (FBD), of fossil desma-bearing which young occurrences, sponges, has that and a the itand old diverse al- have fossils group a in of rich demosponges theaiming and that analysis to continuous form of date fossil rigid the divergence record skeletons events, demosponge time. dating radiation like back We and the to constrain transition themitochondrial the from Cambrian genomes timing marine of (⇠500 of to six Ma), key freshwater desma-bearingrepresentation evolutionary habitats. demosponges genomic from To libraries do size-selected and reduced- so,fossils apply we and a 33 assembled fossilized complete birth-death demospongeof mitochondrial model Demospongiae. genomes including to Our 30 infer study a supportsage dated a phylogeny estimates Neoproterozoic origin for of theboniferous Demospongiae. split and Novel the of previously freshwatersponges assumed and is supported. Recent marine (⇠18 Moreover, sponges wesification Ma) provide dating of diversification detailed Tetractinellidae back age (⇠315 of Ma), estimates to freshwater (⇠120 the for the Astrophorina Ma) a Car- (⇠240 possible and Ma), diver- the thegroups family Spirophorina for Corallistidae (⇠188 dating Ma) thefossil all history. Demospongiae, of due which to are considered their as extraordinary key richKey and words continuous mospongiae, fossilized birth-death model, Porifera, molecular dating Astrid Schuster 1 bioRiv Divergence times in demospongesinsights (Porifera): from first new mitogenomesfossils and in the a inclusion birth-death of clock model USA München, Germany Ludwig-Maximilians-Universtität München, Richard-Wagner Str. 10, D-80333 München, Germany Robert J. Toonen certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available available is made It in perpetuity. the preprint to display a license bioRxiv granted has who author/funder, is the review) by peer certified bioRxiv preprint preprint bioRxiv Preprint bioRiv muelleri, which cf. . (Tetractinellida: Astropho- The copyright holder for this preprint (which was not was (which preprint this for holder copyright The Ephydatia (Calcarea, Calcinea) which en- and Divergence times in demosponges (Porifera) 2 Poecillastra laminaris Clathrina clathrus this version posted July 9, 2017. 2017. 9, July posted version this CC-BY-NC-ND 4.0 International license International 4.0 CC-BY-NC-ND Spongilla lacustris ; under a under gene [11, 12] and the existence of different gene arrangements cox1 https://doi.org/10.1101/159806 doi: doi: codes 37 genes distributed in[13]. six Despite linear chromosomes their ranging somewhatfully 7.6-9.4 unique used kb features, to in mt size infer genomesgathering robust have more demosponge been sponge phylogenies success- mt [3, genomeslution 8, will of 9] improve this our and animal understanding it group. of is the clear evo- that The demosponge order Tetractinellida comprisestribution, 23 of which families eleven of possess world-wide acalled rock-like dis- skeleton desma. built of In interlocking contrast spicules usually to limited most to other loose spicules demosponges,for [e.g. which 14], fossil their most remains tetractinellid well are families preservedthese are known fossils are and the their Corallistidaeknown continuous of at record least which [e.g. since characteristicout the 15]. desmas the Late (dicranoclones) Mesozoic Jurassic Among are and with a Cenozoic continuous [15]. fossilcbd However, record among through- all tetractinellids, only [3]. An extreme examplegenomes of is the the special mt characteristics genome of of sponge mitochondrial were assembled from Illumina (TruSeq)genomes synthetic sequenced long-reads to [7], date all9]. sponge were mt assembled However, Sanger from sequencing sangerticular is if sequencing outdated multiple reads mt regarding [8, genomes costscan are be and pursued. challenging yield, in Additionally, (demo)sponges the in due usegenes, to par- of the long this presence intergenic of method regions extra that protein-coding trons may in include the repetitive sequences [4, 10], in- Schuster et al. 1 Introduction The sequencing of sponge mitochondriallast (mt) decade genomes greatly [1–5]. increasedber in Nevertheless, the of we mitochondrialanalyses are genomes at still suitable the far as levelmospongiae, from a of are a orders basis so and representative for farmoscleromorpha below, num- molecular undersampled. because (106 some phylogenetic species) While key the inare taxa, mt the sequenced, such genomes species-poorest as this class, for De- mt ratio Ho- 14.2% genomes), of is Calcarea the (690 onlyspecies, species species, 0.1-0.5% 1 (15) 38 for mt mt genome), Hexactinellida genomes)https://www.ncbi.nlm.nih.gov and /genomes/OrganelleResource.cgi?taxid=6040). (679 (Organelle Demospongiae species, (8225 Genome 3 ResourceTherefore, database there is in a GenBank; genomes considerable in need sponges for to denser allow taxonomic for sequencing finer-scaled phylogenomic of analyses. mt Despite a few exceptionsrina), like where the mt genomeor was the assembled using freshwater 454 sponges pyrosequencing data [6], certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available available is made It in perpetuity. the preprint to display a license bioRxiv granted has who author/funder, is the review) by peer certified bioRxiv preprint preprint bioRxiv Preprint [8] bioRiv Geodia neptuni . [6], The copyright holder for this preprint (which was not was (which preprint this for holder copyright The Divergence times in demosponges (Porifera) 3 Poecillastra laminaris [9] have been sequenced to date, none of which this version posted July 9, 2017. 2017. 9, July posted version this CC-BY-NC-ND 4.0 International license International 4.0 CC-BY-NC-ND ; under a under sequence and assemble the mitochondrial genomes of six species of Cinachyrella kuekenthali https://doi.org/10.1101/159806 de novo doi: doi: Schuster et al. three complete mt genomes (i.e. are from families of desma-bearingSphaerocladina tetractinellids. is another orderrecord dating of back desma-bearing to demospongessequenced the to with Cambrian date. a [15] fossil standing However, from demosponge this which evolution group as no it is is mtsponges regarded of genome as [16–20], particular the has sister thus importance been group constitutes for tomon freshwater a under- ancestor key of freshwater order and for marine reconstructing sponges. the lastGiven com- the rich fossilgenomes record of of representatives these ofto rock-sponges tetractinellids combine [21–23], and the sequencing Sphaerocladina robustness thethe will of mt rich allow the fossil us phylogenies record inferreddemosponges of that from these can mt be sponge genomes, used groups, with to to better understand provide their aHere, evolutionary dated we history. generated phylogeny size-selected of reducedto representation genomic libraries [24] the orders Tetractinellida (mainly Corallistidae)features and of Sphaerocladina. the Structural six novelgenomes mt and 30 genomes fossil are taxa discussed. ofDemospongiae diverse In using ages total are the 35 used fossilized to demospongeto infer birth-death mt a node (FBD) dated phylogeny calibrated clock of molecular model.’oldest’ known clock In fossil models, ages contrast as whichassignment constraints of only on fossils certain of allow different nodes, users ages theinformation to FBD to about a model clade the set without allows fossils requiring the in morphological suitable the for analysis [25]. groups Thus, consistingdesma-bearing the demosponges of FBD [e.g. model a 21–23]. appears richular Until in and now the the well absence FBD studied of model,times a fossil in in fossil partic- bears record character [25], matrix, such ferns was[28], as [26], used groups tetraodontiform to with fishes estimate fossils divergence [27] extendinghas and back been certain to beeches made the to Mesozoic.such use as However, sponges, no this that attempt method radiated to in the estimate Early the Paleozoic. divergenceOur approach time not of only groups, allowsing us studies to of Porifera, revise but the alsowe results provides of answers confirm to previous a the molecular Neoproterozoic following dat- questions:was origin the Can of split Demospongiae of marine byCan (Sphaerocladina) our the and approach? freshwater suggested sponges recent When (Spongillida)? divergence30] time be of freshwater confirmed sponges by (7-10is our Ma) this [29, analysis? in When congruence was with thecbd the origin first of occurrence Tetractinellida and of tetraxial-like fossil spicules? and certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available available is made It in perpetuity. the preprint to display a license bioRxiv granted has who author/funder, is the review) by peer certified bioRxiv preprint preprint bioRxiv Preprint sp., (New bioRiv Sau3AI Corallistes and sp., . MboI The copyright holder for this preprint (which was not was (which preprint this for holder copyright The v8.1.8 (http://www.geneious.com, sp.), subsampled from the Harbor ® Divergence times in demosponges (Porifera) ⇠2 Mio reads per library) were merged 4 Vetulina sp., collected fresh and preserved at -80°C. Detailed in- this version posted July 9, 2017. 2017. 9, July posted version this CC-BY-NC-ND 4.0 International license International 4.0 CC-BY-NC-ND ; Craniella under a under sp., Cinachyrella alloclada https://doi.org/10.1101/159806 doi: doi: [34]) and a custombrary sequenced, BLAST one database closely forand related used each mitogenome as library was reference downloaded wasgenomes. from genome The build. reference NCBI to genome used map For for each mt each speciescbd reads is li- given against in Supplementary and assemble the mt using the Paired-End reAd mergeR (PEAR)own [33] Galaxy software platform. as A implemented minimumof in overlap our the of assembled 10 sequences bp, ofming a 50 possible of bp minimum low and length a qualityPaired sequences quality parts were score imported (including in threshold Geneious for adaptors the and trim- barcodes) of 20 was used. formation on the samples useddate including and museum vouchers, depths location, is collection with provided AmpureXP in (Agentcourt) theprotocol, beads Supplementary Table 3-5 to 1. times removeValidation according DNA degenerated and was to DNA purified quantification the fragmentsSensitivity was manufacturer and dsDNA performed quantitation secondary using assay metabolites. onular the a Devices, AccuClear SpectraMax M2 Sunnyvale, Ultra plateEngland California). High reader BioLab) (Molec- were Two usedgested to enzymes, products digest were 1.0-1.3 cleaned µg withwater. DNA Ampure for XP The 6 beads Illumina h andaccording KAPA at eluted to Hyper 37°C in the Prep 25 [24]. manufacturer’s µl Kitselection Di- protocol HPLC v1.14 at for (Wilmington, 350-750 library MA) preparation bp(Bioanalyzer was including and and size used quantitative library real-time amplificationpair-end RT-PCR), sequenced [32]. all on six an Upon librariestute Illumina quality of were MiSeq Marine control 300 (Illumina, Biology Inc.) bp (HIMB) Genetics at Core the facility (Hawaii, Hawai’i USA). Insti- Forward and reverse pair-end sequences ( 2.2 Mitochondrial genome assembly Branch Oceanographic Institute (HBOI; USA,men of Florida) collection and one speci- Genomic DNA was extracted usingfrom a frozen standard (-80°C) phenol-chloroform sponge protocol tissue [31] of five species (Corallistes 2 Materials and Methods 2.1 DNA extraction and Illumina library preparation Schuster et al. Are the estimatedtrophorina divergences and recovered the for familyappearance Corallistidae the dates? in suborders accordance Spirophorina, with their As- putative fossil Neophrissospongia certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available available is made It in perpetuity. the preprint to display a license bioRxiv granted has who author/funder, is the review) by peer certified bioRxiv preprint preprint bioRxiv Preprint bioRiv cox1+intron region Cinachyrella alloclada . statistics in phylobayes, of v8.1.8 from the 14 protein The copyright holder for this preprint (which was not was (which preprint this for holder copyright The ® cox1 bpcomp and Divergence times in demosponges (Porifera) v8.1.8. ® 5 tracecomp this version posted July 9, 2017. 2017. 9, July posted version this CC-BY-NC-ND 4.0 International license International 4.0 CC-BY-NC-ND ; (HM032738). Consensus sequences were assembled under a under and mapped against the reference genomes respectively. Mitochondrial https://doi.org/10.1101/159806 Cinachyrella alloclada doi: doi: 2.4 Fossils and theirThe assignments protein alignment wasfile complemented available by at fossil LMU taxa Opention Data and of XXX). their point As ages fossil the ages (nexus taken FBD [25], (see model the requires Supplementary youngest the Table stratigraphicon specifica- 2). age the for node In each ages order fossildifferent of was analyses, to which using review differ different the bysils, parameters possible to the in test influence following for BEAST, parameters: the wemodel 1) sensitivity carried number and of out fossil of the sampling fos- two root density;sphaeroclone 2) desmas age the because and; origin of theforms 3) the homology [see FBD the e.g. of 39] included/excluded those is Paleozoic debatable, spicules and fossils to to with the assesscbd the Mesozoic impact of removing the oldest A concatenated alignment was built using Geneious were checked by blastingof the library database against the 2.3 Protein alignment and phylogenetic reconstruction de novo Schuster et al. Table 1. The entireprotein, custom rRNA, database and was tRNA genes, blastedsible as against contamination, well all as all reference intergenicthe reads genome regions. NCBI were To database: check assembled non-sponge foranalysis. separately fragments, pos- if and any, were The blasted thenence against remaining excluded genome. from sponge Possible the intronic sequences regions were within the mapped again to the refer- genomes were annotated usingORF the finder similarity as annotation implemented in tool Geneious (>75%) and the coding genes extracted from theprotein mt alignment was genomes 3994 of characters 35 long,stant, of demosponge which 285 taxa. 1429 characters characters The were were final con- parsimony parsimony uninformative informative. and 2280 Thisnetic characters alignment were tree was with used to PhyloBayes-MPIconvergence, infer assessed (v1.7) using a [35]. the Bayesian phyloge- Two concurrent chains ran until with the site-heterogeneous CAT-GTR modelset [36]. to 30% Burn-in ofysis was points with sampled. conservatively 1,000 Additionally, a bootstrapthe Maximum best-fitting replicates evolutionary Likelihood was model (ML) (VT+Gamma+I+F) done as anal- 3.4 suggested using by [38]; RAxML ProtTest the v8.0.26 proportionommended [37] of from and invariant the sites RAxMLSupplementary parameter manual Figure [37]. (I) 1. was A excluded summary as tree rec- is provided in the certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available available is made It in perpetuity. the preprint to display a license bioRxiv granted has who author/funder, is the review) by peer certified bioRxiv preprint preprint bioRxiv Preprint bioRiv indet., 298 Ma [41]), . Mastosia wetzleri, 155.5 The copyright holder for this preprint (which was not was (which preprint this for holder copyright The Divergence times in demosponges (Porifera) ).ucldStdev.c As the substitution rates in 6 and this version posted July 9, 2017. 2017. 9, July posted version this CC-BY-NC-ND 4.0 International license International 4.0 CC-BY-NC-ND ; under a under parameter. The diversification rate prior was set to an exponential sp., 162 Ma [40]), freshwater sponges (Spongillina https://doi.org/10.1101/159806 doi: doi: Heteroscleromorpha mt genomes are consideredexponential prior to distribution be with low 95% [3],ucldStdev.c probability density we on assumed values an <1with for the mean equal to(22 1.0 or as 30) the used proportion canthe be of regarded sampling extant as proportion (33 balanced. species) with A and Alpha beta fossil distribution 2.0. was taxa cbd chosen The for default prior ’uniform’ (0,1) was 2.5 FBD model settings The fossilized birth-death model [25,was 45] used as implemented with in the BEAST followingmolecular v.2.4.3 settings [46] clock for both model analyses: wasapplied An chosen uncorrelated on using relaxed the default data settings.mation matrix in No as [47] partitioning it (see was haddata, Table 4 a no Gamma and influence Site Figure model on 3 withspecified. the the in JTT As divergence [47]). amino the time acid For start esti- substitutionmolecular the of model molecular clock [48] the sequence was analyses FBD [49, process900 Ma) (root 50] with of we a the usedMa, lognormal tree), prior two max=624 based (mean=517 on Ma). different Ma, previous ageslognormal standard Two distribution deviation (1000 hyperparameters (ucldMean.c min=471 Ma were and induced for the uncorrelated Schuster et al. fossil on the predicted1) ages and 30 (see (BEAST alsoders analysis Table (Poecilosclerida, Tethyida, 2) 1). Spongillida, taxa Sphaerocladina and belonging Fossilswere Tetractinellida) to of extracted five 22 from different the (BEASTon demosponge literature analysis their or- and suggested affinities linked to to moderninclude taxa extant (Supplementary the species Table 2). oldest or reliable These clades also sia fossils based known to date from Poecilosclerida (Ophiode- Sphaerocladina (Amplaspongia bulba, 456Ma Ma [43]) [42], and or Astrophorina (Dicranoclonellainformation schmidti, for 150.8 all Ma fossils [44]). used,level, Detailed such taxonomic/systematic as museum affinity numbers, towell locality, stratigraphic as modern the taxa, Paleobiology ageare Database (https://paleobiodb.org/) provided range, reference in references number the as ancestor Supplementary or Table as 2. extinctphological sister data Fossil matrix taxa. taxa is can difficultin Because to be compile a nearly seen due representative all to as demosponge e.g.and fossils, either mor- the BI we lack trees of placed microscleres (seetaxa them of Supplementary to both Figure extant the 1). andthe appropriate fossil analysis, namely: sponges subclades Consequently, were Tetractinellida, 10 in Sphaerocladina, constrainedHaplosclerida, defined Poecilosclerida, to the Tethyida, Spongillida, higher be ML monophyletic Astrophorina, for yet Spirophorina, unnamed clade Corallistidae combining Sphaerocladina and and the freshwater sponges. certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available available is made It in perpetuity. the preprint to display a license bioRxiv granted has who author/funder, is the review) by peer certified bioRxiv preprint preprint bioRxiv Preprint Am- bioRiv sequence of cox1 . The copyright holder for this preprint (which was not was (which preprint this for holder copyright The (GW3895) contains a 1,141 bp Divergence times in demosponges (Porifera) 7 gene, which encodes for a homing endonuclease this version posted July 9, 2017. 2017. 9, July posted version this CC-BY-NC-ND 4.0 International license International 4.0 CC-BY-NC-ND ; cox1 Cinachyrella alloclada as previously found in several other species of the genus under a under https://doi.org/10.1101/159806 doi: doi: phimedon queenslandica cbd long group I intron ingene the (HEG) of theis LAGLIDADG inserted family at (Supplementary nucleotide Figure position 3A). 723 This with intron respect to the While this approach has proven usefulians in [54, other taxa 55], such astained here molluscs and from we cnidar- size-selected provide reduced theFor representation all first genomic six complete libraries libraries, mitochondrial ofof we sponges. genomes 50 obtained bp more ob- and than acircular 2 quality and Mio. score vary >20 reads in for length of32.8% all and a reads. to GC-content minimum All 35.7% between length mitochondrial 17,364 respectivelymitogenomes genomes and were (Supplementary of 20,261 other Figure bp Heteroscleromorpha and 3),tain [see which 24 e.g. tRNAs, is 3]. 14 insame protein-coding All line gene genes mitogenomes with and order con- two andmitochondrial ribosomal coding genome RNAs strand of and as have found the in their reference genomes. The 3 Results and Discussion 3.1 Mitochondrial genome organisation – a general comparision Schuster et al. used for the turnover parameter.run Two independent for Markov 400 chain million analysesevaluated were generations, using sampling Tracer v.1.6 everyan [51] 5000 to effective generation. assure sample stationarity Runs sizeof of were (EES) the each for independent Markov allboth runs. chain, analysis parameters were discarded The over as burn-in, first 200,in and 25% LogCombiner the and remaining and of convergence trees summarized were themented combined in sampled in TreeAnnotator the tree (both BEAST programs topologiesposterior package) are density from with (HPD). imple- Before meaning this, divergence the all times FullToExtantTreeConverter fossils tool and were (a 95%Possible removed tool prior highest implemented from influences in the to BEAUti tree the v.2.4.3). specifying us- posterior the distribution sampling estimates from were checked themarized by prior comparison only of and the rerunning turnover,both diversification the runs and analysis. and sampling A the proportion sum- that of priors is the provided number innode of Supplementary ages fossils Figure of 2A-C, used indicating and interest are Sphaerocladina, from sufficient Tetractinellida, both Astrophorina, for Spirophorinadae) BEAST our were and analyses extracted analyses. Corallisti- from (split the combinedthe of log-output-files Additionally, frequency and freshwater histograms distribution sponges showing ofdio the [52, posterior 53] age (script estimates availableposterior at were density openData plotted interval LMU (HPD), in XX), the RStu- indicating mean the and 95% the highest standard deviations. certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available available is made It in perpetuity. the preprint to display a license bioRxiv granted has who author/funder, is the review) by peer certified bioRxiv preprint preprint bioRxiv Preprint C. [8]. (gug) genes sp. is bioRiv Vetulina sp., four Eunapius trnaI is sister to Vetulina nad4/trnH Geodia neptuni . (13bp), ) was located in Neophrissospongia The copyright holder for this preprint (which was not was (which preprint this for holder copyright The sp. The same applies to other (ucg) sp. form a robust clade within ) and has two types of Cinachyrella alloclada nad4L/cox1 Divergence times in demosponges (Porifera) spp. and Vetulina (ucg) 56]. Compared to the closest reference 8 (1bp), sp. forming a robust clade within the subclass Corallistes Axinella corrugata, see [10]), which suggests that Neophrissospongia this version posted July 9, 2017. 2017. 9, July posted version this CC-BY-NC-ND 4.0 International license International 4.0 CC-BY-NC-ND or ; (10bp) as previously reported for ugc Craniella ). The gene order and coding strands in under a under (cau) Ephydatia muelleri)[ spp. and E. subterraneus. Although all freshwater sponges are known trnI and [e.g. 11, 12]. In and with nad6/trnA and sp. is 4,589 bp shorter (total 20,261 bp) in size, shows reduced inter- Corallistes morpha Suberites domuncula (cau) https://doi.org/10.1101/159806 doi: doi: assembled mitogenomes despite of(e.g. their presence in othersuch heteroscleromorphs repeat motifs evolvedintergenic several regions. times independently in sponges with large to possess various repeatgenomes, motifs some (direct, inverted of and whichfeatures palindromes) were form in found repetitive in their hairpin the mt mt structures genome [4], of none of these the same as for The present studybased provides on the the relaxed firstindicate molecular dated a clock Neoproterozoic phylogeny FBD divergence model. of ofsee Heteroscleromorpha/Keratosa The Heteroscleromorpha (node Table two R, 1, analyses Figure performed 1, Suppl. Figurecbd 4). As the origin time of the FBD process 3.3 Implications for divergence time estimates for Heterosclero- Our ML and BI treesder corroborate Sphaerocladina the), (Vetulina sister group whichpossession relationship is of of sphaeroclone morphologically the desmas, marine characterizedplementary or- to by Figure freshwater 1), the sponges hencechondrial (Spongillida) strengthens single (Sup- gene previous data ribosomal [16–20].study, and Of the partial five mito- Tetractinellida sequencedthe in this suborder Astrophorina. Furthermore, 3.2 Phylogenetic analyses gene pairs overlapped (atp8/atp6 kuekenthali Spirophorina (Supplementary Figurecurrently 1). sequenced This mt studysupports genomes previous increases within phylogenies the the of number20, order this 57–59]. order of Tetractinellida based by on five single and genes [see e.g. 19, Schuster et al. Cinachyrella (21bp) and (trnI sp. (Sphaerocladina), the samesubterraneus as found in freshwater sponges (e.g. genome available to dateVetulina (E. subterraneus; 88.5% pairwisegenic regions, sequence lacks identity), one tRNA gene (trnR A further gene-pair overlap of 23 bp (nad5/trnA certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available available is made It in perpetuity. the preprint to display a license bioRxiv granted has who author/funder, is the review) by peer certified bioRxiv preprint preprint bioRxiv Preprint bioRiv 155.5 30 2 . 900.0 The copyright holder for this preprint (which was not was (which preprint this for holder copyright The BEAST analysis orginFBD: oldest Sphaerocladina fossil: number of total fossil: 18 (5, 37) 311 (298, 338) 120 (40, 222) 315 (216,423) 188 (155, 239) 240 (173, 317) 594 (515, 730) Fig. 1 Divergence times in demosponges (Porifera) 9 456.0 22 this version posted July 9, 2017. 2017. 9, July posted version this CC-BY-NC-ND 4.0 International license International 4.0 CC-BY-NC-ND ; 1 under a under 1000.0 28 (7, 53) 483 (467, 517) 166 (52, 304) 391 (232, 564) 185 (163, 246) 279 (178, 396) 875 (606, 1200) BEAST analysis number of total fossil: BEAST 2.4.3 parameters Suppl. Fig. 4 originFBD: oldest Sphaerocladina fossil: Suppl. Table 2, black colored taxa Suppl. Table 2, black & red colored taxa https://doi.org/10.1101/159806 B C D E F R Nodes A doi: doi: should be greater thandistribution the [25], maximum we value obtainedpha/Keratosa of different in divergence the both times root analyses for age(see with Heteroscleromor- Table with 1, the a Figure root 1, log-normal node agespongiae, R). affecting using Previous different the ages estimated software, divergence forbetween clock-model time crown-group settings, 657-872 Demo- and Ma taxonanalyses [30, sets, varied (see 47, Table 50, 1,able 60], fossil Figure representing which 1 crown-group is Demospongiae andthe was in described Suppl. early by the Cambrian [61] (515 range from Figuredoes Ma). of 4, not contradict As both node a this of R). possible fossildeep Neoproterozoic our The only origin divergence first constitutes of of demosponges. a reli- of crown-group minimum This demosponge-specific age Demospongiae biomarkers it concurs (24-ipc(Neoproterozoic) with sterol) and in the today rocks first present datingAdditional appearance in 540-650 paleontological all Ma evidence major for anby demosponge early the clades discovery divergence [49, of of 62]. a spongesas 600 a is stem Ma given group old descendant fossil, for with all poriferan sponges features [63]. and interpreted cbd Schuster et al. Table 1: Divergence time estimatestwo (Ma) different of anlyses. demosponge clades Estimates of95% are interest highest given from posterior for density the interval. mean, and in brackets for the certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available available is made It in perpetuity. the preprint to display a license bioRxiv granted has who author/funder, is the review) by peer certified bioRxiv preprint preprint bioRxiv Preprint bioRiv . The copyright holder for this preprint (which was not was (which preprint this for holder copyright The Divergence times in demosponges (Porifera) 10 this version posted July 9, 2017. 2017. 9, July posted version this CC-BY-NC-ND 4.0 International license International 4.0 CC-BY-NC-ND ; under a under Time calibrated phylogeny of Demospongiae Figure 1: https://doi.org/10.1101/159806 based on paramters of BEAST analysis 2 plotted on stratigraphic chart. New in Table 1. The capital letter R specifies the root age of the dated phylogeny. shaded in light gray. Error bras on node ages are in dark turquoise. Nodes of doi: doi: sequenced species are in dark green and bold. Taxonomic clades of interest are Schuster et al. cbd interest are marked with a capital letters A-F and correspond to node ages listed certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available available is made It in perpetuity. the preprint to display a license bioRxiv granted has who author/funder, is the review) by peer certified bioRxiv preprint preprint bioRxiv Preprint bioRiv ⇠456 Ma) [42] ⇠172 Ma younger . The copyright holder for this preprint (which was not was (which preprint this for holder copyright The , Upper Ordovician Divergence times in demosponges (Porifera) 11 ⇠200 Ma gap in the fossil record [e.g. 64]. Due to this version posted July 9, 2017. 2017. 9, July posted version this CC-BY-NC-ND 4.0 International license International 4.0 CC-BY-NC-ND ; sp. (Sphaerocladina) available, this study represents the under a under Vetulina water sponges https://doi.org/10.1101/159806 doi: doi: cbd resembles species with the same desmathough larger types in as sizes those and found more insplit heavily the between silicified Mesozoic, marine [e.g. al- 64], and ourEarly freshwater analysis Ordovician sponges dates the (⇠483 (Point B, Ma).desma Suppl. Sponges skeleton Figure are with 4) well a to knownlate massively the in Ordovician, silicified the sphaeroclone Middle Paleozoic, Silurian andno and were sphaeroclone common late desmas during Devonian are the Jurassic, [see reported which e.g. from represents 39, the a 64–67]. Carboniferousthis long until But, gap, the it Middle ismologous debatable whether to the those Paleozoic found sphaeroclone in desmassuitable are as the ho- fossil Mesozoic constraint. and If Cenozoicthe Paleozoic [64, sphaeroclone analysis desmas 68], (BEAST are and analysis excluded therefore from 2),freshwater the sponges mean dates age back of(see the to node split the B between Carboniferous marine induring and the and Figure Carboniferous is until 1, thenected Middle Table Jurassic to 1). has the been Permian-Triassic proposed boundary Theto to (PTB) lack the be mass reduction con- of extinction in [39], size agroups which of fossil [69] sponge led spicules, sphaeroclone and the to desmas disappearancelow the of neritic certain habitat sponges environments displacement to ofet deeper several al. sponge bathyal 71] proposed taxa waters thatin from [see the shal- the e.g. observed 70]. Mesozoic decline and resultedpothesis [Maldonado turnover is from of corroborated the the by sponge reductionpast the fauna the of lack PTB of silica mass sphaeroclone extinction insphaeroclones desmas as in the found well the as around oceans. Paleozoic the and to observedMesozoic. smaller This change and from hy- less massive-large silicified sphaeroclones in the Many earlier molecular datingdatasets, studies however, of these were Porifera were hamperedexample by based lacking an on freshwater incomplete mitogenomic andences taxon desma-bearing of sampling, divergence sponges. for timesmarine for Consequently, and infer- key freshwater sponges demosponge could taxa,mitogenome not of be such addressed. as Now, with thefirst the split dated complete between phylogeny that suggestsfreshwater a likely sponges. timeframe Hypothesizing for that the themas split oldest of from marine fossil the and with Paleozoic (Amplaspongia sphaeroclone bulba des- Schuster et al. 3.4 Inferred divergence scenarios for the split of marine and fresh- certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available available is made It in perpetuity. the preprint to display a license bioRxiv granted has who author/funder, is the review) by peer certified bioRxiv preprint preprint bioRxiv Preprint Ephy- bioRiv . Rezinkovia echinata, all and The copyright holder for this preprint (which was not was (which preprint this for holder copyright The Divergence times in demosponges (Porifera) 12 this version posted July 9, 2017. 2017. 9, July posted version this CC-BY-NC-ND 4.0 International license International 4.0 CC-BY-NC-ND ⇠345.7 Ma) provide support for a post-Cambrian ori- ; under a under Eunapius, but absent from Lubomirskiidae [80], our data is consistent and Corallistidae ⇠385 Ma) and [76]( and https://doi.org/10.1101/159806 doi: doi: We estimated a meanerous) origin (node age D, for Table 1, Tetractinellidadistribution Figure of on 1, 315 the BEAST node Ma analysiserous age (Late 2), (Figure origin Carbonif- with 1, is a BEAST normal lateto analysis frequency a 2). for Middle this Indeed, Cambrian a group (⇠514tetraxial-like Carbonif- fossil Ma) considering spicules origin known previous from of the estimates this81]. Middle which clade Cambrian (510-520 Despite in point Ma) these addition [47, Cambrian to[50]( fossil the discoveries, earliest the molecular clock analyses of cbd 3.6 Inferred divergence scenario of Tetractinellida, Spirophorina Lubomirskiidae), all of which areOur known dated to be phylogeny endemic suggests to(⇠3.4 a Lake Ma, divergence Baikal Figure [29, of 1, 79]. this this Node area clade A), (3.2-2.8 to which Ma) the [29, correlatesince Early 79]. the to lower Pliocene the As Cretaceous gemmules known [75],datia are and fossil known are record present from from in the the fossilwith Recent record the spongillids hypothesis thatsessed the gemmules, which most were recent subsequently lost commonas in ancestor several the endemic Lake of lineages Baikal such Spongillida Lubomirskiidae pos- [see discussion in 80]. Schuster et al. 3.5 Inferred timing ofThe extant occurrence freshwater sponge of diversification thePermo-Carboniferous [41] earliest and freshwater constitute sponge theof fossil first freshwater and spicule sponges only is known fromsponges, dated fossil the to record however, Paleozoic. the is28.3 The dated Ma, radiation as of Paleogene, much recent Tableinterpretation younger 1, freshwater as Paleozoic in Node spicules. A). bothas Also, Therefore, of either [72] our our interpreted marine results the analyses ortion findings question of marine (18.0- of [41]’s this [41] influenced, oldestsponges which with described again intact freshwater challenge gemmules sponge. (i.e.reproduction the highly freshwater-sponge interpreta- In resistant specific contrary, buds to fossilwell-known for desiccation, from asexual freshwater freezing the lower and Cretaceous anoxiaRecent [75], [73, thus freshwater supporting 74]) sponges a are before diversificationa of the divergence Paleogene of (66 7-10 Ma). Marelaxed for molecular Yet, clock Recent [30] freshwater approach, suggested whereupon spongesogene the using divergence. study a of node-calibrated [76] indicateThe a Paleogene Pale- record ofthe freshwater sponges Neogene is record known [77,(Baikalospongia to intermedia, 78]. be Lubomirskia more baicalensis Our diverse than analysis includes three freshwater species certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available available is made It in perpetuity. the preprint to display a license bioRxiv granted has who author/funder, is the review) by peer certified bioRxiv preprint preprint bioRxiv Preprint bioRiv . ⇠155 Ma. The only known is described from the Early The copyright holder for this preprint (which was not was (which preprint this for holder copyright The Divergence times in demosponges (Porifera) ⇠188.7 Ma (Lower Jurassic). The node 13 Neophrissospongia this version posted July 9, 2017. 2017. 9, July posted version this CC-BY-NC-ND 4.0 International license International 4.0 CC-BY-NC-ND ; under a under ⇠120 Ma (Late Cretaceous). The frequency distribution on https://doi.org/10.1101/159806 doi: doi: Campanian of Poland [23],back to but the our Middle analysisand Jurassic continuous (Figure indicates fossil 1). a records among Assampling deeper the this sensitivity origin included of family dating taxa, the shows we FBDby one tested clock of 50% this model the (Suppl. clade by richest for increasingpling the Table neither number 2, influenced our of BEAST results fossils stance) analysis positively nor (by 2). negatively, reducing which the corroborates This errorinvestigation other bars findings increase of for of the of in- [25] divergence the andstrengthen ages [26]. fossil the of The Jurassic sam- this origin desma-bearing ofon this possible demosponge calibration clade family constraints and for provides further additional molecular information clock approaches. The tetractinellid suborder Spirophorina (node C,ysis Table 1, 2) Figure 1, is BEAST dated anal- to the node age indicates a slightly5, right-shifted BEAST normal distribution analysis (Suppl. 2).presence Figure of so A called characteristic sigmaspiredescribed diagnostic (S- to a feature C-shaped) C-shaped for microclere microsclere this spiculesDaly [83]. and [Plate group Georgina [84] 24 is Basin in the (Northernwhich Territory 84] he in associated from Australia, to see the "orthocladine" Plate sponges.Spirophorina Middle 24 [85] in Cambrian in suggested the the [84]), Early occurrencethese of Paleozoic, with observations a can possible notspicules Cambrian be are origin, supported also however, by present anyrida in of and other our demosponge Desmacellida, analyses. lineages themight like As discovered not e.g. sigma-like be C-shaped homologous in microsclere to Poeciloscle- described those in ofcbd Spirophorina. [84] Schuster et al. gin of this clade.due These to contradictory their massive results and may thickerspicules have sizes may different the not Cambrian reasons. be tetractinellid First, homologous (tetraxial-like) the to presence post-Paleozoic of forms aster-like [e.g.groups and 39, other monaxon 64]. than spicules Second, the inof Tetractinellida several the may Cambrian recent lead fossil demosponge to spicules.ous the Third, spicule the erroneous types high interpretation in level particularhamper of unambiguous of secondary interpretation microscleres losses of of within their vari- Astrophorina homology. [20, 57, 82] The astrophorid family Corallistidaeby (node dicranoclone E, desmas, Table is 1, here datedage Figure to shows 1), a characterized left-skewedanalysis distribution 2), to which younger correlates ages withJurassic (Suppl. the to current Figure Recent known 5, [15, fossilcluded BEAST 23]. record Recent from tetractinellids Additional the is support late providedphylogeny by for (cox1) a of a node-based [76], Jurassic calibrated who originfossil single-gene dated Corallistidae representative of to of the the in- genus certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available available is made It in perpetuity. the preprint to display a license bioRxiv granted has who author/funder, is the review) by peer certified bioRxiv preprint preprint bioRxiv Preprint bioRiv ⇠188 Ma. ⇠311 Ma, . The copyright holder for this preprint (which was not was (which preprint this for holder copyright The Divergence times in demosponges (Porifera) ⇠315 Ma, the suborders Astrophorina to 14 ⇠120 Ma and the family Corallistidae to this version posted July 9, 2017. 2017. 9, July posted version this CC-BY-NC-ND 4.0 International license International 4.0 CC-BY-NC-ND ; under a under https://doi.org/10.1101/159806 doi: doi: cbd Acknowledgements Funding was provided by theber German DFG Research Foundation ER (DFG)Samples (grant 611/3-1, were num- collected DFG by Wo896/15-1)ing HBOI’s several and Johnson-Sea-Link epeditions. the manned We submersible greatlyon LMU dur- acknowledge library Mentoring Zac preparation H. and Program. Forsman Amyrine for Eggers Biology). discussions for sequencing We are (HawaiiConkling for thankful Institute helping of to in Ma- the Amy collectionHBOI). Wright (Harbor for Branch Florida sharing Atlantic University, samples and Megan Furthermore, we discovered thatthe increasing Corallistidae the fossil indicates samplingsampling that density, by which this corroborates 50% with approach within theNevertheless, is findings of our relatively other estimated studies insensitive [25, divergencetaxa to 26]. times such fossil as of the differentfiner-scaled Astrophorina higher divergence or time tetractinellid Corallistidae estimates can toof be secondarily shed further spicule new used losses light to for on possibleical inferring changing e.g events geochemical/geological the in histor- correlations the past. The split of freshwater sponges andmost marine of Sphaerocladina is which dated correlate to viously with assumed the recent fossil record. (⇠18results, Ma) Additionally, and diversification we in confirmed of particular pre- freshwater thebe sponges. dated used split These as of a freshwaterorder and root to marine get age sponges, a for can betteras further idea the of dated radiation timing e.g. phylogenies in their on different historical ancient freshwater biogeographical lakes. sponges processes such in Schuster et al. 4 Conclusion We here successfully assembledsponge six taxa generated complete by a mitogenomes size-selectedIntegrating reduced of representation these genomic different library. data demo- newly into tested a relaxed molecular novel clock approachvide mitogenome based new on alignment insights the into FBD in the model, evolutionzoic we tandem of origin pro- selected with of Demospongiae. Demospongiae a is The confirmed.cation Neoprotero- of Furthermore, the the Tetractinellida origin is and⇠240 dated diversifi- Ma, to the Spirophorina to certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available available is made It in perpetuity. the preprint to display a license bioRxiv granted has who author/funder, is the review) by peer certified bioRxiv preprint preprint bioRxiv Preprint 1.1 Mol. Spongilla bioRiv Molecular 10 (2010), BMC Evolu- Mitochondrial . The copyright holder for this preprint (which was not was (which preprint this for holder copyright The Molecular Biology and Evolu- Mitochondrial DNA Part B Lubomirskia baicalensis”. In: Divergence times in demosponges (Porifera) BMC Evolutionary Biology 24.7 (2007), pp. 1518–1527. 24 (2007), pp. 19–22. 15 muelleri”. In: 49 (2008), pp. 111–124. cf. (Astrophorida, Vulcanellidae)”. 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