MBE Advance Access published February 23, 2014

Illuminating the base of the annelid tree using transcriptomics

Anne Weigert1, Conrad Helm1, Matthias Meyer2, Birgit Nickel2, Detlev Arendt3, Bernhard Hausdorf4, Scott R. Santos5, Kenneth M. Halanych5, Günter Purschke6, Christoph Bleidorn1,8 and Torsten H. Struck7,8

1

Molecular Evolution and Animal Systematics, University of Leipzig, Talstr. 33, 04103

Leipzig, Germany 2

Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig,

Germany 3

EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany

4

Zoological Museum, University of Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg,

Germany 5

Molette Biology Laboratory for Environmental and Climate Change Studies, University of

Auburn, 101 Rouse Life, AL 36849, USA 6

Department of Zoology and Developmental Biology, University of Osnabrueck, Barbarastr.

11, 49069 Osnabrueck, Germany 7

Zoological Research Museum Alexander Koenig, Adenauerallee 160, 53113 Bonn,

Germany 8

shared senior authors

Corresponding author: Anne Weigert, Molecular Evolution and Animal Systematics, University of Leipzig, Talstr. 33, 04103 Leipzig, Germany; [email protected]; Phone: +49-341-9736743; Fax: +49-341-9736789 © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: [email protected] 1

Abstract

Annelida is one of three animal groups possessing segmentation and is central in considerations about the evolution of different character traits. It has even been proposed that the bilaterian ancestor resembled an annelid. However, a robust phylogeny of Annelida, especially with respect to the basal relationships, has been lacking. Our study based on transcriptomic data comprising 68,750 – 170,497 amino acid sites from 305 – 622 proteins resolves annelid relationships, including Chaetopteridae, Amphinomidae, Sipuncula, Oweniidae, Magelonidae in the basal part of the tree. Myzostomida, which have been indicated to belong to the basal radiation as well, are now found deeply nested within Annelida as sister group to Errantia in most analyses. Based on our reconstruction of a robust annelid phylogeny, we show that the basal branching taxa include a huge variety of life-styles such as tube-dwelling and deposit-feeding, endobenthic and burrowing, tubicolous and filter-feeding, as well as errant and carnivorous forms. Ancestral character state reconstruction suggests that the ancestral annelid possessed a pair of either sensory or grooved palps, bicellular eyes, biramous parapodia bearing simple chaeta and lacked nuchal organs. Since the oldest fossil of Annelida is reported for Sipuncula (520 Mya), we infer that the early diversification of annelids took place at least in the Lower Cambrian.

2

Introduction

Annelids, or segmented worms, comprise over 21,000 recognized species found in marine, freshwater and terrestrial habitats. The group has been central in debates on major transitions in animal evolution such as the development of segmentation, evolution of the nervous system, transitions to a terrestrial lifestyle, and origins and diversifications of larval types (Purschke 1999; Rouse 1999; Seaver 2003; Jekely, et al. 2008). To address such topics a well-supported phylogeny of Annelida is needed to assess ancestral conditions and to allow discrimination between alternative hypotheses. Unfortunately, relationships among basal annelid lineages are still poorly understood (Struck, et al. 2011; Kvist and Siddall 2013), hindering our understanding of early annelid evolution. Since they usually lack hard body parts, the fossil record of the early annelid radiation is sparse and provides little resolution on the early annelid radiation. Although some trace and tube fossils from the Ediacara fauna (635–542 Mya) reportedly resemble polychaetes (Glaessner 1976b, a; Retallack 2007), the oldest accepted annelid fossils are reported from the Maotianshan Shale, Sirius Passet and the Burgess Shale from the Lower Cambrian about 520 Mya (Huang, et al. 2004; Morris and Peel 2008; Vinther, et al. 2011). Several

hypotheses

have

been

proposed

regarding

morphological

characteristics of the last common ancestor of Annelida but currently there is little consensus on this issue. According to Westheide (1997) there are two major types of competing scenarios. The first type of scenario describes evolution of annelids from simple to complex, where the stem species lack head appendages and parapodia have simple chaetae as an adaptation to burrowing (Clark 1969; Westheide 1997). This view was inspired by the traditional division of Annelida into Polychaeta and Clitellata 3

(oligochaetes and leeches) and received support by morphological-cladistic analyses (Rouse and Fauchald 1997). In accordance with this hypothesis the former “Archiannelida”, which were considered a primitive group due to the lack of parapodia and chaetae in several taxa, have been debated as very close to the annelid stem species (Hermans 1969). However, molecular and morphological data show that “Archiannelida” constitute a polyphyletic assemblage of independently evolved putatively progenetic lineages, which converged on a simplified body plan (Fauchald 1977; Purschke and Jouin 1988). The second type of scenario describes the opposite direction of evolution from a more complex errant, epibenthic ancestor with well-developed head appendages, parapodia and chaetae towards more simple forms by modifications, reductions and losses (Storch 1968). Investigating morphological or molecular data, which found Clitellata, Echiura and Siboglinidae nested within Polychaeta and Myzostomida as well as Sipuncula within the annelid radiation, in part substantiated this view (McHugh 1997; Struck, et al. 2008; Bleidorn, et al. 2009; Zrzavy, et al. 2009; Helm, et al. 2012). These analyses indicated that reductions of characters frequently occurred in Annelida (Purschke, et al. 2000; Bleidorn 2007). At present, phylogenetic hypotheses lack consensus as to which annelid taxa are to be placed at the base of the tree (McHugh 2000; Bleidorn, et al. 2003; Rousset, et al. 2007; Struck, et al. 2008; Zrzavy, et al. 2009). Additionally, some analyses struggled to recover a monophyletic Annelida and lacked resolution for deeper nodes perhaps due to using limited numbers of loci. Only recently Struck, et al. (2011) presented a robust backbone of the annelid tree based on a phylogenomic analysis including expressed sequence tag (EST) libraries of several annelid families. In this analysis ancestral character state reconstructions suggested that the last common ancestor possessed only one pair of head appendages (i.e., grooved 4

palps), nuchal organs and parapodia with simple and internalized supporting chaetae. From this ancestor evolution progressed to both more complexly and more simply organized annelids. In that study, the basal part of the annelid tree comprises only three taxa: Sipuncula, Myzostomida and Chaetopteridae. Although congruent with some previous studies (Struck, et al. 2008; Zrzavy, et al. 2009; Dordel, et al. 2010), support for relationships among these basal branching taxa depended on the reconstruction method used (Struck, et al. 2011; Kvist and Siddall 2013). Moreover, crucial taxa with respect to basal relationships of Annelida were lacking, e.g., Oweniidae and Magelonidae (Rousset, et al. 2007; Zrzavy, et al. 2009). To illuminate the basal radiation of Annelida and to test their phylogenetic relationships we extended the taxon sampling by generating transcriptome libraries for 22 additional annelid taxa, focusing on potential basal branching taxa. In total our analyses now comprise 60 annelid species, representing 39 annelid families. This is about one third of the total number of approximately 125 annelid families (Fauchald and Rouse 1997; Jamieson and Ferraguti 2006; Siddall, et al. 2006), covering 6 out of 35 clitellate families and 33 out of 80 polychaete families. Furthermore, we expanded the data matrix to 170,497 amino acid positions derived from 622 genes. So far this is the largest data set available for annelids based on next generation sequencing data to provide a robust annelid phylogeny.

Results

We generated seven data subsets ranging from 68,750 to 170,497 amino acid positions derived from 305 to 622 genes (Table 1). Gene coverage of the matrix increased from 0.378 to 0.547 for the 79-taxa data subsets, from 0.375 to 0.549 in 5

the 77-taxa data subsets and from 0.402 to 0.555 for the 72-taxa data subset (Table 1). Topologies obtained from all seven data subsets are nearly identical except for the positions of Myzostomida and Orbiniidae (Fig. 1, supplementary Fig. S1-S6). Errantia, as well as Sedentaria are recovered in all analyses with Clitellata deeply nesting within Sedentaria. The relationships in the basal part of the annelid tree are robust regardless of data set employed and supported by moderate (BS ≥ 70) to significantly high (BS ≥ 95) bootstrap values, especially in the analyses without Myzostomida (Fig. 1, supplementary Fig. S4 and S5). Sipuncula together with Amphinomidae are sister group to Pleistoannelida. Chaetopteridae is sister to this clade. Magelonidae and Oweniidae form a monophyletic group and together are the sister group of all other annelids. Monophyly of Annelida is recovered with significantly high (BS ≥ 99) support values in all analyses without Myzostomida (Fig. 1, supplementary Fig. S4 and S5). By including Myzostomida monophyletic Annelida are supported by moderate (BS ≥ 70, supplementary Fig. S2) to significantly high (BS ≥ 99, supplementary Fig. S1) support values in the analyses comprising the highest number of genes (Table 1: 79taxa data subsets 1.5 and 1). In these analyses Myzostomida fall within Pleistoannelida as sister taxon of Errantia, which comprises Phyllodocida and Eunicida (supplementary Fig. S1 and S2). However, in the analysis of the 79-taxa data subset 2 (Table 1) comprising 305 genes and 68,903 aa positions, longbranched Myzostomida are closely related to outgroup taxa Cycliophora and Entoprocta (supplementary Fig. S3). The problem of analyzing the phylogenetic position of the long branching Myzostomida has been shown before (Bleidorn, et al. 2007) and is also reflected by their LB scores in our present analyses. In all three 79taxa data subsets, the two myzostomid species as well as the cycliophoran Symbion and the two entoproct species of Pedicellina are among the six taxa with highest LB 6

scores. Leaf stability analyses also reflected the problematic placement of myzostomids (supplementary Table S6). Due to the possible long branch attraction of Myzostomida and outgroups, we created a seventh data set comprising 72 taxa where we excluded all outgroups except Mollusca, Nemertea and Brachiopoda. Since the analysis of the 79-taxa data subset 2 was the only one resulting in paraphyletic Annelida, with Myzostomida nesting within outgroups (supplementary Fig. S3) we applied the same weighting parameter to the 72-taxa data set as to the data set with 79 taxa (MARE α=2) using the same genes (supplementary Table S5) to investigate the influence of LBA on the position of Myzostomida (data set 72-2). As a result we find monophyletic Annelida with significantly high (BS ≥ 95) support and Myzostomida nest within Errantia as sister taxon of Eunicida (supplementary Fig. S6). Within Sedentaria, relationships among taxa stay robust across all seven data sets except for the position of Orbiniidae. The leaf stability indices of the basal branching annelids were above 0.96 for all data sets and, hence, the lower support values observed in the analyses of the data subset 79-1.5 (supplementary Fig. S2) can be attributed to the unstable position of Myzostomida as discussed above. We also tested whether alternative positions for the basal branching taxa (i.e., Sipuncula, Amphinomidae, Chaetopteridae, Oweniidae, and Magelonidae) could be rejected. The tested options were placement of Sipuncula outside Annelida, inclusion of Amphinomida within Errantia as well as Chaetopteridae, Oweniidae, and Magelonidae as members of Sedentaria (supplementary Table S4). All nine tested a priori hypotheses, which are mainly based on the traditional systematic view and on morphology could be significantly rejected (supplementary Table S4). Moreover, we explored if paralogy or xenology, compositional heterogeneity, lack of data or long branches influenced the placement of these taxa. The plot of psL of each constrained analysis in comparison to the best tree showed that the 7

phylogenetic signal for each topology (the constraint and the best tree) is scattered across the whole alignment and not concentrated in certain genes (data dryad: Files S1-S9). Hence, we find no evidence that the support for the position of Oweniidae, Magelonidae, Chaetopteridae, Amphinomidae, and Sipuncula in the basal part of the annelid tree is due to paralogous or xenologous sequences. Wilcoxon-Mann-Whitney tests also revealed no significant differences in the distributions of both RCFV values and proportion of missing sequence data for the “basal” annelid taxa and all other taxa in the tree in all seven data subsets (supplementary Table S3). Thus, the basal position of these annelid taxa is most likely not due to shared compositional heterogeneity or similar degrees of missing data. Since we extended the taxon sampling and certain ancestral character traits of the annelid stem species were not present in all basal branching taxa, like the nuchal organs and grooved palps, we repeated the ancestral state reconstruction of Struck, et al. (2011) including our new taxa and results. The results of the new reconstructions are generally identical to the ones of Struck, et al. (2011) in, among others, the presence of a pair of peristomial palps, bicellular eyes and biramous parapodia bearing simple chaetae (supplementary Table S7, Fig. 3). However, three major differences could be observed: 1.) it is uncertain if the pair of peristomial palps is of the solid or the grooved kind; 2.) internalized supporting chaetae are lacking and 3.) nuchal organs are absent. The other differences were either uncertain characters becoming a certain state or vice versa.

8

Discussion

Annelid relationships Phylogenomic analyses provided robust support for basal annelid relationships including Oweniidae, Magelonidae, Chaetopteridae, Sipuncula, and Amphinomidae (Fig. 2). Interestingly Magelonidae and Oweniidae represent the sister group to all other annelids with strong support. Our results are surprisingly congruent with a hypothesis made by Rieger (1988) in 1988, where he suggested that members of Oweniidae closely resemble the annelid stem species and should be placed near the base of the annelid tree. His view was inspired by the presence of certain morphological characters in Oweniidae, which are absent in other annelid taxa and considered as plesiomorphic rather than as derived: the presence of a largely intraepidermal nervous system (Bubko and Minichev 1972), the mitraria larva possessing monociliated epidermal cells and nephridia with deuterostome similarities (Smart and Von Dassow 2009) as well as presence of monociliated epidermal cells in adults (Gardiner 1978; Westheide 1997). Since monociliated epidermal cells were thought to be absent in Annelida; they can elsewhere be found in members of the lophotrochozoan

groups

Gnathostomulida,

Gastrotricha,

Phoronida,

and

Brachiopoda (Rieger 1976; Gardiner 1978; Nielsen 2002). For Oweniidae they could be confirmed in larvae and adults of Owenia fusiformis (Gardiner 1978) and in adults of the genus Myriowenia (Westheide 1997). Interestingly larvae of Magelona mirabilis (Magelonidae) likewise possess an epidermis made up of monociliated cells (Bartolomaeus 1995). A close relationship of Magelonidae and Oweniidae was also recently proposed by (Capa, et al. 2012) based on a cladistic analyses of morpgological characters. However, care must be taken in evaluating characters as apomorphic or plesiomorphic. For instance, an intra- or basiepidermal nervous 9

system is widespread in annelids and homoplasy cannot be ruled out (Bullock and Horridge 1965; Purschke 2002; Orrhage and Muller 2005). Moreover, retention of a plesiomorphic character does of course in no way support the phylogenetic position of its bearer. Given our results and the scattered appearance of monociliated epidermis cells in Metazoa (Rieger 1976) the former could well be a synapomorphy for the clade of Magelonidae and Oweniidae within Annelida. Moreover, we find important changes regarding the phylogenetic position of certain taxa with respect to the analyses of Struck, et al. (2011), highlighting the importance of extended taxon sampling as well as extended gene coverage to recover a robust phylogeny beyond the basal relationships. By increasing the number of analyzed genes in our analyses Myzostomida fell within Annelida as part of Errantia and are not part of the basal radiation as in Struck, et al. (2011). We find that bootstrap values for monophyly of Annelida and a myzostomid-Errantia sister group relationship increase with the number of analyzed genes. The phylogenetic position of myzostomids was always problematic due to long-branch attraction with outgroup taxa, but several previous analyses using multiple markers and rare genomic changes strongly support Myzostomida as part of the annelid radiation as well (Bleidorn, et al. 2007; Hartmann, et al. 2012; Helm, et al. 2012). In contrast to Struck, et al. (2011) Orbiniidae and Amphinomidae are placed differently. By including more species of each taxon the grouping of Aphroditiformia (Phyllodocida), Orbiniidae and Amphinomidae was not recovered: Instead Orbiniidae grouped within Sedentaria, whereas Amphinomidae branched off in the basal part of the tree as sister to Sipuncula. These changes also resulted in the monophyly of Phyllodocida, which was paraphyletic in Struck, et al. (2011). The previous positions of Aphroditiformia (Phyllodocida), Orbiniidae and Amphinomidae were most likely due to low gene coverage as well as a shared paralog by these three (Struck 2013). Notably, the 10

basal branching position of Amphinomidae had been found in a few previous molecular studies, but received little attention so far (Hausdorf, et al. 2007; Struck, et al. 2008). Also, an early branching of Chaetopteridae, which has been indicated previously (Struck, et al. 2007; Struck, et al. 2008; Bleidorn, et al. 2009; Zrzavy, et al. 2009; Dordel, et al. 2010) was confirmed with high support. Placement of Sipuncula within Annelida is strongly supported by our analyses and substantiates previous studies (Bleidorn, et al. 2009; Mwinyi, et al. 2009; Dordel, et al. 2010). Due to the absence of segmentation, parapodia and chaetae as well as typical head and body appendages the phylogenetic position of Sipuncula was under discussion since their description (Schulze, et al. 2005). Sperling, et al. (2009) recently suggested the absence of segmentation in Sipuncula likely to be primary rather than secondary based on microRNAs and the fossil record. Since the amount of characters used in their analysis was low and the taxon sampling extremely poor (three annelid species), with crucial basal branching taxa lacking, placement of Sipuncula as sister group to the three other annelids was not surprising. On the contrary our analyses, strongly supporting Sipuncula as part of the annelid radiation, suggest that absence of segmentation may be a derived character state, which was indicated in a previously study by immunohistological investigations of the nervous system (Kristof, et al. 2008).

Character evolution in annelids The controversy of the direction of annelid evolution is crucially linked to character states assumed for their last common ancestor (Clark 1969; Westheide 1997). In this discussion the vast morphological diversity and different life modes of annelid taxa have to be taken into account as, for example, the basal branching taxa include tubedwelling,

deposit-feeding

Oweniidae,

endobenthic,

burrowing

Magelonidae, 11

tubicolous, filter-feeding Chaetopteridae, unsegmented, burrowing Sipuncula, as well as errant, carnivorous Amphinomidae. According to Struck (2011), the ancestral annelid was an intermediate form between a simple- or complex-bodied ancestor and represented a “microphagous surface deposit-feeder crawling upon and through soft-bottom habitats”. Although generally in agreement with these conclusions, results of the reconstructions herein showed slight differences to the ones of Struck, et al. (2011) in the uncertainty of the kind of peristomial palps as either solid or grooved, lack of internalized supporting chaetae and absence of nuchal organs. Thus, in comparison to these previous studies (Struck 2011; Struck, et al. 2011) the ancestral annelid was slightly more simply organized. Palps, regardless of whether they emerge from the prostomium or peristomium, are innervated similarly and, therefore, regarded to be homologous (Fauchald and Rouse 1997; Orrhage and Muller 2005). They can be divided into grooved ciliated feeding palps and ventral, tapering sensory palps; the former bi-functional (sensory and feeding) the latter purely sensory. Within the basal radiation sensory palps are found in Amphinomidae only. In Magelonidae a derived type of grooved palps may be present, which develops in ontogeny after shedding of the primary ones (Wilson 1982). Therefore, the palps of the ancestral annelid most likely were of the grooved (ciliated) type having a dual function of gathering food and sensing (Struck 2011), although this hypothesis could not be resolved by ancetral reconstruction. The lack of supporting chaetae is much more in line with the known fossil record of the Cambrian as such chaetae were not observed in these fossils (Vinther, et al. 2011; Eibye-Jacobsen and Vinther 2012). Although almost all annelid taxa possess nuchal organs, their lack in the annelid ground pattern has been proposed before, based on the traditional position of 12

Clitellata as sister to Polychaeta (Rouse and Fauchald 1997; Purschke 2002). However, as Clitellata are deeply nested within polychaetes (Rousset, et al. 2007; Zrzavy, et al. 2009; Struck, et al. 2011), nuchal organs have likely been lost in Clitellata (Purschke, et al. 2000), as well as in Echiura, Myzostomida and Siboglinidae, which also lack nuchal organs. The presence of nuchal organs in Sipuncula is controversially discussed (Purschke 1997) and requires ultrastructural investigations in more species. On the other hand, the lack of nuchal organs in Oweniidae and Magelonidae is most likely a primary absence given the results presented herein. Thus, nuchal organs are likely not an autapomorphy of Annelida (Purschke 2002). Therefore, according to our analyses the ancestral annelid possessed a pair of either sensory or grooved palps, bicellular eyes, biramous parapodia bearing simple chaeta and lacked nuchal organs (Fig. 3, Table S7). Eventually characters like the collageneous cuticle (Purschke 2002), the specific arrangement of the body wall musculature (Purschke 2002), the intraepidermal position of the nervous system (as e. g. found in Oweniidae) have to be considered as part of the annelid ground pattern as well.

Implications on the annelid radiation from the fossil record Investigating the fossil record the oldest annelid fossils are described for Sipuncula from the Lower Cambrian, which seem remarkably similar to recent sipunculids (Huang, et al. 2004). All three specimens were discovered in the Maotianshan Shales and illustrate that the morphological appearance of Sipuncula has only slightly changed in the past 520 million years. Polychaete fossils from the Lower Cambrian are sparsely known. Although Maotianchaeta fuxianella and Facivermis yunnanicus known from the Maotianshan Shales resemble worm-like organisms (Li, et al. 2007), 13

a close relationship with lobopods seems more plausible (Liu, et al. 2006). Despite that, recently described polychaete fossils from the Sirius Passet (518-505 Mya) were hypothesised as stem annelids (Morris and Peel 2008; Vinther, et al. 2011). In addition, various forms of tube-like fossils have been discovered from the Lower and Middle Cambrian, which were presumably constructed by stem group annelids (Skovsted and Peel 2011). Skovsted and Peel (2011) concluded that these fossils had a similar life style as recent Chaetopteridae, based on the absence of attachment structures, vertical orientation of the tubes in the sediment and opening on both ends, which allowed the tube-dwelling worm-like animal to move and filter water. However, these interpretations have to be considered with caution, since no specimen within the tubes has been discovered yet. Nonetheless, the recovered phylogeny and the fossil record of Annelida indicate that lineages leading to recent annelid

groups

such

as

Magelonidae

+

Owenidae,

Chaetopteridae

and

Amphinomidae are as old as Sipuncula. Consequently, these lineages are separated since the Early Cambrian from the rest of Annelida. Considering the age of the early annelid radiation and that since this time several mass extinctions are recorded, including the end-Permian eradication of 90% of all marine species (Jin, et al. 2000), it might come as no surprise that we observe a patchwork of morphologies and life modes of recent taxa branching off from the basal part of the annelid tree. No annelid taxon shows all ancestral conditions, but is a composition of plesiomorphic and apomorphic characters even in the case that the taxon is sister to all other annelids (Crisp and Cook 2005). The increased taxon sampling of annelid lineages clearly improved our picture of annelid evolution. The backbone presented in Struck et al. (2011) was largely supported and some more lineages branching in the basal part of the tree were discovered. Additional inclusion of so far lacking annelid families in future phylogenomic studies will be an important 14

step in the refinement of our view of character evolution in annelids. Based on our analyses we strongly advocate the establishment of additional annelid model systems of species within the basal radiation, which would contribute significantly to the understanding of the annelid ground pattern.

Material and Methods

Sampling, transcriptome library construction and sequencing Table S1 provides information on collection of organisms. All samples were either snap-frozen in liquid nitrogen, fixed immediately in Ambion® RNAlater® (Life Technologies, Carlsbad, CA, USA) or kept in a seawater tank until RNA was extracted. For all species total RNA was extracted using Trizol Reagent (Life Technologies, Carlsbad, CA, USA) and purified with the RNeasy® Mini Kit (Qiagen, Hilden, Germany). Purification of mRNA was performed using the Dynabeads® mRNA Purification Kit (Life Technologies, Carlsbad, CA, USA). Subsequent fragmentation was carried out using the Ambion® RNA Fragmentation Reagent to obtain fragments around 200-250 bps. First strand synthesis of cDNA was conducted with SuperScript® II reverse transcriptase (Life Technologies, Carlsbad, CA, USA) using random hexamer primers followed by second strand synthesis using DNA Polymerase I and RNase H (Life Technologies, Carlsbad, CA, USA). Sequencing libraries were prepared following the protocol of Meyer and Kircher (2010) using double indices as published in Kircher, et al. (2012). The concentration and purity of RNA, cDNA and sequencing libraries was determined with Nanodrop and additionally on a Bioanalyzer 2100 (Agilent Technologies, CA, USA). Most libraries were either sequenced on the Illumina Genome Analyzer IIx or on the Illumina MiSeq with 76 15

cycles paired end following the manufacturer’s protocols. Libraries of Magelona berkeleyi, Paramphinome jeffreysi, and Phyllochaetopterus sp. were sequenced following the standard Tru-Seq Illumina protocols and sequenced as 100bp paired end runs on a Illumina Hi-Seq 2000. Magelona berkeleyi and P. jeffreysi were run at Hudson

Alpha

Genomics

Service

Lab

(Huntville,

Alabama,

USA)

and

Phyllochaetopterus sp. was sequenced at the Emory Genetics Laboratory (Decatur, GA, USA). Libraries for Ophelia rathkei and Eurythoe complanata were generated at GeneCore (EMBL, Heidelberg, Germany) following the standard protocol from Illumina for sequencing of mRNA and sequenced on the Illumina Genome Analyzer IIx with 105 cycles paired end. All sequence data is deposited in the NCBI sequence read archive.

Processing raw data and sequence assembly Bases were called with IBIS 1.1.2 (Kircher, et al. 2009), adaptor and primer sequences were removed and reads with low complexity as well as false paired indices were discarded. Raw data of all libraries were trimmed by applying a filter of 15, discarding all reads with more than 5 bases below a quality score of 15. The quality

of

all

sequences

was

checked

(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).

using

FastQC

Subsequently

all

libraries, except libraries of Ophelia rathkei and Eurythoe complanata, were assembled de novo using the CLC Genomics Workbench 5.1 (CLC bio, Århus, Denmark) with the following settings: mismatch cost 3; insertion cost 3; deletion cost 3; length fraction 0.5; similarity fraction 0.8; minimum contig length 200; automatic word size; automatic bubble size; and contig adjustment by mapped reads. Data used for the assembly of Myzostoma cirriferum comprised reads which were generated previously (Supplementary Table S1) as well as reads of an additional run 16

of the identical library on the Illumina Genome Analyzer IIx with 76 cycles paired end. Libraries of Ophelia rathkei and Eurythoe complanata were assembled using Velvet v. 1.1.04 (Zerbino and Birney 2008) and OASES v. 0.1.21 (Schulz, et al. 2012) with the default parameters. All assembled libraries were checked for possible (cross) contamination by local Blast with 18S and several putatively single copy ribosomal proteins and were rechecked on NCBI.

Obtaining and processing data from other taxa Additional data for 36 annelids, 3 nemerteans, 4 molluscs, 3 brachiopods, 2 phoronids, 2 bryozoans, 2 entoprocts, and 1 cycliophoran were obtained from public resources of NCBI (National Center for Biotechnology Information (NCBI) run by the National Institutes of Health), including the Expressed Sequence Tags database, the Sequence Read Archive and the Trace archive. Data for Capitella teleta, Helobdella robusta and Lottia gigantea were extracted from the Joint Genome Institute (supplementary Table S2). Raw Illumina data were assembled with CLC Workbench 5.1 using identical parameters as described above. 454 data was assembled with MIRA v. 3.4.0 (http://www.chevreux.org) using the default settings. Data obtained from Struck, et al. (2011) as well as all outgroup taxa except Hanleya sp. and Lingula anatina were processed as described in Hausdorf, et al. (2007).

Orthology assignment Orthology prediction was performed using HaMStR version 8b with the representative option

(Ebersberger,

et

al.

2009).

The

applied

core-ortholog

set

lophotrochozoa_hmmer3, which was generated using proteomes from seven primer taxa (Helobdella robusta, Capitella teleta, Lottia gigantea, Schistosoma mansoni, Daphnia pulex, Apis mellifera and Caenorhabditis elegans) and comprises 2,339 17

orthologous genes, is deposited in data dryad. Each potential candidate-ortholog was then checked with reciprocal blast against the reference-taxon Helobdella robusta, and was discarded if it did not match to the expected ortholog from the referencetaxon. Redundant sequences were eliminated using a custom Perl script, which checked for the presence of redundant sequence identifiers in the assigned orthologous genes for each taxon. Additionally, we checked our putative orthologous sequences for contamination with protist sequences using local BLAST against the Apicomplexa proteome. We chose Apicomplexa for comparisons as gregarine parasites have been described for several annelids. Orthologous sequences which were more similar to Apicomplexa than to the reference-taxon Helobdella robusta were discarded.

Sequence alignment, masking and concatenating and matrix reduction Three data matrices were generated for phylogenetic analyses: Data set 79 comprising 79 taxa, data set 77, which excludes the long-branching myzostomids resulting in a matrix of 77 taxa and data set 72 comprising 72 taxa due to the exclusion of all outgroups except Mollusca, Nemertea and Brachiopoda (Table 1). For all data sets, single gene alignments of each orthologous gene were generated separately using MAFFT (Katoh, et al. 2002) and gaps as well as highly diverse amino acid positions were removed from the data matrix with REAP (Hartmann and Vision 2008). All masked single gene alignments were concatenated into a supermatrix using a custom Perl script. To investigate the influence of missing data on the topology of the tree we generated different data subsets from all three data sets. To accomplish that, we enhanced the information content by increasing matrix density using the program MARE, which examines phylogenetic signal as assessed by quartet mapping (MARE v0.1.2-rc, http://www.zfmk.de). Three different weighing 18

parameters (α=1, 1.5 or 2) were applied to generate 3 data subsets differing in gene coverage and number of genes for the 77- and as well for the 79-taxa data sets (Table 1), resulting in 6 data subsets. For the 72- taxa data set only the weighting parameter α=2 was applied. To retain all taxa within a given dataset, the parameter – c was applied for all species. In total 7 data subsets were generated (Table 1). For all data subsets generated by MARE, base composition heterogeneity as well as proportion of missing sequence data was calculated for each taxon (Table 1) using BaCoCa (Kück and Struck 2014). To assess base composition heterogeneity we employed taxon-specific RCFV values that determine the absolute deviation of the base composition of a taxon from the mean across all taxa (Zhong, et al. 2011). All alignments are available at data dryad.

Phylogenetic analyses and testing ProtTest2.4 was used to select the best fitting amino acid substitution model (Abascal, et al. 2005), suggesting the LG+Γ+I model. For all seven data subsets Maximum Likelihood analyses were conducted with RAxML v. 7.3.1 (Stamatakis 2006). Rapid bootstrapping (BS) was applied with 500 bootstrap replicates each under the CAT approximation and searches for the best tree were performed ten times for each data set. Leaf stability indices were determined using Phyutility v. 2.2 (Smith and Dunn 2008) and long-branch taxa were identified using the LB score implemented in TreSpEx v.b042 (Struck T.H., 2013, www.annelida.de). The LB score determines the percentage deviation of the average pair-wise patristic distance of a taxon to all other taxa in the tree relative to the average pair-wise patristic distance of all taxon to each other. Thus, this score is a tree-based measurement, which is independent of the root of the tree in contrast to tip-to-root measurements. To test if the best trees differs significantly from different a priori hypotheses mainly based on 19

morphology (supplementary Table S3), per-site log-likelihoods of the best tree from the unconstrained, as well as constrained analyses of the data set comprising 77 taxa and MARE settings of α = 1/1.5/2 were computed using RAxML v. 7.3.1 (Stamatakis 2006). For hypothesis testing, an AU-test was conducted using CONSEL (Shimodaira and Hasegawa 2001). Moreover, differences in per-site log-likelihoods (psL) of each constrained analysis in comparison to the best tree were also used to explore if the presence of paralogous or xenologous sequences were responsible for the topological differences observed in the two compared trees (Smith, et al. 2011; Struck 2013). To make this assessment, psL was plotted against amino acid positions to determine if the phylogenetic signal for each topology was present across partitions in the alignment or concentrated into a single or few genes (data dryad: Files S1-S9). Signal present in just a few genes indicates follow-up analyses maybe warranted to identify paralogy or xenology.

Ancestral character state reconstruction Ancestral state reconstructions using both the likelihood and the parsimony criterion were conducted as described in Struck, et al. (2011) based on a family tree derived from the trees shown in Fig. S1. The morphological data matrix from the study of Struck, et al. (2011) was expanded to include Acrocirridae, Eunicidae, Magelonidae, Nephytidae, Oweniidae, Polynoidae, Sabellaridae, Sabellidae, and Tomopteridae. Character states were based on Zrzavy, et al. (2009), but slightly modified by updating/changing the coding of characters related to “shape of parapodia”, “pygidial cirri”, “uncini”, “hooks” and “presence of eyes” according to Struck, et al. (2011) to ensure comparability with the results of the study of Struck, et al. (2011) (data dryad: Files S10 and S11).

20

Acknowledgements

We would like to thank Marie-Theres Gansauge for technical assistance and Anne-C. Zakrzewski and Harald Hausen for collecting specimens of Spiochaetopterus sp.. Special thanks to Natascha Hill and Alexander Donath for the help with installing the HaMStR software and with the Perl scripts used in the pipeline. The authors are also grateful to Tomas Larsson, Michael Gerth and Lars Hering for help in data processing and suggestions and Carlos Sanchez Ortiz for providing us a picture of Eurythoe complanta var. mexicana. We thank the “Station Biologique de Roscoff” for specimen supply and providing of laboratory facilities. This study was funded by DFG grants (STR-683/7-1, STR-683/8-1, BL787/5-1 and in part Pu84/6-2, Pu84/6-3),the National Science Foundation USA (DEB-1036537) and by the EU due to ASSEMBLE grant agreement no. 227799 (http://www.assemblemarine.org).

Supplementary Material Supplementary materials include seven tables (supplementary table S1 – S7) and seven figures (supplementary fig. S1 – S7).

Figure legends

Fig. 1: Best ML-tree of the RAxML analysis using the LG+I+G model of the data set comprising 77 taxa and including 421 genes with 104,410 amino acid positions (MARE settings α=1.5). Only bootstrap values above 50 are shown. 21

Fig. 2: Overview and phylogenetic placement of the five basal branching lineages in the annelid tree: A) Cladogram of the phylogenetic relationships within Annelida without myzostomids based on all seven analyses (with Orbiniidae as sister group to Siboglinidae and Cirratuliformia as in six of the seven analyses). B) Sipuncula: Phascolosoma scolops from Sydney, Australia. C) Amphinomidae: Eurythoe complanta var. mexicana from the Sea of Cortez, Mexico. Photo: Carlos Sanchez Ortiz

(Universidad

Autónoma

de

Baja

California

Sur)

D)

Chaetopteridae:

Chaetopterus variopedatus from Roscoff, France. E) Oweniidae: Owenia fusiformis from Saint-Efflam, France. Photo: F) Magelonidae: Magelona johnstoni from SaintEfflam, France.

Fig. 3: Ancestral reconstructions of body and parapodial characters of the last common ancestor of Annelida. A: Annelida, derived from using the parsimony and likelihood reconstruction option. B: Annelida, derived from Struck et al. (2011). Body characters are shown on the left and parapodial characters on the right. Question marks indicate that the state of the character is uncertain. Abbreviations: bie bicellular eyes; doc - dorsal cirrus; grp - grooved palps; isc - internalized supporting chaetae; nuo - nuchal organ; sic - simple chaetae; sop - solid palps; vec - ventral cirrus.

Table Legends

Table 1: Data sets generated with MARE and analyzed in this study with additional information on number of genes, amino acid positions, gene coverage and 22

information content for each data subset. On the two unreduced data sets comprising either 77 or 79 taxa, three different weighing parameters (MARE, α=1, 1.5 or 2) were applied resulting in six data subsets. On the unreduced data set comprising 72 taxa only one weighing parameter (MARE, α=2) was applied. Overall seven data subsets were generated.

Data set

No. of taxa

No. of genes

aa positions

Gene coverage

Information Content

unreduced

79

2339

751.842

0.378

0.090

Subset 1

79

620

169.332

0.489

0,193

Subset 1.5

79

419

104.410

0.520

0,227

Subset 2

79

305

68.903

0.547

0,255

unreduced

77

2339

746.984

0.375

0.090

Subset 1

77

622

170.497

0.487

0,193

Subset 1.5

77

421

103.420

0.519

0,227

Subset 2

77

308

68.750

0.549

0,256

unreduced

72

2339

756.207

0.402

0.096

Subset 2

72

305

69.157

0.555

0,261

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Fig. 1

Fig. 2

Fig. 3