The making of a branching annelid: An analysis of complete mitochondrial genome and ribosomal data of Ramisyllis multicaudata

Abstract

amisyllis multicaudata is a member of Syllidae (Annelida, Errantia, Phyllodocida) with a remarkable
branching body plan. Using a next-generation sequencing approach, the complete mitochondrial
genomes of R. multicaudata and Trypanobia sp. are sequenced and analysed, representing the first
ones from Syllidae. The gene order in these two syllids does not follow the order proposed as the
putative ground pattern in Errantia. The phylogenetic relationships of R. multicaudata are discerned
using a phylogenetic approach with the nuclear 18S and the mitochondrial 16S and cox1 genes.
Ramisyllis multicaudata is the sister group of a clade containing Trypanobia species. Both genera,
Ramisyllis and Trypanobia, together with Parahaplosyllis, Trypanosyllis, Eurysyllis, and Xenosyllis
are located in a long branched clade. The long branches are explained by an accelerated mutational
rate in the 18S rRNA gene. Using a phylogenetic backbone, we propose a scenario in which the
postembryonic addition of segments that occurs in most syllids, their huge diversity of reproductive
modes, and their ability to regenerate lost parts, in combination, have provided an evolutionary
basis to develop a new branching body pattern as realised in Ramisyllis.

Full text

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SCIENTIFIC RepoRts | 5:12072 | DOI: 10.1038/srep12072
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The making of a branching
annelid: an analysis of complete
mitochondrial genome and
ribosomal data of Ramisyllis
multicaudata
M. Teresa Aguado
1
, Christopher J. Glasby
2
, Paul C. Schroeder
3
, Anne Weigert
4,5
&
Christoph Bleidorn
4
Ramisyllis multicaudata is a member of Syllidae (Annelida, Errantia, Phyllodocida) with a remarkable
branching body plan. Using a next-generation sequencing approach, the complete mitochondrial
genomes of R. multicaudata and Trypanobia sp. are sequenced and analysed, representing the rst
ones from Syllidae. The gene order in these two syllids does not follow the order proposed as the
putative ground pattern in Errantia. The phylogenetic relationships of R. multicaudata are discerned
using a phylogenetic approach with the nuclear 18S and the mitochondrial 16S and cox1 genes.
Ramisyllis multicaudata is the sister group of a clade containing Trypanobia species. Both genera,
Ramisyllis and Trypanobia, together with Parahaplosyllis, Trypanosyllis, Eurysyllis, and Xenosyllis
are located in a long branched clade. The long branches are explained by an accelerated mutational
rate in the 18S rRNA gene. Using a phylogenetic backbone, we propose a scenario in which the
postembryonic addition of segments that occurs in most syllids, their huge diversity of reproductive
modes, and their ability to regenerate lost parts, in combination, have provided an evolutionary
basis to develop a new branching body pattern as realised in Ramisyllis.
Annelids are a taxon of marine lophotrochozoans with mainly segmented members showing a huge
diversity of body plans
1
. One of the most speciose taxa is the Syllidae, which are further well-known
for their diverse reproductive modes. ere are two reproductive modes in Syllidae, called epigamy
and schizogamy, and both modes involve strong anatomical and behavioural changes. Epigamy is con-
sidered the plesiomorphic reproductive mode. It results in signicant morphological and behavioural
changes in the benthic, sexually mature adults
2
, which undergo enlargement of anterior appendages
and eyes and development of swimming notochaetae in midbody-posterior segments. Notochaetae are
absent in non-reproductive syllids, which only bear neurochaetae for locomotion. Transformed indi-
viduals actively ascend to the pelagic realm where spawning occurs. Aer spawning, the animals usu-
ally die, though some are able to reverse these changes and go back to the benthic realm for further
reproductive activity. Schizogamy, the putatively derived mode, produces reproductive individuals or
1
Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid,
Spain.
2
Museum and Art Gallery of the Northern Territory, GPO Box 4646, Darwin, N.T., Australia.
3
School of
Biological Sciences, Washington State University, Pullman, Washington 99163-4236, USA.
4
Molecular Evolution
and Systematics of Animals, Institute of Biology, University of Leipzig, Talstraße 33, D-04103 Leipzig, Germany.
5
Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig, Germany. Correspondence
and requests for materials should be addressed to M.T.A. (email: maite.aguado@uam.es) or C.B. (email:
bleidorn@uni-leipzig.de)
Received: 17 March 2015
Accepted: 12 June 2015
Published: 17 July 2015
OPEN

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stolons from newly produced posterior segments. e stolons develop eyes and their own anterior
appendages, as well as special swimming (noto-) chaetae, while they maintain attached to the paren-
tal body. When they are completely mature, they are detached from the parental stock for swimming
and spawning
2–5
. Finally, the stolons die aer spawning. Meanwhile, the stock remains in the benthic
realm, thereby avoiding the dangers of swimming into the pelagic zone, and will be able to reproduce
more than once. Schizogamy can be subdivided into scissiparity (only one stolon is developed in a
reproductive cycle), or gemmiparity (several stolons are developed simultaneously during the same
reproductive cycle)
3–5
. Some gemmiparous syllids, like Myrianida Milne Edwards, 1845 (a member of
Autolytinae), are able to produce a series of stolons, one aer another (Fig.1A), with the last one being
Figure 1. Dierent modes of gemmiparity in Syllidae and budding in Syllis ramosa. A. Sequential
gemmiparity in Myrianida sp. (modied aer Okada, 1933
12
); B. Colateral budding in Trypanosyllis
gemmipara Johnson, 1901 (modied aer Johnson, 1902
8
); C. Collateral budding in Trypanosyllis crosslandi
Potts, 1911 (modied aer Potts, 1911
9
); D. Successive budding in Trypanobia asterobia (modied aer
Okada, 1933
12
); E. Development of a lateral branch in S. ramosa (drawing by MT Aguado from the
holotype); F. Branches and stolon in S. ramosa (modied aer McIntosh, 1885
20
). All gures used for
modications are part of the public domain.

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the most developed and the rst to be detached
6
. Another type of gemmiparity has been described
for some species of Syllinae: Trypanosyllis Claparède, 1864, Trypanobia Imajima & Hartmann, 1964
(Recently erected from subgenus to genus level), and Parahaplosyllis Hartmann-Schröder, 1990
7–17
.
ese animals are able to produce numerous stolons in collateral or successive budding, i.e. growing
dorsally or laterodorsally from the last segment or from several posterior most segments (Fig.1B–D). In
addition, Trypanosyllis and Trypanobia have been reported to live in a symbiotic association with other
animals, like echinoderms and sponges. Among them, the genus Trypanobia is the best known symbi-
otic form, with species such as T. asterobia (Okada, 1933) living within the body of the sea star Luidia
quinaria von Martens, 1865 and developing the stolons within the host
12
.
Remarkably, within Syllinae, two species have been described with a morphology that makes them
unique among all so far ~20,000 described annelids: Ramisyllis multicaudata Glasby, Schroeder and
Aguado, 2012 and Syllis ramosa McIntosh, 1879. ese animals are the only two branching annelids
18–20
and they live in strict symbiosis within sponges (Fig.2A,B). ey have one “head” but multiple branches
(Fig.2C,D); each of them goes into a canal of their host, growing within the sponge by producing new
branches and enlarging the existing ones. is body pattern and biology has astonished biologists and
the general public since they were rst described. e feeding behaviour providing nutrition for these
large syllids is unknown and it remains unclear if they prey on their host. Ramisyllis multicaudata and S.
ramosa also reproduce by stolons that are developed at the end of terminal branches (Fig.1E,F). Initial
phylogenetic analyses found that R. multicaudata is related to the genera Parahaplosyllis, Trypanosyllis,
Eurysyllis Ehlers, 1864, and Xenosyllis Marion & Bobretzky, 1875
18
. ese genera, together with Trypanobia,
show a dorsoventrally attened or ribbon-shaped body. e analysis performed by Glasby et al. (2012)
18
only included the sequences of 16S and a fragment of the 18S, which was dicult to amplify for
Figure 2. A. Living sponge, Petrosia sp. with posterior ends of Ramisyllis multicaudata emerging from
surface pores and moving actively on the sponge surface; B. Detail of R. multicaudata posterior ends on
the surface of the sponge; C. R. multicaudata, non-type, SEM of branch points, mid-body region; D. R.
multicaudata, non-type, SEM, detail of one midbody branch point. All photographs were taken by CJ Glasby
and PC Schroeder by SEM with methods as described previously
18
.

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R. multicaudata using standard primers. However, the phylogenetic placement of this branching annelid
is a prerequisite to understand the evolution and character transformations leading to this unique body
plan.
Mitochondrial genomes are valuable sources of phylogenetically informative markers. e mitochon-
drial genome is, in most animals, a circular duplex molecule of DNA, approximately 13–17 kb in length,
with 13 protein coding genes (nad1-6, nad4L, cox1-3, cob, atp6/8), 22 tRNAs (trnX), two rRNAs (rnS,
rnL), and one AT-rich non-coding region, the control region (CR), which is related with the origin of
replication
21
. e order of these genes is subject to several types of rearrangements, such as inversion,
transposition, and tandem duplication random loss, which is a duplication of several continuous genes,
followed by random loss of one copy of each of the redundant genes
21
. e high number of combinations
found in Metazoa suggests that the order of genes is relatively unconstrained
22
.
Mitochondrial gene order has been used for phylogenetic inference in several animal taxa at dif-
ferent taxonomic levels
23,24
. Because of high substitution rates of nucleotides and amino acids between
taxa, and biases in nucleotide frequencies, the usefulness of mitochondrial sequences for deep phylog-
enies seems to be restricted
25
. However, they are well suited to recover phylogenetic relationships of
younger divergences
25
. Complete mitochondrial genomes of annelids are only available for a limited
number of taxa and, as far as we know, all genes are transcribed from the same strand
26
. e gene
order is considered as generally conserved in this taxon and most investigated annelids dier only in a
few rearrangements of tRNAs, which are usually regarded as more variable
21
. To date, around 40 com-
plete mitochondrial genomes have been published, representing three of the main lineages (Errantia,
Sedentaria, Sipuncula) within the group
27
. No mitochondrial genomes have been published so far for
the taxon Syllidae (Errantia).
e advent of next generation sequencing techniques has simplied the generation of mitochondrial
genomes, which now can be assembled as a by-product from whole genome sequencing. Due to the
high copy number of mitochondria per cell, even low coverage genome data enabled the reconstruction
of complete organelle genomes
28
. Hence we used short read Illumina sequencing for the generation of
whole genome shotgun (wgs) data for two syllid species. Based on genome assemblies we extracted the
mitochondrial and, additionally, nuclear ribosomal data, for phylogenetic analysis. Based on this data,
we resolve the phylogenetic relationships of R. multicaudata using a molecular phylogenetic approach
and extensive taxon sampling. As initial analyses suggested a possible close relationship with the genus
Trypanobia, we also generated wgs data for this taxon. Moreover, the complete mitochondrial genomes
of R. multicaudata and Trypanobia sp. are compared and the derived phylogeny is used to develop an
evolutionary scenario leading to a branching annelid.
Results
Phylogenetic analyses. e rst noticeable result to be indicated is that the 18S sequence obtained
herein from the sequencing library of Ramisyllis multicaudata does not match with the one amplied
by PCR and sequenced in 2012 (Genbank Acc. n° JQ292795)
18
. e 18S from our genome assem-
bly is considerably longer than those of many other syllids (> 2200 bp) and shows large insertions in
the V2- and V5 region. However the sequences from 16S obtained herein from the mt genome and
the one already published (Genbank JQ313812) were identical. Analysing carefully the small piece of
18S sequence JQ292795 and also the sequence of its proposed sister group, Parahaplosyllis brevicirra
Hartmann-Schröder, 1990 (Genbank JF903679), we found suspicious similarities with the 18S sequence
of another syllid, Syllis alternata Moore, 1908 (Genbank JF903649). We consider these as possible con-
taminations since they were sequenced at the same time. Hence, the sequence from 18S obtained herein
was used to replace the previous one (JQ292795), which together with P. brevicirra (JF903679) were
excluded from the analyses of the 18S partitions.
e ML analysis of the rst, most inclusive data set (large 18S, Fig. 3), shows R. multicaudata as
sister to Trypanobia sp. and Trypanobia depressa (Augener, 1913) (100% bootstrap), and related to other
genera, such as Trypanosyllis, Xenosyllis and Eurysyllis, the same relatives as proposed by Glasby et al.
(2012)
18
. All these taxa are in a maximally supported clade (100% bootstrap) that shows a conspicu-
ously long branch. In order to dismiss possible Long Branch Attraction eects (LBA) and make the
alignments easier, a second group of data sets was analysed focussing on a smaller taxon sampling (the
trimmed 18S, 16S and cox1, respectively). e trees for each independent analysis show congruent topol-
ogies (Supplementary Figure 1A–C). Ramisyllis multicaudata, Trypanobia sp. and T. depressa are closely
related and nested in a larger clade together with Trypanosyllis, Eurysyllis, Xenosyllis, and Parahaplosyllis
(16S and cox1 trimmed partitions, Supplementary Figure 1B,C). e 18S topology (Supplementary
Figure 4A) reveals again a long branch for this clade, while in 16S and cox1 it is not longer than others
(Supplementary Figure 1B,C). In 16S and cox1 partitions, P. brevicirra is close to its previously proposed
relatives
18
. e combined data set (trimmed 18S + 16S + cox1) recovers R. multicaudata closely related
to Trypanobia sp. and T. depressa (100% bootstrap) (Fig.4A). e Ramisyllis-Trypanobia clade is sister
to a clade containing Eurysyllis tuberculata Ehlers, 1864 and Xenosyllis scabroides San Martín, Aguado &
Hutchings, 2008. Parahaplosyllis brevicirra is sister to Trypanosyllis sp. and Trypanosyllis zebra (Grube,
1860) (94% bootstrap). Trypanosyllis coeliaca Claparède, 1868 is not located within this latter group. In
all analyses, the genus Trypanosyllis is paraphyletic.

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Figure 3. Maximum likelihood tree obtained when analysing the complete 18S partition. Bootstrap
support values are above nodes. Picture of Ramisyllis multicaudata with stolons taken by CJ Glasby.

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Figure 4. A. ML tree obtained when analysing the combined trimmed data set (18S + 16S + cox1).
Reproductive modes in the ribbon clade are shown. e reproductive mode found in some species is
assumed for the genera they belong. Collateral budding is dubious in Trypanosyllis since the gemmiparous
species might be related to any or both groups (T. zebra-Trypanosyllis sp. and T. coeliaca). B. Reproductive
modes in Syllidae. Phylogenetic relationships based on the analysis of the gene 18S (Fig.2). Reproductive
modes are assumed at generic level.

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Mitochondrial and nuclear genomes. e assembly of the R. multicaudata whole genome shotgun
data resulted in 278,762 contigs with an N50 of 769 bp and an average GC content of 32%. e assembly
of Trypanobia sp. resulted in 40,225 contigs with an N50 of 676 bp and an average GC content of 38%.
As the coverage turned out to be too low for analysing k-mer abundances, an approximate genome size
estimation was inferred from estimating the coverage of single copy ribosomal protein genes. Based on
the average coverage of these genes we estimate a size ~500 mbp for Trypanobia sp. and a genome size of
~1500 mbp for R. multicaudata. BLAST-searches for putative contigs of the sponge host genome revealed
no signs of possible contamination from host tissue, as for example due to feeding.
BLAST-searches identied the complete mitochondrial genomes of both investigated syllids as a sin-
gle contig, which in both cases represented the longest contig of the low coverage genome assembly. e
complete mitochondrial genome of R. multicaudata is 15,748 bp long and is assembled with a coverage of
105x; the one of Trypanobia sp. is 16,630 bp long and has a coverage of 70x. e two genomes are AT-rich
(67% in R. multicaudata, 69% in Trypanobia sp.); A is the most common base (34% in R. multicaudata,
36% in Trypanobia sp.), and G the least common (12% and 11%, respectively). In both genomes, the
coding strand has a strong skew of G vs. C (− 0.31 in R. multicaudata, − 0.29 in Trypanobia sp.), whereas
the AT skew is positive (0.02, 0.04, respectively). Both mt genomes contain the same 37 genes as found
in most other annelids and typically present in bilaterian mt genomes: 13 protein-coding genes, two
genes for rRNAs, and 22 genes for tRNAs (Supplementary Table 2, 3). As in the case for all annelids so
far studied, all genes are transcribed from the same strand (referred to as plus-strand). However, huge
dierences were found in the gene arrangement of R. multicaudata and Trypanobia sp. when compared
with the other known annelids
26
(Fig.5) and the putative ground pattern of Bilateria
25
. Additionally, none
of the conserved blocks for Bilateria proposed by Bernt et al. (2013)
25
have been found in the syllids.
e CREx analyses (Supplementary Figure 2,3) showed that the dierences between Lumbricus ter-
restris Linnaeus, 1758 (representing Sedentaria) and Platynereis dumerilii (Audouin & Milne Edwards,
1834) (representing Errantia), each with R. multicaudata could be explained by 5 tandem duplication
random losses (tdrl), respectively. Dierences between L. terrestris and P. dumerilii, each with Trypanobia
sp. could be explained by 4 tdrl and 1 transposition, respectively. Finally, dierences between R. multi-
caudata and Trypanobia sp. could be explained by 1 transposition and 4 tdrl; and dierences between
L. terrestris and P. dumerilii by 1 transposition and 3 reversals. In addition, the matrix of a gene order
similarity measure (Supplementary Table 4) reveals that the gene order between R. multicaudata and
Trypanobia sp. is more dierent than the order of L. terrestris and P. dumerilii. Considering the order
of only protein coding genes, R. multicaudata and Trypanobia sp. dier in one transposition (Fig. 5).
In summary, the two mitochondrial genomes of the closely related syllids here investigated show more
dierences between themselves than exist between L. terrestris and P. dumerilii, the latter which might
be separated by some hundred million years.
e putative control region in R. multicaudata is 702 bp in length and anked by nad6 and trnL1
(Supplementary Table 1, Fig.5). In Trypanobia sp., the control region is 782 bp and it is located between
Figure 5. Mitochondrial gene order in Ramisyllis multicaudata and Trypanobia sp. Putative ground
patterns for Annelida aer Golombek et al. (2013)
26
.

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trnS2 and trnL1 (Supplementary Table 2, Fig.5). In R. multicaudata, besides the control region, 27 other
non-coding regions are dispersed over the whole genome, ranging from one to 147 base pairs; the largest
one located between trnG and rrnL (Supplementary Table 1). In Trypanobia sp., there are 34 non-coding
regions in addition to the putative control one, ranging from 1 to 265 base pairs, the largest one between
trnY and trnM (Supplementary Table 2).
Start codons in protein-coding genes are highly biased towards ATG (Supplementary Table 3). In
R. multicaudata, ATG is observed in 10 of 13 coding genes; other start codons are ATA and ATT. In
Trypanobia sp., ATG is observed in 11 of the 13 coding genes; other start codons are ATT and TTG.
In R. multicaudata, the dominant stop codon is TAA, except 1 gene ending in TAG, and one putatively
incomplete stop codon ending in only T (Supplementary Table 1). In Trypanobia sp., the most used stop
codon is also TAA, except 1 gene were it is TAG (Supplementary Table 2). ere is also a codon usage
bias in both genomes (Supplementary Table 3). In general, NNG codons are the least used, while espe-
cially NNA, followed by NNT codons are the most common codon types.
e typical 22 tRNAs were found in both mt genomes. ey mostly possess the common cloverleaf
structure, with an acceptor arm, anticodon arm, TΨ C arm, DHU arm, and associated loop regions
(Supplementary Figure 4,5). In R. multicaudata, the DHV stem is missing in trnC and trnR; in Trypanobia
sp. the DHV stem is missing in trnR. Ramisyllis multicaudata and Trypanobia sp. have trnS1 and trnS2
with a shortened DHV stem. e sizes of the ribosomal RNAs are, in R. multicaudata, rrnL: 1008 bp,
rrnS: 787 bp; and in Trypanobia sp., rrnL: 1007 bp, rrnS: 789 bp. e two genes are not separated by any
tRNA, only by an intergenic spacer of 20 and 27 bp in R. multicaudata and Trypanobia sp., respectively
(Supplementary Table 1, 2).
Discussion
Our phylogenetic analyses nd Ramisyllis multicaudata and Trypanobia sp. within Syllidae closely related
to other genera (Parahaplosyllis, Trypanosyllis, Xenosyllis and Eurysyllis) in a long branched clade (Figs3
and 4, Supplementary Figure 1). e genera Parahaplosyllis, Trypanosyllis, Eurysyllis, Xenosyllis, and
Trypanobia share a distinct dorsoventrally attened body, referred to as ribbon-like shaped
29
. is fea-
ture might represent a synapomorphy of this clade, though there is a reversal in R. multicaudata, which
exhibits a cylindrical body pattern. is characteristic is used here to name the long branched clade
including R. multicaudata as the “ribbon” clade (Fig.4A).
e phylogenetic results shown herein are broadly congruent with a preliminary analysis
18
, even
though the 18S sequence of R. multicaudata in that publication seems to be a contamination from
another syllid species. Highly covered ribosomal sequences from the whole genome shotgun approach
showed derived 18S sequences, when compared with other syllids. As such, the 18S of R. multicaudata
is considerably longer, containing several long insertions. ese dierences may explain the failure when
trying repeatedly to sequence 18S from R. multicaudata in 2012. Analyses of single gene partitions show
that the 18S gene in particular greatly contributes to the observed long branches of the ribbon clade.
Analyses solely based on the mitochondrial genes reveal congruent topologies, but with considerably
shorter branch lengths. Interestingly, a previous study mentioned a big eort when sequencing 18S from
R. multicaudata, with sequences from the host sponge recovered repeatedly
18
. However, BLAST searches
against high copy number genes of the sponge (e.g., ribosomal genes, mitochondrial genes) did not
identify any sponge related contigs in our whole genome assembly. We therefore suggest that R. multi-
caudata is not feeding on its host, even though the way these worms support their large body mass with
nutrition remains unclear.
e analysed wgs data do not only reveal highly derived ribosomal sequences, leading to long branches
in our analyses. Similarly, the mitochondrial gene order found in R. multicaudata and Trypanobia sp.
also diers considerably in comparison with the gene order of Sedentaria (L. terrestris) and Errantia (P.
dumerilii) (Supplementary Figure 2,3). Annelids of both these major groups analysed so far showed a
remarkably conserved gene order, suggesting a common ground pattern (Fig.5). Indeed, the dierences
between the gene order of Errantia, Sedentaria, the basal branching annelid lineages (represented by
sipunculids), and the putative ground pattern of Spiralia (Bernt et al., 2013) are less drastic than those
between any of these patterns and the analysed syllids (Fig. 5, Supplementary Table 4). In the light of
these results, we conclude that the gene order in the mt genome of (Pleisto-) Annelida is more diverse
than expected. Our results indicate that there might be some constraints that maintain the gene order,
reected in the relatively conserved patterns of annelid lineages; but once these constraints are violated,
many changes seem possible, as revealed by the patterns in R. multicaudata and Trypanobia sp. However,
since these are the rst two mitochondrial genomes from syllids, it is not possible to assess if Syllidae
in general, or only members of the ribbon clade including Ramisyllis and Trypanobia, show these huge
dierences.
Other mitochondrial genome features of R. multicaudata and Trypanobia sp. seem to be more in line
with those of other annelids. Both species share with other annelids a similar pattern of codon usage
bias (NNA and NNT the most common types, while NNG the least used)
30
. A negative GC-skew is also
found in most of the mitochondrial genomes known from annelids. Regarding the tRNAs, DHU stems
are lacking in many metazoan mitochondrial tRNA genes
22
. e sizes of the ribosomal RNAs in R.
multicaudata and Trypanobia sp. are within the size range of other invertebrates including molluscs and
annelids. e two genes are not separated though usually, in many animals, trnV is found in the middle;

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among annelids only echiurans (Urechis caupo Fisher & MacGinitie, 1928) and myzostomids (Myzostoma
seymourcollegiorum Rouse & Grygier, 2005) share this condition
31
.
Low coverage, whole genome analyses of the two syllids allowed several interesting ndings. Both taxa
not only share an unusual biology, but also show derived ribosomal sequences and a strongly changed
mitochondrial gene order. Rough estimations of the nuclear genome size from mapping sequence reads
on putatively single copy ribosomal proteins indicates a genome size between 500 mbp and 1.5 gbp for
both these taxa. In the case of Ramisyllis this size seems to be considerably larger than that of most other
syllid annelids, which ranges between 100 and 500 mbp
32
. In summary, these results suggest a labile
genomic architecture of the ancestral lineages of these taxa, putatively driven by genetic dri which could
point to several past population size bottlenecks
33
.
e branching body pattern of Ramisyllis might be the result of the combination of processes that are
closely related: post-embryonic development and regeneration. In many groups of marine annelids, seg-
ment formation occurs during both larval and juvenile development and is the result of the activity of a
posterior growth zone located immediately anterior to the pygidium
34
. e posterior growth zone has been
also referred to as segment addition zone (SAZ)
35
, and this latter term will be followed herein. Usually, in
marine annelids with an unlimited number of segments bodies grow indeterminately, adding segments
throughout their whole life
35
. e SAZ may contain teloblasts, which are progenitor cells that divide to
produce new segments
36
. In these dividing cells, the cleavage plane must be perpendicular to the main
axis (antero-posterior axis or A-P axis), to proliferate according to bilateral symmetry. In addition to
the continuous segment formation, many groups within annelids are able to regenerate lost segments.
Posterior regeneration processes imply the generation of a new SAZ
36
. On the molecular level, gene
expression during adult growth and regeneration shows several similarities, suggesting a shared mech-
anism
37,38
. Interestingly, the occasional occurrence of annelids with two tails have been recorded both
in nature, and, more frequently, in worms in which regeneration has been studied experimentally
39–42
.
ese aberrant forms might be the result of punctual “mistakes” or induced experiments during the
regeneration process; and there are no reports of these animals living further for reproductive activity.
Considering the occurrence of these aberrant branching forms, branching in Ramisyllis might be the
result of a similar unknown event, which could have been established as a regular growth pattern.
Ramisyllis is a member of Syllidae, which might suggest that an additional process could be involved.
Syllids are animals with a high capacity of regeneration of lost parts; they can regenerate anteriorly as
well as posteriorly, and they show a derived mode of reproduction called schizogamy that involves this
regeneration ability
5,14
. Schizogamy implies the ability to produce reproductive individuals or stolons
from newly produced segments, and also the ability to regenerate the posterior end aer detachment of
stolons
5
. e regeneration of the parental stock begins with a pygidium and a new SAZ. erefore, the
life cycle in schizogamous syllids is a combination of segment addition during adult life, reproduction
and regeneration.
e recovered phylogeny shown herein (Fig.4A,B) can be used to develop a scenario of major char-
acter transformations leading to a branched annelid species. e herein analysed R. multicaudata and
Trypanobia sp. are both members of Syllinae, characterized, among other features, by a schizogamous
reproductive mode. In addition they are both members of the ribbon clade, where species reproduce
by gemmiparity; i.e. the development of simultaneous stolons during the same reproductive cycle
3–5
(Fig.4A). ere is one report of Parahaplosyllis as gemmiparous
16
, one report of Trypanobia, and several
of Trypanosyllis species
7–17
. Other species in these genera reproduce by scissiparity or their reproductive
mode has not been identied yet. Additionally, it is unknown if gemmiparity and scissiparity are exclu-
sive of each species or if a species can alternate between the two modes.
In gemmiparous species of Trypanosyllis and Parahaplosyllis the stolons are developed from the paren-
tal body through the same posterior SAZ (Fig.1B,C). However, stolons in T. asterobia are produced by
separate consecutive parental segments (Fig.1D). is evidence implies that Trypanobia develops succes-
sive SAZ simultaneously. In both, collateral budding (in Trypanosyllis and Parahaplosyllis) and successive
budding (Trypanobia), the newly produced segments are developed from each SAZ at dierent angles
from the A-P axis, being most evident in Trypanobia (Fig. 1D). e stolon attachment to the parental
stock dorsally or dorsolaterally from the stock might suggest that the cleavage plane of division in the
proliferating cells is somehow rotated; however, this hypothetical process is completely unknown to date.
A similar process might be responsible for development of the body pattern in R. multicaudata, since
the asymmetrical branches in its body suggest dierent active SAZs; however, time for proliferation
might be longer and instead of producing the stolons directly from the newly produced segments, these
arise only at the tip of terminal branches. Additionally, in Ramisyllis the development of SAZs is random,
while in Trypanobia they are developed in successive posterior segments. In Ramisyllis, branches occur
asymmetrically and laterally from the A-P main axis. e reproductive mode of R. multicaudata and S.
ramosa could be also considered a particular type of gemmiparity, since they are able to produce several
stolons simultaneously; at the tip of terminal branches of its body
18–20
(Figs1F and 2A,B).
Summarizing all these observations, and assuming that the reproductive modes found in some species
is representative for the genera they belong, a possible scenario might be proposed in which an ances-
tor of the ribbon clade reproduced by collateral gemmiparity (as seen in species of Parahaplosyllis and
Trypanosyllis) (Fig.1B,C). is condition could have been later modied in the ancestor of Trypanobia
and Ramisyllis, which might also maintain symbiotic relationships with other organisms (Fig. 2A,B).

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SCIENTIFIC RepoRts | 5:12072 | DOI: 10.1038/srep12072
is hypothetical ancestor might have been able to develop several simultaneous SAZs, each producing
an asymmetrical branch of new segments. e newly developed segments are directly transformed into
stolons in Trypanobia (Fig.1D), or more segments are added in a growing large chain in R. multicaudata
(Fig. 1E,F), and only the distal ones are transformed into stolons; the latter strategy appears to be an
adaptation to life in the complex canal system of a sponge. Within the ribbon clade, gemmiparity reverses
into scissiparity in Eurysyllis and Xenosyllis (Fig.1A,B).
An alternative hypothesis may explain the branching pattern as an independent process from the
schizogamous reproductive mode, and hence, considering the phylogenetic results, an autopomorphy of
Ramisyllis. Future comparative studies on the genetic basis of the stolonization processes and branching
body patterns of these species could give further support to any of these scenarios.
Conclusions
In this study, we provide the rst complete mitochondrial genomes for Syllidae and present a scenario
for the evolution of the unique morphology of the branching syllid Ramisyllis multicaudata. We nd that
R. multicaudata and Trypanobia are sister taxa closely related to Parahaplosyllis, Trypanosyllis, Eurysyllis,
and Xenosyllis and herein called the ribbon clade. e two closely related investigated species reveal
strongly rearranged mitochondrial gene orders, unique for annelids. Both a comparatively large nuclear
genome and strongly rearranged mitochondrial genomes could indicate past population size bottlenecks
as such labile genomic architectures are more likely to be driven by genetic dri. We recognize that
major phenotypic changes in syllid annelids are known from experimental studies on regeneration. e
combination of undetermined postembryonic addition of segments, high ability to regenerate lost parts
and gemmiparity might represent the basis for the development of a branching body pattern.
Macroevolutionary questions require complex answers integrating processes from multiple scales,
ranging from within genomes to among species
43
. e available evidence obtained in this study suggests
that R. multicaudata represents an example of major phenotypic transitions occurring by saltatational
evolution
44,45
. Future analyses unravelling the genetic basis for the development of this unique body
plan will provide further evidence for or against this hypothesis and hence we will be able to discern if
Ramisyllis is indeed a hopeful monster.
Materials and methods
Genome sequencing and analyses. Sample collection from Darwin Harbour (Australia) and prepa-
ration of Ramisyllis multicaudata was previously described
18
. Scanning Electron Microscope (SEM) images
were taken with a Hitachi S-570 microscope. Trypanobia sp. was collected at Lizard Island (northern Great
Barrier Reef, Queensland, Australia) during a Polychaete Workshop in 2013. DNA was extracted from a
single individual of each R. multicaudata and Trypanobia sp. by proteinase K digestion and subsequent
chloroform extraction. For Illumina sequencing, double index sequencing libraries with average insert
sizes of around 300 bp were prepared as previously described
46
. e libraries were sequenced as 125 bp
paired-end run for R. multicaudata and 96 bp paired-end run for Trypanobia sp., both on an Illumina
Hi-Seq 2000. Base calling was performed with freeIbis
47
, adaptor and primer sequences were removed
using leeHom
48
, and reads with low complexity and false paired indices were discarded. Raw data of
all libraries were trimmed by removing all reads that included more than 5 bases with a quality score
below 15. e quality of all sequences was checked using FastQC (http://www.bioinformatics.babraham.
ac.uk/projects/fastqc/, last accessed February 17th, 2015). De novo genome assemblies were conducted
with IDBA-UD 1.1.046
49
, using an initial k-mer size of 21, an iteration size of 10 and a maximum
k-mer size of 81. N50 and average GC-content of genome assemblies were evaluated using QUAST
50
. All
sequence data were submitted to the National Centre for Biotechnology Information (NCBI) sequence
read archive under accession numbers SRR2006110 for R. multicaudata and SRR2006109 for Trypanobia
sp. Assembled and annotated mitochondrial genomes can be found on NCBI Genbank under accession
numbers KR534502 for R. multicaudata and KR534503 for Trypanobia sp. Approximate genome size was
estimated by mapping reads of single copy ribosomal proteins and subsequent coverage estimation using
the soware segemehl under default options
51
. e coverage of mitochondrial genomes was estimated
by mapping sequence reads back to the contig comprising the mitochondrial genome using the same
soware. Possible contaminations of the R. multicaudata assembly due to its sponge host species (Petrosia
sp.) were searched for using blastn searches. ree high copy number genes (18S, 28S, cox1) published for
Petrosia (Strongylophora) strongylata iele, 1903 (KF576656, KC869619) and Petrosia ciformis (Poiret,
1789) (JX999088) served as queries.
Phylogenetic analyses. e nuclear 18S rRNA gene was retrieved from assemblies of R. multicau-
data and Trypanobia sp. and was used in the phylogenetic analyses. Other terminals (Supplementary
Table 1) were mostly included in previous analyses
2,52,53
. Two more species, Trypanobia depressa and
Trypanosyllis sp. were included for the rst time in these analyses; both were also collected in Lizard
Island. e genes 18S, 16S and cox1 from these two species were obtained using DNA extraction, prim-
ers, and amplication, and Sanger based sequencing procedures as specied previously
2,54
. Alignments
were performed using the program MAFFT
55
with the iterative renement method E-INS-i, and default
gap open and extension values. Several data sets were examined: 1. One set of 18S sequences from a large
number of syllids (195 terminals, Supplementary Table 5); 2. Trimmed sets of the partitions 18S, 16S and

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SCIENTIFIC RepoRts | 5:12072 | DOI: 10.1038/srep12072
cox1 with a reduced taxon sampling (51, 48 and 21, respectively) were used in order to simplify the align-
ments and dismiss possible Long Branch Attraction eects (LBA); 3. A combined data set of the trimmed
partitions (18S + 16S + cox1) (52 terminals). Each partition (large 18S and trimmed 18S, 16S and cox1
genes) was analysed independently, as was the combined molecular trimmed data set (18S + 16S + cox1).
Concatenation of partitions for the combined data set was conducted with FASconCAT
56
. Maximum
Likelihood (ML) analyses were conducted using RAxML version 8.1.2
57
with the GTR + I + G model.
Bootstrap support values were generated with a rapid bootstrapping algorithm for 1000 replicates in
RAxML.
Mitochondrial genome annotation and analyses. AT and GC skews were determined for the
complete mitochondrial genomes (plus strand) according to the formula AT skew = (A− T)/(A + T)
and GC skew = (G− C)/(G + C), where the letters stand for the absolute number of the corresponding
nucleotides in the sequences
58
. Characterization of codon usage bias was calculated with the program
DAMBE5
59
.
e mitochondrial (mt) genomes of R. multicaudata and Trypanobia sp. were annotated using the
MITOS webserver
60
with the invertebrate mitochondrial code (NCBI code). is server also provided
the secondary structure of tRNAs and rRNAs. To compare mitochondrial gene orders, we included the
complete mt genomes of the earthworm Lumbricus terrestris and the nereidid polychaete Platynereis
dumerilii, downloaded from GenBank with the accession numbers NC_001673 and AF178678, respec-
tively. ese two species were chosen as representatives of the Sedentaria and Errantia respectively, the
two lineages comprising most of the species diversity of Annelida
27
. e mt genomes of L. terrestris
and P. dumerilii were also annotated using the MITOS webserver
60
with the invertebrate mitochondrial
code (NCBI code). Finally, all automatic annotations were manually edited. We used CREx
61
to con-
duct pairwise comparisons of the mitochondrial gene order of R. multicaudata and Trypanobia sp. with
L. terrestris and P. dumerilii, respectively. CREx determines the most parsimonious genome rearrange-
ment scenario between the gene order of each pair of taxa including transpositions, reverse transposi-
tions, reversals, and tandem-duplication-random-loss (tdrl) events.
Note added in proof: Trypanobia (previously a subgenus of Trypanosyllis) has been recently erected
to genus level
62
.
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Acknowledgments
We thank Michael Gerth and Robert Bücking for supporting bioinformatic analyses, and Gabriel Renaud
for processing the Illumina raw data. is study has been partly supported by a Geddes visiting fellowship
from the Australian Museum and received funding from the DFG (BL787/5–1). Material was collected
during the Polychaetes Workshop held in Lizard Island, 2013 funded by e Lizard Island Research
Foundation, Permit number G12/35718.1 issued by the Great Barrier Reef Marine Park Authority. We
acknowledge support from the German Research Foundation (DFG) and Universität Leipzig within the
program of Open Access Publishing.

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SCIENTIFIC RepoRts | 5:12072 | DOI: 10.1038/srep12072
Author Contributions
M.T.A. and C.B. designed the study; M.T.A., C.J.G., P.C.S. and A.W. performed experiments; M.T.A.
and C.B. analysed the data and wrote the manuscript. All authors read and contributed to the nal
manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Aguado, M. T. et al. e making of a branching annelid: an analysis of
complete mitochondrial genome and ribosomal data of Ramisyllis multicaudata. Sci. Rep. 5, 12072; doi:
10.1038/srep12072 (2015).
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