# Hidden diversity of Acoelomorpha revealed through metabarcoding

Alicia S. Arroyo, David López-Escardó, Colomban de Vargas, Iñaki Ruiz-Trillo

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## To cite this version:

Alicia S. Arroyo, David López-Escardó, Colomban de Vargas, Iñaki Ruiz-Trillo. Hidden diversity of Acoelomorpha revealed through metabarcoding. Biology Letters, 2016, 12 (9), pp.845-871. 10.1098/rsbl.2016.0674. hal-01376566

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**Cite this article:** Arroyo AS, López-Escardó D, de Vargas C, Ruiz-Trillo I. 2016 Hidden diversity of Acoelomorpha revealed through metabarcoding. *Biol. Lett.* **12**: 20160674.
http://dx.doi.org/10.1098/rsbl.2016.0674

Received: 16 August 2016

Accepted: 6 September 2016

**Subject Areas:**

evolution, ecology

**Keywords:**

Xenoacoelomorpha, metabarcoding, molecular diversity, origins of Bilateria, acoels

**Author for correspondence:**

Iñaki Ruiz-Trillo

e-mail: inaki.ruiz@ibe.upf-csic.es

Electronic supplementary material is available online at https://dx.doi.org/10.6084/m9.figs share.c.3469881.

THE ROYAL SOCIETY
PUBLISHING

Evolutionary biology

# Hidden diversity of Acoelomorpha
revealed through metabarcoding

Alicia S. Arroyo<fnref n="1" />, David López-Escardó<fnref n="1" />, Colomban de Vargas<fnref n="2" />,<fnref n="3" /> and Iñaki Ruiz-Trillo<fnref n="1" />,<fnref n="4" />,<fnref n="5" />

<figure><img src="image_5.png" /></figure>

IR-T, 0000-0001-6547-5304

Animals with bilateral symmetry comprise the majority of the described species within Metazoa. However, the nature of the first bilaterian animal remains unknown. As most recent molecular phylogenies point to Xenacoelomorpha as the sister group to the rest of Bilateria, understanding their biology, ecology and diversity is key to reconstructing the nature of the last common bilaterian ancestor (Urbilateria). To date, sampling efforts have focused mainly on coastal areas, leaving potential gaps in our understanding of the full diversity of xenacoelomorphs. We therefore analysed 18S rDNA metabarcoding data from three marine projects covering benthic and pelagic habitats worldwide. Our results show that acoels have a greater richness in planktonic environments than previously described. Interestingly, we also identified a putative novel clade of acoels in the deep benthos that branches as sister group to the rest of Acoela, thus representing the earliest-branching acoel clade. Our data highlight deep-sea environments as an ideal habitat to sample acoels with key phylogenetic positions, which might be useful for reconstructing the early evolution of Bilateria.

## 1. Introduction

The vast majority of the described animal species are bilaterally symmetrical [1]. The establishment of two orthogonal body axes provided the basis for enormous structural complexity compared with radially symmetrical animals, which allowed a more diverse evolutionary outcome [2]. However, how bilaterians evolved and the nature of the first bilaterian animal remains elusive.

Bilaterian animals are separated into four major groups: Acoelomorpha, Ecdysozoa, Lophotrochozoa (or Spiralia) and Deuterostomia [1,3,4]. Although there has been some disagreement, it now seems clear that Xenacoelomorpha is the sister group to the rest of Bilateria (also known as Nephrozoa [5]) [6–8]. Thus, Xenacoelomorpha is a key taxon to compare with the rest of the bilaterians and reconstruct the nature of the last bilaterian common ancestor, namely Urbilateria.

Members of Xenacoelomorpha, which is formed by Acoela, Nemertodermatida and *Xenoturbella*, are morphologically quite simple: the digestive system only has one opening, they lack circulatory, respiratory and excretory systems, and also lack a body cavity between the gut and the epidermis [8,9]. Xenacoelomorphs live in benthic habitats, and the majority of described species have come

© 2016 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

<footnote n="1">¹Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Passeig Marítim de la Barceloneta, 37-49, 08003 Barcelona, Spain</footnote>

<footnote n="2">²CNRS, UMR 7144, Station Biologique de Roscoff, Place Georges Teissier, 29680 Roscoff, France</footnote>

<footnote n="3">³Sorbonne Universités, Université Pierre et Marie Curie (UPMC) Paris 06, UMR 7144, Station Biologique de Roscoff, Place Georges Teissier, 29680 Roscoff, France</footnote>

<footnote n="4">⁴Departament de Genètica, Microbiologia i Estadística, Universitat de Barcelona, Avinguda Diagonal 643, 08028 Barcelona, Spain</footnote>

<footnote n="5">⁵ICREA, Pg. Lluís Companys 23, 08010 Barcelona, Spain</footnote>
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<table><caption>**Figure 1.** Worldwide distribution of Xenacoelomorpha OTUs. Top: distribution of Acoelomorpha across sampling sites and depth. Bottom: sequencing platforms and sampling information for the projects where the data were collected.</caption><thead><tr><th>project</th><th>18S rDNA region</th><th>NGS platform</th><th>sampling site</th><th>depth</th></tr></thead><tbody><tr><td>TaraOceans</td><td>v9</td><td>Illumina</td><td>worldwide (open water and coast)</td><td>pelagos</td></tr><tr><td>BioMarks</td><td>v4</td><td>Roche 454</td><td>Europe (coast)</td><td>pelagos, benthos</td></tr><tr><td>DeepSea</td><td>v1-v3<br>v7-v8</td><td>Roche 454</td><td>Atlantic and Pacific Ocean (deepsea)</td><td>benthos</td></tr></tbody></table>

from sediments, mainly in coastal areas [10–12]. This morphological simplicity of Xenacoelomorpha seems to support the planuloid–acoeloid hypothesis proposed by Von Graff [13] and Hyman [14], which envisaged Urbilateria to be a simple, benthicacoelomate organism exhibiting direct development [2,15].

However, the full diversity and morphological disparity of Xenacoelomorpha is not yet known, because it has never been approached in a systematic, high-throughput manner. It is therefore possible that there are unobserved or unsampled xenacoelomorph lineages with more complex morphologies or lifestyles, in different habitats, or occupying earlier phylogenetic positions in the Xenacoelomorpha tree. For example, some studies have describedacoel morphospecies in freshwater [16,17], brackish water [18] and planktonic habitats [19]. Thus, any attempt to understand the nature and ecology of Urbilateria will require a more global and systematic analyses of Xenacoelomorpha diversity.

## 2. Material and methods

Clustered operational taxonomic units (OTUs) were obtained from public repositories or directly from the authors. The reference tree was constructed from 255acoelomorph 18S rDNA GenBank sequences (from herein RefTree). Alignment was carried out using the E-INS-I option from MAFFT v. 7.271 [20] and manually trimmed. The maximum-likelihood (ML) tree was built using RAxML v. 8.0.0 [21] considering a GTR-GAMMA substitution model. Nodal support was obtained through 1000 bootstrap replicates. We selected the OTUs through RAxML-EPA [22] and chose those whose abundance was greater than 10 reads.

A final ML tree using both the RefTree sequences and our OTUs was inferred using RAxML [21], with the same conditions as above. A Bayesian tree was built using MrBayes v. 3.2.6 [23] using a GTR + I + Γ model of evolution. Pplacer v. 1.1 [24] was used to perform a phylogenetic placement of the OTUs into the RefTree. Novelty blast percentages were obtained running a blastn 2.2.31 [25] against our curated Acoelomorpha-GenBank database.

A more detailed description of Materials and Methods can be found in the electronic supplementary material.

## 3. Results and conclusion

Here, we use a comprehensive metabarcoding approach with 18S rDNA to assess xenacoelomorph diversity in marine environments. The aim was to search for potential novel lineages that may be of interest to understand the ancestral xenacoelomorph body plan, as well as to identify the environments in which it would be possible to find them. To this end, we analysed the most complete marine eukaryotic metabarcoding datasets to date, comprising both benthic and pelagic marine environments and from diverse global samplings. In particular, we analysed three major metabarcoding projects (figure 1): (1) BioMarks, with benthic and pelagic samples from European coastal areas (biomarks.eu), (2) Tara Oceans, with pelagic samplings from all over the world (oceans.taraexpeditions.org) and (3) a deep-sea project (hereafter DeepSea), with benthic samples from great depths (more than 3000 m) in both North Pacific and North Atlantic Oceans [26].

We found a total of 101 Xenacoelomorpha environmental OTUs (figure 1 and Material and Methods; see electronic
<figure><img src="image_7.png" /><figcaption>**Figure 2.** Molecular novelty in Acoelomorpha. (a) Blast identity of 101 acelomorph OTUs against the Acoelomorpha 18S GenBank database in known well-described families [27]. Note the high percentage of richness with low sequence similarity to Acoela. (b) Maximum-likelihood tree inferred from 101 Acoelomorpha OTUs and RefTree GenBank sequences (see Materials and Methods). Nodal support indicates 1000 ML bootstrap replicates and posterior probabilities. Coloured OTUs represent novel molecular linages within Acoela. (c) Phylogenetic placement of Acoelomorpha OTUs using pplacer software (see Materials and Methods). Our data show that a large number of OTUs cannot be assigned to a sequenced acelomorph species in the reference tree. Deep sea clades are shown in red, with arrows pointing out to the phylogenetic placements. LWR (likelihood weight ratio) of each placement is displayed near each node.</figcaption></figure>

supplementary material, S1 and S2 for raw data). Of those, 97 OTUs corresponded to Acoela and four to Nemertodermatida. We did not recover any *Xenoturbella* OTUs. Interestingly, a high percentage (74%) of those sequences show a low blast identity (less than 90%) against the Acoelomorpha 18S rDNA data present in NCBI (figure 2a). This indicates that most of the sequences we recovered are molecularly quite different to the acelomorphs sequenced so far, even though extensive sampling efforts have been undertaken for acelomorphs in the last decade [10–12,27].

In order to relate the Acoelomorpha novelty with their phylogeny, we performed a phylogenetic placement of all our OTUs against our Acoelomorpha reference tree using pplacer (see Material and Methods). The more internally an OTU is located by pplacer in the tree, the more molecularly different this OTU is compared with the known reference database.

Interestingly, more than half of the acelomorph OTUs (68%) appeared phylogenetically located in internal rather than external nodes of the Acoelomorpha tree (figure 2c). Therefore, our data indicate that the genetic diversity of Acoelomorpha is much broader than previously thought.

To identify the exact phylogenetic position of our OTUs, we performed ML and Bayesian inference phylogenetic trees (figure 2b; electronic supplementary material, figure S1). Our trees confirmed that some of the new molecular diversity was found in pivotal positions as sister group to major clades. Two OTUs were especially noteworthy, because they probably represent completely new lineages. This is the case of the OTU_DS_13115-11580 (which we name as 'deep sea Acoela clade 2'), which appears as the sister group of the Crucimusculata group [27]. Even more important is the finding of a new clade ('deep-sea Acoela clade 1', from OTU_DS_4335-14605) that represents, with high statistical
support, the sister group to the rest of Acoela. This novel
acoel clade branches earlier than Diopisthoporidae, an acel
family thought to be the earliest off-shoot and suggested to
possess many ancestral characters [27]. Interestingly, both
OTUs representing novel clades were found in very deep
environments, where the physico-chemical conditions differ
from those of shallow coastal areas. While deep sea Acoela
2 was found in fine mud at 4878 m depth in the North Atlan-
tic Ocean, deep sea Acoela clade 1 was found at a depth of
3678 m in the North Pacific Ocean, around 170 km offshore
from Monterey Bay, California. This finding suggests that
deep benthos is an ideal habitat in which to search for new
acoelomorph taxa that may provide important information
about the full genomic and morphological diversity of this
group. It is perhaps not surprising, then, that the most
recently described *Xenoturbella* species were also identified
in that habitat [7].

Having identified the most appropriate habitats for
sampling of key acel lineages, we then analysed the full
diversity of our OTUs among all samples. These data
revealed interesting biogeographic patterns in acoels. For
example, some acel OTUs appear to be cosmopolitan and
very abundant in pelagic environments. This is surprising
given that only a few acel species had been described as
planktonic [19]. These species have ecological capabilities
that distinguish them from sedimentary acoels, such as
strong endosymbiont relationships with algae and mixo-
trophy strategies [19] that could help them to cope with the
oligotrophic condition in open marine waters. Thus, our
high-throughput analysis indicates that there is a greater
complexity in the ecology and lifestyle of acoels than pre-
viously suspected (see electronic supplementary material

for an extended discussion of the differences between Nemer-
todermatida and Acoela diversity).

Overall, our data reveal substantial hidden molecular
diversity in Acoelomorpha, especially within acoels, than
shown in previous morphological studies. In particular,
we show that plankton harbours a huge diversity of
unsampled acoels, although within known families, while
deep-sea sediments have the potential to uncover key novel
taxa, including the here reported putative sister group to
the rest of acoels. As Hejnol & Pang [8] pointed out, 'strategic
sampling is essential for understanding the evolution of
major traits'. We believe that our data could help to design
future projects with the specific goal of finding new morpho-
species from phylogenetically relevant lineages in which the
study of anatomical, morphological and molecular evolution
could be carried out.

**Data accessibility.** Sequences here reported and the phylogenetic alignment are available as electronic supplementary material.

**Authors' contributions.** I.R.-T. designed and coordinated the study. C.d.V. and D.L.-E. provided some of the data. A.S.A. undertook the analysis and prepared the figures. D.L.-E., A.S.A. and I.R.-T. interpreted the data, and finally A.S.A. and I.R.-T. wrote the manuscript. All authors agreed to be held accountable for the content of the manuscript and approved the final version to be published.

**Competing interests.** We have no competing interests.

**Funding.** This work was supported by a grant from Ministerio de Economía y Competitividad (MINECO) (BFU2014-57779-P) and a European Research Council Consolidator Grant (ERC2012Co616960) to I.R.-T., the former co-funded by the European Regional Development Fund. We acknowledge support from Secretaria d'Universitats i Recerca de la Generalitat de Catalunya (Project 2014 SGR 619).

**Acknowledgements.** We thank Javier del Campo, Dan Ritcher and Alexey Kozlov for their suggestions.

References

1. Dunn CW, Giribet G, Edgecombe GD, Hejnol A. 2014. Animal phylogeny and its evolutionary implications. *Annu. Rev. Ecol. Evol. Syst.* **45**, 371–395. (doi:10.1146/annurev-ecolsys-120213-091627)

2. Baguñà J, Riutort M. 2004. The dawn of bilaterian animals: the case of acelomorph flatworms. *BioEssays* **26**, 1046–1057. (doi:10.1002/bies.20113)

3. Hejnol A et al. 2009. Assessing the root of bilaterian animals with scalable phylogenomic methods. *Proc. R. Soc. B* **276**, 4261–4270. (doi:10.1098/rspb. 2009.0896)

4. Cannon JT, Vellutini BC, Smith J, Ronquist F, Jondelius U, Hejnol A. 2016. Xenacoelomorpha is the sister group to Nephrozoa. *Nature* **530**, 89–93. (doi:10.1038/nature16520)

5. Jondelius U, Ruiz-Trillo I, Baguñà J, Riutort M. 2002. The Nemertodermatida are basal bilaterians and not members of the Platyhelminthes. *Zool. Scr.* **31**, 201–215. (doi:10.1046/j.1463-6409.2002. 00090.x)

6. Ruiz-Trillo I, Riutort MJ, Littlewood DT, Herniou EA, Baguña J. 1999. Acoel flatworms: earliest extant bilaterian Metazoans, not members of Platyhelminthes. *Science* **283**, 1919–1923. (doi:10. 1126/science.283.5409.1919)

7. Rouse GW, Wilson NG, Carvajal JI, Vrijenhoek RC. 2016. New deep-sea species of *Xenoturbella* and the position of Xenacoelomorpha. *Nature* **530**, 94–97. (doi:10.1038/nature16545)

8. Hejnol A, Pang K. 2016. Xenacoelomorpha's significance for understanding bilaterian evolution. *Curr. Opin. Genet.* **39**, 48–54. (doi:10.1016/j. gde.2016.05.019)

9. Haszprunar G. 2015. Review of data for a morphological look on Xenacoelomorpha (Bilateria incertae sedis). *Org. Divers. Evol.* **16**, 1–27.

10. Meyer-Wachsmuth I, Curini Galletti M, Jondelius U. 2014. Hyper-cryptic marine meiofauna: species complexes in Nemertodermatida. *PLoS ONE* **9**, e107688. (doi:10.1371/journal.pone. 0107688)

11. Zauchner T, Salvenmoser W, Egger B. 2015. A cultivable acel species from the Mediterranean, *Aphanostoma pisae* sp. nov. (Acoela, Acoelomorpha). *Zootaxa* **3941**, 401–413. (doi:10. 11646/zootaxa.3941.3.6)

12. Curini-Galletti M et al. 2012. Patterns of diversity in soft-bodied meiofauna: dispersal ability and body size matter. *PLoS ONE* **7**, 1–13. (doi:10.1371/ journal.pone.0033801)

13. Graff LV. 1882 *Monographie der Turbellarien I. Rhabdocoelida*. Leipzig, Germany: Verlag von Wilhelmi Engelman: I. I-lx, 1–442.

14. Hyman LH. 1940 *The invertebrates: Protozoa through Ctenophora*: vol. 1. New York, NY: McGraw-Hill:

15. Nakano H, Lundin K, Bourlat SJ, Telford MJ, Funch P, Nyengaard JR, Obst M, Thorndyke MC. 2013. *Xenoturbella bocki* exhibits direct development with similarities to Acoelomorpha. *Nat. Commun.* **4**, 1537. (doi:10.1038/ncomms2556)

16. Nastasescu M, Popescu-Marinescu V. 2004. Turbellaria spreading within iron gates area existing in benthic and phytophile fauna. *Revue Roumaine de Biologie Serie de Biologie Animale* **47**, 97–10.

17. Vila-Farré M, Álvarez-Presas M, Achatz JG. 2013 First record of *Oligochoerus limnophilus* (Acoela, Acoelomorpha) from British waters. *Arx. Miscel-lània Zoològica* **11**, 153–157.

18. Ax P, Dörjes J. 1966. *Oligochoerus limnophilus* nov. spec. ein kaspisches Faunenelement als erster Süßwasserverstreter der Turbellaria Acoela in Flüssen Mitteleuropas. *Internationale Revue der Gesamten Hydrobiologie* **51**, 15–44. (doi:10.1002/iroh. 19660510104)
19. Stoecker D, Swanberg N, Tyler S. 1989. Oceanic mixotrophic flatworms. *Mar. Ecol. Prog. Ser.* **58**, 41–51. (doi:10.3354/meps058041)

20. Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. *Mol. Biol. Evol.* **30**, 772–780. (doi:10.1093/molbev/mst010)

21. Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. *Bioinformatics* **30**, 1312–1313. (doi:10.1093/bioinformatics/btu033)

22. Berger SA, Krompass D, Stamatakis A. 2011. Performance, accuracy, and web server for evolutionary placement of short sequence reads under maximum likelihood. *Syst. Biol.* **60**, 291–302. (doi:10.1093/sysbio/syr010)

23. Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. *Bioinformatics* **19**, 1572–1574. (doi:10.1093/bioinformatics/btg180)

24. Matsen FA, Kodner RB, Armbrust EV. 2010. pplacer: linear time maximum-likelihood and Bayesian phylogenetic placement of sequences onto a fixed reference tree. *BMC Bioinform.* **11**, 538. (doi:10.1186/1471-2105-11-538)

25. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. 2009. BLAST plus: architecture and applications. *BMC Bioinform.* **10**, 1. (doi:10.1186/1471-2105-10-421)

26. Bik HM, Sung W, De Ley P, Baldwin JG, Sharma J, Rocha-Olivares A, Thomas WK. 2012. Metagenetic community analysis of microbial eukaryotes illuminates biogeographic patterns in deep-sea and shallow water sediments. *Mol. Ecol.* **21**, 1048–1059. (doi:10.1111/j.1365-294X.2011.05297.x)

27. Jondelius U, Wallberg A, Hooge M, Raikova Ol. 2011. How the worm got its pharynx: phylogeny, classification and bayesian assessment of character evolution in Acoela. *Syst. Biol.* **60**, 845–871. (doi:10.1093/sysbio/syr073)