The phylogenetic position of Acoela as revealed by the complete mitochondrial genome of Symsagittifera roscoffensis
© Mwinyi et al; licensee BioMed Central Ltd. 2010
Received: 20 May 2010
Accepted: 13 October 2010
Published: 13 October 2010
Acoels are simply organized unsegmented worms, lacking hindgut and anus. Several publications over recent years challenge the long-held view that acoels are early offshoots of the flatworms. Instead a basal position as sister group to all other bilaterian animals was suggested, mainly based on molecular evidence. This led to the view that features of acoels might reflect those of the last common ancestor of Bilateria, and resulted in several evo-devo studies trying to interpret bilaterian evolution using acoels as a proxy model for the "Urbilateria".
We describe the first complete mitochondrial genome sequence of a member of the Acoela, Symsagittifera roscoffensis. Gene content and circular organization of the mitochondrial genome does not significantly differ from other bilaterian animals. However, gene order shows no similarity to any other mitochondrial genome within the Metazoa. Phylogenetic analyses of concatenated alignments of amino acid sequences from protein coding genes support a position of Acoela and Nemertodermatida as the sister group to all other Bilateria. Our data provided no support for a sister group relationship between Xenoturbellida and Acoela or Acoelomorpha. The phylogenetic position of Xenoturbella bocki as sister group to or part of the deuterostomes was also unstable.
Our phylogenetic analysis supports the view that acoels and nemertodermatids are the earliest divergent extant lineage of Bilateria. As such they remain a valid source for seeking primitive characters present in the last common ancestor of Bilateria. Gene order of mitochondrial genomes seems to be very variable among Acoela and Nemertodermatida and the groundplan for the metazoan mitochondrial genome remains elusive. More data are needed to interpret mitochondrial genome evolution at the base of Bilateria.
Acoels are marine, soft-bodied, unsegmented worms without hindgut and anus - the mouth opens to a central digestive parenchyma, a gut lumen is absent. Acoels move with their multiciliated epidermis although many are 'surprisingly muscular' . Most of the species are free-living, some are ectocommensals. Several species from the subtaxa Sagittiferidae and Convolutidae form obligate symbioses with green algae , making them functional photoautotroph organisms. In traditional systematics the Acoela were considered to be representatives of the Platyhelminthes, due to their 'flatworm-like' features such as the ciliated epidermis, the frontal organ, neoblasts, hermaphroditic reproduction, biflagellate sperm, and a lack of body cavities (acoelomate structure), hindgut and anus [3, 4]. Based on the ultrastructural characteristics of cilia, and the hypothesized reduction of gut and protonephridia, Ehlers (1985) combined Acoela with Nemertodermatida to form the Acoelomorpha. In his system the Catenulida form the sister group to all other Platyhelminthes (Euplatyhelminthes), which comprise the sister groups Acoelomorpha and Rhabditophora. However, the monophyly of the Platyhelminthes was soon questioned because of the weakness of these morphological characters [5, 6]. Subsequent ultrastructural studies have demonstrated numerous differences between Acoelomorpha and Platyhelminthes, particularly amongst characters once thought to be homologous. For example, frontal organ morphology [6, 7], sperm ultrastructure , and patterns in the nervous and muscular systems [9–11] all demonstrate the uniqueness of acoelomorphs.
Early molecular systematic studies using ribosomal RNA genes strongly suggested that Acoela and Nemertodermatida were distinctly separate from the Platyhelminthes [12–14]; this result remains coherent even in the light of more taxa, more sequence data and more sophisticated models of phylogenetic analysis . In the last 10 years several phylogenetic studies with molecular sequences have suggested a phylogenetic position of acoels as sister group to all other Bilateria [16–19]. Studies with broad taxon sampling of both Acoela and Nemertodermatida supported paraphyly of Acoelomorpha, with Acoela forming the sister group to the remaining Bilateria (Nemertodermatida + Nephrozoa) [19–21]. Presuming a position as sister group to all other Bilateria and considering the comparably simple body organisation, the morphological features of acoels may provide insights concerning the bodyplan of the 'last common bilaterian ancestor' , the ancestor of extant acoels, protostomes and deuterostomes . Thus, acoels came into the focus of studies in evolutionary developmental biology as a possible window into the deep past of bilaterians [23–27].
In spite of these advances there is still controversy about the phylogenetic position of Acoela, and at the same time Nemertodermatida. Despite overwhelming molecular evidence against a platyhelminth affinity, some authors discuss the stem cell system of Acoela and Rhabditophora as a potential synapomorphy of these taxa . However, data about stem cells from other invertebrate taxa are very sparse, so this character is in need of a broad comparative study. Recent phylogenomic studies do not recover platyhelminth affinities for Acoela, but show quite different results due to the varying amount of genes and taxa covered. A phylogenomic analysis of EST data from Isodiametra pulchra  found no relevant nodal support for any sister group relation. However, the best tree from this analysis clustered I. pulchra together with the deuterostomes. Another EST study, including the acoel species Neochildia fusca and S. roscoffensis also failed to support any convincing relationship with another metazoan phylum or lineage . Thus, the authors omitted acoels from subsequent analyses due to their low leaf stability. Finally, a recent increase in taxon sampling incorporated in the latter study, with additional sampling of acoels and including nemertodermatids, supported Acoelomorpha (Acoela + Nemertodermatida) as a monophylum with bootstrap support of 70% and 90% in two datasets of different sizes . Acoelomorpha were the sister group to Xenoturbella in that study, but with only moderate bootstrap support from one of the two analysed datasets. Xenoturbella and Acoelomorpha together formed the sister group to all other Bilateria (= Nephrozoa), once again with merely moderate nodal support.
To evaluate the phylogenetic position of acoels using an independent set of molecular data we present the first complete sequence of a mitochondrial genome of a member of the Acoela, Symsagittifera roscoffensis (Graff, 1891). We describe gene content and tRNA secondary structure, compare the mitochondrial gene order to other taxa and show the results of a phylogenetic analysis with sequence alignments from mitochondrial protein-coding genes.
Results and discussion
Organisation of the genome and genes
Mitochondrial genome organisation of Symsagittifera roscoffensis.
(start - end)
1 - 1551
1591 - 1651
1688 - 2428
2449 - 2499
2533 - 2594
2603 - 3394
3396 - 3451
3459 - 4160
4169 - 4237
4241 - 4303
4304 - 4375
4395 - 4456
4462 - 4527
4555 - 4621
4624 - 4688
4689 - 5452
5453 - 5514
5534 - 7309
7336 - 7400
7409 - 7475
7476 - 8417
8418 - 8478
8483 - 8546
8554 - 8602
8616 - 9095
9106 - 9170
9174 - 10523
10661 - 11650
11721 - 12881
12918 - 13787
13893 - 14162
14167 - 14234
14262 - 14654
14674 - 14739
Overlaps between genes were not detected, except for the remaining possibility that trnL1 and trnL2 are positioned within nad5 and rrnL, respectively. All protein genes terminate with the codon TAA, except for cox3 ending with TAG. Existing start codons are more variable: ATT is found in cytb, nad3, cox2 and cox3; nad1 and nad4 start with ATG; atp6 begins with ATA and nad6 with ATC. Only cox1 with GGT and nad2 with CAT are exceptions from the commonly used start codons in mitochondrial genomes. Another uncommon feature is a repeat region of 42 bp found in nad6 (5' TGA GAA ATT TAC AAT CAA ATT TTA ACT ATT TCT CCT AGA TTT 3').
Due to these findings and the preliminary analyses we suspected that it was predominantly Xenoturbella which had an unstable position in the phylogenetic trees. Therefore we conducted additional analyses with a dataset without Xenoturbella, and a second analysis without Acoela and Nemertodermatida. In both variants four independent chains were run. In all four chains with Xenoturbella omitted, Acoela and Nemertodermatida form a monophylum which is the sister group to the remaining Bilateria (with significant support in three of the four chains; additional file 1, Fig. S2). In the analysis without Acoela and Nemertodermatida, Xenoturbella was found either as sister to Deuterostomia (with support values of 0.65 and 0.99) or as sister to Ambulacraria (with support values of 0.87 and 1.0)(Additional file 1, Fig. S3). Thus, in the absence of acoels Xenoturbella has a more unstable position in the bilaterian tree than acoels have in the absence of Xenoturbella. Acoels remain a critically important taxon to place within the Metazoa.
Mitochondrial gene order of the complete mitochondrial genome of the acoel S. roscoffensis is highly divergent from that of other bilaterian animals, including the partial mitochondrial genome of Paratomella rubra. Even computational approaches of gene order comparison like minimal breakpoint analysis and common interval analysis did not favour any affinity of S. roscoffensis to another taxon. Phylogenetic analyses of mitochondrial amino acid sequences give support for acoels forming a clade with nemertodermatids. But the limited available dataset representing the Nemertodermatida gives this result a rather preliminary nature. The dataset of Nemertoderma westbladi is incomplete as it consists of sequences from only three complete and two partial genes, thus covering only 34.8% of the final alignment. For a better evaluation of monophyly versus paraphyly of Acoelomorpha we are in need of more complete mitochondrial genome sequences from Nemertodermatida and Acoela.
Altogether we see more evidence for a position of Acoela and Nemertodermatida branching off early from the bilaterian tree rather than being grouped with deuterostomes or protostomes. The position of Xenoturbella cannot be fixed with this dataset, but its affinity to deuterostomes is greater than to Acoela and/or Nemertoderma.
Our trees also demonstrate limitations of the CAT-BP model with the bilaterian mitochondrial protein dataset. Nematoda are still clustering with the similarly long-branched Platyhelminthes and Syndermata, instead of forming a monophylum with other ecdysozoans (in this case arthropods, a priapulid and an onychophoran). Almost all other molecular datasets support Ecdyszoa (including Nematoda), so this must be an artifact probably due to selection. A similar problem is described from snakes, where selection seems to act on mitochondrial protein-complexes under special physiological conditions . The long branches in the bilaterian tree are found in both parasitic and in free-living species of nematodes and platyhelminths. Thus, a parasitic life style does not seem to contribute to this accelerated evolutionary change.
If Acoela and Nemertodermatida represent ancient clades which split off early from the bilaterian tree, while Xenoturbella splits off later than these two, probably as sister group of the deuterostomes [38, 40], then we can easily interpret morphological features shared by both as plesiomorphic character states, shared with the last common ancestor of Bilateria. As Telford  noted, Acoela and Xenoturbella share the following features: an acoelomate bodyplan with ventral mouth and absence of anus ; a unique tapering shelf at the ciliary tip and other similarities in the ciliary rootlets ; the nervous system is non-centralized and intra-epidermal in some Acoela and in Xenoturbella . Recently, Nielsen  pointed out that the genomes of Xenoturbella and acoels have a significantly reduced Hox gene complement [25, 26, 52]. The more complex set of hox genes in the remaining bilaterians would be a valuable apomorphic character supporting Nephrozoa excl. Xenoturbella, a topology as in Fig. 5 (noting that there is only insufficient data from Nemertodermatida).
But this plesiomorphic feature cannot support a relationship between Xenoturbella and acoels. If Xenoturbella is part of the deuterostomes (as suggested by the tree in Fig. 4), the hox complement of Xenoturbella must be secondarily reduced, as there are many similarities in the hox complement of the remaining deuterostome and protostome taxa. Nuclear genome data of the complete hox clusters seem to be indispensible for a comprehensive evaluation of the evolution of hox genes at the base of Bilateria.
With regard to comparative analysis of mitochondrial genomes, especially gene order, more data are definitely needed, e.g. complete mitochondrial genome sequences from more than one acoel species, since the comparison of S. roscoffensis and P. rubra has shown that gene order in acoels seem to differ radically. Thus, single "representative" species are by no means sufficient to characterise or represent taxa . Similarly, nemertodermatid genomes also require further and exhaustive evaluations. There is still no complete mitochondrial sequence of this group available, preventing meaningful, genome-based phylogenetic analyses.
Specimen, DNA extraction, PCR and cloning
Primer pairs and annealing temperatures successfully used for primer walking.
CTT ATG TTT TTT TTA GTT TGC GAC CTC
GGG GGA GTG ATT GCT TTG TTG C
CAA CAG GGT TTC ACG GAA TAC ACG
GAG GTC GCA AAC TAA AAA AAA CAT AAG
TGT TGG AGA TCT AAC AGA ATA AGC AC
CTT TCT GAG ATA AAA GTA GGT CCT GG
Sequencing and data assembly
Initial sequencing of amplified PCR fragments was carried out in the Berlin lab, using a CEQ™8000 capillary sequencer (Beckmann-Coulter, USA) and the CEQ DCTS Quick Start kit (Beckmann-Coulter) according to the standard protocol, except for using half volumes for setup of the sequencing reaction (10 μl). Final sequencing was performed by the professional sequencing service of AGOWA (Berlin, Germany). Following sequencing, BLAST programs on the NCBI server were used to determine rRNA- and protein-encoding genes. ClustalW and the cap-contig program, both integrated in BioEdit version 7.0.5 , were used for sequence assembly and comparison. The final sequence was compared with previously published mitogenomic sequences of other taxa recovered from GenBank and OGRe . For comparison and evaluation of gene boundaries we built alignments from genes of several metazoan species (predominantly including Platyhelminthes and other Lophotrochozoa, as well as Xenoturbella bocki). In addition, our sequence was compared to the recently published EST data of S. roscoffensis from the NCBI trace archive to get an independent confirmation of gene boundaries. The putative secondary structures of all tRNAs were either detected in a combined approach using tRNAscan-SE , ARWEN  or by extensive inspection of intergenic regions by eye. The complete mt genome sequence of S. roscoffensis is deposited at the NCBI database with accession number [GenBank: HM237350]. CREx  was used to determine common intervals and breakpoint distances in pair wise comparisons of gene orders. AT and GC skew were calculated according to the following formula: AT skew = (A%-T%)/(A%+T%); GC skew = ( G%-C%)/(G%+C%), as described in .
Names, taxonomic classifications and GenBank accession numbers of the species used in our phylogenetic analyses (asterisks point to partial genome data; percental coverage in the final alignment is indicated for these taxa).
GenBank accession number
Annelida - Clitellata
Annelida - "Polychaeta"
Platyhelminthes - Turbellaria
Platyhelminthes - Trematoda
Platyhelminthes - Trematoda
Platyhelminthes - Monogenea
Platyhelminthes - Monogenea
Platyhelminthes - Cestoda
Platyhelminthes - Cestoda
Brachionus plicatilis (part 1)
Brachionus plicatilis (part 2)
Chelicerata - Xiphosura
Myriapoda - Chilopoda
Hexapoda - Pterygota
Crustacea - Phyllopoda
Crustacea - Malacostraca
Mollusca - Polyplacophora
Mollusca - Gastropoda
Mollusca - Cephalopoda
Chordata - Cephalochordata
Chordata - Craniata
Echinodermata - Crinoidea
Echinodermata - Asteroidea
Echinodermata - Holothuroidea
Porifera - Demospongia
Porifera - Demospongia
Cnidaria - Anthozoa
Cnidaria - Scyphozoa
- atp 6/8 :
ATPase subunit 6/8 genes
- cob :
cytochrome b gene
- cox 1-3 :
cytochrome oxidase subunit I-III genes
- nad1-6 and nad4L:
NADH dehydrogenase subunit 1-6 and 4L genes
- rrnS/rrnL :
small/large rRNA subunit genes
- trnX :
tRNA gene X ('X' replaces the one-letter amino acid code of the respective tRNA)
polymerase chain reaction
AM and LP greatly appreciate Thomas Bartolomaeus for his support during this work. AM and LP were supported by German research foundation (DFG), grants BA 1520/10-1,2 (LP); PO 765/4-3 (LP, AM), both from priority programme 1174 "Deep Metazoan Phylogeny"; A short term research stay from AM was supported by DAAD; XB received funding from Europôle Mer, a research consortium on marine science and technology in Brittany http://www.europolemer.eu/en/; DTJL was supported by NHM/GIA funds. UJ and SB were supported by the Swedish Research council.
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