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Recommended Citation Kayal, Ehsan, "The ve olution of the mitochondrial genomes of calcareous sponges and cnidarians" (2012). Graduate Theses and Dissertations. 12621. https://lib.dr.iastate.edu/etd/12621
This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. The evolution of the mitochondrial genomes of calcareous sponges and cnidarians
A dissertation submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major: Ecology and Evolutionary Biology
Program of Study Committee Dennis V. Lavrov, Major Professor Anne Bronikowski John Downing Eric Henderson Stephan Q. Schneider Jeanne M. Serb
Iowa State University
Copyright 2012, Ehsan Kayal ii
TABLE OF CONTENTS
ABSTRACT ...... V
CHAPTER 1. GENERAL INTRODUCTION...... 1
Introduction ...... 1
Thesis Organization ...... 5
References ...... 6
Introduction ...... 12
Materials and methods...... 15
Results ...... 18
CHAPTER 3. EVOLUTION OF LINEAR MITOCHONDRIAL GENOMES IN MEDUSOZOAN CNIDARIANS...... 51
Introduction ...... 52
Materials and methods...... 55
Results ...... 58
References ...... 80
CHAPTER 4. CNIDARIAN PHYLOGENETIC RELATIONSHIPS AS REVEALED BY MITOGENOMICS...... 84
Results ...... 89
Conclusions ...... 99
CHAPTER 5. THE ERATIC EVOLUTION OF ANIMAL MITOCHONDRIAL DNA...... 118
Introduction ...... 119
Mitochondrial genome structure and organization...... 121
The size of animal mitochondrial genome is not uniform ...... 123
Changing the code is not limited to bilaterian animals ...... 129
Concluding remarks...... 131
References ...... 133
CHAPTER 6. GENERAL CONCLUSIONS...... 146
Conclusions ...... 146
Broader perspectives and future directions...... 147
References ...... 149
APPENDIX A. SUPPLEMENTARY MATERIALS FOR CHAPTER 2 ...... 152
APPENDIX B. SUPPLEMENTARY MATERIALS FOR CHAPTER 3 ...... 161
APPENDIX B. SUPPLEMENTARY MATERIALS FOR CHAPTER 4 ...... 163
The mitochondrial DNA (mtDNA) in animals (Metazoa) is a favorite molecule for phylogenetic studies given its relative uniformity in both size and organization. Yet, as the depth coverage of representative animal groups increases sharply thanks to recent advances in sequencing technology, some clades remain stubbornly under sampled, if even represented at all. Difficulties associated with data collection from problematic taxa can arise from highly derived sequences, fragmented genomes, unusual structure, or any combination of these.
Particularly illustrative examples are found in non-bilaterian animals (placozoans, sponges, cnidarians, comb jellies) where the mtDNA is more variable in size and structure. The present dissertation provides several case studies of what is considered “unusual” mtDNA for animals.
First, we describe some unusual characteristics of the mitochondrial genomes found in calcareous sponges (Calcarea, Porifera), where one, potentially two, novel genetic codes are inferred, transfer RNAs (tRNAs) are edited, and ribosomal RNA (rRNA) genes are in pieces.
We also hypothesize that the mtDNA is linear and multipartite. Then, we explore the evolution of the mtDNA in medusozoan cnidarians (Medusozoa, Cnidaria). The mtDNA in Medusozoa is linear, and encodes two extra protein genes (lost in one clade) putatively involved in the maintenance and replication of the linear chromosomes. In addition, secondary segmentalization has occurred independently in some hydras (Hydridae) and box jellies
(Cubozoa). Using the sequences from these mito-genomes, we propose a new phylogeny for
Cnidaria, providing additional support for the clade [Medusozoa + Octocorallia], rendering
Anthozoa (Hexacorallia + Octocorallia) paraphyletic. Finally, this dissertation concludes by a mini review stating the current state of knowledge of metazoan mtDNA and some of the
vi pitfalls in the field of mitogenomics. In particular, the new findings further challenge the classical idea of a uniform mtDNA organization (frozen genome) in animals, and question any directional explanation of the evolution of the mtDNA in animals.
CHAPTER 1. GENERAL INTRODUCTION
Deoxyribonucleic acid (DNA) is the main carrier of genetic information in cells. In eukaryotes, the majority of the genetic information is found in the nucleus (nDNA), where duplication and transcription take place. But genetic material is not restricted to the nucleus as other cell compartments can also harbor and transmit DNA in the form of plastid DNA
2009) from α-proteobacteria-like ancestor in the case of the mitochondrion (Thrash et al.,
2011), and of a photosynthetic cyanobacterium in the case of plastids (Chan, Gross, Yoon, &
Bhattacharya, 2011; Janouskovec, Horák, Oborník, Lukes, & Keeling, 2010) are considered to be at the origin of eukaryotic organelles. Following the original endosymbiotic event, transfer of genetic material has occurred to various degree between the organelle and either another organelle or the nucleus (Adams & Palmer, 2003; Archibald, 2009; Martin, 2003; Timmis,
Ayliffe, Huang, & Martin, 2004). Consequently, none of the organelles carry genomes similar to those of their prokaryotic ancestor, i.e. a compact circular chromosome with a complete set of genes (Burger, Gray, & Lang, 2003; Hikosaka et al., 2010; Nash, Nisbet, Barbrook, &
Howe, 2008; Sesterhenn et al., 2010; Valach et al., 2011; Vlcek, Marande, Teijeiro, Lukes, &
By comparison to other organisms, animal mitochondrial genome is relatively stable in size and composition (Gissi, Iannelli, & Pesole, 2008). Structural variation of animal mtDNA encompass single or multi-partite linear or circular molecules, and recent sequencing efforts
provide additional evidence regarding the plasticity of animal mtDNA structure (Armstrong,
Blok, & Phillips, 2000; Doublet, 2010; Doublet et al., 2012; Gibson, Blok, Phillips, et al.,
2007; Gibson, Blok, & Dowton, 2007; Kayal et al., 2012; Kayal & Lavrov, 2008; Marcadé et
al., 2007; R. Shao, Kirkness, & Barker, 2009; Smith et al., 2012; Suga, Mark Welch, Tanaka,
Sakakura, & Hagiwara, 2008; Voigt, Erpenbeck, & Wörheide, 2008; Wei et al., 2012), further
challenging an earlier "frozen genome" view (J L Boore, 1999; Saccone et al., 2002). In
animals (phylum Metazoa), the mtDNA is a hot spot of translational novelties as illustrated by
the occurrence of frameshifting (Haen, Lang, Pomponi, & Lavrov, 2007; Rosengarten,
Sperling, Moreno, Leys, & Dellaporta, 2008), at least 13 codon reassignments (Abascal,
Posada, & Zardoya, 2012; Knight, Freeland, & Landweber, 2001), and still counting. The
exploration of obscure taxa (e.g. interstitial phyla, myxozoa, nematomorphs) might bring
additional evidences on the organizational elasticity of animal mtDNA. This also raises some
questions about the directionality of mtDNA evolution in animals as proposed earlier (Lavrov,
Recent advances in sequencing technology provide increasing amounts of molecular
data at relatively low prices (Glenn, 2011), revolutionizing many research areas in biology
(Egan, Schlueter, & Spooner, 2012; Kamb, 2011; Lerner & Fleischer, 2010; Mardis, 2008,
2011; Morozova & Marra, 2008; Raffan & Semple, 2011; Straub et al., 2011; Weinstock,
2011). As genomic studies are becoming widespread, the urge for acquiring more mtDNA data might start waning away. At the same time, the application of massively parallel next- generation sequencing platforms to mito-genomics (the study of mitochondrial genomes) (Jex,
Littlewood, & Gasser, 2010; Mason, Li, Helgen, & Murphy, 2011; Zaragoza, Fass, Diegoli,
Lin, & Arbustini, 2010) renders traditional protocols (e.g. Burger, Lavrov, Forget, & Lang,
2007) obsolete. In fact, for an equivalent effort input, next generation sequencing platforms produce >100 times the amount of mitochondrial genome sequence that traditional methods used to provide. These new technologies can also help deciphering the mtDNA of the so-called obscure animal taxa (see above) for which no mitochondrial sequence is available yet.
One can expect an increasing tide of sequence data in the coming years, some of which will without doubt improve our understanding of the evolution of animal mtDNA. Yet, most of these data will most likely come from a handful of model organisms, hardly contributing to our understanding of mtDNA diversity. As the field of mitogenomics reinvents itself in the light of next generation sequencing, particular attention is needed on those forgotten groups that either fell out of favor or never achieved to capture the interest of the scientific community. For instance, non-bilaterian animals (sponges, cnidarians, ctenophorans and placozoans) display the highest diversity in mitogenome organization, and are of primary importance in understanding the evolutionary history of early metazoans (Lavrov, 2007).
In that context, this thesis focuses on two groups of non-bilaterian animals for which
scarce (medusozoan cnidarians) or no mitochondrial sequences (calcareous sponges) was
available at the start of the program. Unlike most metazoans, the mtDNA of medusozoan
cnidarians are organized into one, two or eight mitochondrial chromosomes (Kayal et al., 2012;
Kayal & Lavrov, 2008; Park et al., 2012; Z. Shao, Graf, Chaga, & Lavrov, 2006; Smith et al.,
2012; Voigt et al., 2008). In Metazoa, the only other case of linear mtDNA has been found in
some crustacean isopods where the mtDNA consists of a circular ~28-kb dimer and a linear
~14-kb monomer (Doublet et al., 2012; Marcadé et al., 2007; Raimond et al., 1999). In order to
understand the evolution of linear mtDNA in medusozoan cnidarians, we determined the
partial or complete mtDNA sequences for 24 species belonging to all four medusozoan classes
using a combination of traditional and next generation sequencing approaches (Chapter 3).
Mitochondrial genomes provide one of the most commonly used molecular markers in
organization levels (Jeffrey L Boore & Fuerstenberg, 2008; Lavrov, 2007; Moret & Warnow,
2005). Thanks to our recent sampling of the mtDNA from representative species from all medusozoan classes, we were able to reconstruct the phylogenetic relationships within Cnidaria
[Medusozoa + Octocorallia] is not an artifact of reconstruction due to bias taxon sampling as suggested earlier (Collins, 2009; Park et al., 2012).
Within the past few years, sponge mitogenome sampling has greatly increased
(Belinky, Rot, Ilan, & Huchon, 2008; Ereskovsky & Lavrov, 2011; D Erpenbeck et al., 2007;
Dirk Erpenbeck, Voigt, Wörheide, & Lavrov, 2009; Gazave et al., 2010; Haen et al., 2007;
Lavrov, 2010; Lavrov, Forget, Kelly, & Lang, 2005; Lukić-Bilela et al., 2008; Rosengarten,
Sperling, Moreno, Leys, & Dellaporta, 2008; Wang & Lavrov, 2007, 2008) and many more are to be expected in the framework of the Assembling the Porifera Tree of Life project
(www.portol.org/). Yet, one class of sponges (Calcarea) remains stubbornly absent from the list. Here we determined partial mitochondrial sequences of three calcareous sponges (Chapter
We also propose that the mtDNA is fragmented into several linear molecules. While still incomplete, these sequences shed lights on the particular evolutionary path followed by the mtDNA of these sponges.
This dissertation consists of this general introduction (Chapter 1), two case studies describing the mtDNA of calcareous sponges (Chapter 2) and medusozoan cnidarian (Chapter
3), a phylogeny of Cnidaria based on mito-genomic data (Chapters 4), a mini review stating the current state of knowledge of metazoan mtDNA (Chapter 5), and a general conclusion that summarizes the major results of the thesis and recommends future developments (Chapter 6).
Chapter 2 describes the partial mtDNA of several calcareous sponges, where two novel genetic codes, tRNA editing, gene and genome fragmentation are reported. Chapter 3 looks at the evolution of linear mtDNA in medusozoan cnidarians. Chapter 4 redefines the phylogenetic relationships within Cnidaria using the extended mito-genomic dataset of Chapter 3. Chapter 3 has been published in Genome Biology and Evolution (2012) and Chapter 4 was submitted to
BMC Evolutionary Biology. Modified versions of Chapter 2 and 5 will be submitted for publication in Molecular Biology and Evolution and Mitochondrial DNA respectively.
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CHAPTER 2. CALCAREOUS SPONGES REDEFINE THE LIMITS OF
MTDNA EVOLUTION IN THE ANIMAL KINGDOM
A version of this manuscript to be submitted to Molecular Biology and Evolution
Ehsan Kayal, Oliver Voigt, Gert Wörheide, Lise Forget, Franz B. Lang, Dennis V. Lavrov
Although animal mitochondrial DNA (mtDNA) is often depicted as a remarkably
uniform molecule, recent sampling particularly from non-bilaterian animals has challenged this view. For instance, mitochondrial genomes from the three major lineages in the phylum
Porifera (Demospongiae, Homoscleromorpha, and Hexactinellida) revealed distinct
evolutionary trajectories. Remarkably, to date no genuine mitochondrial sequence has been
reported from Calcarea, the fourth class within the phylum. Here we report partial
Leucetta chagosensis (Leucettidae) from the subclass Calcinea, and Petrobiona massiliana
(Lithonida), subclass Calcaronea. Our analysis of calcarean mtDNA revealed a unique
combination of unusual mitochondrial features, including a multi-partite genome with potential
linear organization, at least one, potentially two novel genetic code(s) used in protein synthesis
that involves the first known reassignment of CGN codon family in mtDNA, template-
dependent editing of tRNAs, and highly fragmented rRNA genes. In addition, the rate of
mitochondrial sequence evolution in this group is several orders of magnitude higher than in
other animals. The combination of unusual mitochondrial features found in this class of
12 calcareous sponges puts into a new perspective our understanding of the extent of possible mtDNA evolution in Animalia.
Calcareous sponges (Calcarea) represent one of the four recognized classes within the phylum Porifera (Gazave et al., 2011; Hooper & Soest, 2002) and the only class of sponges with a skeleton made entirely of calcite, a form of calcium carbonate (CaCO3). The majority of
Calcarea species are small, colorless, and inconspicuous organisms found mostly in cryptic marine habitats (e.g. beneath rocks and inside cavities). Currently, there are ~675 described species for this class subdivided into two monophyletic subclasses Calcinea and Calcaronea
(Van Soest et al., 2012), which are well supported by the analyses of multiple independent characters (see (M Manuel, 2006)). However, despite several recent revisions conducted at different taxonomic levels (e.g. (Borojevic, Boury-Esnault, & Vacelet, 1990, 2000; Klautau &
Borojevic, 2001; Klautau & Valentine, 2003; M Manuel, 2006)), the phylogenetic relationships within these subclasses are far from being resolved. In addition, the phylogenetic position of
Calcarea relative to other sponges remains highly controversial (reviewed in (Erpenbeck &
Wöorheide, 2007)). Traditionally, calcareous sponges were placed as a sister group to either
Demospongiae (e.g. Cellularia, (Reiswig & Mackie, 1983)) or Demospongiae + Hexactinellida
(e.g. Silicea, (Gray, 1867)). By contrast, most molecular studies using rRNA (C Borchiellini,
Manuel, Alivon, Vacelet, & Parco, 2001; Cavalier-Smith, Allsopp, Chao, Boury-Esnault, &
Vacelet, 1996; Michael Manuel, Borchiellini, Alivon, Le Parco, & Boury-Esnault, 2003;
Medina, Collins, Silberman, & Sogin, 2001; Zrzavy, Mihulka, Kepka, Bezdek, & Tietz, 1998) and nuclear housekeeping genes (E. a Sperling, Peterson, & Pisani, 2009) reconstructed
Porifera as a paraphyletic group with calcareous sponges being more closely related to
Eumetazoa than to other sponges. Recent phylogenomic analyses based on ESTs data from a large number of animal species recovered support for the monophyly of the phylum Porifera, where Calcarea is placed as the sister taxa to Homoscleromorpha (Philippe et al., 2009, 2011;
Pick et al., 2010).
The uncertainties in both the phylogenetic position of Calcarea and the phylogenetic relationships within this group are mainly due to the current relative obscurity of the group to the sponge world. In fact, calcareous sponges were subject to numerous studies and monographs during the 19th century, as illustrated by their instrumental role in Haeckel’s development of the Gastrea theory (Haeckel, 1874). However, they fell out of favor during the
20th century, with most biologists becoming oblivious to the “beauty and fragrance of the
calcareous sponges” (Burton 1963 in (Hooper & Soest, 2002)). Consequently, relatively few studies explored the phylogeny of the group, and those that did primarily used nuclear rDNA sequences as molecular markers. Yet, resolving the phylogenetic position of Calcarea is crucial for determining the validity of Porifera as a clade and better understanding early animal evolution (E. a. Sperling, Pisani, & Peterson, 2007). This task calls for the use of several independent molecular markers (nuclear and mitochondrial) for phylogenetic reconstruction.
Animal mtDNA is a favorite molecule for phylogenetic, population genetic, and biogeographic studies. In fact, the relatively easy access to mitogenomes relative to nuclear sequences in most animal groups has propelled sequencing of complete mtDNAs into the frontline of genomic studies. While earlier studies considered animal mtDNA as a remarkably uniform genome (Saccone et al., 2002), recent sampling has uncovered its substantial
14 organizational diversity (Dennis V. Lavrov, 2007). In particular, complete mtDNA sequences from the other sponge lineages (Demospongiae, Hexactinellida, and Homoscleromorpha) have revealed a variety of unusual features such as extra protein genes, additional or missing tRNA genes, novel genetic code, translational frameshifting, and large non-coding regions (Belinky,
Rot, Ilan, & Huchon, 2008; Erpenbeck, Voigt, Wörheide, & Lavrov, 2009; Haen, Lang,
Pomponi, & Lavrov, 2007; Dennis V Lavrov, 2010; Dennis V Lavrov, Forget, Kelly, & Lang,
2005; Lukić-Bilela et al., 2008; Rosengarten, Sperling, Moreno, Leys, & Dellaporta, 2008;
Wang & Lavrov, 2007, 2008). However, to date there is a scarcity of mtDNA sequences from
Calcarea in public databases. Furthermore, six mitochondrial sequences available in the
GenBank database assigned to this group (Accession numbers AF035266, AF035267,
AF362006, AF362010, AF362015, AF362018) appear to be contamination from the demosponges (Figure S1, see also (Carole Borchiellini et al., 2004)).
Here we report partial mitochondrial sequences from four calcareous sponges, two
belonging to the subclass Calcinea: Clathrina clathrus (Clathrinidae) and Leucetta chagosensis
(Leucettidae), and one from the subclass Calcaronea: Petrobiona massiliana (Petrobionidae).
For Clathrina clathrus we PCR-amplified genomic sequence of three fragments of 7.5, 6.8 and
5.6 kbp in size using a Long-PCR approach (Burger, Lavrov, Forget, & Lang, 2007). For
Leucetta chagosensis we identified sequences of four mitochondrial protein genes from an EST database (Philippe et al., 2009). Nearly identical sequences have been PCR amplified from
another specimen of L. chagosensis collected elsewhere. For Petrobiona massiliana, we
identified mitochondrial gene from total genome sequencing reads. We then extended the
mitochondrial sequences of P. massiliana by PCR amplification using specific primers to
15 obtain three contigs of 3.5, 1.8 and 0.8 kbps. Our results provide a starting point for determining additional mt-genomes from calcareous sponges, which should reveal the full range of mtDNA sequence diversity in this group. They also reveal a derived the extensive accumulation of unusual features in calcareous sponge mtDNA that puts into a new perspective our understanding of the extent of possible mtDNA evolution in the Animal Kingdom.
Materials and methods
Obtaining mitochondrial sequences
A specimen of Clathrina clathrus was collected by Michael Nickel off Isola del Giglio
(Italy) and preserved in an 8M-guanidium chloride solution. A rock encrusted by a specimen of
Petrobiona massiliana was collected off the coasts of Marseille and preserved in an 8M-
guanidium chloride solution. Total DNA was prepared by phenol-chloroform extraction following proteinase K digestion. For C. clathrus, regions of cob, cox1 and rnl were amplified
and sequenced using conserved primers for animals (Burger et al., 2007). Sequences were
extended using a modified step-out protocol developed in our lab (Burger et al., 2007) and re-
amplified by standard PCR. Long PCR products were sheared and cloned using TOPO TA
Cloning Kit from Invitrogen. Total DNA from P. massiliana was sheared, tagged and
submitted for shallow depth total DNA sequencing on a GAIIX Illumina platform (1/3 for a
lane, 75 cycles). We used two different methods for the identification of mitochondrial protein
gene in P. massiliana. On the one had, short reads were assembled using MIRA v.3 (Chevreux
et al., 2004) and mitochondrial genes were identified by comparison to sequences from C.
clathrus. Sequences were extended by PCR as described above. Amplicons were used as
templates to map short Illumina reads using MIRA v.3. On the other hand, we used Neighbor
Joining (NJ) trees to pin down a list of putative mitochondrial genes. We extended these sequences by several rounds of mapping using Geneious Pro v5.5.6 (Drummond et al., 2011).
Sequencing reactions were processed at the Iowa State University DNA Facility. The EST library for Leucetta chagosensis came from the database reported in Philippe et al. (Philippe et al., 2009). cDNA library construction and ESTs sequencing were performed at the Max Planck
Institute for Molecular Genetics (Germany). Another specimen of L. chagosensis has been collected in the Red Sea (while the ESTs were generated from Australian L. chagosensis) and partial sequences for cob, cox1, cox3 and nad1 were PCR amplified and sequenced (primer sequences given in table S1).
RNA extractions and cDNA synthesis
Total RNA was extracted from a specimen of Clathrina clathrus collected by DVL at the Station marine d'Endoume, Marseille, France using TRIZOL extraction protocol
(Chomczynski & Mackey, 1995). Total RNA was treated with DNase and an aliquot was circularized using T4 RNA ligase. cDNA was produced using SuperScript® III from
Invitrogen using instructions from the manual, and several tRNAs and partial subunits of mitochondrial small and large ribosomal RNAs (rRNAs) were PCR amplified using specific primers designed based on DNA sequences (Table S1). PCR products were either directly sequenced (rRNA regions) or cloned (tRNAs) using the TA Cloning Kit from Invitrogen. At least eight colonies were analyzed per tRNA gene.
Gene annotation, RNA folding and genome description
Mitochondrial sequences were assembled with the STADEN software suite (Staden,
1996). Open reading frames in the nucleotide sequences were identified using the standard genetic code and minimally derived code (with TAG = Trp) characteristic for demosponge mitochondria (Dennis V Lavrov et al., 2005). Deviations from these genetic codes were found by analyzing 100% conserved positions in amino acid alignments of atp6, cob, cox1, nad1-4 genes for Clathrina clathrus, cob, cox1, cox3 and nad1 genes for Leucetta chagosensis, and cob, cox1, cox3, nad4-5 for Petrobiona massiliana, with homologues from representatives of
other non-bilaterian taxa. Gene annotation and gene boundaries were assigned by comparing
the translated sequences to homologous sequences from other non-bilaterian animals. rRNA
genes were identified by comparison to other rRNA sequences from sponges and cnidarians
and verified based upon their secondary structure. tRNA-like genes were identified with the
tRNAscan-SE and ARWEN programs (Laslett & Canbäck, 2008; Lowe & Eddy, 1997) and
We aligned the amino acid sequences of the genes atp6, cob, cox1, cox3, and nad1-5
from the calcarean species Clathrina clathrus, Leucetta chagosensis and Petrobiona
massiliana to homologues from 57 species representating other non-bilaterian groups (Table
S2). Sequences were aligned using the MUSCLE plugin of Geneious Pro v.5.5 (Drummond et
al., 2011) with default parameters. Individual gene alignments were filtered by Gblocks
(Castresana, 2000) with default parameters and allowing all gap positions. The final
concatenated data set was 2835 amino acids in length (Supplementary material). Maximum
likelihood (ML) and Bayesian (BI) analyzes were conducted on the concatenated data sets
18 using RAxML v.7.2.6 (Stamatakis, 2006) and PhyloBayes v. 3.2f (Lartillot, Blanquart, &
Lepage, n.d.). ML runs were performed for 1000 bootstraps iterations under the GTR model of
sequence evolution with the number of categories defined by a gamma (Γ) distribution and the
CAT approximation. BI analyzes consisted of four chains over 25,000 generations using the
CAT model (maxdiff<0.2), and sampled every 10th tree after the first 100 burn-in cycles
Multipartite architecture of calcareous sponge mitochondrial genomes
1- Clathrina clathrus (Calcinea)
We obtained the sequences for three fragments of the mitochondrial DNA (mtDNA) of
Clathrina clathrus (7.5, 6.8 and 5.6 kbp in size) (Figure 1A). We tried to determine whether C. clathrus sequences were parts of a larger single molecule or belonged to different mitochondrial chromosomes. To do so, we performed a Southern Blot (SB) analysis using probes specific to each of the three sequenced fragments. Each probe hybridized to two distinct sized bands. The size estimates for the lower bands were ~1.5 to 3 kbp larger than those for the corresponding determined contigs (Figure 1B), while the upper bands were approximately twice the size of the lower bands. Restriction enzymes digestion of the total DNA followed by
SB analysis displayed complex hybridization patterns difficult to interpret that reject neither a linear or circular organization of the mtDNA in C. clathrus.
The largest contig of C. clatrhus mtDNA (7.5kbp sequenced) corresponds to a chromosome ~10-11 kbp in size (“chrcl-cox1” hereafter) and contains four protein genes
(nad3, nad2, cox1, atp6) and at least two tRNA genes (trnN, trnR). The second largest contig
(6.8 kbp sequenced) corresponds to a chromosome ~9 kbp in size (“chrcl-cob”) and contains
three protein genes (nad4, nad1, and cob). The smallest contig (5.6 kbp sequenced)
corresponds to the smallest chromosome (“chrcl-rnl”, ~7 kbp) and encodes fragments of rns
and rnl as well as at least two tRNAs (Figure 1A). The remaining genes typically present in
animal mtDNA were not found and might be located either on different chromosomes or have
transferred to the nucleus. Yet, the presence of cox3 in Leucetta chagosensis and Petrobiona
massiliana mtDNAs (see below) suggests that the mtDNA in C. clathrus is organized into at
least four linear molecules. The arrangement of identified genes in Clathrina clathrus mtDNA
is very different from those in other non-bilaterian animals. In fact, we found only three shared
gene boundaries between C. clatrhus and other taxa: rns-rnl with some demosponges, trnQ-rns
with glass sponges, and nad1-cob with octocorals.
All three mitochondrial chromosomes in Clathrina clathrus are similarly compact with
near absence of intergenic region (IGR). We found genes located in one part of the
chromosome, while another part contains large non-coding repetitive sequences regions
(Figure 1A). In particular, a 190 bp-long, GC-rich sequence, which we called rep1, is present at
the one end of cox1 and cob chromosomes. The two copies of this sequence are 92.3%
identical in sequence and have a potential to fold into a complex secondary structure (Figure
2). We also amplified a region at one end of the rnl-chromosome with the combination of rnl-
rep1 primers, suggestion the presence the rep1 structure on this chromosome. Another
sequence, which we called rep2, is found at the other end of the cox1- and rnl-chromosomes.
The two copies of this sequence are 353 nucleotides in length and share 70.3% of sequence
20 identity. No evident secondary structure was found for rep2. We were not able to find sequence
similar to rep2 in the terminal region of the cob-chromosome (Figure 1A); instead we found
one strong GC-rich stem-loop structure. Interestingly, amplifications between rep1-rep2 give a
single band of ~6kb. Such pattern also advocates for fragmented chromosomal organization of
C. clathrus mtDNA.
The partial sequence of Clathrina clathrus mtDNA has a low A+T content (56.7%) and
displays small AT- and GC-skews between the two strands (0.03 and -0.08 respectively). The
AT-skew of the partial rRNA genes sequence is about 0.05 and GC-skew of 0.23. Strong
negative AT and GC-skews are found in C. clathrus protein-coding sequences (-0.11 and -0.25
respectively). In comparison, the coding regions in Leucetta chagosensis are less GC rich than
those in C. clathrus (27 vs. 42 %), with negative AT- and no GC-skews (-0.15 and 0.00
respectively). The presence of GC-rich repeated sequences is most likely responsible for higher
total GC values in the mtDNA of C. clathrus.
2- Petrobiona massiliana (Calcaronea)
Using C. clathrus mtDNA sequences as queries to mine the assemblies obtained from
shallow sequencing of Petrobiona massiliana total DNA, we identified several putative
mitochondrial protein genes that do not harbor internal stop codon according to the minimally
derived genetic code used for non-bilaterians (TGA=tryptophan). We identified a list of eight
sequences for atp6, cob, cox1, cox3, nad1, and nad4-5 (Figure S2). We used several gene-
specific primers for PCR-amplification and between these genes. cob, nad4 and nad5 could be
amplified into a single large PCR amplicon (chrpe-cob, 3.5 kbp). However, when we used this
21 large contig as a reference for assembling the short reads from the total DNA sequencing, no reads mapped in the regions between these genes, suggesting that the amplicon is actually a
PCR-generated artifact. Such artifacts are common features of multipartite genomes with terminal repeats (TRs) where the polymerase jumps between the TRs.
We found cob and nad5 genes on the largest contig (chrpe 1, 2.5 kbp). The other genes
were found on individual contigs: chrpe 2 (1.5 kbps) harbors cox1; chrpe 3 (1.2 kbps) contains
part of nad1; chrpe 4 (1.1 kbps) nad4; chrpe 5 (0.8 kbps) has encodes part of cox3. We were
not able to identify other mitochondrial genes with significant confidence. There is no shared
gene boundary between the mitochondrial gene arrangement in P. massiliana and other non-
bilaterian animals. We found various sized non-coding regions (NCR) at the end of most
contigs, with the largest ones found on chrpe 3 (345 bps) upstream of nad1 and on chrpe 2 (131
bps) downstream of cox1. Some of these regions can be folded into stem-loop structures. The
partial mitochondrial sequences of Petrobiona massiliana are more AT rich (53.3 %), and
display higher AT- (-0.14) and GC-skew (0.14) values between the two strands than their
Eccentric protein coding sequences in calcareous sponge mtDNA.
We identified seven mitochondrial protein genes (atp6, cob, cox1, nad1-4) in Clathrina
clathrus, four (cob, cox1, cox3, nad1) in Leucetta chagosensis, and six (cob, cox1, cox3, nad1,
nad4-5) in Petrobiona massiliana. These genes are on average of the same size as their
homologues in other non-bilaterian animals but share with them low sequence identities (Table
1). In C. clathrus mtDNA, ATG is inferred as the start-codon for three genes (atp6, nad2 and
nad3), while ATT inferred for two (cob and nad1), and TTA and CTT for one each (cox1 and
nad4, respectively). ATG is inferred as the start-codon of nad1 and nad5 in P. massiliana,
TTA inferred for cox1 and cox3. We were not able to infer a start codon for the other genes
(cob and nad4) due to missing data. TAA is the only stop codon inferred for C. clathrus and L.
chagosensis protein-coding genes, but both TAA and TAG are the stop codons inferred for P.
massiliana (see below). We also found an unidentified ORF on the rnl-chromosome of C.
Our data suggest that two different genetic codes are used in mitochondrial protein synthesis in calcareous sponges. In both Calcinea species, inferred mitochondrial protein-
coding sequences have inframe UAG codons, which usually code for translation termination in
all animal mtDNAs published so far. We found 33 internal UAG codons in C. clathrus coding
sequences and 23 UAG codons in those of L. chagosensis. We excluded mRNA editing as an
explanation for the presence of these internal termination codons because we found inframe
UAG codons in both PCRs and cDNA-based mitochondrial sequences obtained from L.
chagosensis and in cDNA-based cox1 sequences obtained from C. clathrus. We compared
pattern of codon usage for the positions that are completely conserved in inferred amino acid
sequences of encoded mitochondrial proteins between our sequences and other non-bilaterian
animals. These comparisons revealed that eight UAG codons in our sequences occurred at the
positions conserved as tyrosine in other non-bilaterian animals. In addition these sequences show an unexpected pattern of occurrence for CGN codons, specified as arginine in the standard genetic code. None of the CGN codons in calcareous sponge coding sequences are present in 100% conserved arginine position, but 49 codons in C. clathrus and 18 codons in L.
chagosensis are found in 100% conserved glycine positions. Finally, several internal TGA codons occurred at the positions conserved as tryptophan.
In the Calcaronea Petrobiona massiliana, the TGA codon encodes 52% of tryptophan
residue. We did not find any indication of the use of calcinean genetic code. In fact, no internal
TAG codon was found; TAG was the inferred termination codon for cob. In addition, we found
ATA codons in P. massiliana for 6 of the 100% conserved methionine positions for animals (3 in cox1, 1 in nad4 and 2 in nad5). ATA codons encode 60% of the methionine residues in the mitochondrial protein genes of this species (Table 2). These findings suggest that two independent changes of the genetic code occurred in calcareous sponges: the reassignment of
CGN codon family from arginine to glycine, and the UAG codon from termination to tyrosine in Calcinea; the reassignment of ATA codon to methionine in Calcaronea. Calcareans share with all animals as well as multiple outgroups the reassignment of the UGA codon from termination to tryptophan (Knight, Freeland, & Landweber, 2001).
Template-dependent editing in Clathrina clathrus mt-tRNAs
Using tRNAscan and ARWEN searches we identified several tRNA-like sequences in
the mtDNA of C. clathrus (Figure 1A). The inferred secondary structure of these motifs
resemble mtDNA-encoded tRNAs in bilaterian animals by the lack of conservation in the DHU
and TΨC arms and multiple mismatches in the amino-acyl acceptor arm. Six of these tRNAs
have well-conserved sequences of anticodon, D- T-arm loops, but multiple mismatches in the
stems (Figure 3). We investigated the nature of these tRNA-like sequences by a RT-PCR
approach (D V Lavrov, Brown, & Boore, 2000). For four of them ( , ,
and ), we found what appears to be mature tRNAs with the CCA sequence added at the 3’ end (Chens, Joyce, Wolfe, Steffen, & Martinn, 1992), and with both the T-arm and the 3’ end of the acceptor stem edited to form perfect Crick-Watson
complementary pairs (Figure 3). We were unable to RT-PCR amplify the remaining tRNA-like
structures and the nature of these sequences needs further investigation. The process of tRNA
editing in C. clathrus mitochondria resembles those described in the centipede Lithobius
forficatus (D V Lavrov et al., 2000) and onychophorans (Segovia, Pett, Trewick, & Lavrov,
(Beckenbach & Joy, 2009). However, unlike bilaterian mt-tRNA editing cases, tRNA genes are
not truncated in C. clathrus mtDNA, which advocates for an alternative mechanism of editing.
Fragmented rRNA genes
We identified several fragment of the large and small subunits of ribosomal RNA (rns
and rnl) in the mitochondrial sequences of C. clathrus (Figure 1A). These fragments are
distributed on the rnl-chromosome of C. clathrus mtDNA and, when assembled, harbor
conserved structures of animal mitochondrial rRNA genes, including conserved stem loops
required for rRNA functions. We found three segments of rns that are organized in a non-
sequential order and interrupted by (Figure 4). Similarly, the two inferred
fragments of rnl (rnl-p1 and rnl-p2) are interrupted by (Figure S3). As shown
above, both and appear to code for functional tRNAs. Although it is
possible that these rRNA fragments are pseudogenes or partially duplicated sequences, the
25 pattern of their conservation (with conserved sequences in functionally important regions), their presence in cDNA libraries, and the ability to form functional rRNA subunits all together argue for their being functional genes.
Phylogenetic inferences using Calcarea mtDNA.
We created an alignment of 2835 amino acid sequences of ten mitochondrial protein- coding genes from 57 species representing all non-bilaterian groups, but excluding the ctenophore sequences. We then conducted both Bayesian and Maximum Likelihood
phylogenetic analyzes under the GTR and CAT models of sequence evolution. Both Cnidaria
and Porifera were polyphyletic in all our phylogenetic trees (Figure 5). We found the groupings
both CAT and GTR models of sequence evolution. Interestingly, the CAT model appears to
overestimate the number of substitutions (illustrated by longer branches) for Calcarea and
Hexactinellida, compared to the GTR model (Figure 5). Comparatively, the GTR model was
more sensitive to missing data, where Leucetta chagosensis with the fewer number of
sequences was the longer branch in Calcarea (Figure 5A). The tree topologies were identical
under both the GTR+Γ and GTR+CAT models of sequence evolution, with lower bootstrap
values (BV) in the former (Figure 5A, Figure S4). In these trees, we found the long branch taxa
(Hexactinellida and Calcarea) regrouping together, and Hexacorallia the sister taxon to
[Demospongia + Homoscleromorpha] (BV=93/96 under GTR+Γ and GTR+CAT,
respectively). Under the CAT model we found [Octocorallia + Medusozoa] (PP=75), and a
composite group made of monophyletic Demospongia (PP=0.91), Hexacorallia (PP=0.6),
Calcarea (PP=0.99), and Hexactinellida (PP=0.99), and polyphyletic Homoscleromorpha
A unique combination of unusual mitochondrial features in Calcarea
For a long time sampling of animal mitochondrial genomes has been biased by two different factors: a prevailing scientific interest in a few taxa and technical difficulties associated with working with others. While the first of these biases is slowly lifting thanks to a better understanding of animal diversity, the second, often caused by atypical mtDNA architecture and/or sequence evolution, is only now starting to be addressed. Thus, one can expect that the few remaining metazoan lineages with no available mitochondrial sequence to have the most unusual mt-genomes. The mitochondrial genomic sequences from the calcareous
sponges Clathrina clathrus, Leucetta chagosensis, and Petrobiona massiliana determined in
this study are among the most remarkable in animals and comparable to the extreme
mitogenomes found in some protists (Burger, Jackson, & Waller, 2012). Here we discuss four
uncommon mitochondrial features revealed by these sequences: apparent multipartite and
linear genome architecture, novel genetic codes, edited tRNAs, and highly fragmented rRNAs.
Animal mtDNA is commonly depicted as a single, small, circular molecule, yet derived
forms of mitogenomes have been described in several taxa. For instance, it has been known for
more than 20 years that Medusozoa, one of the two major lineages in the phylum Cnidaria, has
linear and, in some cases, multipartite genomes (Kayal et al., 2012; Smith et al., 2012). In
bilaterian animals, some terrestrial isopods harbor two mtDNA “isoforms” composed of ~14
27 kb linear monomers and ~28 kb head-to-head circular dimers (Doublet, 2010; Doublet et al.,
2012; Marcadé et al., 2007; Raimond et al., 1999). Our study suggests that the mtDNA of C. clathrus is also multipartite, potentially linear, composed of at least three chromosomes of various sizes, each harboring a subset of mt genes and repeated identical regions. The results of the SB analyses, while difficult to interpret, might represent the two conformations
(supercoiled and relaxed forms) of the circular mtDNA molecules. This is reinforced by our inability to assemble P. massiliana sequences into a single contig. The presence of homologous sequences on various mitochondrial chromosomes was also reported in the multipartite mtDNA of the rotifer Brachionus plicatilis (Suga, Mark Welch, Tanaka, Sakakura, &
Hagiwara, 2008), the potato cyst nematode Globodera pallida (Armstrong, Blok, & Phillips,
2000), the human body louse Pediculus humanus (Shao, Kirkness, & Barker, 2009), and the booklice Liposcelis bostrychophila (Wei et al., 2012). In medusozoan cnidarians, inverted repeats at the ends of the linear molecules are thought to play a role in the integrity of the mitochondrial chromosomes (Smith et al., 2012). These similarities suggest that multipartite and potentially linear mitochondrial genome organization has also evolved in C. clathrus and, possibly, in all calcareous sponges. Unfortunately, our failure to obtain the complete mitochondrial sequences using traditional approaches hindered any attempt to definitively settle on the linear or circular nature of these mtDNA molecules.
The second unusual feature of the calcareous sponge mitochondria reported here is the presence of at least one, potentially two different and novel genetic codes inferred for mitochondrial protein synthesis. These include unique reassignments of the UAG codons from termination to tyrosine and of the CGN codon family from arginine to glycine in calcineans
Clathrina clathrus and Leucetta chagosensis, and putatively of the ATA codon from isoleucine
to methionine in the calcaronean Petrobiona massiliana. We also found the reassignment of the
UGA codon from termination to tryptophan in both subclasses that is shared with other animals
and some fungi (Knight et al., 2001). While the CGN codons were previously identified as
“mutable in their meaning” (Söll & RajBhandary, 2006) and unassigned in yeast mitochondria
(Clark-Walker & Weiller, 1994), our study is the first to report their actual reassignment.
Similarly, the reassignment of the UAG codon to tyrosine is novel, although changes of the
UAG codon to leucine/alanine and glutamine have been found in mtDNA of green plants and
nuclear genomes of ciliates, respectively (Knight et al., 2001). Interestingly, a convergent
change for another termination codon (UAA) to tyrosine has been recently reported in a
nematode (Jacob, Vanholme, Van Leeuwen, & Gheysen, 2009). The reassignment of ATA
codon to methionine is typical of bilaterians and some fungi (Knight et al., 2001). If confirmed,
the change of the ATA codon becomes a second convergence of codon change between
Bilateria and sponges, similar to the change in AGR codons in glass sponges (Haen et al.,
2007). But, unlike what has been suggested earlier (Haen et al., 2007), given the position of
sponges in the most recent animal phylogenies (Pick et al., 2010), these changes do not appear
to “represent an intermediate state in the reassignment of” these codons “in animal mtDNA”
(Haen et al., 2007), but rather episodic events in the erratic evolution of the mtDNA in
metazoans. Changes in the genetic code are often associated with the presence of reassigned
tRNAs in the mitochondria that are either encoded by the mtDNA and/or imported from the
cytoplasm (Lang, Lavrov, Beck, & Steinberg, 2012). Our data does not allow ruling out either
Another unusual feature of calcareous mitochondrial genomes is fragmented rRNA genes. Fragmented rRNA genes are seldom in animal mtDNA and so far have been found only in oysters Crassostera gigas and C. virginica, where lrRNA is encoded in two pieces (C. A.
Milbury & Gaffney, 2005; C. a Milbury, Lee, Cannone, Gaffney, & Gutell, 2010), and in
Placozoa (Signorovitch, Buss, & Dellaporta, 2007) where lrRNA is encoded in two or tree pieces separated by as much as 20 kbp (Burger, Yan, Javadi, & Lang, 2009; Signorovitch et al.,
Kloareg, & Goër, 1995; Nedelcu, 1997). Extreme rRNA segmentation has been documented for mitochondrial rRNA genes of apicomplexan and dinoflagellates (Jackson et al., 2007;
Waller & Jackson, 2009). Yet, the high degree of rRNA fragmentation in C. clathrus mitochondria is unique in animals. Assuming that these rRNA-like fragments generate functional rRNA molecules, it would be very interesting to explore the mechanisms involved in their processing, particularly for the recognition of individual pieces, and their interaction inside the ribosome.
Our calcarean mitochondrial sequences encode truncated tRNA molecules, which appear to undergo a post-transcriptional RNA editing. Several different types of mitochondrial tRNA editing has been reported to date, including C-to-U editing in mitochondria of marsupials (Börner, Mörl, Janke, & Pääbo, 1996; Janke & Pääbo, 1993), trypanosomes
(Alfonzo, Blanc, Estévez, Rubio, & Simpson, 1999) and plants (e.g. (Fey et al., 2002;
Maréchal-Drouard, Ramamonjisoa, Cosset, Weil, & Dietrich, 1993)); insertion editing in slime molds (Antes, Costandy, Mahendran, & Miller, 1998); 5’-end editing in amoebozoans (K.
Lonergan & Gray, 1993; K. M. Lonergan & Gray, 1993) and lower fungi (Laforest, Roewer, &
Lang, 1997); or a combination of these (Gott, Somerlot, & Gray, 2010). The 3’-end editing similar to that found in our study has been reported in bilaterian animals (D V Lavrov et al.,
2000; Reichert, Rothbauer, & Mörl, 1998; Segovia et al., 2011; Tomita, Ueda, & Watanabe,
1996; Yokobori & Pääbo, 1995, 1997) and a jacobid flagellate (Leigh & Lang, 2004). Further
studies are needed to determine whether the tRNA editing found in Clathrina clathrus is a
recent acquisitions that is specific for this species/group of species, as is common in animals
(e.g. (Brennicke, Marchfelder, & Binder, 1999)) or it is a feature common to all calcareous
Finally, the mitochondrial sequences of calcareous sponges determined for this study
display rapid and unique evolutionary patterns that evocate the very unusual mtDNAs found in
some dinoflagellates (Jackson et al., 2007; Waller & Jackson, 2009). These sequences also
exhibit an extremely high rate of sequence evolution, hampering our attempts to recover the
position of Calcarea within Metazoa based on mtDNA sequence data. In previous studies, the phylogenetic positions of the highly derived mtDNA sequences from Ctenophora (Kohn et al.,
2011; Pett et al., 2011) have been problematic, resulting in LBA artifacts between these species and bilaterian animals. Similarly, we also found the artifactual topology [Ctenophora +
Calcarea] the sister taxon to Bilateria (data not shown). Therefore, we decided to focus our efforts on the position of Calcarea within non-bilaterian animals, and particularly within sponges, by removing sequences from bilaterian and ctenophore species. Under the GTR model of sequence evolution, we recovered [[Demospongia + Homoscleromorpha] +
Hexacorallia], a result already found in previous studies (e.g. (Dennis V Lavrov, Wang, &
Kelly, 2008)). GTR analyses also showed the grouping of the long branch clades Calcarea and
Hexactinellida (Figure 5A). Given the limitations of the GTR model, notably a homogenous distribution of amino acids among positions, we assume such topology is the result of LBA artifacts (Lartillot, Brinkmann, & Philippe, 2007). The CAT model performed better regarding
LBA artifacts, where the long branch Calcarea and Hexactinellida did not group together, but resulted in a polytomy between Porifera and Hexacorallia (Figure 5B). The use of more complex models of sequence evolution, such as the site-heterogeneous CATGTR+G model, might overcome some of the systematic errors present in the mitochondrial sequence data, but their implementation requires more sequences data from a wider range of taxa in order to accurately estimate all needed parameters (Philippe & Roure, 2011).
The lack of phylogenetic signal is not surprising given the highly derived nature of the mitochondrial sequences for Calcarea. Put into a broader perspective, our analyses provide additional evidences regarding the limitations of mtDNA data for resolving deep phylogenetic
nodes within early diverging animals. By comparison, recent EST-based studies have
recovered monophyletic Porifera and Cnidaria, where Calcarea is the sister group to
Homoscleromorpha (Philippe et al., 2009; Pick et al., 2010). Yet, the relatively high rates of sequence evolution in calcareous sponges suggest that mitochondrial sequences can be valuable tools for the study of higher taxonomic ranks (e.g. (Voigt, Eichmann, & Wörheide,
2011)). In addition, if calcareous and homoscleromorph sponges are indeed related, these taxa would represent an interesting system for the study of mitochondrial genome evolution in sister taxa as they display very different trends in mt-genome evolution.
In the second half of 19th century, calcareous sponges were a subject of numerous
studies, resulting in several monographs on their taxonomy and development. Although, this
sponge group fell out of favor during the 20th century, we expect that the new genomic era will
reignite the interest in “these remarkable animals” (Hooper & Soest, 2002). Given that the
mitochondrial genomes presented here are incomplete, and that we only explored two
representatives from the subclass Calcinea and one from Calcaronea out of the ~675 described
species in Calcarea, we expect that future studies of calcareous sponge mtDNA, will uncover
additional unusual features in their mtDNA organization and evolution. Such studies will shed
light on the evolution of the mitochondrial genome in Calcarea, and in animals in general.
Supplementary figures S1-4 and supplementary tables 1 and 2 are available at
Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
This work was supported by the National Science Foundation (DEB-0828783 to DVL)
and the German Science Foundation (WO896/3 and WO896/6 to GW).
We thank Micheal Nickel and Dan Jackson for collecting and providing Clathrina
clathrus and Leucetta specimens used for mtDNA sequencing and cDNA work, respectively.
We also thank Mohsen Kayal for his insightful comments on an earlier version of the
33 manuscript. GW and OV thank the German Science Foundation (DFG) for funding (WO896/3, and through the DFG Priority Programme SPP1174 "Deep Metazoan Phylogeny", project
WO896/6). E.K. wishes to acknowledge the funding through the Smithsonian Predoctoral
Fellow grant, as well as people from the Wendell lab for funding part of this project.
Table 1: Amino acid similarity for the mitochondrial protein coding genes atp6, cob, cox1, cox3, nad1-5 between the calcareous sponges used in this study (Clathrina clathrus, Leucetta
chagosensis and Petrobiona massiliana) and complete mitochondrial genomes from
demosponges, homoscleromorphs, glass-sponges and cnidarians.
Homoscleromorpha Demospongia Hexactinellida Cnidaria Placozoa
Clathrina 33.9±0.4 34.3 ±0.7 30.7 ±0.7 33.5±1.0 30.0 ±0.5 clathrus
Leucetta 46.4 ±0.7 46.4 ±0.8 44.4 ±0.5 45.9±1.8 43.2 ±0.1 chagosensis
Petrobiona 24.7±0.4 24.7±0.3 23.4±0.1 24.9±1.1 22.3±0.1 massiliana
Table 2: Codon usage table used in the mtDNA of the calcareous sponges Clathrina clathrus
(C.c.), Leucetta chagosensis (L.c.), and Petrobiona massiliana (P.m.). Inferred derivations
from the standard genetic code are highlighted in bold. TGA1: from termination codon (TER) to tryptophan (Trp) in all metazoan groups; TAG2: from termination codon to tyrosine (Tyr) in calcineans; CGN3: from arginine to glycine in calcineans; ATA4: from isoleucine (Ile) to
methionine (Met) in the calcaronean P. massiliana.
19 16 45 41 17 11 29 13 27 24 42 62 39 26 64 77 P.m
7 1 7 7 1 2 1 3 2 33 22 27 25 31 30 12 L.c.
8 5 4 12 51 17 26 68 11 37 36 36 20 21 33 11 C.c.
3 3 1 3
TGT TGC TGG AGT GGT AGC GGC AGA AGG GGA GGG TGA CGT CGC CGA CGG
Ser Trp Gly Arg Arg Cys TER
2 1 27 35 32 11 14 18 33 15 19 19 21 13 25 20 P.m.
9 4 5 5 6 5 4 40 23 28 15 63 40 27 28 11 L.c.
7 49 53 33 31 16 29 13 33 51 33 46 27 17 35 33 C.c.
TAT TAC CAT TAA AAT GAT CAC CAA CAG AAC GAC AAA AAG GAA GAG TAG
His Tyr Gln Lys Glu Asn Asp TER
35 26 39 33 33 23 20 15 27 28 31 13 35 40 50 24 P.m.
2 2 8 1 8 1 39 13 18 23 11 16 26 26 24 11 L.c.
4 4 3 0 51 60 4 43 18 32 36 59 25 13 41 67 32 C.c.
TCT TCC CCT TCA TCG ACT GCT CCC CCA CCG ACC GCC ACA ACG GCA GCG
Ser Pro Thr Ala
3 33 76 65 44 29 6 58 38 34 61 40 37 16 60 58 102 P.m.
6 1 3 1 24 14 35 38 76 22 88 19 37 18 120 144 L.c.
69 35 38 63 29 94 44 61 35 49 56 25 141 107 159 137 C.c.
TTT TTC CTT TTA TTG ATT GTT CTC CTA CTG ATC GTC ATG G GTG ATA
Ile Val Phe Leu Leu Met
nad2 cox1 atp6 rep2
chrcl-cob rep1 cob-loop
nad1 cob nad4
chrcl-rnl rep1 r n s 1 - 2 r n s 1 - orf rep2
P S rns3 M rns1 rnl-p2 rnl-p1 rns2
1 2 3
Figure 1: A. Genetic map of the partial mitochondrial “chromosomes” of the calcareous sponge Clathrina clathrus (Calcinea). All genes above the contig are transcribed from left to right and those under from right to left. tRNA-like genes are identified by the one-letter code for their corresponding amino acid. Underlined tRNAs and rRNA pieces correspond to those confirmed at the RNA level. Abbreviations are as follows: atp6, subunits 6 of ATP synthase; cob, apocytochrome b; cox1, subunit one of cytochrome oxidase; nad1-4, NADH dehydrogenase subunits 1 to 4; rnl and rns, large and small ribosomal RNAs respectively; orf is an unidentified open reading frame. rep1 and rep2 correspond to repeated regions present in the mt-genome, with a subjective orientation (black arrow); cob-loop is a large stem-loop structure found on chrcl-cob. Smaller stem-loops can be folded into structures resembling
tRNAs. B. Southern blot analysis of the mtDNA of Clathrina clathrus. Lanes 1, 2 and 3 were
hybridized with probes corresponding to cob, rnl and cox1 respectively. Lower bands have size
estimates similar to the sequenced contigs (see text).
G T G rep1 T T A A C-G A-T A-T C G T A T T GTGTTG C AGCAGAAG .||||| G T ||||||| TACAAC C G CGTCTTC G G A T T-A T.G T A G A-T A-T G.T G-C C-G G-C G.T A-T C-G C-G A C G-C T T A G G T A A A G A C-G G-C T-A C-G C-G T-A C-G T T C c T C T C C A-T C G A T-A T-A G G T-A A-T trnN end A-T C A-T A G-CAGTCCAAACTCCTTTTCC GCCCCTATGAAGATTCTTGATACAAATTCCAAT
A T A C G G C C T T loop2 C C G T G A-T C G T A rep1 C-G T T G-C A A C-G C-G A T T A-T G T C C-G A-T C G T GTGTTG C A-T AGCAGAAG T .||||| G C-G T ||||||| TACAAC A A-T G CGTCTTC G T-A C-G T.G A T G- T A C G-C A-T A-T C-G G.T G-C A A loop1 C-G G-C C-G G.T A-T A-T C-G C-G C-G A A C A C G-C T CGG A G C-G A T T A A C-G C ||| A G T A GC-G A T-A G AGCC T A-T T G-C T-A T-A C-G GC-G C-G C-G G-C T-A C-G T-A T-A T-A G-C T-A T C-G T-A T C T-A T-A T C T T.G T-A C T A-T GT.G C C T-A C A-T T T G-C A A G-C A T-A T-A C T T-A G-C G G C T-A A-T T-A A-T C-G T-A A-T C T T-A C G-C T A-T C G-C A-T A A A C-G C A A G T-A T-A A- T G-C A G C C T-A G- C A-T T-A T- A A A T-A T- A nad4 end cob end T-A G-C 134 nucleotides T- A C-G T.G T- A C A TACACGC G-C TTTTTTGTGTT [...... ] CCTAGTAATA- T CCTTTTCCAG [...... ] TGGGAGACCTTTT-ATCCTACGTGTGAA
Figure 2: Repeated elements in the mitochondrial genome of Clathrina clathrus. A. Linear map of the partial mitochondrial sequences of C. clathrus displaying the repeated elements rep1 and rep2 and some putative stem-loops. A black arrow illustrates the suggested
orientation of these elements. B. Putative stem-loop structures and repeats found in the non-
coding regions of C. clathrus mtDNA. Bold sequences correspond to the repeated region rep1
found in the stem-loop structure at one end of both cob- and cox1-contigs. Arrows in the
39 complex stem-loops structures are discrepancies (either substitution or insertion) between the repeats. Adjacent gene boundaries are given with their orientation.
A A C-G GxA TxT Asparagine C-G Arginine (N) C (R) C TxC A C TxT A C AxC A T-A A CxC C-G CxA G-C CxA T-A C-G C-G C-G AxA T TACCC T TA T-A T C T-A A-T T GGACC A T-A A ||||| G A ||x|| G C C-G C-G ATGGG T T CCGGG C C C-G T T T C-G G C-G G A-T T T T C T TACCC A T GGCCC A A A A ||||| A G T TTTA G A A T ||||| G ATGGG C C TTG CCGGG C .||| C T T G ||| T T G GAAT G T A A G TAAC G C T G T A T A T-A GxA A C-G T-A G-C A-T T-A G-C A-T CxT C A C G T A T A GTT TCT
T T AxA Methionine A-T Proline CxT (M) CxC (P) GxA TxG C C GxT A C TxT A C AxG A G-C A G-C A-T A-T A-T A-T TxG T A C-G C-G A AATCC A T GAGTC T CA T-A G G-C |x| || A G-C A x|||| A T-A TGAGG ATCAG C G-C A T T C A-T T T T G-C TG A-T A-T A T-A T A T C A A A AC TCC T TAGTC A A A G || ||| A A A A A G CG T TTTG ||||| TGAGG T C ATCAG C G || A T G .||| T T T G GC T G T A GAAC G A T A A A A A TG G.T C G.T C-G G-C A-T T-A G-C G-C A-T T-A T C T A T A T G CAT TGG
Figure 3: Inferred secondary structures for Clathrina clathrus , ,
and at both DNA (on the left) and RNA level (on the right). Grey
letters correspond to the DNA sequence and black letters are changes found at the RNA level.
Signatures for mature (functional) tRNAs (ACC on the acceptor arm) are shown.
. . U A U U A ...... A C U ...... C U U U C G G G A ...... A G A AA A A G C U C U . G . G C U C . . G C A U ...... A U . . A U . . . . . A U G G G G U . . U A G G A U A U . . A A G . . A U G C rns G . . p2 C G C . . U A . . C G A C G G G . . . G C A C U C A U . A U G U U C C U G A C . . A U G G . A U U . A G C G A G G A U U U G . A U A A A . . A U C U . A U U U G rnl . U A p1 . . UG U A C . . A A G C . . G C G G U C . . A U U U G C U U C . U G G . G U G C A trnM . A . G U A A . G G C A G . G A G G U G A U U A C U G C U G A C G U U A A A U A A A A G C G A C G U A U C CA C C G G C U C U A C G A U U U U A G G G U A C G U A G U G C A A U C U C U A A C A C . . G C U A G A A A A U A . . A G C C U G C U C U A A A . . A A A A U G C A G C G A U C U C A G G rns . . U C U A G U A . U A U p1 . . G A G G C U T . U . G U A C G . C U G U G G . . G C G C G C A U U A . . C G A U U . . G . U A G A . G G C A U C . . . A C C UA A . A . . U C G G A G C G G C U A UC . A U . . . C G U G A A . . . . . A G C A G A A G A C U U G A UGC . . A A A UA . . U C G G . A A U . . . A U A G . . . U C . . A U U G GU . . . U A A G U . G A A GG . . . A A A A . G U A A U U . . . . . UCC AG C G U U U G . . U U C U G G U G U U U A C U A U C A A . . . . U G A C . . U A A . AGG U C U A G C . . . . U G U U G C A G A U G A U A G A U . . AA U C A U C A U A U U A G . . U C A G C G U . G G U U . G U G U . . . G G G G G A G . A . . A U C U U C . . G A . G G A U A A . . . AU G G . . A A . . o 5' A . . . . U U ...... U G . U G C A U G G U C A U A G U A G U . U A C A G . C G A A A ...... A C U G G U G U C G U C A C C C G A . . . . C G . A G C . U U A . U G C A U U C G A UG A C G A . U A G C A GA U C AA o . A A 3' . . . G U . . U A . C U U A G G . . . UU A A A G G . . C C C A G A G rnsp3 . . U GA U U U A U . U . . G G G U C U CG A A . . A U A G A U G A G . . . . G . . . G C . . . . U G G G ...... G G . . . U A ...... A A ...... G G . . C G . . . . AG . A . . . . C G . . . . . U C . . U A ...... U A . . U A . . C G ...... G U . . U A . . G C . . . . U . . . A U ...... G C . . . U G . . . . G A . . . A ...... M S P ...... 2 . . . . B . . . . 1 3 2 1 2 ...... rns rnl . .
Figure 4: A. Inferred secondary structure of the partial small mitochondrial ribosomal RNA subunit (rns) in Clathrina clathrus based on the skeleton of Oscarella sp rns. Sequences correspond to Clathrina; black dots are based on Oscarella rns. rnsp1, rnsp2 and rnsp3 are partial
sequences of rRNA named in the same order than folded complete ribosomal RNA. Black
41 dashed lines denote breaks in the coding sequences compared to the "complete" RNA structure.
B. Order of the different pieces of rRNA found at DNA level. Underlined tRNAs correspond to those confirmed at the RNA level. Note that the rns sequence is incomplete and that additional sequence may be present in the unsequenced portion of the genome.
A) * Placozoa Linuche unguiculata Coronatae Alatina moseri Cubozoa * Haliclystus sanjuanensis Lucernaria janetae Staurozoa Cubaia aphrodite * Clava multicornis Laomedea flexuosa * Millepora platyphylla Hydrozoa Pennaria tiarella * Ectopleura larynx Hydra magnipapillata * Cyanea capillata * Chrysaora sp * Pelagia noctiluca Aurelia aurita Discomedusae * Cassiopea andromeda Catostylus mosaicus Renilla mulleri * Keratoisidinae sp Briareum asbestinum Dendronephthya gigantea Octocorallia Sarcophyton glaucum Savalia savaglia * Metridium senile Chrysopathes formosa Acropora tenuis * Ricordea florida Hexacorallia * Lophelia pertusa * Astrangia sp Madracis mirabilis Igernella notabilis Ircinia strobilina Keratosa (G1) Aplysina fulva * Halisarca dujardini Myxospongiae (G2) * Callyspongia plicifera Xestospongia muta marine Haplosclerida (G3) * Ephydatia muelleri * Lubomirskia baicalensis Axinella corrugata Geodia neptuni Topsentia ophiraphidites Democlavia (G4) Suberites domuncula * Tethya actinia Oscarella carmela * Corticium candelabrum * Plakina monolopha Homoscleromorpha (G0) Plakinastrella cf. onkodes * Aphrocallistes vastus * Iphiteon panicea Hexactinellida Sympagella nux * Clathrina clathrus * Leucetta chagosensis Petrobiona massiliana * Placozoa B) 0.88 Linuche unguiculata Coronatae Alatina moseri Cubozoa * Haliclystus sanjuanensis 0.59 Lucernaria janetae Staurozoa Cubaia aphrodite 0.99 0.98 Clava multicornis 0.99 Laomedea flexuosa 0.93 Millepora platyphylla 0.99 Hydrozoa Pennaria tiarella 0.99 0.99 Ectopleura larynx 0.99 Hydra magnipapillata 0.75 0.99 Cyanea capillata Chrysaora sp 0.99 Pelagia noctiluca 0.99 Aurelia aurita Discomedusae 0.99 Cassiopea andromeda 0.99 Catostylus mosaicus
0.99 Renilla mulleri Keratoisidinae sp 0.99 Briareum asbestinum Octocorallia 0.88 Dendronephthya gigantea 0.99 Sarcophyton glaucum
0.6 Savalia savaglia Metridium senile Chrysopathes formosa 0.61 0.99 Acropora tenuis 0.82 Ricordea florida Hexacorallia 0.95 Lophelia pertusa 0.99 Astrangia sp 0.99 Madracis mirabilis 0.99 Clathrina clathrus 0.99 Leucetta chagosensis Petrobiona massiliana Corticium candelabrum * Plakina monolopha * Plakinastrella cf. onkodes Homoscleromorpha (G0)
0.99 Aphrocallistes vastus 0.59 Iphiteon panicea Hexactinellida 0.99 Sympagella nux 0.99 Igernella notabilis Keratosa (G1) 0.94 Ircinia strobilina Aplysina fulva 0.99 Halisarca dujardini Myxospongiae (G2) 0.91 Callyspongia plicifera 0.99 Xestospongia muta marine Haplosclerida (G3) 0.97 0.99 Ephydatia muelleri 0.98 Lubomirskia baicalensis Axinella corrugata 0.92 Topsentia ophiraphidites 0.99 Democlavia (G4) Geodia neptuni 0.96 0.99 Suberites domuncula Tethya actinia Oscarella carmela Homoscleromorpha (G0)
Figure 5: Phylogenetic analysis of concatenated amino acid sequences from representative non-bilaterian species for atp6, cob, cox1, cox3, and nad1-5 genes under the GTR+Γ (A) and
CAT+Γ (B) models of sequence evolution using RAxML v.7.2.6 and PhyloBayes v. 3.2f
respectively. A star shows maximum support for that node. Calcareous species have been
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CHAPTER 3. EVOLUTION OF LINEAR MITOCHONDRIAL
GENOMES IN MEDUSOZOAN CNIDARIANS
A paper published in Genome Biology and Evolution
Ehsan Kayal, Bastian Bentlage, Allen G. Collins, Mohsen Kayal, Stacy Pirro, and Dennis V.
In nearly all animals, mitochondrial DNA (mtDNA) consists of a single circular molecule that encodes several subunits of the protein complexes involved in oxidative phosphorylation as well as part of the machinery for their expression. By contrast, mtDNA in species belonging to Medusozoa (one of the two major lineages in the phylum Cnidaria) comprises one to several linear molecules. Many questions remain on the ubiquity of linear mtDNA in medusozoans and the mechanisms responsible for its evolution, replication, and transcription. To address some of these questions, we determined the sequences of nearly complete linear mtDNA from 24 species representing all four medusozoan classes: Cubozoa,
Hydrozoa, Scyphozoa, and Staurozoa. All newly determined medusozoan mitochondrial genomes harbor the 17 genes typical for cnidarians and map as linear molecules with high degree of gene order conservation relative to anthozoans. In addition, two ORFs, polB and
ORF314, are identified in cubozoan, schyphozoan, staurozoan, and trachyline hydrozoan mtDNA. polB belongs to the B-type DNA polymerase gene family, while the product of
ORF314 may act as a terminal protein (TP) that binds telomeres. We posit that these two ORFs
are remnants of a linear plasmid that invaded the mitochondrial genomes of the last common
ancestor of Medusozoa, and is responsible for its linearity. Hydroidolinan hydrozoans have lost
the two ORFs and instead have duplicated cox1 at each end of their mitochondrial
chromosome(s). Fragmentation of mtDNA occurred independently in Cubozoa and Hydridae
(Hydrozoa, Hydroidolina). Our broad sampling allows us to reconstruct the evolutionary
history of linear mtDNA in medusozoans.
Sequence data from this article have been deposited in GenBank under the accession numbers: JN700934-JN700988
The mitochondrial genome is one of the most commonly used molecular markers in
animal phylogenetics, with informative characters at the nucleotide, amino acid and gene organization levels (Moret and Warnow 2005; Lavrov 2007). As a consequence, the metazoan
mitogenomic dataset has benefited from a wide sampling effort across the animal kingdom. In
most animals, mitochondrial DNA (mtDNA) is a single, small (usually less than 20 kbp)
circular molecule that contains a nearly constant set of genes. With a few exceptions, these genes are orthologous across animals (Wolstenholme 1992; Boore 1999). Most animal
mitochondrial genomes encode 12-14 protein genes and up to 28 RNA genes (Gissi et al.
2008). While complete or nearly complete mitochondrial genomes for more than 2400 animal
species are available on the Organelle Genome Resources database in GenBank
(ncbi.nlm.nih.gov/genomes/OrganelleResource.cgi?taxid=33208), only 97 represent non-
53 bilaterians: 5 for Placozoa, 42 for Porifera, 49 for Cnidaria and one for Ctenophora. This paucity of completely sequenced non-bilaterian mtDNAs is unfortunate because non-bilaterian mitochondrial genomes represent a large portion of the variability in both sequence and genome organization among animals (Lavrov 2007; Gissi et al. 2008).
The phylum Cnidaria represents a “hot spot” of animal mitochondrial genomic diversity, with mtDNA displaying variation in both gene content and genome organization.
Variation in the gene content is illustrated by the loss of all but one or two tRNA genes, a feature also found in some demosponges and in all chaetognaths (Helfenbein et al. 2004;
Papillon et al. 2004; Faure and Casanova 2006; Wang and Lavrov 2008; Miyamoto et al.
2010). In addition, introns (e.g. Beagley et al., 1998; Chen et al. 2008), duplicated genes (Kayal and Lavrov 2008; Voigt et al. 2008) and extra protein genes (mutS in octocorals, polB in
Scyphozoa, unidentified ORFs in several hexacorals) have been found in several cnidarian classes (Pont-Kingdon et al. 1998; Medina et al. 2006; Shao et al. 2006). Furthermore, the structure of cnidarian mtDNA is also variable: a single circular molecule is found in Anthozoa
(hexa- and octocorals), while medusozoan mtDNA consists of one or several exclusively linear molecules (Warrior and Gall 1985; Bridge et al. 1992; Ender and Schierwater 2003). Working with linear mtDNA presents unique challenges for both PCR amplification and sequencing. As a potential reflection of these challenges, only six medusozoan linear mitochondrial genomes have been determined to date, those of the jellyfishes Aurelia aurita and Chrysaora quinquecirrha (Scyphozoa), and the three hydra species Hydra magnipapillata, H. oligactis, and H. vulgaris (Hydrozoa) (Shao et al. 2006; Kayal and Lavrov 2008; Voigt et al. 2008; Park et al. in press), while 43 complete circular mitochondrial genomes are available for anthozoans.
Linear mtDNA is common outside animals and has been extensively studied in fungi, plants, and several groups of unicellular eukaryotes (Bendich 1993; Burger et al 2003; Nosek and Tomáska 2003; Hikosaka et al 2010; Valach et al 2011). The linear form of these molecules is thought to affect their stability, mechanisms of DNA replication, and expression of the genes they harbor. Unlike circular DNA molecules, linear chromosomes are less stable due to their higher susceptibility to exonuclease activity. In addition, replication of linear genomes has to overcome what is known as "the challenge of the ends", namely the shortening of the linear molecule after each replication cycle (Nosek et al. 1998). In the nucleus, the ends of the linear chromosomes are capped by repetitive sequences known as telomeres that both protect the molecule from degradation and allow its replication in a faithful manner (i.e. without the loss of essential sequences). Similarly, linear mtDNAs in algae and fungi have overcome linear instability with an array of telomere structures (Nosek et al. 1998). Recent studies of several yeast species from the genus Candida have suggested the concomitant involvement of the product of (1) a B-type DNA polymerase gene encoded by the mtDNA for
5’ end DNA extension and (2) terminal proteins (TPs) that bind the single stranded end of the linear molecules (Fricova et al 2010 and reference therein). Thus far, the small number of available linear mitochondrial genomes for medusozoans has hindered attempts to assign any of the available models for the maintenance of chromosome ends proposed for non-animal groups (Nosek et al. 1998; Nosek and Tomaska 2002), or to reconstruct the evolutionary history of linear mtDNA in medusozoans.
Here we present a description of 24 nearly complete mitochondrial genomes from a phylogenetically diverse set of species representing each of the four medusozoan classes, as
(Hydroidolina and Trachylina), Scyphozoa (Coronatae and Discomedusae), and Staurozoa
(Cleistocarpida and Eleutherocarpida). Our study represents the most in-depth exploration of
linear mtDNA in Cnidaria, and includes the first mitochondrial genomes from the classes
Cubozoa and Staurozoa.
Materials and methods
Animal sampling, DNA extractions, and PCR amplification
We collected several specimens of Cassiopea andromeda (Scyphozoa) and Millepora
platyphylla (Hydrozoa) on the site of Tiahura in Moorea (French Polynesia). We collected
specimens of Carybdea xaymacana (Cubozoa), Cassiopea frondosa and Chrysaora sp.
(Scyphozoa), and Cubaia aphrodite (Hydrozoa) at Bocas del Toro (Panama). The staurozoan
Craterolophus convolvulus was collected off the island of Helgoland (Germany) and
Haliclystus “sanjuanensis” was collected off the coast of Washington state (USA). The
Chiropsalmus quadrumanus was collected on the east coast of the USA, near the border between North and South Carolina. We purchased specimens of Cyanea capillata (Scyphozoa),
Clava multicornis, Ectopleura larynx, Laomedea flexuosa, and Pennaria tiarella (Hydrozoa)
from the Marine Biological Laboratory at Woods Hole (www.mbl.edu), and Nemopsis bachei
from the Gulf Specimen Marine Lab (www.gulfspecimen.org). Tissues from Catostylus
mosaicus, Linuche unguiculata, and Rhizostoma pulmo (Scyphozoa) were kindly provided by
Dr. Michael Dawson, individuals of Obelia longissima (Hydrozoa) by Dr. Annette F.
Govindarajan, a specimen of Pelagia noctiluca (Scyphozoa) collected at the Station Marine d'Endoume (Marseille, France) by Dr. Alexander Ereskovsky, and a DNA aliquot for
Lucernaria janetae from the East Pacific Rise by Dr. Janet Voight. The identities of hydrozoan species were confirmed morphologically by Dr. Peter Schuchert and assessed for all species by comparing partial cox1 and rnl sequences to the NCBI database. We extracted total DNA using phenol-chloroform extraction followed by proteinase K digestion.
We amplified and sequenced parts of cob, cox1, cox2, nad5, rnl, and rns using conserved primers for animals (Burger et al. 2007). We used the sequences obtained from these amplifications to design species-specific primers for long PCR amplifications. We amplified nearly complete mitochondrial genomes either in a single piece between cob-cox1 or cob-cox2, or in two parts between rnl-nad5 and rns-cox1. We extended our sequences using modified step-out protocol (Burger et al. 2007) and re-amplified the contigs using standard PCR. We then sheared long PCR products and processed them for multiplex sequencing (Meyer et al.
2008) using either 454 high-throughput or Illumina Sequencing platforms, carried out at the
Center for Genomics & Bioinformatics of Indiana University and the Office of Biotechnology
DNA Facility of Iowa State University, respectively. For C. convolvulus, H. “sanjuanensis”, and L. unguiculata, we constructed random clone libraries from the purified PCR product using the TOPO Shotgun Subcloning Kit from Invitrogen (see Burger et al. 2007 for details) and sequenced at the Office of Biotechnology DNA Facility of the Iowa State University.
We identified mitochondrial sequences from partial genomic reads of Alatina moseri
generated by the Iridian Genomes project. We used specific primers and step-out protocol on a
tissue sample from another individual of A. moseri to extend our sequences toward the end of
57 the mitochondrial chromosomes. Because of the high degree of segmentation of cubozoan
mtDNA, we PCR amplified nearly complete coding sequences using standard PCRs (for rns-
nad1 and cox2-cox3) and step-out protocols (for cob, cox1, nad4, nad2-nad5, ORF314-polB,
and rnl), and cloned the products into TOPO vectors. Plasmid preparation and sequencing were
processed at the Iowa State University Office of Biotechnology DNA facility. We used
enzymatic digestion of total DNA with the methylation-sensitive restriction enzyme HpaII to
confirm that PCR products amplified in this study are genuine mitochondrial sequences
(Kumar and Bendich 2011).
Genome assembly, gene annotation, and analysis
We assembled all sequences using the STADEN software suite (Staden 1996). We used
both the tRNAscan-SE and ARWEN programs (Lowe and Eddy 1997; Laslett and Canback
2008) to identify tRNA genes. We reconstructed the secondary structures of and
by comparison with their homologues in other cnidarian species. Other genes were
identified by similarity searches in local databases using the FASTA program (Pearson 1994).
We predicted the boundaries of rRNA genes by aligning them to rRNA sequences from
published cnidarians and checking the alignments manually. We also aligned individual protein
genes with ClustalW 1.82 (Thompson et al. 1994) using default parameters. We manually
verified all alignments based on either amino-acid alignments (for protein coding genes), or
inferred rRNA or tRNA secondary structures (for RNA-coding genes) to Aurelia aurita and
Hydra oligactis (Shao et al. 2006; Kayal and Lavrov 2008). For the analysis of sequence
similarities, we calculated sequence identities using local scripts based on BioPerl modules
(Stajich et al. 2002). We estimated codon usage with the CUSP program in the EMBOSS
package (Rice et al. 2000).
The linear mitochondrial genomes of Medusozoa
We amplified and sequenced mitochondrial (mt) genomes of eight species representing the two clades of Scyphozoa (Coronatae: Linuche unguiculata; Discomedusae: Cassiopea
andromeda, C. frondosa, Catostylus mosaicus, Chrysaora sp, Cyanea capillata, Pelagia
noctiluca and Rhizostoma pulmo (partial)), eight species representing the two clades of
Hydrozoa (Hydroidolina: Clava multicornis, Ectopleura larynx, Laomedea flexuosa, Millepora platyphylla, Nemopsis bachei, Obelia longissima, and Pennaria tiarella; Trachylina: Cubaia
aphrodite), and three species representing both orders of Staurozoa (Cleistocarpida:
Cratolophus convolvulus; Eleutherocarpida: Haliclystus “sanjuanensis” and Lucernaria janetae). In addition, we obtained substantial mitochondrial sequences from five species belonging to the two major cubozoan clades (Carybdeida: Alatina moseri, Carukia barnesi, and Carybdea xaymacana; Chirodropida: Chironex fleckeri and Chiropsalmus quadrumanus).
The mtDNA of all scyphozoan, staurozoan, and hydrozoan species sampled for this
study mapped into single linear molecules (Figure 2A). Cubozoan mtDNA was composed of
eight mitochondrial chromosomes, each encoding one to five mitochondrial genes with the
same transcriptional orientation. This is in agreement with earlier DNA hybridization
experiments, which suggested that cubozoan mtDNA was composed of several up to 4 kb
linear chromosomes (Ender and Schierwater 2003). All cubozoan mitochondrial genes were
similar in size to their homologues in other medusozoans, and contain neither internal stop
codons (TAA or TAG) nor frameshifts. In addition, incubation of Alatina moseri total DNA
with methylation-sensitive enzyme HpaII (method described in Kumar and Bendich 2011)
inhibited PCR amplifications of the contigs harboring the restriction sites (Supplementary Fig.
S1). Together, this evidence strongly suggested that our cubozoan contigs were genuine mitochondrial sequences, as opposed to nuclear pseudogenes (NUMTs).
All medusozoan mtDNA contained a set of genes for 13 protein subunits conserved in animals and involved in the electron transport chain of oxidative phosphorylation (atp6-8, cob, cox1-3, nad1-6), two ribosomal RNAs (rRNA) (rnl and rns), and two transfer RNAs (tRNA)
(trnM(cau) and trnW(uca)). trnW(uca) was missing from the partial mitochondrial genome of the coronate Linuche unguiculata and no tRNA genes were found in the partial sequences from cubozoans (see below). We also found some level of class-specific conservation at the ends of the linear mitochondrial chromosomes: duplicated cox1 in most hydrozoans (Hydroidolina), polB and an extra ORF in cubozoans, scyphozoans, staurozoans, and the trachyline hydrozoan
Cubaia aphrodite (see below).
Medusozoan mitochondrial genomes displayed a compact organization with few, if any intergenic regions, and several pairs of overlapping genes. We found overlapping nad3-nad6 genes in the mtDNA of all hydrozoans, scyphozoans, and staurozoan species (11±3, 35±17, and 9±2 nucleotides respectively). In addition, nad4L-nad3 overlapped by 10 nucleotides in all scyphozoan species, nad1-nad4 overlapped by 10±3 nucleotides in all hydrozoans, atp6-atp8 overlapped by 7 nucleotides, and nad2-nad5 by 13 nucleotides in all staurozoans. Similarly, nad2-nad5 overlapped by 26 and 44 nucleotides in the staurozoans Haliclystus “sanjuanensis” and Lucernaria janetae, respectively. Finally, in the aplanulate Ectopleura larynx we found cob-cox1 overlapping by 44 nucleotides. The same two genes overlapped by 8 nucleotides in
Hydra magnipaillata and H. vulgaris (Voigt et al. 2008), which are also part of the Aplanulata clade (Collins et al. 2005).
We compared the variation in the mtDNA nucleotide composition among different cnidarian classes (Table 1). The linear mtDNA in medusozoans displayed GC-skew values close to zero: cubozoans, hydrozoans, and scyphozoans all shared a small positive GC-skew
(on average 0.04 SD=0.03) between the two strands of the coding sequences, while
staurozoans had a slightly negative GC-skew (-0.05 SD=0.02). In comparison, the circular
mtDNA in anthozoans displayed a positive GC-skew (0.20 SD=0.07 in hexacorals and 0.09
SD=0.01 in octocorals). AT-skews in all studied medusozoan species were negative for the
coding strand (between -0.11 and -0.21) and similar to those in anthozoans. The A+T content
of coding regions varied substantially between different medusozoan classes. Hydrozoan
mtDNA had the highest A+T content (74% SD=4), and cubozoan mtDNA had the lowest (61%
SD=3). Scyphozoans and staurozoans had intermediate %A+T values (67% SD=4 and 67%
SD=2 respectively) similar to those in anthozoans. We found a strong correlation (R2=0.84)
between nucleotide composition and the proportion of GC-rich amino acids (the “GARP”
amino acids: glycine, alanine, arginine, and proline) (Figure 1). This result was consistent with
previous studies, which found that nucleotide composition can serve as a proxy for amino acid
compositions of protein sequences and can be an important source of systematic errors in
phylogenetic inference (Delsuc et al 2005 and references therein).
Gene arrangements in medusozoan mtDNA
Analysis of the newly determined mitochondrial sequences revealed a well-conserved
mitochondrial gene order within Medusozoa (Figure 2A). Eleven representatives of both
discomedusan Scyphozoa and Staurozoa sampled for this study had a completely conserved
gene order, identical to that found in Aurelia aurita (Shao et al 2006). In these groups, the
cluster formed by cox1-rnl-ORF314-polB had a different transcriptional orientation to the rest
61 of the genes (Figure 2A). The two clusters were separated by a relatively large non-coding
region of about 80 nucleotides, potentially involved in the control of mitochondrial
transcription (see below). In the coronate Linuche unguiculata, trnW was not found either in its
conserved position between cox2 and atp8 or in the rest of the genome. We were also unable to
identify sequences for polB and ORF314 in this species. However, given that the sequence of
L. unguiculata is incomplete, it is possible that these genes are present in the mitochondrial
genome, downstream of cox1. The gene arrangement was also conserved in all cubozoan
species, despite the presence of multiple chromosomes (Figure 2A). Single gene chromosomes
harbored cob, cox1, nad4, and rnl genes. Other chromosomes contained gene pairs nad2-nad5
and ORF314-polB, and gene clusters cox2-atp8-atp6-cox3 and rns-nad6-nad3-nad4L-nad1.
When placed together, the gene order across the eight chromosomes in cubozoan mtDNA is
nearly identical to that in most scyphozoans and staurozoans except for the absence of tRNA
The mitochondrial genome organization within Hydrozoa was more variable, with at
least four different gene orders. The genome organization in the trachyline Cubaia aphrodite
was identical to that in discomedusan Scyphozoa and Staurozoa (Figure 2A). All species from
the clades Capitata (Millepora platyphylla and Pennaria tiarella), Filifera III (Clava
multicornis) and VI (Nemopsis bachei), and Leptothecata (Laomedea flexuosa and Obelia
longissima) had a genome organization resembling that of C. aphrodite, with the exception of a
duplicated cox1 downstream of cob and the loss of ORF314-polB (Figure 2A; see Cartwright et
al. 2008 for description of hydrozoan subclades). The genome organization in the aplanulate
Ectopleura larynx was similar to H. oligactis, where rnl was inverted and both tRNA genes
(trnM and trnW) moved compared to other hydrozoans. Interestingly, all non-trachyline
62 hydrozoan species sequenced in this study had two identical copies of cox1, one at each end of the mitochondrial chromosome. In comparison, the three previously published Hydridae
mitochondrial genomes (Hydra magnipapillata, H. oligactis, and H. vulgaris) were reported to
have only one complete copy of cox1 downstream of cob, with the other copy(ies) being
partially degenerated and lacking the 5’ end of the gene (Kayal and Lavrov 2008; Voigt et al.
2008). Furthermore, in H. magnipapillata and H. vulgaris, the mtDNA was split into two
nearly equal sized chromosomes, a feature also reported for several other species belonging to
the family Hydridae (Warrior and Gall 1985; Bridge et al. 1992; Ender and Schierwater 2003).
Overall, the mitochondrial gene order in medusozoans is well conserved, with only four
unique gene orders found so far (ignoring genome fragmentation and cox1 degeneration in
Hydridae). We inferred the evolution of mitochondrial genome organization in medusozoans
using the currently accepted cnidarian phylogeny (Figure 2B; Collins et al. 2006; Cartwright et
al. 2008). Gene order similar to the scyphozoan A. aurita is found in all four of the recognized
medusozoan classes. Given the reportedly low rate of genome rearrangement in animals, it is
most parsimonious to reconstruct the mitochondrial ancestral medusozoan gene order (AMGO)
as identical to the gene order found in A. aurita (Figure 2B).
Protein gene similarity in medusozoan mtDNA
Sizes of orthologous protein genes in medusozoan mtDNA showed little variation (0-
4%), as compared to anthozoans (0-11%). By contrast, the overall amino acid pairwise identity
of protein genes for Medusozoa was highly variable, ranging between 24-96% (average 49%
SD=12) (Table 1). Such variability indicates that mtDNA proteins can be good markers for
reconstructing the evolutionary history of medusozoans at lower taxonomic levels.
Interestingly, the amino acid pairwise identity of protein genes between coronates and other
63 scyphozoans (50% SD=2) were similar to the values for all other cnidarian classes (50% and
51% with hexa- and octocorals, 38% with cubozoans, 47% with hydrozoans, and 48% with staurozoans), with a mean of 49% SD=4 to all cnidarians.
The minimally derived genetic code (TGA=tryptophan) was inferred for mitochondrial protein synthesis in all the medusozoan genomes analyzed in this study. Codon usage bias estimated as the effective number of codons Nc (Wright 1990) was highly correlated with the
GC content of the mtDNAs (R2=0.97), where the AT-rich hydrozoan mtDNA displayed the
highest codon usage bias (35 SD=4), and the lowest values were found in cubozoans and
staurozoans (47 SD=4 and 47 SD=1 respectively). Within a given codon family, codons ending
with A or T were favored over those ending with C or G. CGN codons were nearly absent from
the mtDNA of cubozoans, hydrozoans, and scyphozoans, where they represented <15% of
arginine residues with no CGG codon used in hydrozoans. In contrast, CGN codons encoded
about 50% of arginine residues in staurozoans.
ATG was inferred as the initiation codon for most mitochondrial coding sequences, yet,
we inferred GTG as a start codon for two genes in Hydrozoa (cob in L. flexuosa and Obelia longissima, and cox1 in E. larynx) and five genes in Scyphozoa (atp8 in L. unguiculata, nad1 in P. noctiluca, nad3 in all but two species, nad4L in C. mosaicus, and nad5 in Chrysaora sp)
(Table 1). GTG was also the inferred initiation codon in at least 5 out of the 13 protein genes in
Cubozoa, and mostly present in the order Chirodropida. Both stop codons TAA and TAG were
found in all medusozoan classes (80% and 20% of the genes respectively, where the former
value represents both TAA and incomplete TA stop codons). Incomplete stop codons TA were
inferred for several protein genes in medusozoan mtDNA, predominantly in atp6, atp8, nad3, and nad4L (Table 1). In the aplanulatan Ectopleura larynx the nearest stop codon for nad5 is
64 located 30 nucleotides past the 5’ end of rns. Interestingly, no stop codon was inferred for nad5 in the scyphozoan P. noctiluca, as the nearest stop codon is found 77 nucleotides within rns.
RNA genes in medusozoan mtDNA
All complete medusozoan mt-genomes analyzed in this study contained four RNA
genes typical for Cnidaria: two genes coding for large and small subunit rRNA (rnl and rns), and two for methionine and tryptophan tRNAs (trnM(cau) and trnW(uca)). The genes for the small and large ribosomal RNA subunits were on average 17% smaller in size and displayed less size variation in medusozoans compared to their homologues from anthozoans (Table 1).
The nucleotide composition of RNA genes was slightly more AT-rich than that of the protein coding regions.
Both medusozoan mt-tRNA genes were more variable in primary sequence and
inferred secondary structure than their homologues in anthozoans and demosponges (Wang and
Lavrov 2008). The initiator was on average 70 nucleotides long, ranging from 68 in
staurozoans to 71 in hydrozoans and scyphozoans. The shorter in Staurozoa lacked
two nucleotides at positions 16 and 17 of the D-loop (Figure 3). The anticodon arm in all
hydrozoan and staurozoan had an extra base pair typical to type 7 tRNA (Watanabe
et al. 1994; Steinberg and Cedergren 1995; Steinberg et al. 1997), resulting in a shorter variable
loop (three nucleotides) in the hydrozoans, but a standard four-nucleotides long variable loop
in staurozoans. The of all medusozoans but Hydra species had two nucleotides (8
and 9) in the connector region between the acceptor and the D-arm, which makes the loss of
nucleotide 9 in mitochondrial a synapomorphy for the Hydridae family. Finally, all
hydrozoans but Cubaia aphrodite and all staurozoans lacked nucleotide 16 in the D-loop of
. The partial cubozoans mitochondrial sequences determined for this study harbored no tRNA genes.
Intergenic and putative control regions in medusozoan mtDNA
We found a single, relatively large (60 to 94 nucleotides) and AT-rich (>70%)
intergenic region (IGR) between cox1 and cox2 in all scyphozoan and staurozoan species, and in the trachyline hydrozoan Cubaia aphrodite mtDNA (Figure 2A). This IGR delimits two sets of genes with opposite transcriptional polarity. A large portion of this region can be folded into a conserved stem loop motif with up to 13-base pairs in the stem, most of them (23 bases) completely conserved in all scyphozoans sampled in this study and some (12 bases) also conserved in staurozoans and in the hydrozoan C. aphrodite (Figure 4A). In most other hydrozoan groups (Filifera, Capitata, and Leptothecata), the largest IGR was located between cox2 and rnl, two genes with opposite transcriptional polarities (Figure 2A). This IGR also harbored a 20 nucleotide-long sequence that, while displaying low sequence conservation between hydrozoan species, could be folded into a strong stem-loop element (Figure 4A).
In aplanulatan hydrozoans, we found a relatively large (42-52 nucleotides) non-coding
region between cox3 and nad2 that displayed up to 70% similarity with trnW sequences.
Finally we did not find major intergenic regions in cubozoan mtDNA.
The “ends” of medusozoan mtDNA: extra ORFs versus duplicated genes
All species of Staurozoa, discomedusan Scyphozoa (the sequence of the corresponding
region in the coronate scyphozoan Linuche unguiculata remains undetermined), and the
trachyline hydrozoan Cubaia aphrodite contained two ORFs at one end of the molecule. In
Cubozoa, analogous ORFs were found on a separate mitochondrial chromosome. The
sequences of the larger ORF were similar with polB described in Aurelia aurita (Shao et al.
2006) and formed a monophyletic group in a phylogenetic analysis that included polB from other organisms (Supplementary Fig. S2). The sequence of the smaller ORF (ORF314) was poorly conserved within Medusozoa, with only 2 identical amino acid residues between the three classes. This ORF encoded about 100 amino acids and was always located upstream of polB. While preliminary analysis using 3D modeling of the tertiary structure of the putative product of ORF314 suggests similarities with elongation factors and a potential DNA-binding activity (data not shown), further studies are needed to investigate the role of this gene.
The mtDNA of all hydrozoans but the trachyline Cubaia aphrodite lacked the two extra
ORFs (polB and ORF314) (Figure 2A). Furthermore, we were not able to identify copies of
these genes in the nuclear genome of H. magnipapillata (Chapman JA et al. 2010). Instead, we
found a complete copy of cox1 at each end of the mitochondrial chromosome (Figure 2A). The two copies of cox1 had opposite transcriptional polarities and formed part of inverted terminal
repeats (ITRs, Figure 2A). In addition, we found a relatively conserved 116 base pair sequence
downstream of cox1 in Aurelia aurita and Cassiopea spp mtDNAs that could be folded into a
complex stemloop structure (Figure 4B). The presence of inverted terminal repeats (ITRs) is a
hallmark of linear mitochondrial genomes and has been reported in several distantly related
organisms (Valach et al 2011 and references therein).
Linear mitochondrial DNA has been well documented in several non-metazoan groups
such as plants and fungi. In yeast, where linear mtDNA has been extensively studied, the
distribution of linear and circular mitochondrial genomes has no apparent phylogenetic signal,
with closely related species having either linear or circular mtDNA (Nosek et al 1998; Nosek
67 and Tomaska 2008). In animals, strictly linear mtDNA has been described only in Medusozoa
(Warrior and Gall 1985; Bridge et al. 1992), for which only six genomes have been published
to date (Shao et al. 2006; Kayal and Lavrov 2008; Voigt et al. 2008; Park et al. in press). The
only other report of linear mtDNA in animals comes from the crustacean isopod Armadillidium
vulgare, where the mtDNA consists of a circular ~28-kb dimer and a linear ~14-kb monomer
(Raimond et al 1999; Marcadé et al 2007). Our study represents the first thorough survey of the
evolution of linear mtDNA in Medusozoa. In order to better understand the evolutionary
history of mtDNA linearity in this group, we surveyed species belonging to all medusozoan
classes, as well as their primary subclades.
We analyzed medusozoan mitochondrial genomes in a phylogenetic framework and
propose the following hypotheses for the evolution of linear mtDNA in this group: (1) a single
origin for the linear mtDNA in the lineage leading to Medusozoa, most likely by the integration
of a linear plasmid; (2) loss of polB and ORF314, and duplication of cox1 at each end of the
linear mt chromosome after the divergence of Trachylina from the rest of hydrozoans
(Hydroidolina); (3) gene rearrangement at the stem of Aplanulata; (4) partial loss of cox1
sequence in Hydridae and split of the mitochondrial chromosome in several species within this
clade; (5) high level of mtDNA fragmentation in the branch leading to Cubozoa; (6)
displacement of trnW in the coronate Linuche unguiculata (Figure 2B). It is worth noting that
fragmentation of the mitochondrial molecules appears to be an irreversible process as no
rearranged mitochondrial genomes have been observed in taxa where mtDNA is fragmented, as
one would expect if different pieces would fuse randomly.
Fukuhara and collaborators suggested a positive correlation between mitochondrial
gene order conservation and linear as opposed to circular structure of yeast mtDNA (Fukuhura
68 et al. 1993). Our study revealed a similar trend. If we ignore genome fragmentation and cox1 duplication, only four unique gene orders can be distinguished in medusozoan mtDNA: the
AMGO is found in all four medusozoan classes, with modified gene orders found in the
coronate Linuche unguiculata, Hydroidolina, and Aplanulata (Fig. 2A). By contrast, at least
nine unique gene orders (more if presence/absence of ORFs is considered) have been reported
in the circular mtDNA of anthozoans (Uda et al. 2011). Furthermore, most of the differences in
the medusozoan mitochondrial gene orders can be explained by a few simple gene
rearrangement events, while multiple and/or complex rearrangements events are needed to
explain differences in anthozoan gene orders. These observations suggest that linearization has
favored stabilization of the mitochondrial genome organization in Medusozoa. On the other
hand, this result may to some extent reflect the difference in the species richness in Anthozoa
vs. Medusozoa (about twice as many species in the former; Daly et al. 2007) and/or the number
of mt-genomes that have been studied in each group (43 vs. 30).
One other difference between anthozoan and medusozoan mtDNA is the amount and
distribution of non-coding, intergenic DNA. Anthozoan mtDNA usually has several relatively large intergenic regions that sometimes contain repetitive sequences and/or ORFs (Park et al.
2011). By contrast, medusozoan mitochondrial genomes are compactly arranged with few if any intergenic nucleotides and, sometimes, overlapping genes. Most mitochondrial genomes investigated in this study contained only a single moderately large (<100 bp) IGR separating two regions of mtDNA with opposite transcriptional polarities. The conservation of sequence and inferred secondary structure of this region suggests that it may be involved in the control of mtDNA transcription and/or replication in Scyphozoa, Staurozoa, and trachyline Hydrozoa.
Similarly, the largest IGR in other hydrozoans (situated between trnM and the cox1 in
Aplanulata, and between cox2 and rnl in other hydroidolinans) may be involved in the control of transcription as suggested earlier (Voigt et al. 2008). Compactly arranged mtDNA with a single large intergenic region involved in the initiation of replication (Desjardin and Morais
1990) and transcription (L'Abbé et al. 1991) is also a feature of bilaterian animals.
Interestingly, the maturation of a polycistronic pre-mRNA in bilaterian mitochondria involves excision of tRNA sequences located between most coding sequences in the genome, resulting in monocistronic mRNAs (Ojala et al. 1980). Based on the near absence of intergenic regions, one can assume that the transcription of medusozoan mtDNA is also polycistronic. However, the loss of all but two tRNA genes necessitates a different mechanism for mRNA maturation in medusozoan mitochondria. Finally, the absence of large IGRs and the conservation of unidirectional transcriptional orientation of all genes in cubozoan mtDNA suggest that the initiation of replication and transcription occurs at the end(s) of each of the mitochondrial chromosomes.
We found two distinctive genetic elements at the end of medusozoan mitochondrial chromosomes: duplicated cox1 in the hydroidolinan hydrozoans, and two extra ORFs (polB and ORF314) in all the other medusozoan classes (Cubozoa, discomedusan Scyphozoa,
Staurozoa, and trachyline Hydrozoa). In an earlier study, Shao and collaborators showed that polB from the scyphozoan Aurelia aurita mtDNA shares several conserved motifs characteristic of the polymerase domain in family B DNA polymerases (Shao et al. 2006). The authors also attributed the origin of the two extra protein genes to an ancient invasion event by a linear plasmid that resulted in the linearization of the mtDNA in Medusozoa. Similar polB- like sequences are found in the linear mtDNA of several non-animal groups such as fungi and algae, where they are hypothetically associated with the integration of linear plasmids in the
70 mitochondrial genome (Mouhamadou et al. 2004). The grouping of medusozoan polB sequences into a monophyletic clade suggests a single invasion event early in the evolutionary history of the group that coincided with the origin of its linear mtDNA at the stem of the medusozoan tree. Yet the low level of sequence conservation in polB-like sequences between medusozoan and non-animal species hinders our attempts to predict the source of the invading element. Nevertheless, the conservation of both sequence length and position of the two ORFs suggests some level of selection pressure for their maintenance in the mtDNA of most medusozoans.
Short inverted terminal repeats (ITRs) have been previously reported in the mtDNA of
A. aurita (Shao et al. 2006) and the hydrozoans Hydra magnipapillata, H. oligactis, and H.
vulgaris (Kayal and Lavrov 2008; Voigt et al. 2008). ITRs are one out of the few telomeric
structures found in linear mtDNAs (Nosek et al. 1998; Nosek and Tomaska 2003), and
involved in the maintenance and replication of linear chromosomes. In yeast, short ITRs are
associated with type III linear mtDNA, where a terminal protein (TP) covalently binds at the
single-stranded 5’ end of the linear molecules (Nosek and Tomaska 2008). The linear
molecules generally encode TPs, either as part of a DNA polymerase gene, or by a yet
unidentified ORF (Fricova et al. 2010 and references therein). In medusozoan mtDNA, the
putative product of ORF314, if shown to have DNA-binding properties, may act as a TP.
Consequently, assuming polB and ORF314 are functional genes, we predict that the mtDNA in
cubozoans, scyphozoans, staurozoans and trachyline hydrozoans uses mechanisms of
maintenance and replication similar to type III linear mtDNA as found in yeasts, linear
plasmids, and adenoviral DNA. This assertion is further supported by the similarity of polB
sequences between medusozoan and linear plasmids. As described above, both polB and
ORF314 are absent from non-trachyline hydrozoan mtDNAs, and no homologues could be
found in the completely sequenced nuclear genome of Hydra magnipapillata (Hydridae,
Hydrozoa). Thus, unlike what has been suggested by an earlier study (Voigt et al. 2008),
hydrozoans appear to employ an alternative mechanism for the maintenance and replication of
their mtDNA. However, recruitment of a nuclear encoded TP for the maintenance of linear
mtDNA in these hydrozoans cannot be ruled out. In addition, the duplication of cox1 at each end of the mitochondrial chromosome(s) is limited to the lineage leading to Hydroidolina, suggesting that a novel mechanism evolved after the divergence of Trachylina and the rest of hydrozoans. Future studies that explore the end conformations of medusozoan mtDNA will shed light on the maintenance and processing of these linear molecules.
Despite our efforts, we were not able to verify the expression of polB and ORF314 at
the RNA level, most likely as a consequence of poor RNA conservation in the preserved tissue
samples. We were also unable to investigate the processes involved in transcription and
replication of the mtDNA in medusozoans. The analysis of a dataset containing ESTs from the
scyphozoan Cyanea capillata was also unsuccessful, highlighting the difficulties associated
with working with RNA in this group. Interestingly, in EST assemblies obtained from the
staurozoan Haliclystus “sanjuanensis” we found a single mitochondrial hit that spans from the
3’-end of cox1 to the 5’-end of rnl, suggesting that these genes are transcribed in a
polycistronic manner. Yet, additional studies are needed to shed light on the role of the two
extra ORFs (polB and ORF314) and their putative products in the maintenance of linear
mitochondrial chromosomes in medusozoans, and on the mechanisms of mitochondrial gene
expression in the group.
Medusozoan mtDNA is typically a small compact genome composed of one or several linear chromosomes with a highly conserved gene order, and inverted terminal repeats (ITRs) at the end of the molecule(s). The linearity of medusozoan mtDNA is most likely the product of a unique invasion event by a linear plasmid-type element harboring a polB-like and a putative terminal protein (TP) gene, with secondary loss of the two extra ORFs within hydrozoans. Medusozoan mitochondrial genomes lack large IGRs, contain several overlapping genes, and harbor shorter protein and ribosomal genes than representatives from other non- bilaterian clades (Signorovitch et al. 2007; Wang and Lavrov 2008). Besides their strictly linear mitochondrial genomes, a unique feature in animals, the medusozoan mtDNA display characteristics that can be described as intermediary between bilaterian and non-bilaterian animals. On the one hand, medusozoans use a minimally derived genetic code for mitochondrial protein synthesis and show relatively low evolution rate of their mitochondrial genes, as found in most non-bilaterian animals. On the other hand, the compactness of their genomes and a relatively stable gene order both mirror the evolution of bilaterian mtDNA. Put into a broader perspective, our results show that the evolution of animal mitochondrial genomes does not follow a single linear pathway as proposed in an earlier study (Lavrov 2007).
This is well illustrated in the cnidarian clade where both bilaterian-like and non-bilaterian-like mitochondrial features are found. Future studies that focus on undersampled animal taxa may further expose the rather unpredictable evolutionary pattern of genome evolution in animal mitochondria, revealing a more complex picture of mtDNA evolution in Metazoa. In addition, we predict that a larger survey of linear mtDNA in medusozoans may uncover original/new
solutions to the "the challenge of the ends" (Nosek et al. 1998).
This work was supported by a grant from the National Science Foundation to DVL
[DEB-0829783] and by funding from Iowa State University.
We would like to thank Dr. Michael Dawson and Dr. Alexander Ereskovsky for their contributions to the sampling effort of Scyphozoa, Dr. Janet Voight for providing us with DNA from the staurozoan Lucernaria janetae, and Dr. Annette F. Govindarajan for providing us with the hydrozoan Obelia longissima. We also thank Dr. Casey Dunn and the Cnidarian Tree of Life project (DEB-0531779 to Paulyn Cartwright and AGC) for granting access to EST data from the scyphozoan Cyanea capillata and Haliclystus “sanjuanensis”. We are very grateful to
Dr. Peter Schuchert for the identification of our hydrozoan samples. BB wishes to acknowledge funding through US NSF Doctoral Dissertation Grant DEB 0910237. EK wishes to acknowledge funding through the Smithsonian Predoctoral Fellow grant.
Table 1: Gene properties in the mtDNA of Medusozoa. The average size in nucleotides (and standard deviation), AT composition (and standard deviation), and putative start and stop codons are reported for each of the Medusozoa subclasses.
Cubozoa Hydrozoa Scyphozoa Staurozoa
size1 %AT2 start3 end4 size %AT start end size %AT start end size %AT start end 708 704 75 704 708 63 atp6 63 ±2 AG * A A* 69 ±4 A A* A A ±7 ±1 ±4 ±3 ±0 ±1 210 206 83 208 204 62 atp8 64 ±4 AG AG* A A* 73 ±4 AG AG* A A ±2 ±3 ±5 ±7 ±0 ±4 1148 73 1146 60 cob 1149 62 ±2 A G AG A 66 ±2 A AG 1068 A ? ±12 ±3 ±8 ±2 67 1580 1578 61 cox1 1569 58 ±3 A A 1569 AG AG 64 ±3 A AG A A ±3 ±7 ±0 ±1 737 744 73 746 747 62 cox2 61 ±2 A AG* A AG 67 ±4 A AG* A AG ±2 ±10 ±4 ±8 ±0 ±1 786 786 72 786 786 61 cox3 59 ±3 A AG A AG 64 ±3 A AG A AG ±0 ±0 ±4 ±0 ±0 ±1 987 989 73 972 987 59 nad1 62 ±3 AG AG A AG 66 ±4 AG A* A A ±8 ±4 ±4 ±5 ±0 ±0 1328 79 1323 1346 59 nad2 1341 63 ±7 A G* A AG* 70 ±5 A AG A A ±32 ±5 ±13 ±2 ±4 351 355 77 357 354 65 nad3 62 ±4 AG * A * 69 ±4 AG A* A A* ±0 ±4 ±4 ±6 ±0 ±4 1458 76 1441 1461 61 nad4 1446 59 A G A AG* 68 ±5 A AG* A AG* ±2 ±4 ±2 ±0 ±3 290 299 79 303 299 64 nad4L 67 ±3 A G* A * 72 ±5 AG A* A * ±2 ±2 ±4 ±1 ±2 ±1 1832 76 1830 1860 60 nad5 1824 62 ±1 AG G A AG* 68 ±5 AG A* A AG ±2 ±4 ±19 ±13 ±2 542 556 79 564 553 62 nad6 64 ±3 A G* A AG* 70 ±5 A AG* A A ±4 ±8 ±5 ±12 ±2 ±2 313 ORF314 315 64 A A 291 78 A G 73 ±8 A A 288 62 A A ±7 polB 873 58 G A ? ? ? ? 969 70 ±8 A A 1119 58 ATG ? 1746 76 1818 57 rnl 769 57 NA NA NA NA 69 ±5 NA NA 1830 NA NA ±9 ±4 ±34 ±1 910 74 950 914 57 rns 672 62 NA NA NA NA 69 ±3 NA NA NA NA ±21 ±2 ±10 ±1 ±1 71 69 69 53 trnM - - NA NA NA NA 71 ±0 64 ±5 NA NA NA NA ±1 ±2 ±0 ±2 70 65 71 52 trnW - - NA NA NA NA 70 ±0 64 ±5 NA NA NA NA ±1 ±3 ±0 ±2
1Average size in nucleotides, with standard deviation
2AT composition, with standard deviation
3Start A and G stand for ATG and GTG start codons respectively
4Stop A and G stand for TAA and TAG stop codons, respectively; a star corresponds to an incomplete stop codon (see text).
Compositional properties of cnidarian mitochondrial coding sequences 26
R2 = 0.84 22
Hexacorallia Octocorallia 18 Scyphozoa
% G+A+R+P Staurozoa 16 Hydrozoa Cubozoa
10 15 20 25 30 35 40 45 50 % GC
Figure 1: Compositional properties of cnidarian mitochondrial coding sequences. The total G-C content of mitochondrial protein genes is plotted against the percentage of amino
acids encoded by G- and C-rich codons (glycine, alanine, arginine, and proline [G + A + R +
P]). The trend line and correlation coefficient are displayed. Hydrozoa are characterized by
very A-T rich genomes and relatively fewer [G + A + R + P] codons. Staurozoan have
relatively higher GC content similar to anthozoans.
A polB orf1 rnl cox1 cox2 atp8 atp6 cox3 nad2 nad5 rns nad6 nad3 nad4L nad1 nad4 cob Cubozoa
cox1 cox2 atp8 atp6 cox3 M nad2 nad5 rns nad6 nad3 nad4L nad1 nad4 cob Linuche (Coronatae)
polB orf1 rnl cox1 cox2 W atp8 atp6 cox3 M nad2 nad5 rns nad6 nad3 nad4L nad1 nad4 cob other Scyphozoa + Staurozoa
polB orf1 rnl cox1 cox2 W atp8 atp6 cox3 M nad2 nad5 rns nad6 nad3 nad4L nad1 nad4 cob AMGO
polB orf1 rnl cox1 cox2 W atp8 atp6 cox3 M nad2 nad5 rns nad6 nad3 nad4L nad1 nad4 cob Cubaia (Trachylina)
Filifera + Capitata cox1 rnl cox2 W atp8 atp6 cox3 M nad2 nad5 rns nad6 nad3 nad4L nad1 nad4 cob cox1 + Leptothecata H y d r o z a
cox1 M rnl W cox2 atp8 atp6 cox3 nad2 nad5 rns nad6 nad3 nad4L nad1 nad4 cob cox1 A p l a n u t H y d r i a e cox1-p M rnl W cox2 atp8 atp6 cox3 nad2 nad5 rns nad6 nad3 nad4L nad1 nad4 cob cox1
cox1-p M rnl W cox2 atp8 atp6 cox3 nad2 nad5 cox1-p cox1-p M rns nad6 nad3 nad4L nad1 nad4 cob cox1
Hydrozoa B Acraspeda Aplanulata Scyphozoa
rachyolintaher Hydroidolina Anthozoa Staurozoa Coronatae Cubozoa T Hydridae
Figure 2: Evolution of mitochondrial genomes in Medusozoa (Cnidaria) based on phylogenetic relationships according to Collins et al. (2006), Collins et al. (2008), and
Cartwright et al. (2008). A) Comparison of mitochondrial gene orders between various medusozoan clades and the putative mitochondrial Ancestral Medusozoan Gene Order
(AMGO). Breaks in contigs mark the ends of linear mitochondrial chromosomes. Genes are transcribed from left to right unless underlined. cox1-p corresponds to the partial copy of cox1
at the end of the mtDNA molecule(s) in the Hydridae family. polB is an inferred member of the
B DNA polymerase gene family and ORF314 is an unidentified ORF located upstream of it.
Lightning bolts represent breaks in the mitochondrial chromosomes. Black arrows depict
directions of inferred genome rearrangements. B) Evolution of genome organization of the
linear mtDNA in Medusozoa. The phylogeny is based on Collins et al. (2006). Major genomic
rearrangements are labeled. (1) Linearization of the mtDNA by insertion of a linear plasmid of
unknown source; (2) loss of polB and ORF314, and duplication of cox1 at each end of the
linear chromosome within Hydrozoa after the divergence of Trachylina; (3) displacement of
tRNAs and inversion of rnl in Aplanulata; (4) genome segmentalization of the mtDNA in
several Hydridae species with consequent trnM and cox1 duplication; (5) high level
segmentalization of the mtDNA in cubozoan; (6) displacement of trnW in the coronate Linuche
Methionine w Tryptophan G h-W R-Y (M) R-Y (M) R-Y H-D R-Y R-Y G-Y R-Y R-Y R-Y R-Y R-Y B-V T YHBYCYNA T YDCVVTDA N A N ||||| R A A M ||||| R N TCAR A TTRR C RDNRGTT N RHBGGTT G |||| D |||| T G R G A AGTT A W A RAYY H R V N R D D Y-R r.y Y-R R-Y A-T W-W G-C D-Y R-Y R-Y Y H Y-R T R C A CAT T A TCA
Figure 3: Consensus secondary structure of tRNAs encoded by the mtDNA of Hydrozoa,
Scyphozoa, and Staurozoa. Bold letters with black arrows correspond to position that are missing in some taxa: in , two nucleotides at positions 16 and 17 of the D-loop in
Staurozoa, and positions 1 and 71 in the acceptor stem of the hydrozoan Nemopsis bachei; in
, position 9 of the connector region between the acceptor and the D-arm in the
Hydridae family, position 16 of the D-loop in staurozoan and all hydrozoan but the trachyline
Cubaia aphrodite, and position 42 of the variable loop in all hydrozoan. Bold letters without arrow correspond to an extra base pair in the anticodon arm of in Hydrozoa and
Staurozoa, typical to type 7 tRNAs.
A A T-A .. G-C . .. W W . . A-T . . T D . . W R A T-A AA N-M W W AT C-B W-W A A-T y-a T-D A-T W-T T-A A-T T-H T-A A-T W-W C-G A-T t-a A-T A.A T-A W-T T-A T-A R-T G-C R-T t y T-A T-A W-A A-T c-G S-B A-T n(N)T-A (N)n n(N) (N)n n(N) (N)n
Scyphozoa + Staurozoa + Trachylina Capitata + Filifera Leptothecata
B Scyphozoa ...... G-Y T-A T-A T-A T-A T-A . . T . G-C C...... C . . T R Y W-R T CA T-A R T R T A-T T-A A-T T-A A-T T-A T-A A-T A-T W-T A-T T-A A-T
Figure 4: Putative control region and terminal stem-loop found in medusozoan mtDNA.
A) Consensus sequence of the putative control regions (CR) found in the mtDNA of Hydrozoa,
Scyphozoa, and Staurozoa. This CR corresponds to the largest intergenic region (IGR) that
delimits two sets of genes with opposite transcriptional polarity. B) Putative stem-loop
structure found at the end of scyphozoan mtDNA.
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CHAPTER 4. CNIDARIAN PHYLOGENETIC RELATIONSHIPS AS
REVEALED BY MITOGENOMICS
A paper submitted to BMC Evolutionary Biology
Ehsan Kayal, Béatrice Roure, Hervé Philippe, Allen G. Collins, Dennis V. Lavrov
Cnidaria (corals, sea anemones, hydroids, jellyfish) is a phylum of relatively simple aquatic animals characterized by the presence of one particular type of cell (cnidocyst). Species within Cnidaria have life cycles that involve one or both of two distinct body forms, a typically benthic polyp, which may or may not be colonial, and a typically pelagic medusa. The currently accepted taxonomic scheme subdivides Cnidaria into two main assemblages:
Anthozoa (Hexacorallia + Octocorallia) – cnidarians with a reproductive polyp and the absence of a medusa stage – and Medusozoa (Cubozoa, Hydrozoa, Scyphozoa, Staurozoa) – cnidarians that usually possess a reproductive medusa stage. However, the monophyly of Anthozoa has been questioned by mitogenomic-based studies, where Octocorallia emerges as the sister group
We expanded the sampling of cnidarian mitochondrial genomes, particularly from
Medusozoa, to reevaluate phylogenetic relationships within Cnidaria. Our phylogenetic
85 analyses based on a mito-genomic dataset reject the monophyly of Anthozoa and indicate that the Octocorallia + Medusozoa relationship is not the result of sampling bias, as proposed earlier. Our analyses also reject Acraspeda [Cubozoa + Coronatae + Discomedusae] and
Scyphozoa [Discomedusae + Coronatae]. Furthermore, we use the new dataset to evaluate previously proposed phylogenetic relationships within medusozoan classes and to infer the evolution of key morphological characters within Cnidaria.
Cnidarian mitochondrial genomic data contain a strong phylogenetic signal, which can be used to understand the evolution in this phylum. Mitogenomic-based phylogenies reject the
monophyly of Anthozoa, providing further evidence for the polyp first hypothesis. They also
reject traditional Acraspeda and Scyphozoa hypotheses, suggesting that the shared
morphological characters in these groups are plesiomorphic, originating in the branch leading
to Medusozoa. We expect that the increase in mitogenomic data along with improvements in
phylogenetic inference methods and use of additional nuclear markers will further enhance our
understanding of the phylogenetic relationships within Cnidaria.
The phylum Cnidaria currently encompasses five classes: Anthozoa subdivided into the
subclasses Hexacorallia (hard corals and sea anemones) and Octocorallia (soft corals, sea pens,
and gorgonians), Staurozoa (stalked jellyfish), Scyphozoa (true jellyfish), Cubozoa (box jellies
or sea wasps), and Hydrozoa (hydras, hydroids, hydromedusae, and siphonophores). Non-
anthozoan classes are grouped into the subphylum Medusozoa [1, 2]. Cubozoa (Werner 1975)
and Staurozoa (Marques and Collins 2004) were originally included in the class Scyphozoa
(Götte 1887), but have each been promoted to the taxonomic status of class, leaving three
orders Coronatae, Rhizostomeae and Semaeostomeae in Scyphozoa . The relationships
among and within medusozoan classes remain contentious. Although traditionally box jellies
were considered to be closely related to true jellyfish (Acraspeda; Gegenbaur 1856), some
studies have suggested the grouping of [Cubozoa + Staurozoa] and [Hydrozoa +
[Rhizostomeae + Semaeostomea (Discomedusae)]] [3, 4].
Recent research efforts, in particular the Cnidarian Tree of Life (CnidToL) project
(cnidtol.com), have generated large molecular datasets, primarily ribosomal DNA (rDNA)
sequences, from a diverse sampling of cnidarian species. Analyses of these datasets lead to
substantial progress in understanding phylogenetic relationships within the cnidarian classes
but also challenged some traditional clades. For example, some studies have nested the
monophyletic rhizostome jellyfish within a paraphyletic Semaeostomeae [5–8], a view supported by the most recent phylogenetic study using 18S and 28S sequences . Other relevant findings include the sister group relationships between Trachylina and Hydroidolina within Hydrozoa , paraphyletic “Filifera” (Kühn 1913) within Hydroidolina [10, 11]; monophyletic stony corals (Scleractinia) within Hexacorallia [12, 13] in opposition to earlier studies ; and, two robust clades Carybdeida and Chirodropida composing box jellyfish
Resolving phylogenetic relationships for the phylum Cnidaria is a prerequisite for the reconstruction of the evolutionary history of key morphological novelties in this group, e.g. life history and morphological characters [6, 10, 16], medusan morphospace and swimming ability
87 evolution is the ancestral state of the adult life stage, polyp or medusa, which has been a matter of controversy for over a century [6, 19]. All anthozoans (hexacorals and octocorals) lack a
free-living medusa stage, while cubozoans and most scyphozoans contain both polyp and
medusa stages. Hydrozoans display the widest range of diversity in their life cycle, with the
absence of a sessile polyp in Trachymedusae (which may be diphyletic ) and many species
of Narcomedusae, and a highly reduced or absent medusa in other lineages (e.g. some clades
within Leptothecata and Aplanulata, including the freshwater hydras (Hydridae))[1, 5, 10, 21,
22]. While some earlier studies have proposed loss of the medusa form in Anthozoa, the
current view holds the medusa is an apomorphy (derived character) for Medusozoa [1, 6]).
Consequently, today it is generally considered that (1) a sessile polyp-like form was the
ancestral life form of the phylum Cnidaria and, (2) an adult pelagic medusa phase evolved (one
or several times) in Medusozoa [1, 6]. However, the latter view has only slight phylogenetic
support in the currently accepted cnidarian phylogeny, where the most parsimonious scenario
involves the gain of the polyp form in the ancestral Cnidaria and of the medusa form in
Medusozoa. Alternatively, both the polyp and the medusa forms could have been acquired in
the ancestral cnidarian, and the medusa form subsequently lost in the branch leading to
Anthozoa. The multiple losses of the medusa stage in different medusozoan lineages suggest
that this character is indeed evolutionary labile.
Medusozoa (sometimes referred to as Tesserazoa) is supported by a combination of
characters both morphological (e.g. presence of an adult pelagic stage, cnidocils (cilia of
cnidocytes lacking basal rootlets), and microbasic eurytele nematocysts) and molecular (e.g.
rDNA, mitochondrial protein genes, linear mitochondrial DNA) [1, 6, 19]. Similarly, several
morphological characters have been suggested as synapomorphies for Anthozoa, including the
88 presence of an actinopharynx (a tube leading from the mouth into the coelenteron), mesenteries and possibly siphonoglyphs (ciliated grooves longitudinally extending along the pharynx), although the latter are absent in some anthozoan lineages . Anthozoa [23–26] has also received support in molecular based rRNA data, although only one of them (Collins (2002) included a dense sampling of both anthozoan and medusozoan taxa (see Figure 1A). By contrast, studies based on mitochondrial DNA data do not support Anthozoa and instead group octocorals with medusozoans [27–30] (Figure 1B). This alternative phylogenetic hypothesis, if true, would necessitate reinterpretation of morphological characters shared by anthozoans as symplesiomorphies (retained from the common ancestor) rather than synapomorphies. In addition, rDNA-based studies have also exposed some disparities between classical taxonomy and molecular phylogenies for some groups  and were unable to resolve phylogenetic relationships within others, e.g. Hydroidolina [6, 11] and alcyonacean octocorals . Thus, additional markers are necessary to achieve a better understanding of cnidarian relationships.
Mitochondrial DNA (mtDNA) has traditionally been a popular molecular marker for understanding the phylogenetic relationships in animals. Recent technological advances in sequencing complete mtDNA sequences have provided easier access to mitogenomic data for phylogenomic studies [32–34]. Some suggested advantages of mtDNA over nuclear DNA
(nDNA) in phylogenetics are the orthology of all genes  and the small genome structure being relatively conserved, which provides organizational characters such as gene order
(considered as Rare Genetic Changes or RGC) [36, 37]. In non-bilaterian animals, recent increase in the number of completely sequenced mtDNAs has helped to resolve deep phylogenetic nodes within sponges [28, 38] and Hexacorallia within Cnidaria [14, 39]. Yet, the very poor sample size of medusozoan taxa in previous studies raises some question about the
89 validity of phylogenetic results based on them. Indeed, it is known that inadequate taxon sampling and systematic errors can override genuine phylogenetic signal, resulting in flawed phylogenetic reconstructions [40, 42].
We assembled a more taxonomically balanced mitogenomic dataset to investigate the
evolutionary history of cnidarians. Our database contains newly published mitochondrial
sequences from 24 representative species of all medusozoan classes, including three orders of
Scyphozoa, both orders of Cubozoa, and six out of the nine orders of Hydrozoa . We also
included sequences from two octocoral orders Penatulacea and Helioporacea, and two
hexacoral orders Antipatharia and Ceriantharia. Our analyses suggest that the paraphyly of
Anthozoa does not result from poor taxon sampling. We also found the groupings
[Discomedusae + Hydrozoa] and [Coronatae + [Cubozoa + Staurozoa]], contradicting the
current rDNA-based phylogenetic hypothesis within Medusozoa.
The impact of sequence alignment and models of sequence evolution on phylogenetic analyses
Two different strategies were used for data alignment and filtering. The first alignment
(AliS; 3111 amino acids) was created using a combination of the ClustalW and SOAP
programs. The second alignment (AliMG; 3485 aa) was produced by MUSCLE and Gblocks
programs. Both alignments were used for phylogenetic inferences (ML and BI) to assess the
impact of the aligning procedure and yielded nearly identical results. The few discrepancies
included the phylogenetic position of the blue coral Heliopora coerulea, which was impacted
by alignment method but with low support. Given the results from the AliS alignment were
90 more in agreement with traditional phylogenies within Cnidaria, we decided to concentrate on this dataset in this paper.
We also evaluated how the site-heterogeneous CAT and CATGTR models perform compared to the reference site-homogeneous model GTR under BI by using cross-validation.
According to pair-wise difference of log-likelihood scores, we found that the CATGTR model
more accurately explained our data for both alignments (293.87 +/- 22.923 for AliS and 330.78
+/- 40.071 for AliMG); the CAT model was the worst of the three models for our alignments (-
97.65 +/- 51.928 for AliS and -111.61 +/- 63.337 for AliMG).
Phylogenetic relationships among cnidarian classes
Phylogenetic relationships among cnidarian classes were analyzed under both Bayesian
(BI) and Maximum Likelihood (ML) frameworks using the AliS alignment. We also used two
different models of sequence evolution (GTR, and CATGTR) for Bayesian inferences. We
found maximum support (bootstrap values of 100, posterior probabilities of 1) for the
monophyly of Medusozoa, Cubozoa, Staurozoa, Hydrozoa, and Discomedusae in all analyses.
A few differences in topology emerged when using two different models of sequence
evolution, most of them limited to poorly supported branches. For instance, we found a
monophyletic Cnidaria (posterior probability PP=0.99) and a monophyletic Hexacorallia but
with no support (PP=0.47), and the placement of the coronate Linuche unguiculata as the sister
taxa to the clade [Cubozoa + Staurozoa] (PP=0.84) under the CATGTR model (Figure 2). On
the other hand, we found paraphyletic Cnidaria and Hexacorallia in all GTR trees, where the
position of Ceriantheoptsis americanus was unstable. We also observed that L. unguiculata
was the first diverging medusozoan clade in GTR (BI) analyses (PP=0.89) but the sister taxa to
[Cubozoa + Staurozoa] in GTR (ML) without support (bootstraps BV=12). Removing the
91 coronate L. unguiculata and those species with missing data (the blue coral Heliopora coerulea and the tube anemone C. americanus) did not impact cnidarian paraphyly under GTR (ML) model (Figure S3).
All analyses supported the clade [Octocorallia + Medusozoa] (CATGTR: PP=0.99;
GTR: PP=0.72 BV=42), although support values were lower when GTR model was used.
Within Medusozoa, we recovered the monophyly of Cubozoa, Staurozoa, and Hydrozoa with maximum support (BV=100 PP=1), but Scyphozoa [Coronatae + Discomedusae] was not recovered as a monophyletic group in any analyses (Table 1). The class Hydrozoa always grouped with Discomedusae (CATGTR: PP=1; GTR: PP=1 BV=73), and Cubozoa was the sister taxon to Staurozoa (CATGTR: PP=0.56; GTR: PP=1 BV=81). As we removed the single species coronate L. unguiculata, the support values for the clade [Cubozoa + Staurozoa] slightly increased (BV=85), while support values for the clade [Hydrozoa + Discomedusae] decreased (BV=66).
Within Discomedusae, our data reject the monophyly of Semaeostomeae (Table 1) with the rhizostome Rhizostoma pulmo as the sister taxon to the semaeostome Aurelia aurita, and
Cyaneidae grouping with Pelagiidae. Cubozoa is divided into the two monophyletic clades
Carybdeida and Chirodropida as found earlier , but our data does not resolve the relationships between the three carybdeids. In Hydrozoa, our analyses supported the dichotomy
Trachylina (Cubaia aphrodite) – Hydroidolina (the rest of the hydrozoans) with high support values (PPs=1; BVs=100; Figure 2). The aplanulatan Hydra spp and Ectopleura larynx and the capitates Millepora platyphylla and Pennaria disticha formed a monophyletic clade (PPs=1;
BV=90). The species Clava multicornis (clade Filifera III) and Nemopsis bachei (clade Filifera
IV) also formed a monophyletic clade as the sister group to the rest of Hydroidolina with maximum support values. The position of the leptothecate Laomedea flexuosa was different between GTR and CATGTR analyses. The GTR trees placed Leptothecata as the sister taxon to the clade [Filifera III + Filifera IV] (PPs=1; BV=88) similar to ML analyses using nuclear and mitochondrial rRNA genes with the GTRMIX model of sequence evolution . By contrast, Bayesian analysis using the CATGTR model strongly supported the leptothecate hydrozoan as the sister taxon to the clade [Aplanulata + Capitata] (PP=0.99).
Our data suggest Ceriantharia (tube anemones) is the sister taxon to the rest of
Hexacorallia, but this relationship was not well supported (Figure 2). Ceriantharia aside,
Zoanthidea (zoanthids) was the earliest diverging lineage, followed by Actiniaria (sea anemones), Antipatharia (black corals), and the clade [Corallimorpharia + Scleractinia]. The order Scleractinia (stony corals) was monophyletic in CATGTR analyses with low support values (PP=0.79), but paraphyletic under GTR framework, where one clade of scleractinians was the sister taxa to corallimorphs (PP=1;BV=84). Finally, despite the addition of two new sequences from Pennatulacea (Renilla muelleri and Stylatula elongata) and partial data from H. coerulea, our analyses did not resolve the phylogenetic relationships within Octocorallia.
Testing evolutionary hypotheses
We used several phylogenetic tests to evaluate the support for traditional taxonomic hypotheses of our datasets under the ML framework. We used the Approximately Unbiased
(AU) test, the Kishino-Hasegawa (KH) test, and the Shimodaira-Hasegawa (SH) test with the
GTR+Γ model (Table 2). We found that the order Semaeostomeae is significantly rejected
(AU=0; KH=0; SH=0.2), but not the inclusion of staurozoan species as part of an extensive
“Scyphozoa” clade (AU=0.1; KH=0.1; SH=0.2). We also found that despite the absence of
93 support for the monophyly of both Hexacorallia and Scleractinia in GTR trees, hypothesis tests do not reject these clades (Table 2). GTR-based analyses do not significantly reject the validity of Anthozoa (AU=0.7; KH=0.4; SH=1), Scyphozoa (AU=0.2; KH=0.1; SH=0.5/0.4), or the clade Acraspeda [Cubozoa + Scyphozoa] (AU=0.8; KH=0.6; SH=1), even after removal of the long-branch coronate L. unguiculata (AU=0.9; KH=0.8; SH=1). By comparison, under BI we found no support for the validity of Anthozoa (PPs=0; BV=0), Acraspeda (PPs=0; BV=0),
Scyphozoa (PPs=0; BV=3/0), and Semaeostomeae (PPs=0; BV=0) in any of our trees under both GTR and CATGTR models in a Bayesian framework.
We reevaluated cnidarian phylogenetic relationships using a dataset of mitochondrial protein genes from a systematically more balanced sample of species that encompasses classes, including cubozoans and staurozoans , which were absent in previous works [3, 29]. We decided to exclude sequences from bilaterian animals from our analyses because they form long branches in mitochondrial phylogenomic trees that attract other long branches, resulting in
Long Branch Attraction artifacts [43, 44]. In the future, the inclusion of bilaterians in mtDNA- based phylogenies can be tested when better models of sequence evolution are available. Our cnidarian-rich dataset reduced one major source of potential error that could derive from biased and insufficient taxon sampling for the groups of interest. We also tested our choice of the model of sequence evolution. Our findings suggest that CATGTR is the best model to describe mitochondrial protein data under a Bayesian framework. In fact, this site-heterogeneous model recovered the monophyly of Cnidaria and Hexacorallia, both supported by other molecular datasets and morphological characters [3, 45–49]. By contrast, these clades were paraphyletic
94 in both Maximum Likelihood and Bayesian analyses based on GTR model of sequence evolution. This is not surprising given that a main assumption underlying the GTR model, i.e. homogeneity of the substitution pattern across sites, is violated by most molecular data, rendering the correct capture the phylogenetic signal present in our alignments more difficult
. The resulting topologies from CATGTR analyses differed significantly from the current
consensus view of cnidarian phylogeny [1, 2]. Using the best estimate as a working hypothesis
for cnidarian relationships, we can propose a putative reconstruction of character evolution for
The mitogenomic point of view of cnidarian systematics
The current systematics view based on nuclear genes under the GTR model subdivides
the phylum Cnidaria into Anthozoa and Medusozoa [1, 2], but mitochondrial protein genes
have consistently supported anthozoan paraphyly with Octocorallia being the sister taxon to
Medusozoa [27–30]. It has been argued that the paraphyly of Anthozoa reported in earlier
mitogenomic studies resulted from poor taxon sampling [2, 29]. Here we show that
paraphyletic Anthozoa does not result from unbalanced taxon sampling. In addition, a principal
component analysis of the amino acid composition (Figure S4) suggests that compositional
bias is also an unlikely explanation. The amino acid composition of Hexacorallia is rather
divergent, but not similar to those of the two outgroups (Porifera and Placozoa); in fact, the
composition similarity between Octocorallia and the outgroups would favor the alternative
possibility of Anthozoa paraphyly [Medusozoa + Hexacorallia], which is not observed in our
analyses. To further test the possible impact of compositional bias, we analyzed our alignments
using the CATGTR+G model with a Dayhoff recoding strategy, despite the fact that it implies
a loss of signal, increasing the stochastic error. Interestingly, Octocorallia remained sister-
95 group of Medusozoa, even if the statistical support is reduced (data not shown). Consequently, according to our analyses, mitochondrial protein genes support the clade [Octocorallia +
Medusozoa] (Figure 2). In contrast, the amino acid composition of Hydrozoa and
Discomedusae is similar (Figure S4) and the Dayhoff recoding recovered Discomedusae as sister-group of Cubozoa (a reduced version of Acraspeda), although with low support,
suggesting that the monophyly of the clade [Hydrozoa + Discomedusae] might be due to an
amino acid composition artifact. In fact, preliminary analyses including the very fast evolving
and compositionally biased Bilateria using the site-heterogeneous CATGTR+G model
supported the unlikely grouping of Bilateria and Cubozoa (data not shown). This suggests that
the use of a complex model of sequence evolution and of a rich taxon sampling is not sufficient
to overcome all the systematic errors in the mitochondrial genomes. With the current increase
in genome sequencing efforts, it will be soon possible to corroborate, or not, our phylogenetic
tree (Figure 2) with large nuclear DNA datasets.
Within Medusozoa, Staurozoa is considered the first diverging clade, and the sister
taxon to [Acraspeda (Cubozoa + Scyphozoa) + Hydrozoa] (reviewed in ). While the position
of Linuche unguiculata is still ambiguous in our trees, we found no support for either the
inclusion of coronates in Scyphozoa (PP=0; BV=3), or for Acraspeda [Cubozoa + Scyphozoa]
(PP=0; BV=0). Instead, we found high support for the clades [Hydrozoa + Discomedusae]
(CATGTR: PP=1; GTR: PP=1 BV=73) and [Cubozoa + Staurozoa] (0.56/1/81). This suggests
that Scyphozoa is polyphyletic, although both Scyphozoa and Hydrozoa display similar amino
acid composition. Additional sequences from coronates can test the phylogenetic relationships
Recent molecular studies have refined our understanding of hydrozoan relationships, particularly the sister group relationship between Hydroidolina and Trachylina [5, 6, 10, 11, 21,
51]. However, relationships within Hydroidolina have been very difficult to resolve based on either nuclear or mitochondrial rDNA genes. Here we have been able to sample five important hydroidolinan clades: Aplanulata, Capitata, Filifera III and IV, Leptothecata, and
Limnomedusae. Mitochondrial protein genes provide good resolution for order-level
relationships within Hydrozoa, although our sampling was limited in scope (only 10 species
representing 6 out of 11 orders; Figure 2). We recovered monophyletic Hydroidolina as the
sister group to our single representative from Trachylina. We also found monophyletic
Aplanulata and Capitata, and paraphyletic Anthoathecata as suggested earlier [6, 10]. On the
other hand, we found a consistent grouping of capitate and aplanulate hydrozoans in all our
trees (PPs=1; BV=90), which is in contradiction with earlier rDNA-based studies [5, 10, 11,
20, 21, 49, 51]. The high support values here suggest that even better resolution of
Hydroidolina may be achieved with an increase in the number of complete mtDNAs for
representative taxa within this difficult clade.
Based on mitochondrial genome data, the monophyly of stony corals (Scleractinia) has
recently been put into question , but more thorough studies employing alternative datasets
have rejected this hypothesis [13, 52]. Our analyses support the monophyly of Scleractinia
under the preferred CATGTR model (PP=0.80; AU=0.2; KH=0.1; SH=0.5). Our phylogenetic
analyses did not resolve relationships within octocorals. This was predictable given the low-
level of variation of mitochondrial genes in octocorals (see ), a pattern attributed to the
activity of the mtMutS gene they encoded. It is noteworthy mentioning that our alignments do
not encompass sequences from the mtMutS gene, absent in all other cnidarian and animal taxa,
97 and which displays a comparatively higher rate of sequence evolution to other genes .
While future molecular studies of the evolutionary history of octocorals may be tempted to
limit their investigation to only a portion of the mtDNA , complete mitogenomes provide
additional genomic features such as gene order and the composition of intergenic regions
(IGRs) that could be valuable to systematic studies . Furthermore, the combination of
mitogenomic sequences with nuclear data will likely provide even better phylogenetic
resolution for this group.
Morphological evolution in cnidarians
Unlike the dichotomous Anthozoa-Medusozoa, our strongly supported finding that
Anthozoa is paraphyletic further supports the idea that bilateral symmetry, a step of foremost
importance in metazoan evolution as it is exhibited as part of most animal body plans
(bauplan), was anciently acquired prior to the divergence between Cnidaria and Bilateria. In
fact in Cnidaria, increasing evidence supports the presence of bilateral symmetry in corals and
sea anemones, where it is represented by the siphonoglyph [56, 57], while most medusozoans
do not exhibit any bilateral symmetry [19, 58], with siphonophores being an exception .
The tree topologies provided here strengthen the view that the ancestral cnidarian displayed a
bilateral symmetry from which the radial tetrameral symmetry (body divided into four identical
parts) of most medusae and many polyps of medusozoans derived (Figure 3). Previous studies
have already suggested occurrences of derivation from a bilateral bauplan in several animal
groups, such as siphonophores , myxozoans  and some bilaterians [61, 62]. Bilateriality
has evolved very early in animal evolution near the root of the metazoan tree , and our data
support the view that such a step was taken before the divergence of Cnidaria. Future studies
98 that resolve the position of cnidarians within the metazoan tree of life will shed light on the origin of bilateral symmetry in animals.
Based upon the basal position of Staurozoa in previous phylogenies, Collins and collaborators  proposed that the medusa of Hydrozoa, Cubozoa and Scyphozoa derived from a stauromedusa-like ancestor. However, if the mitogenomic hypothesis of Cnidaria is true
(Figure 3), then the free-living medusa form likely evolved in the branch leading to monophyletic Medusozoa, with subsequent independent losses of this life stage occurring in the lineages leading to Staurozoa and several hydrozoan clades (Figure 3). This scenario is consistent with earlier studies that originated the name "stalked jellyfish" for staurozoans, and concluded that these species represented "degenerated" jellyfish descended from ancestors with a pelagic medusa [6, 8, 64, 65]. Simplification or losses of the medusa form has also been documented in several Hydrozoa clades . The acquisition of a pelagic form is significant given that a free-swimming medusa allows a higher degree of offspring dispersion than by gametes and larvae alone .
Earlier studies have suggested several synapomorphies for the extended Acraspeda clade (Cubozoa + Scyphozoa + Staurozoa), namely radial tetrameral symmetry: medusa formation located at the apical end of the polyp, polyps with canal system and gastrodermal musculature organized in bunches of ectodermal origin (4 inter-radial within the mesoglea in
Staurozoa and Scyphozoa, but not limited to 4 in Cubozoa), the presence of rhopalia or rhopalioid-like structures, medusa with gastric filaments and septa in the gastrovascular cavity
[19, 58]. Paraphyletic Acraspeda as suggested by our analyses implies that either these characters were acquired several times independently in Cubozoa, Coronatae and
Discomedusae, or that they have been inherited from the ancestral medusozoan and lost in
Hydrozoa. Given the complexity of these characters, it is unlikely that they have re-derived
independently in various lineages. Rather, the most parsimonious scenario is their presence in
the last common ancestor of medusozoans, with subsequent loss(es) in hydrozoans. Similarly,
Scyphozoa is defined by the presence of ephyrae and simple rhopalia, and production of
medusae through strobilation of the polyp [1, 6, 19, 58]. If Scyphozoa is paraphyletic as
suggested by our trees, these characters can be either convergent or plesiomorphic. For
instance, rhopalia-like structures are present in all medusozoan clades but Hydrozoa.
According to the mitogenomic view of cnidarian phylogeny, the most parsimonious scenario
suggests that the ancestral medusozoan possessed some sort of rhopalium, maybe similar to
those present in discomedusans and coronates. According to this scenario, simplification of
rhopalia must have accompanied the degeneration of the medusae in Staurozoa, while they
were completely lost in Hydrozoa. By contrast, the rhopalia in Cubozoa have evolved into
complex structures with multiple, complex eyes. Marques and Collins (2004) have suggested a
clade formed by [Cubozoa + Staurozoa] based on cladistic analysis of a set of morphological
and life history characters . They suggested the presence of y-shaped septa and a quadrate
or square symmetry of horizontal cross-section as synapomorphies for this clade. Mitogenomic
data also support such a grouping, providing additional evidence for the validity of these
characters as synapomorphies for the clade [Cubozoa + Staurozoa].
We used an extended dataset of mitochondrial protein genes to reevaluate the
phylogeny of Cnidaria, paying attention to common biases in phylogenetic reconstructions
resulting from insufficient taxon sampling and using simplistic models of sequence evolution.
Our phylogenetic analyses suggest the grouping of Octocorallia and Medusozoa to the exclusion of Hexacorallia, resulting in Paraphyletic Anthozoa. We also recovered the
[Discomedusae + Hydrozoa] and [Coronatae + [Cubozoa + Staurozoa]] relationships within
Medusozoa. It should be noted, however, that although our data provide little or no support for the clades Anthozoa, Acraspeda and Scyphozoa, they are not rejected with statistically significant support in the likelihood framework. Using the new mito-phylogenomic view, we reconstructed the evolution of several morphological characters in medusozoans. In particular, we provided additional evidence for the “polyp first” theory, where the ancestral cnidarian was a bilateral polyp-like organism, and that a radially symmetrical and vagile medusae evolved in the branch leading to Medusozoa. Our analyses support the view that the ancestor of cnidarians and bilaterians (UrEumetazoa) possessed bilateral symmetry . According to our working hypothesis, synapomorphies traditionally associated with Acraspeda such as the presence of gastric filaments in the medusae and gastrodermal musculature organized in bunches of ectodermal origin were most likely acquired early in the evolution of Medusozoa, and later lost in the branch leading to Hydrozoa.
Mitochondrial sequence acquisition and alignment
We determined complete or nearly complete mitochondrial sequences from 24 medusozoan species  and analyzed them with 51 sequences previously available in
GenBank, four placozoan and 22 poriferan species (Figure S1). Our dataset covered most of the cnidarian diversity with representatives from all medusozoan classes, including representatives from the three orders of Scyphozoa, both orders of Cubozoa, and six out of the
eleven clades of Hydrozoa, and three species from both Staurozoa clades . In addition, we also incorporated sequences from the black coral Cirripathes lutkeni (JX023266), the sea pens
Renilla muelleri (JX023273) and Stylatula elongata (JX023275), the alcyonarian Sinularia sp
(JX023274), as well as partial sequences for the cerianthid Ceriantheopsis americanus
(JX023261-JX023265) and the octocoral Heliopora coerulea (JX023267-JX023272) as described previously . The resulting dataset contains 75 cnidarian species.
Different alignment programs use different algorithms for retaining or removing sites in alignments. First, we created preliminary alignments to correct potential frameshifts in our sequences. We then created two sets of alignments (AliS and AliMG) using two commonly used approaches to test the impact of alignment algorithm on phylogenetic analyses for our dataset. We aligned individual genes using ClustalW v1.82  and MUSCLE plugin in
Geneious Pro v5.5.6 . For ClustalW we created three alignments with different
combinations of opening/extension gap penalties: 10/0.2, 12/4 and 5/1. Individual gene
alignments were concatenated and the three resulting alignments were compared using SOAP
 and only positions that were identical among the three alignments were retained in the
final alignment (AliS). For MUSCLE alignment, individual genes were aligned using default
parameters and the aligned genes were concatenated. We removed poorly aligned regions with
Gblocks online (Castresana Lab, molevol.cmima.csic.es/castresana/) with the options allowing
gap for all positions and 85% of the number of sequences for flanking positions (AliMG). We
quickly read both alignments and brought minor changes where sequences shown signs of
framshifts (both alignments are available as additional materials). The final SOAP (AliS) and
MUSCLE (AliMG) alignments comprised 3111 and 3485 amino acids, respectively.
We used the program Net from the MUST package  to estimate the amino-acid
composition per species in each of the alignments by assembling a 20 X 106 matrix containing
the frequency of each amino acid. This matrix was then displayed as a two-dimensional plot in
a principal component analysis, as implemented in the R package.
We conducted phylogenetic analyses under Maximum Likelihood (ML) and Bayesian
(PB) frameworks using RAxML v7.2.6 and PhyloBayes v3.3, respectively [50, 72–77]. PB
analyses consisted of two chains over more than 11,000 cycles (maxdiff<0.2) using CAT,
GTR, and CAT+GTR models, and sampled every 10th tree after the first 100, 50 and 300 burn- in cycles, respectively for CAT, GTR and CAT+GTR. ML runs were performed for 1000 bootstrap iterations under the GTR model of sequence evolution with two parameters for the number of categories defined by a gamma (Γ) distribution and the CAT approximation. Under the ML framework, both analyses using the CAT approximation and Γ distribution of the rates across sites models yield nearly identical trees, suggesting that the GTR+CAT approximation does not interfere with the outcome of the phylogenetic runs for our dataset. In order to save computing time and power, we therefore opted for the CAT approximation with the GTR model for further tree search analyses under ML. We assessed the effect of missing data on cnidarian phylogenetic relationships in our trees by removing the partial sequences of C. americanus and H. coerulea. We also removed the coronate Linuche unguiculata given its
problematic position and that it is the only representative of its clade, which could introduce a
systematic bias. We then performed additional GTR analyses under the ML framework on the
reduced, 103 taxa alignment.
Evaluating phylogenetic inferences
We tested several relationships that had been earlier hypothesized under ML by
calculating the per-site log likelihood values with RAxML v7.2.6 under the GTR model. We
then used three commonly used tests (the Approximately Unbiased (AU) test, the Kishino-
Hasegawa (KH) test, and the Shimodaira-Hasegawa (SH) test, see ) to assess the validity of
several key phylogenetic relationships according to our data. To do so, we compared whether
our data significantly rejected the best trees conforming to each of the a priori hypotheses
(Table 2) using the CONSEL software .
For Bayesian inferences, a cross-validation was performed to find the model with the
best fit to the data. Each alignment is randomly split in two unequal parts: a “learning dataset”
with nine-tenth of the original positions and a “testing dataset” with one-tenth; ten replicates
are performed. The parameters of the model are estimated on the learning datasets for each
model, where a fixed topology is inferred with each model (the number of cycles is variable
between various models) and with a burnin corresponding to 1/10th of the total number of cycles required for convergence (maxdiff > 0.2). The estimated parameters are then used on the testing dataset to compute the likelihood of the topology, averaged over the ten replicates.
Finally, we sampled ten morphological characters we considered important from
previously published morphological matrices [10, 19, 58, 80] and mapped them on the
CATGTR based tree. To do so we used PAUP 4.0b10 for Unix  using DELTRAN and
ACCRAN character-state optimization models. The scoring of each character is detailed in
additional materials (Supplementary material).
EK participated in the design of the study, carried out ML phylogenetic analyses and
statistical tests, reconstructed the evolution of morphological characters, and drafted the
104 manuscript. BR carried out BI phylogenetic analyses and extracted the support values, performed the cross validation. HP and DL helped in the design of the study. AGC provided guidance on some analyses and the interpretation of the morphological characters. All authors contributed to the final version of the manuscript.
We would like to thank Matthew Palen for his wonderful drawings of cnidarians. We would also like to thank Niamh Redmond for their help and Henner Brinkmann for his highly insightful comments on this manuscript. This work was supported by a grant from the National
Science Foundation to DVL [DEB-0829783] and by funding from Iowa State University. EK wishes to acknowledge funding through the Smithsonian Predoctoral Fellow grant. Some samples used in this study came from work supported by the NSF-funded Cnidarian Tree of
Life Project (EF-0531779) to P. Cartwright, D. Fautin and AGC.
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Anthozoa Medusozoa Anthozoa Medusozoa H e x a c o r l i O c o t r a l i S t a u r o z C u b o z a S c y p h o z a H y d r o z a H e x a c o r l i O c o t r a l i S c y p h o z a H y d r o z a
Figure 1 - Alternative hypotheses of the cnidarian tree of life.
A. Current view of cnidarian evolutionary history based on rRNA phylogenies. B. Hypothesis of cnidarian relationships obtained using mitochondrial protein coding genes.
* Placozoa 0.93/1/99 Porifera * Zoantharia * * Actiniaria 1/1/79 * Antipatharia 1/1/89 * H * Corallimorpharia 1/1/96 e * * x 1/1/84 * a * * c
a 0.79/-/16 * l * Scleractinia l
1/1/99 * a * *
* * 1/1/92 1/1/70 1/1/99 Ceriantharia 1/1/95 Alcyonacea 0.99/-/7 0.88/-/22 Pennatulacea O 1/1/95 Helioporacea * * c 0.62/0.93/32 t * o
0.99/0.39/20 o 1/1/98 r Alcyonacea a *
1/1/99 l 0.9/0.99/55 i
a 1/1/98 0.99/0.72/42 0.48/-/- 0.99/1/84 0.84/0.10/12 Coronatae * Stauromedusae 0.99/0.95/56 Staurozoa 0.56/1/81 0.99/1/96 Carybdeida * 0.58/-/- Cubozoa * Chirodropida H
* Limnomedusae y * 0.99/1/100 Filifera III
Filifera IV d * Leptothecata 1/1/99 r
0.99/-/- Capitata o 1/1/90
e a s u d e m o c s i * D
1/1/73 * Aplanulata o a
* * 1/1/99 Semaeostomeae * * 0.77/0.99/88 * * Rhizostomeae 0.5 1/1/99
Figure 2 - Cnidarian phylogenetic hypothesis based on mitochondrial protein genes.
Phylogenetic analyses of cnidarian protein coding genes under the CATGTR model with
PhyloBayes for the AliS alignments (3111 positions, 106 taxa) created using ClustalW+SOAP.
Support values correspond to the posterior probabilities for the GTR(BI) and bootstraps for
GTR(ML) analyses. Stars denote maximum support values. A dash denotes discrepancy between the results obtained when assuming different models.
1b Alcyonacea 10g Helioporacea Pennatulacea Octocorallia
4 6 7 Coronatae Coronatae
2- Stauromedusae Staurozoa 9 Carybdeida Cubozoa 7? Chirodropida 1r Limnomedusae 2+ Hydrozoa 5+ 3+ Filifera III 10e 5- 10n Filifera IV
Leptothecata 7 Capitata 8
Semaeostomeae 4 6 Rhizostomeae Discomedusae
Figure 3 - Morphological character reconstruction in Cnidaria.
Reconstruction of key morphological characters in Cnidaria based on the phylogenetic hypothesis based on mitogenomic data. (1) symmetry: b = bilateral, r = radial or biradial; (2) gain (+) and loss (-) of free-swimming medusoid stage (many hydrozoans have secondary lost the medusoid phase); (3) velum (lost in some leptothecate hydrozoans); (4) strobilation; (5) gastric filaments; (6) ephyrae; (7) radial canal (scored uncertain in Cubozoa); (8) circular canal;
(9) square symmetry of horizontal cross section; (10) gastrodermal muscles: e = in bunches of ectodermal origin, g = in bunches of gastrodermal origin, n = not organized in bunches.
Morphological characters are taken from Marques and Collins (2004) and Cartwright and
Nawrocki (2010) studies. Drawings were provided by Matthew Palen.
Table 1 - Support values for topologies in the mtDNA-based phylogeny of cnidarians.
Bootstraps and BI values for several clades traditionally recognized in Cnidaria.
AliS AliMG Taxon GTR-ML GTR-BI CATGTR GTR-ML GTR-BI CATGTR Cnidaria 7 0 0.99 12 0 1 Anthozoa 0 0 0 0 0 0
Hexacorallia 16 0.24 0.48 12 0.22 0.57 Antipatharia 100 1 1 100 1 1 Actiniaria 100 1 1 100 1 1 Corallimorpharia 100 1 1 100 1 1 Scleractinia 16 0 0.80 12 0 0.51 Zoantharia 100 1 1 100 1 1
Octocorallia 100 1 1 100 1 1 Alcyonacea 3 0.02 0 7 0 0.01 Pennatulacea 95 1 1 96 1 1
Medusozoa 100 1 1 100 1 1 Acraspeda 0 0 0 0 0 0 Cubozoa 100 1 1 100 1 1 Carybdeida 96 1 0.99 99 1 1 Chirodropida 100 1 1 100 1 1 Hydrozoa 100 1 1 100 1 1 Aplanulata 100 1 1 100 1 1 Capitata 99 1 1 95 1 1 Scyphozoa 3 0 0 0 0 0 Discomedusae 100 1 1 100 1 1 Rhizostomeae 12 0.01 0.22 11 0 0.14 Semaeostomeae 0 0 0 0 0 0 Staurozoa 100 1 1 100 1 1
Placozoa 100 1 1 100 1 1 Porifera 99 1 0.94 100 1 0.88 Homoscleromorpha 100 1 1 100 1 1 Demospongiae 100 1 1 100 1 1 Keratosa 100 1 1 100 1 1 Myxospongiae 100 1 1 100 1 1 marine Haplosclerida 100 1 1 100 1 1 Democlavia 100 1 1 100 1 1
Table 2 - The use of several statistical tests verifying the validity of some groups in cnidarians.
Probability values for the AU, KH and SH tests and BI values for several clades traditionally recognized in Cnidaria.
AliS AliMG AU KH SH AU KH SH Cnidaria 0.46 0.23 0.91 0.27 0.15 0.86 Anthozoa 0.69 0.42 0.98 0.63 0.41 0.93 Hexacorallia 0.39 0.21 0.84 0.48 0.32 0.86 Scleractinia 0.16 0.09 0.52 0.25 0.17 0.53 Acraspeda* 0.84 0.58 1.00 0.80 0.59 0.99 Rhisostoma 0.16 0.91 0.55 0.24 0.17 0.64 Scyphozoa 0.20 0.14 0.50 0.10 0.08 0.42 Scyozoap + Cubozoa + Hydrozoa* 0.01 0.01 0.42 0.12 0.14 0.41 Scyphozoa + Cubozoa + Staurozoa* 0.02 0.02 0.20 0.04 0.01 0.27 Scyphozoa + Staurozoa* 0.06 0.06 0.24 0.01 0.04 0.25 Semaeostoma 0.01 0.01 0.18 0.01 0.02 0.18
* similar results have been obtained for a smaller dataset that does not contain the sequences from the coronate Linuche unguiculata, the tube anemone Ceriantheopsis americanus and the blue octocoral Heliopora coerulea.
CHAPTER 5. THE ERATIC EVOLUTION OF ANIMAL
A paper to be submitted to Mitochondrial DNA
Since the original endocytosis event of the α-proteobacteria ancestor, the endosymbiotic mitochondrion has experienced various degrees of genome reduction in different eukaryotic lineages. In animals, the general tendency appears to be a small and fairly uniform sized mitochondrial DNA (mtDNA), with nearly absent intergenic regions and non- coding DNA, a relatively stable number of RNA and protein genes. Such pattern has led to the minimalistic view of “frozen” mitogenome in metazoans. Yet, an increasing amount of studies, particularly from disregarded taxa, have documented exceptional patterns of genome evolution in several animal groups. Here, I summarize major deviations from the “typical” animal mtDNA with respect to their size, gene content, and genome organization. The overall picture of such survey appears to be of a more plastic genome than previously suggested. This also hinders any future attempt to define a single evolutionary trend, also known as a “unifying theory”, for the evolution of animal mtDNA.
Mitochondria are prokaryote-like organelles found in nearly all eukaryotes and involved in a variety of cellular functions, the most ubiquitous being oxidative phosphorylation. The mitochondrial ancestor is thought to have possessed a wide array of genes encoding the range of metabolic pathways necessary for a free-living α-proteobacteria- like organism. The endosymbiosis of ancestral mitochondria has then triggered the loss and/or transfer to the nucleus of most prokaryotic genes, with the subsequent import of the proteins needed for mitochondrial (mt) functions . Yet, the mitochondria in all extent organisms possess a genome, the mitochondrial DNA (mtDNA), that encodes at least for part of the regulatory genes involved in its expression, and of several subunits of the complexes of molecules involved in the oxidative phosphorylation chain . Various eukaryotic lineages display different trends in the evolution of their mtDNA with respect to their size, gene content, genome architecture and sequence evolution. The size of the mt genome ranges from a few kb in some protists to >2Mbp in some plants . Genome compactness accounts for most of the disparity in mtDNA size variation, influenced by the distribution of non-coding DNA such as intergenic regions, introns, and repeated sequences . On one side of the size spectrum stands the "typical" animal mtDNA (see below) with a nearly constant and small number of genes, virtually absent intergenic regions, and the lack of introns and repeats. On the other side of the size spectrum stand plant mtDNAs with a higher number of intron-rich protein and rRNA genes separated by long stretches of non-coding DNA.
In addition, the number of protein genes encoded by the mtDNA varies from three
(smallest number found to date) in the apicomplexan human parasites Plasmodium spp.  and dinoflagellates [6, 7], to <100 in the jakobid protist Reclinomonas americana  which has
120 been suggested to resemble the ancestral mtDNA. Finally, eukaryotes mtDNAs display a large assortment of architectures, ranging from a single circular prokaryotic-like genome in many plants, protists and most animals, to linear chromosomes of various sizes in ciliates, green algae, yeast, one cnidarian clade and some crustacean isopods [2, 9–18], with intermediary
“lasso-like” structures as found in the large grass Pennisetum typhoides , to the complex kinetoplasts DNA (kDNA) found in Kinetoplastida .
In animals, the typical mtDNA is a small (~17kb) single circular molecule that encodes a total of 37 genes, 13 of which are protein genes involved in the oxidative phosphorylation pathway, and 24 are RNAs involved in mitochondrial protein synthesis. The 13 protein genes are subunits 6 and 8 of ATP synthase (atp6 and atp8), apocytochrome b (cob), three subunits of cytochrome oxidase (cox1-3), and subunits 1-6 and 4L of the NADH dehydrogenase complex
(nad1-6 and nad4L). RNA genes encode the small and large ribosomal RNA (rRNA) subunits
(rns and rnl), and 22 transfer RNA (tRNA) genes (one per amino acids plus two additional ones for both leucine and serine) [21, 22]. Such apparent conservation in size and organization of animal mtDNA is in part responsible for propelling it into the front line of animal phylogenetics. In fact, the relatively easy access to complete genomes in most metazoan groups, higher rate of sequence evolution with respect to nuclear sequences , overall conservation of the genome structure providing organizational characters such as gene order
(considered as Rare Genetic Changes or RGC) [24, 25] have all favored the sequencing of complete mitogenomes from more than 2500 animal species, 2/3 of which belong to vertebrates and more than 400 to arthropods (Figure 1).
The current understanding of animal mtDNA is highly influenced by vertebrates and arthropods mtDNAs, together representing ~83% of the total number of published
121 mitogenomes to date. As a consequence, the reference animal mtDNA resembles vertebrate, and more particularly mammalian mtDNA. Such bias can be traced back to the history of mitogenomics where the first completely sequenced mtDNAs were from humans . Yet, recent sampling outside Deuterostomia, and in particular from non-bilaterian animals, has revealed additional levels of complexity in the structure and organization of animal mtDNA
. These studies suggest that animal mtDNA is not "frozen genome"  and mammalian mitogenomes are not good representatives of the diversity of mtDNA organizations in animals.
Here I review the current understanding of animal mitogenomes with respect to genome structure and organization, and gene content. Overall, the pattern of mtDNA evolution shows no clear correlation with animal evolutionary history, even less with size and tissue complexity.
In addition, given the incomplete picture of animal mtDNA diversity, more deviations from the typical animal mitogenome shall be expected as additional metazoan taxa are explored with higher scrutiny.
Mitochondrial genome structure and organization
Mitochondrial genomes are commonly mapped into a single circular plasmid (i.e. a typically circular double stranded DNA molecule independent from genomic DNA) structure.
Yet an increasing amount of studies have been reporting deviation from such monochromosomal “plasmidic” view of the mtDNA in various organisms across the tree of life
[9, 28]. In animals, multi chromosomal mtDNA has been described in the rotifer Brachionus plicatilis , in some medusozoan cnidarians [13, 15, 30], and some nematode species [31–
33]. In nematodes, mitochondrial mini-circles have also been reported in species that possess monochromosomal mtDNAs, a potential intermediary state toward multipartite mtDNA .
In arthropods, several parasitic lice species (order Psocodea) have also evolved multipartite
mtDNA composed of up to 18 minicircles [35, 36]. Recently, researchers have shown that the
mitochondrial genome of the booklice Liposcelis bostrychophila is in two circular molecule of
nearly identical size but of unequimolar representation: one chromosome is twice as abundant
as the other one .
In the phylum Cnidaria, the mtDNA of medusozoans (Cubozoa, Hydrozoa,
“Scyphozoa”, and Staurozoa) is organized into one or several linear molecules [13, 15, 17, 18,
38–43]. To date, the only other case of linear mtDNA in animals has been found in a group of
crustacean isopods where the mtDNA consists of a circular ~28-kb dimer and a linear ~14-kb
monomer [10–12, 14]. Finally, preliminary data from calcareous sponges also hint for the
possibility of multipartite, and most likely linear mtDNA organization in this group (this
thesis). Outside animals, linear mtDNA has been documented in a wide array of organisms
such as fungi, algae, and ciliates [44–50].
Unlike circular molecules, linear mtDNA has to overcome the “challenges of the ends”
, namely a higher susceptibility of the linear molecules to exonuclease activity and their
shortening after each replication cycle. In the nucleus, the ends of the linear chromosomes are
capped by telomeres that both protect the molecule from degradation and allow its replication
in a faithful manner. Given the lack of telomerase-dependent telomeres in the mitochondrion,
various organisms have innovated alternative mechanisms that nearly always involves repeated
sequences with opposite orientation at each end of the linear molecule [9, 47, 51]. One interesting mechanism reported in some yeast species, is the concomitant presence of a terminal protein binding the 5’ends of the linear mitochondrial molecule(s) and a B-type DNA
polymerase (polB-like) gene encoded in the genome (e.g. ). polB has been linked to the
123 incorporation by the mtDNA of a linear fungal plasmid, where the invasion of by the linear plasmid linear resulted in the linearization of the mitochondrial molecule . Recent discovery of polB-like genes in the mito-genomes of some medusozoan species [13, 15, 17, 18] provides further support for the horizontal transfer hypothesis, potentially after infection by a fungi that possessed the linear plasmid.
The size of animal mitochondrial genome is not uniform
The most recent review of animal mitochondrial genomes emphasized an overall conservation in mtDNA size for most taxa . Animal mtDNA size ranges from just above
10 kbp in ctenophorans to >43 kbp in the placozoan Trichoplax adhaerens . It has been suggested that "key transitions in animal evolution" (multicellularity and bilateral symmetry) are reflected by the evolution of their mitogenomes , where the advent of bilateral symmetry has been accompanied with several genetic novelties in the mtDNA, such as changes in the genetic code and loss of tRNA genes. For instance, bilaterian mtDNAs are generally smaller in size compared to non-bilaterian counterparts, due to the more compact nature of their genome and smaller sized genes. In contrast, non-bilaterians mitogenomes are considered more permissive to non-coding DNA, where larger-sized genes are interspaced by intergenic regions (IGRs) and introns that can account for up to 34% of the genome in sponges [54, 55] and 45% in placozoans . However, this pattern does not hold in the light of the most recent studies of non-bilaterians. The recently sequenced mitogenomes from the ctenophorans
Mnemiopsis leidyi  and Pleurobrachia bachei , typical non-bilaterian animals, are the smallest for metazoan to date. Likewise, all published mitogenomes from medusozoans (one of the two classes within Cnidaria) resemble bilaterians, with the quasi absence of non-coding
124 regions and small-sized genes [13, 15–18, 30]. Presently, it is not clear whether these small mtDNAs are evolutionary curiosities among an otherwise homogenous set of sponge-like non- bilaterian mitogenomes. Yet an increasing number of evidence suggests that the typically large mtDNA is not a good representative for all non-bilaterian animals. Similarly, tiny mtDNAs found in chaetognaths [59–62] and at least one nematode  also suggest some level of size- plasticity in bilaterian.
In contrast, large size mtDNAs have been documented amid several bilaterian taxa. In mollusks, large mitogenomes resulted from partial segmental duplication of coding regions
[64–67]. In nematodes, large non-coding regions found in several species were generated after, and as a result of, the segmentation of their mtDNA (see below) [31–33, 36]. Some instances of high variation in the size of bilaterian mtDNA appear to be artifacts from either PCR amplification or sequencing. Recent examples of such experimental errors include the apparent
“tandem duplication-random loss” mechanism proposed for explaining the partial duplication of genes in the oyster Crassostrea hongkongensis [68, 69] and the particularly large mitogenome of the mite species Metaseiulus occidentalis . In nematodes, authors have verified the validity the duplication responsible for the large size mtDNA of the enoplean
Romanomermis culicivorax . Overall, mitochondrial genome “gigantism” resulting from partial duplication is rather rare in animals, and suspicious cases such as the brachiopod
Lingula anatine , and other enoplean nematodes [73, 74] necessitate to be investigated thoroughly. Overall, animal mtDNA does not necessary appear to tend toward the size variation of. Instead, the big picture might most likely resemble a randomly distributed size range that does not match the metazoan tree of life, and most importantly, the size does not decrease with the increase in animal body and organization complexity.
A wide array of mitochondrial genetic content
As mentioned earlier, the "typical" (mammalian-based) animal mtDNA harbors 13 intron-less protein genes, 22 tRNA genes and 2 rRNA genes. Such generalization does not apply to non-bilaterian animals where additional genetic material (such as additional protein genes and ORFs, introns, and tRNAs) can be found. In the published mtDNAs of Placozoa and sponges, an additional gene for subunit 9 of ATP synthase (apt9) is also present [54–56, 75–
85], while a subunit for twin-arginine translocase (tatC) has been found in some homoscleromorphs [77, 85], and a lineage-specific ORF has been found in the mtDNA of some marine mussel species . atp9 is found in the unicellular relatives of animals, the choanoﬂagellate Monosiga brevicollis and the ichthyosporean Amoebidium parasiticum , while it has been transferred to the nucleus in all other animal groups. Consequently, the presence of atp9 in the mtDNA of sponges and placozoans is considered an ancestral state for metazoan. Another ancestral feature of animal mtDNA is the presence of the self-splicing group I intron in cox1 that usually harbors a putative homing endonuclease responsible for its mobility, so far reported only in placozoan [56, 87], and some representatives from demosponges and hexacorals [80, 85, 88, 89]. No group I intron has been found in glass sponges [76, 79] or any other animal group, suggesting a tendency to be lost. This is illustrated by loss of the intron in several hexacorals mtDNA where an unidentified ORF can be found upstream or downstream of cox1. Another intron is present within nad5 in all hexacorals (e.g.
[90–92]) and placozoans [56, 87]. This feature is shared with the choanoﬂagellate M. brevicolis, but has been lost in all other cnidarians, sponges, and other animal groups. Two occurrences of a second type of intron (group II intron), have been recently reported in the
126 annelid Nephtys sp  and the placozoan Trichoplax adhaerens  that putatively originated from a horizontal transfer from either a viral or bacterial vector.
The mtDNA of non-bilaterian animals can also contain additional protein genes of extra-mitochondrial origin. In octocorals, some studies have suggested a nuclear origin for the putative mismatch repair protein mutS [43, 94, 95], illustrating the first potential transfer of nuclear gene into animal mtDNA. A DNA-dependent DNA polymerase polB has also been described in one strain of placozoans and the jellyfish Aurelia aurita [13, 15, 17, 18, 56].
Overall non-bilaterian mtDNA seems to have been at least to some extent porous to invasion of alien materials.
The loss of genetic information is by far the most widespread deviation from a typical animal mtDNA. Subunits of the ATP synthase (atp8 and in a lesser extend atp6) have been lost independently in animal species, most of which are characterized by a high rate of mtDNA sequence evolution. atp8 was lost in all three groups parasitic flatworms (Cestoda, Monogena and Trematoda) [22, 96, 97], the acoelomorph Symsagittifera roscoffensis , and the free living planarians Dugesia japonica (NC_016439) and Schmidtea mediterranea (unpublished data), the bryozoan Tubulipora flabellaris , several bivalve species [100, 101], and in all nematodes with the notable exception of Trichinella spiralis [31, 32, 34, 102–106]. atp8 appears to be a disposable gene in animal mtDNA, with recurrent losses across the metazoan tree of life (Figure 2).
Other instances of protein gene loss from the NADH dehydrogenase complex have been reported in three vertebrate species (reviewed in ) and the turbellarian Dugesia japonica (for nad4L), but additional investigations are needed to rule out cases of PCR artifacts
. Analysis of complete nuclear DNA (nDNA) will provide additional insights on the
potential transfer on these genes from the mitochondrion to the nucleus. Two extreme cases of
mtDNA reduction are found in ctenophorans and in chaetognaths, where both atp6 and atp8 genes have been lost [57–62]. Not surprisingly, atp6 has been detected in the nDNA of both ctenophorans [57, 58]. mtDNA-encoded RNAs are prone to a high level of variation in animals
Protein translation in animal mitochondria utilizes a repertoire of RNAs that are typically encoded by their mtDNA . Such repertoire is constituted of two genes for the large and small subunits of the ribosomal RNAs (rnl and rns), and up to 24 tRNA genes.
Unusual rRNA genes are found in placozoans and bivalves, where rnl may be encoded by two or more DNA fragments [56, 68, 109]. High levels of gene segmentation of both subunits of rRNA have been recently demonstrated in the calcareous sponge Clathrina clathrus (this thesis). It is noteworthy mentioning that fragmented rRNA genes have been documented in many non-animal groups, such as bacteria, green algae and some protists [7, 110–115].
Putative functional small rRNA genes have been inferred in parasitic flatworms (i.e. )
where despite a decrease in gene sizes, the secondary structure is well conserved. Extremely
small rRNAs were inferred in the recently sequenced mtDNA from the ctenophoran
Mnemiopsis leidyi [57, 58]. However, it is unclear whether these RNA genes are functional or
pseudogenes. For instance, fragmented rRNA genes could explain the lack of conserved
secondary structure in these sequences. Empirical evidences from ESTs and RT-PCR based
techniques are likely to shed light on the function of these reduced putative RNA genes.
The typical animal mtDNA contains a whole set of tRNAs (one tRNA per codon
family), each of which corresponding to a given amino acid residue, with the exception of two
tRNAs for two codon families encoding leucine and serine. Yet, variation in the number of
mitochondrial encoded tRNAs has been reported in several animal groups. In chordates, Gissi
and collaborators reviewed six instances of tRNA loss and at least 20 duplicated tRNA genes
. Loss of tRNA genes is most widespread in non-bilaterian animals, as reported in some
demosponges and all cnidarians mtDNAs (e.g. [13, 15, 22, 80]). Recent analysis of the ctenophoran M. leidyi mtDNA suggests the total absence of tRNA genes accompanied by apparent parallel losses of all nuclear encoded mitochondrial aminoacyl-tRNA synthetases (mt-
aaRS) . Yet, a second completely sequenced mtDNA from the ctenophoran Pleurobrachia
bachei encodes sequences similar to two tRNAs . It is still unclear whether the absence of
mt-aaRS is a genuine feature of ctenophoran genomes or the reflection of what is so far an
unfinished analysis of the nuclear genome. Future analysis of the expression and the tRNA-like
genes in P. bachei and of the alternative import of nuclear tRNAs in the mitochondrion shall
shed light on the evolution of ctenophoran mtDNA.
Extreme cases of tRNA loss has also been found in the bilaterian clade Chaetognatha,
with only one tRNA gene (trnM) present in all five species sequenced to date [60, 61].
Interestingly, in the first description of Spadella cephaloptera mtDNA, the authors suggested
the complete loss of all tRNAs in . But a more recent study reanalyzed the three mtDNAs
from chaetognaths available at the time and showed the presence of trnM in S. cephaloptera
mtDNA . Similarly, whether the apparent loss of all tRNAs in ctenophoran is real or an
artifact resulting from the limitations of the purely bioinformatics methods used still have to be
Duplicated tRNA genes have also been reported in enoplea nematodes, bivalves and the
cirripede Pollicipes polymerus . In addition to tRNA gene loss, animal mitochondrial
129 tRNAs show some atypical features not found in non-metazoan taxa. Whilst most mt-tRNAs have very similar inferred secondary structure to their nuclear counterparts, some tRNAs are truncated of part or a complete arm. For instance, tRNA-Ser I displays a shorter sequence with the loss of the dihydrouridine (DHU) arm in most animals. The TΨC arm is lost for several tRNAs in the highly derived mtDNA of nematodes [116, 117], the acanthocephalan
Leptorhynchoides thecatus , and some arthropods.
Edited tRNAs have been reported in several animal taxa : for example in arthropods (e.g. ), mollusks (e.g. [121, 122]), and marsupials (e.g. [123, 124]). An extreme case of degenerated tRNAs is found in onychophorans (velvet worms) mtDNA where some authors have suggested the loss of most mitochondrial tRNA genes [125–127]. Yet a more comprehensive survey of these and other mtDNAs from velvet worms revealed that all tRNAs are present but incomplete, with the TΨC arm lacking at DNA level, which are edited at RNA level to become functional [128, 129]. Finally, recent survey of one calcareous sponge mtDNA suggested template-dependent tRNA editing (this thesis).
Changing the code is not limited to bilaterian animals
The standard genetic code, also known as the "canonical code", used for protein synthesis in the nucleus and organelles has been altered in various living organisms. Animal mtDNA displays the highest variation in their genetic code (Figure 2), with at least 13 instances documented to date [76, 79, 130–133]. The loss of tRNA genes in bilaterians has been tentatively associated with changes in the genetic code in bilaterian animals . Yet, compensatory changes in the genetic code are not paralleled with most instances of tRNA loss, suggesting independence between the two events. For instance, cnidarians use the minimally
130 derived genetic code G4 (TGA Stop Trp), yet they have lost nearly all their mitochondrial encoded tRNA genes. In parasitic flatworms, previous studies have shown at least two changes in the genetic code (AUA Met Ile and AAA Lys Asn), however the complete set of 22 tRNA genes are found in their mtDNA. Similarly, the minimally derived genetic code G4 was inferred for mitochondrial protein synthesis in ctenophoran where most  or all  tRNAs are missing from the mtDNA. Finally in chaetognaths, the mtDNA encodes only methionine, yet the genetic code for protein synthesis is the invertebrate mt-code G5 (AGR Arg Ser;
AUA Met Ile; TGA Stop Trp) [60–62].
Changes in the genetic code seem to be correlated with a relative increase in the rate of sequence evolution in animals. Such pattern appears particularly evident when comparing the three major classes (Demospongia, Hexactinellida, and Calcarea) within the phylum Porifera
(sponges). The relatively slow evolving Demospongia (demosponges) use the minimally derived genetic code and their mtDNA typically encodes for 24 tRNAs (as described by the canonical code codon families). In Hexactinellida (glass sponges), recently sequenced mt- genomes showed a change in the mitochondrial genetic code (AGR Arg Ser) convergent between these sponges and bilaterian animals [76, 79]. Recent survey of two calcarean species strongly suggests the use of at least one, potentially two novel genetic code(s) for mitochondrial protein synthesis, where UAG Stop Tyr and CGN Arg Gly in the order
Calcinea and putatively AUA Ile Met in Calcaronea (this thesis). Interestingly, mt sequences from both glass sponges and calcarean display very high rate of sequence evolution similar (for the former) or higher (for the latter) than bilaterian animals.
Here I surveyed the range of available mtDNA sequences from animal, highlighting major deviations from the typical animal mitochondrial genome. The overall picture displays a wide range of evolutionary trajectories that challenges the classical view of a "frozen genome"
. This is exemplified by the presence of various mtDNA organizations in closely related species. Recent studies of under represented taxa have revealed an assortment of novel genomic features of animal mtDNA. Future studies that explore "small" phyla such may provide insights into the evolutionary potentials of the mitochondrial genome in animals. The combination of the new sequencing technologies both at DNA and RNA levels provides unique tools for dealing with such task. One safe prediction is that no single theory can explain the pattern of mtDNA evolution in animals.
This work also underscored the need for verifying findings of unusual features (e.g. tRNA losses in onychophorans and potentially ctenophorans). In such cases, it is important to propose satisfactory mechanisms that would explain the evolution and maintenance of such pattern. In addition, with our current technological advances, limiting the analysis of lost genetic features to solely bioinformatics searches is unsatisfactory, particularly when a single case is considered, and empirical evidences shall be required before making any evolutionary statement. For instance, a recent study has proposed the parallel loss of nuclear-encoded mt aminoacyl-tRNA synthetases (aaRS) with their respective mt-encoded tRNAs in cnidarians
. Yet the authors failed to explain the presence of a mitochondrial tRNA-Met while the aaRS seems to be lost in the nuclear DNA. This case is illustrative of the current trend in the field of molecular biology. With the ongoing increase in the number of genome sequencing studies, a real shift is underway from empirical experimentation toward more speculative
132 bioinformatics-based predictions. As a consequence, all newly sequenced genomes are poorly annotated. Today more than ever there is a need for independent corroboration of computation- based hypotheses.
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Panarthropoda Priapulida Sipuncula Porifera Placozoa Cnidaria Ctenophora Platyhelminthes Nematoda Acanthocephala Vertebrata Rotifera Chaetognatha Acoela Xenoturbellida Tunicata Echinodermata Hemichordata Cephalochordata
Figure 1: Distribution of sequenced metazoan mitochondrial (mt) genomes. The list of complete or nearly complete mt-genomes were gathered at the Genome Resources of the
National Center for Biotechnology Information (NCBI) website
(www.ncbi.nlm.nih.gov/genomes/ORGANELLES/mztax_short.htmlat) on April the 8th 2012.
Each phylum is represented by the percentage of sequenced mt-genomes relative to the total
number of genomes available (2579).
t+ e g+ g- f Annelida Mollusca c4,c12 t- g- Platyhelminthes t-? m Rotifera g- Bryozoa e Onychophora Tardigrada c10 t+ e m l Arthropoda t- t+ m Nematoda g- c2,c5 t- i- Acanthocephala t- g- Chaetognatha c12 c4 Echinodermata c10,c11 Hemichordata Xenoturbellida g- Acoelomorpha
c6 C c1 Urochordata h o r d a t c9 c7 Cephalochordata c8 t- e g- Vertebrata i+ f Placozoa t- m l g+ g- i- C Medusozoa n i d a r t- t- g+ Octocorallia t+ g+ i- Hexacorallia t- g- i- Ctenophora t- i- Demospongia P
c5 i- o r i f e a Hexactinellida g+ g- i- Homoscleromorpha c2,c3?,c13 e m? l? i-? f Calcarea
Figure 2: Evolution of the mtDNA in Metazoa. The phylogeny is based on Dunn et. al (2008),
Philippe et. al (2009), Pick et. al (2010), Rota-Stabelli et. al (2010), and Philippe et. al (2011). c corresponds to changes in the genetic code where codons have been reassigned (based on
Knight et al. 2001): c1 UGA Stop Trp; c2 UAG Stop Tyr; c3 AUA Ile Met; c4 AUA
Met Ile; c5 AGR Arg Ser; c6 AGR Ser Gly; c7 AGR Ser ?; c8 AGR ? Stop; c9
AGA ? Ser; c10 AGG Ser Lys; c11 AAA Lys ?; c12 AAA Lys Asn; c13 CGN
Arg Gly. t- corresponds to loss(es) of mt-tRNAs; t+ are duplicated tRNAs; e corresponds to edited tRNAs. m corresponds to multi-partite mtDNA. l corresponds to linear mt molecule. g- protein gene loss; g+ horizontal transfer of protein gene. i- loss of group I intron; i+ gain of group II intron. f fragmented rRNA genes.
CHAPTER 6. General Conclusions
The increase in completely sequenced mito-genomes has further challenged the view the animal mitochondrial DNA (mtDNA) is a frozen genome. However, most sequencing efforts are directed toward bilaterian, and particularly vertebrate and arthropod species. This is unfortunate given that a large part of mtDNA diversity has been documented in non-bilaterian animals (e.g. [1–9]). For instance, entirely linear mtDNA has been documented only in medusozoan cnidarians [6, 7, 10–13], while some arthropods possess both linear and circular mitochondrial molecules [14–16]. In this dissertation we presented our investigation of linear mtDNA in Medusozoa. We showed that all medusozoan classes possess linear mt molecules that harbor two genes (except in Hydroidolina where these have been secondary lost): polB which is similar to B-type DNA polymerase gene family and an unidentified open reading frames (ORF314) . In addition, we found that Cubozoa species have experienced high level of segmentalization of their mtDNA into eight small and linear chromosomes. While multipartite mtDNA has been documented in nematodes [18–20], rotifers  and arthropods
, linear multipartite mitogenomes present additional challenges for the maintenance and replication of the molecules [23, 24]. We also found that unlike what has been suggested in yeast , the linear mtDNA in medusozoan appear unable to return into a circular conformation.
Mitogenomic data is a useful marker for inferring animal phylogenetic relationships
(e.g. [26, 27]). Recent mitogenomic-based phylogenetic studies (phylomitogenomics) have consistently rejected Anthozoa [Hexacorallia + Octocorallia] in favor of the clade [Medusozoa
+ Hexacorallia] [6, 28, 29]. New analyses with a dataset that contains more balanced taxon
sampling have confirmed anthozoan paraphyly. While independent analyses using genomic
data are needed to confirm, or reject, the mitogenomic view of deep cnidarian phylogeny, the
relatively high rate of sequence evolution of medusozoan mtDNA makes it a powerful too for
resolving higher taxonomic rank relationships.
Recently sequenced mtDNAs from three classes of sponges have unveiled an array of
unusual features, including the presence of an extra protein gene in Homoscleromorpha [30,
31], invasive repetitive elements  and tRNA loss in Demospongia , frameshifting and the
use of a novel genetic code in Hexactinellida [4, 32]. Yet, no mtDNA sequence from the fourth class (Calcarea) was available. Our investigation of partial mt sequences from several representatives of calcareous sponges suggests that calcarean mtDNA is multipartite and linear, encodes fragmented ribosomal RNA (rRNA) and edited transfer RNA (tRNA) genes, uses at least one, potentially two novel genetic codes for protein synthesis, and displays an exceptionally high rate of sequence evolution. These findings reveal a unique combination of unusual features never found in animal mitogenomes, and only documented in some protozoans (e.g. ).
Broader perspectives and future directions
Thanks to recent improvements in sequencing technology, the field of animal mito- genomics has reached its apotheosis. But as the cost in sequencing decreases  and better methods of genome assembly are becoming available , there is more incentives to shift
away from organelles in favor of nuclear genomes. However, as illustrated in this dissertation,
massively parallel sequencing technologies are invaluable tools for exploring genomes that
148 resist traditional approaches. For instance, sequencing the complete genome of the box jellyfish
Alatina moseri has given access to the complete sequences of its octo-chromosomal mtDNA
. Similarly, mt sequences from two out of three calcarean species presented in this dissertation were first identified from ESTs (Leucetta chagosensis) and total DNA (Petrobiona massiliana) reads. Given that several “minor” animal phyla have been completely neglected to date, one can predict that future studies that investigate the mtDNA of clades such as
Gastrotricha or Kinorhyncha using novel sequencing technologies will uncover even more unusual and interesting features.
Barcoding is an easy and inexpensive tool for species identification. In Metazoa, a region of the cytochrome c oxidase I (COI) gene is amplified and sequenced using conserved primers [36, 37]. With the steep decrease in sequencing costs, the whole mtDNA can become the favorite barcode marker for animals.
1. Gissi C, Iannelli F, Pesole G: Evolution of the mitochondrial genome of Metazoa as exemplified by comparison of congeneric species. Heredity 2008, 101:301-20. 2. Lavrov DV: Rapid proliferation of repetitive palindromic elements in mtDNA of the endemic Baikalian sponge Lubomirskia baicalensis. Molecular biology and evolution 2010, 27:757-60. 3. Lavrov DV: Key transitions in animal evolution: a mitochondrial DNA perspective. Integrative and Comparative Biology 2007, 47:734-743. 4. Haen KM, Lang BF, Pomponi S a, Lavrov DV: Glass sponges and bilaterian animals share derived mitochondrial genomic features: a common ancestry or parallel evolution? Molecular biology and evolution 2007, 24:1518-27. 5. Wang X, Lavrov DV: Seventeen New Complete mtDNA Sequences Reveal Extensive Mitochondrial Genome Evolution within the Demospongiae. PLoS ONE 2008, 3:11. 6. Kayal E, Lavrov DV: The mitochondrial genome of Hydra oligactis (Cnidaria, Hydrozoa) sheds new light on animal mtDNA evolution and cnidarian phylogeny. Gene 2008, 410:177-86. 7. Voigt O, Erpenbeck D, Wörheide G: A fragmented metazoan organellar genome: the two mitochondrial chromosomes of Hydra magnipapillata. BMC genomics 2008, 9:350. 8. Smith DR, Kayal E, Yanagihara A a, Collins AG, Pirro S, Keeling PJ: First complete mitochondrial genome sequence from a box jellyfish reveals a highly fragmented, linear architecture and insights into telomere evolution. Genome Biology and Evolution 2012, 4:52-58. 9. Signorovitch AY, Buss LW, Dellaporta SL: Comparative Genomics of Large Mitochondria in Placozoans. PLoS Genetics 2007, 3:7. 10. Bridge D, Cunningham CW, DeSalle R, Buss LW: Class-level relationships in the phylum Cnidaria: molecular and morphological evidence. Molecular Biology and Evolution 1995, 12:679-689. 11. Bridge D, Cunningham CW, Schierwater B, DeSalle R, Buss LW: Class-level relationships in the phylum Cnidaria: evidence from mitochondrial genome structure. Proceedings of the National Academy of Sciences of the United States of America 1992, 89:8750-3. 12. Warrior R, Gall J: The mitochondrial DNA of Hydra attenuata and Hydra littoralis consists of two linear molecules. Archives des Sciences, Genève 1985, 38:439-445. 13. Ender A, Schierwater B: Placozoa Are Not Derived Cnidarians: Evidence from Molecular Morphology. Molecular Biology and Evolution 2003, 20:130-134. 14. Marcadé I, Cordaux R, Doublet V, Debenest C, Bouchon D, Raimond R: Structure and evolution of the atypical mitochondrial genome of Armadillidium vulgare (Isopoda, Crustacea). Journal of molecular evolution 2007, 65:651-9. 15. Raimond R, Marcadé I, Bouchon D, Rigaud T, Bossy JP, Souty-Grosset C: Organization of the large mitochondrial genome in the isopod Armadillidium vulgare. Genetics 1999, 151:203-10.
16. Doublet V, Raimond R, Grandjean F, Lafitte A, Souty-grosset C, Marcadé I: Widespread atypical mitochondrial DNA structure in isopods (Crustacea, Peracarida) related to a constitutive heteroplasmy in terrestrial species. 2012, 244:234-244. 17. Kayal E, Bentlage B, Collins AG, Kayal M, Pirro S, Lavrov DV: Evolution of linear mitochondrial genomes in medusozoan cnidarians. Genome Biology and Evolution 2012, 4:1-12. 18. Gibson T, Blok VC, Dowton M: Sequence and characterization of six mitochondrial subgenomes from Globodera rostochiensis: multipartite structure is conserved among close nematode relatives. Journal of molecular evolution 2007, 65:308-15. 19. Armstrong MR, Blok VC, Phillips MS: A multipartite mitochondrial genome in the potato cyst nematode Globodera pallida. Genetics 2000, 154:181-92. 20. Gibson T, Blok VC, Phillips MS, Hong G, Kumarasinghe D, Riley IT, Dowton M: The mitochondrial subgenomes of the nematode Globodera pallida are mosaics: evidence of recombination in an animal mitochondrial genome. Journal of molecular evolution 2007, 64:463-71. 21. Suga K, Mark Welch DB, Tanaka Y, Sakakura Y, Hagiwara A: Two circular chromosomes of unequal copy number make up the mitochondrial genome of the rotifer Brachionus plicatilis. Molecular biology and evolution 2008, 25:1129-37. 22. Wei D-D, Shao R, Yuan M-L, Dou W, Barker SC, Wang J-J: The Multipartite Mitochondrial Genome of Liposcelis bostrychophila: Insights into the Evolution of Mitochondrial Genomes in Bilateral Animals. PloS one 2012, 7:e33973. 23. Nosek J, Tomáska L, Fukuhara H, Suyama Y, Kovác L: Linear mitochondrial genomes: 30 years down the line. Trends in genetics : TIG 1998, 14:184-8. 24. Nosek J, Tomáska Ľ: Mitochondrial Telomeres: An Evolutionary Paradigm for the Emergence of Telomeric Structures and Their Replication Strategies. In Origin and Evolution of Telomeres. edited by Nosek J, Tomáska Ľ Austin, Texas: Landes Bioscience; 2008:163-171. 25. Valach M, Farkas Z, Fricova D, Kovac J, Brejova B, Vinar T, Pfeiffer I, Kucsera J, Tomaska L, Lang BF, Nosek J: Evolution of linear chromosomes and multipartite genomes in yeast mitochondria. Nucleic acids research 2011:1-18. 26. Philippe H, Brinkmann H, Copley RR, Moroz LL, Nakano H, Poustka AJ, Wallberg A, Peterson KJ, Telford MJ: Acoelomorph flatworms are deuterostomes related to Xenoturbella. Nature 2011, 470:255-8. 27. Rota-Stabelli O, Kayal E, Gleeson D, Daub J, Boore JL, Telford MJ, Pisani D, Blaxter M, Lavrov DV: Ecdysozoan Mitogenomics: Evidence for a Common Origin of the Legged Invertebrates, the Panarthropoda. Genome Biology and Evolution 2010, 2:425-440. 28. Park E, Hwang D-S, Lee J-S, Song J-I, Seo T-K, Won Y-J: Estimation of divergence times in cnidarian evolution based on mitochondrial protein-coding genes and the fossil record. Molecular Phylogenetics and Evolution 2012, 62:329-345. 29. Shao Z, Graf S, Chaga OY, Lavrov DV: Mitochondrial genome of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa): A linear DNA molecule encoding a putative DNA-dependent DNA polymerase. Gene 2006, 381:92-101.
30. Wang X, Lavrov DV: Mitochondrial genome of the homoscleromorph Oscarella carmela (Porifera, Demospongiae) reveals unexpected complexity in the common ancestor of sponges and other animals. Molecular biology and evolution 2007, 24:363-73. 31. Gazave E, Lapébie P, Renard E, Vacelet J, Rocher C, Ereskovsky AV, Lavrov DV, Borchiellini C: Molecular phylogeny restores the supra-generic subdivision of homoscleromorph sponges (Porifera, Homoscleromorpha). PloS one 2010, 5:e14290. 32. Rosengarten RD, Sperling E a, Moreno M a, Leys SP, Dellaporta SL: The mitochondrial genome of the hexactinellid sponge Aphrocallistes vastus: evidence for programmed translational frameshifting. BMC genomics 2008, 9:33. 33. Nash E a, Nisbet RER, Barbrook AC, Howe CJ: Dinoflagellates: a mitochondrial genome all at sea. Trends in genetics : TIG 2008, 24:328-35. 34. Glenn TC: Field guide to next-generation DNA sequencers. Molecular Ecology Resources 2011, 11:759-769. 35. Baker M: De novo genome assembly: what every biologist should know. Nature Methods 2012, 9:333-337. 36. Hebert PDN, Cywinska A, Ball SL, deWaard JR: Biological identifications through DNA barcodes. Proceedings. Biological sciences / The Royal Society 2003, 270:313-21. 37. Savolainen V, Cowan RS, Vogler AP, Roderick GK, Lane R: Towards writing the encyclopedia of life: an introduction to DNA barcoding. Philosophical transactions of the Royal Society of London. Series B, Biological sciences 2005, 360:1805-11.
APPENDIX A. Supplementary Materials for Chapter 2
Table S1: List of primers used for long and short PCR amplification, sequencing and first strand cDNA synthesis for Clathrina clathrus and Leucetta chagosensis. f and r correspond to forward and reverse primers respectively.
Primer for PCR-amplification of partial Leucetta chagosensis mt genes Gene f/r Primer name Sequence (5'3') Reference cob f Leuc_cha_cob_fw AGCTACAGTTATTATTAATTTWCTWAG this work cob r Leuc_cha_cob_rv GTATCTATGGYTCTTCAATAGGTA this work cox1 f C1-J2165-fwd GAAGTTTATATTTTAATTTTACCDGG this work cox1 r COX1_1157rv-HDTYYV ACIACRTARTAIGTRTCRTG Erpenbeck et al. 2002. cox3 f Leuc_cha_cox3_fw CTATTTATCTTATCTGAAGT this work cox3 r Leuc_cha_cox3_rv AAATAATCATACAACATCAAC this work nad1 f Leuc_cha_nd1_fw ATCTATCCAACATCATC this work nad1 r Leuc_cha_nd1_rv GAAACTAATTCAGATTCTC this work Primer for PCR-amplification of partial Clathrina clathrus mt genes cob f cl_cob-f1 ACAAATCCTCTTTCAATCAGGTC this work cob r cl_cob-r1 GAAGAGTACGAACTCTAGGAGC this work cox1 f cl_cox1-f1 GAACCTATTCTGTTGATCCATGC this work cox1 r cl_cox1-r1 TAGAATGTAGACTTCAGGATGAC this work nad4 f cl-nad4-f1 GTTTCATCTTACATGAGTTAGGC this work nad4 r cl-nad4-r1 AAACTATTCCCATATGAGC this work rnl r cl-rnl-r1 CCGTTTATCTGATCTTGTAGCAG this work Primer for cDNA synthesis of Clathrina clathrus RNA genes rnl f this work cl_rnl-p1-f1 ACTGATAGAGGAACTGGAATGAG rnl f this work cl_rnl-p2-f1 ATGTCTTTAGTAGCGCACTAGCC rnl r this work cl_rnl-r0 TACGTATACCCAAGAGTCATCCG rnl r this work cl_rnl-r2 GTTCCGTTACCCAGATTC rnl r this work cl_rnl-r3 CCGTTTCATGAGCTTCAC rnl r this work cl_rnl-r4 ATCGTCATCCCATGATCC f this work rns cl_rns-f0 TAGGAATCTTGATTCATGACCTG f this work rns cl_rns-f2 TATAACGTATGGCTGTGGAGCAC f this work rns cl_rns-f3 TCGTAACATGGTCATAGTAGTGG rns f this work cl_rns-f4 ATGGTAATCCGAGTATTCTGCAC f this work rns cl_rns-f5 TAGAGTGGAGGCTACCTTCGTG r this work rns cl_rns-r1 TCCTACGAAAGTGTCCTTCCAAG f this work trnM cl_trnM-f2 ATGAGGTTCAAATCCTCATCTCC r this work trnM cl_trnM-r1 GAGTATGAATCTGCTGCTATCCC f this work trnP cl_trnP-f1 CACTTGGTATCAGTTCAACTCTG r cl_trnP-r1 this work trnP GTTCCCAAAACACCTGTTCTACC f cl_trnR-f1 this work trnR ACTGTAGCCTTCTAGTCTAAAAG r cl_trnR-r1 this work trnR AGGCTACAGTTATACCGTTAAAC trnN f cl_trnN-f1 TACTGTTAATACGATGGCATGGG this work trnN r cl_trnN-r1 CGTATTAACAGTACGAGATTCTTC this work
Table S2: Accession numbers of the sequences used in this study. The sequences for Cantharellus cibarius and Capsaspora owczarzaki were downloaded from http://megasun.bch.umontreal.ca/People/lang/FMGP/proteins.html.
Table S2: Accession numbers of the155 sequences used in this study.
Species name GenBank Accession Number
BZ10101 NC_008832 BZ243 NC_008834
Trichoplax adhaerens NC_008151
Alatina moseri JN700951-JN700958
Haliclystus sanjuanensis JN700944
Lucernaria janetae JN700946
Linuche unguiculata JN700939
Cassiopea andromeda JN700934
Catostylus mosaicus JN700940
Aurelia aurita NC_008446
Chrysaora sp JN700941
Cyanea capillata JN700937
Pelagia noctiluca JN700949
Acropora tenuis NC_003522
Astrangia sp NC_008161
Chrysopathes formosa NC_008411
Lophelia pertusa NC_015143
Madracis mirabilis NC_011160
Metridium senile NC_000933
Ricordea florida NC_008159
Savalia savaglia NC_008827
Keratoisidinae sp NC_010764
Sarcophyton glaucum AF064823 and AF063191
Briareum asbestinum NC_008073
Renilla mulleri JX023273
Dendronephthya gigantea NC_013573
Hydra magnipapillata NC_011220-NC_011221
Ectopleura larynx JN700938
Cubaia aphrodite JN700942
Millepora platyphylla JN700943
Pennaria tiarella JN700950
Clava multicornis JN700935
Laomedea flexuosa JN700945
Axinella corrugata NC_006894
Callyspongia plicifera NC_010206
Lubomirskia baicalensis NC_013760
Ephydatia muelleri NC_010202
Geodia neptuni NC_006990
Halisarca dujardini NC_010212
Ircinia strobilina NC_013662
Igernella notabilis NC_010216
Aplysina fulva NC_010203
Oscarella carmela NC_009090
Plakina monolopha NC_014884
Plakinastrella cf. onkodes NC_010217
Corticium candelabrum NC_014872
Tethya actinia NC_006991
Xestospongia muta NC_010211
Suberites domuncula NC_010496
Topsentia ophiraphidites NC_010204
Iphiteon panicea EF537576
Sympagella nux EF537577
Aphrocallistes vastus NC_010769
Clathrina clathrus This study
Leucetta chagosensis This study
Petrobiona massiliana This study
Figure S1: Phylogenetic analyses of the calcareous sponge sequences available in GenBank.
(A) Partial rnl sequences from Leucilla nuttingi, Grantia sp and Clathrina sp sequences
(Accession numbers AF035266, AF035267 and AF362006). (B) Partial atp6, cox2 and cox3 sequence for Clathrina sp (Accession numbers AF362015, AF362018 and AF362010).
Support values for the nodes have been shown for both ML and Bayesian analyses using
Treefinder and PhyloBayes respectively. Stars correspond to posterior probability > 0.95 and bootstraps > 95.
0.64/82 Clathrina sp Grantia sp Callyspongia plicifera 0.87/- Xestospongia muta Topsentia ophiraphidites 1/- Ephydatia muelleri Plakortis angulospiculatus * Oscarella carmela 1/- 0.53/- Tethya actinia Iotrochota birotulata 0.94/80 *
* Ptilocaulis walpersi Demospongiae Ectyoplasia ferox 1/- * Geodia neptuni Cinachyrella kuekanthali
Ephydatia muelleri Topsentia ophiraphidites 0.93/56 * Ptilocaulis walpersi * 0.65/69 Ectyoplasia ferox
* Geodia neptuni G4 Cinachyrella kuekanthali 100/86 Agelas schmidti 0.81/75 Tethya actinia 0.76/86 Demospongiae Iotrochota birotulata 0.92/87 Clathrina sp Xestospongia muta * Callyspongia plicifera 0.94/73 G3 Amphimedon compressa
* Halisarca dujardini * Chondrilla nucula G2 0.54/- Aplysina fulva Sympagella nux * Hexactinellida Iphiteon panicea Plakortis angulospiculatus 0.55/100 Oscarella carmela G0
Figure S2: Partial mitochondrial genome organization of the Calcaronea Petrobiona massiliana. The numbers represent the length of each contig in nucleotides. Genes are transcribed from left to right. Dark bars are non-coding regions.
Figure S3: Inferred secondary structure of the partial 3'-end of the large mitochondrial ribosomal RNA subunit (rnl) in Clathrina clathrus. The helices are named as in other sponge rRNA sequences (Wang and Lavrov, 2007). rnlp1 and rnlp2 are two pieces of the 3'-end of rRNA found in "correct" transcriptional order in Clathrina mtDNA interrupted by at DNA level (highlighted in grey). Note that additional sequences for rnl can be present in the partial genome of C. clathrus but were not found.
U A A C G U G G A G C G U A U U A U U 8 4 C U C C U A G A G U A C G C C G C U A G U U A G G C C A U A A U U A C 8 5 U U U G U G t r n S 2 U C A 7 9 A A U G A A A A G G G A G U U A U G U U U G U U A C G G C A A C A A U U A U A 7 6 S 2 A G G U U G U G C A A U A A C A A U A A A A A A A A A U C G G A G A A U A C C C U G G C U A C A A U U G G A C C U G U U 8 3 A G G C G A C A 7 A A A G G A G C A U G A G U G G G C A S 3 G U G G C G G A U G A U A G A G 8 1 7 5 A U A U G A G S 1 A G U G U C G G G A A G U C 8 0 U U G C A G C C G U A G U A G A C C C G U A U C C A U U C U U A U U C A G U A U U U U G C C A G U U G C C G A A C C A G G C A G U G A A G U U U A A G G C U U A U U U S 4 G A U A 7 8 A A G U G C A A G G U A A U G U C U C A G G C A U G A A A U G A G G U U C C C U G C G A G G N U A U A G G G A A A G U C C N A N G N G N N G U U C G U A U U U A U A C G G A G C A C G A A A A 7 0 G 6 9 A U U U N U C G 7 2 C A C A G C A A A G G A A U C A U A C U A C U A U A C C A G G G U C U A G G G A G 7 1 G U U A C A G C A A A G C C U A U U A A U G A G A A A C G U C A A G C U C U U C A U C U C A U A A U A C C G G G G G A U G A C G G A U G U G G A G U U A G G A G A A G G G C G U U G U C U G U G C C G A U G U U U G A U A C U U G A A U A A C A G U A A G G A C A G A U G U 6 8 6 7 C A G U U U G A A A A A A G A A A G U C A C A C A C G G U C U C C U U U C U C A A U C C A U C C G A A G U A U G C U G C G G C U U G U A G U U C U A C U C G C U A G G G G A A A G G A A U U C G G U U A G G G G G G U A A A C A G C A A U U U C U U A A U U A 6 1 U 6 6 4 A A U G G G C G C G A G A U C G A 6 5 U A U A G G C C A U U G C A A A 6 3 _ 1 A G A A U C A G A A C A C A A C U G C 6 2 C A U C G A A A G A A U G U A U C U A U U C U A C A A U G C U U C A G U C A A U U C G C C G U C C A G G G U U C U G A A T 4 C A C C A G U G A G U A A U C A U U G U c 5 U A G A G A G T 1 G U G A C U G U C U U U A U A A U U U G A C A U A C G C G U A G A G G U A U G G G A G A C U U U T 3 U A U U U U C A U G A A A C A G G G A A G G A G G U G C U G G U G A A U U G G A A U U C U A G A A A T 2 U A U G G C G U U U c 1 A G A t r n P A A A C U A C c 4 A G c 2 U U U U A U A U G U C A C U A G A C A U A A A U A U G U G A U G U C C A G U A G C A A A C A C C A U A C G c 3 A A A A C U U G G C G U U U N G G A A G U U C A G U A U A N A A C A G A A C A U G A A C A A U A U U A C C G A 5 9 . 1 G G A A G A U U A A A A G G A U A U A U U G A A A G G A N U G A G G U A A U A C U A A U C C G C U U A U U A C C G A A G U G G G A C A U G U G 4 9 U G 4 8 A C A A A A A A G C A A A G G A G A A C U C 4 9 _ 1 U G A 5 2 C C A U N A U C A A G C A A G 5 0 G U C A G A A U C U A U U U G U U C A G G A C A A C U G G G G G U A C A A U U C U A G U U U A A C U G G A A G A C U G C N A C U A G A 4 6 A U G A A U G U U G G U A U A A G G G A A G C G A G A A A C C C U A A U A A A C G U U G U A A C A U G A
Figure S4: Phylogenetic analysis of concatenated amino acid sequences from representative non-bilaterian species for atp6, cob, cox1, cox3, and nad1-5 genes under the GTR+CAT models of sequence evolution using RAxML v.7.2.6. A star shows maximum support for that node. Calcareous species have been underlined.
Linuche unguiculata Coronatae Alatina moseri Cubozoa * Haliclystus sanjuanensis Lucernaria janetae Staurozoa Cubaia aphrodite * Clava multicornis Laomedea flexuosa * Millepora platyphylla Hydrozoa Pennaria tiarella * Ectopleura larynx Hydra magnipapillata * Cyanea capillata * Chrysaora sp * Pelagia noctiluca * Aurelia aurita Discomedusae * Cassiopea andromeda Catostylus mosaicus Renilla mulleri * Keratoisidinae sp Briareum asbestinum Dendronephthya gigantea Octocorallia Sarcophyton glaucum Savalia savaglia * Metridium senile Chrysopathes formosa Acropora tenuis * Ricordea florida Hexacorallia * Lophelia pertusa * Astrangia sp Madracis mirabilis * Igernella notabilis Ircinia strobilina Keratosa (G1) Aplysina fulva Halisarca dujardini Myxospongiae (G2) * Callyspongia plicifera Xestospongia muta marine Haplosclerida (G3) * * Ephydatia muelleri Lubomirskia baicalensis Axinella corrugata Geodia neptuni Topsentia ophiraphidites Democlavia (G4) * Suberites domuncula Tethya actinia Oscarella carmela * Corticium candelabrum * Plakina monolopha Homoscleromorpha (G0) Plakinastrella cf. onkodes * Aphrocallistes vastus * Iphiteon panicea Hexactinellida Sympagella nux Clathrina clathrus * Leucetta chagosensis Petrobiona massiliana
APPENDIX B. SUPPLEMENTARY MATERIALS FOR CHAPTER 3
Supplementary Fig. S1: Enzymatic digestion of Alatina moseri DNA with the methylation-sensitive restriction enzyme HpaII. Digestion of A. moseri total DNA with HpaII followed by PCR amplification of two mitochondrial contigs containing up to four restriction sites. Lane A and B have been digested for three days with HpaII prior to PCR; lane C and D were obtained with control non-digested DNA. Digestion by HpaII prevents PCR amplification (lane A and B), while DNA methylation in the nucleus shall prohibit digestion of the DNA by the enzyme, resulting in successful PCR amplifications (lane C and D).
Supplementary Fig. S2: Phylogenetic relationships of polB sequences found in medusozoan mtDNAs. Phylogenetic reconstruction of polB sequences under Maximum Likelihood framework with TREEFINDER v. March 2011 using mtREV+GI model of sequence evolution. All medusozoan polB sequences (in blue) form a monophyletic clade. Node supports are bootstrap values for 5000 replicates.
APPENDIX B. SUPPLEMENTARY MATERIALS FOR CHAPTER 4
Additional figure 1 – Species list
List of species and accession numbers used for this studies. Bold species are from this study.
Phylum Subphylum Class Subclass Order species accession number Cnidairia Anthozoa Hexacorallia Actiniaria Metridium senile NC_000933 Nematostella sp NC_008164 Antipatharia Chrysopathes formosa NC_008411 Cirripathes lutkeni JX023266 Leiopathes glaberrima FJ597643-FJ597644 Corallimorpharia Discosoma sp NC_008071 Rhodactis sp NC_008158 Ricordea florida NC_008159 Scleractinia Acropora tenuis NC_003522 Agaricia humilis NC_008160 Anacropora matthai NC_006898 Astrangia sp NC_008161 Colpophyllia natans NC_008162 Lophelia pertusa NC_015143 Madracis mirabilis NC_011160 Montastraea annularis NC_007224 Montastraea faveolata NC_007226 Montastraea franksi NC_007225 Montipora cactus NC_006902 Mussa angulosa NC_008163 Pavona clavus NC_008165 Pocillopora damicornis NC_009797 Pocillopora eydouxi NC_009798 Porites porites NC_008166 Seriatopora caliendrum NC_010245 Seriatopora hystrix NC_010244 Siderastrea radians NC_008167 Stylophora pistillata NC_011162 Zoantharia Palythoa sp DQ640650 Savalia savaglia NC_008827 Ceriantharia Ceriantheopsis americanus JX023261-JX023265 Octocorallia Alcyonacea Acanella eburnea NC_011016 Briareum asbestinum NC_008073 Corallium konojoi NC_015406 Dendronephthya gigantea NC_013573 Dendronephthya castanea GU047877 Dendronephthya mollis HQ694725 Dendronephthya putteri HQ694726 Dendronephthya suensoni GU047878 Echinogorgia complexa HQ694727 Euplexaura crassa HQ694728 Keratoisidinae sp NC_010764 Paracorallium japonicum NC_015405 Pseudopterogorgia NC_008157 Sarcophyton glaucum AF064823 & AF063191 Scleronephthya gracillimum GU047879 Sinularia sp JX023274 Helioporacea Heliopora coerulea JX023267-JX023272 Pennatulacea Renilla mulleri JX023273 Stylatula elongata JX023275 Medusozoa Cubozoa Carybdeida Alatina moseri JN700951-JN700958 Carukia barnesi JN700959-JN700962 Carybdea xaymacana JN700977-JN700983 Chirodropida Chironex fleckeri JN700963-JN700968 Chiropsalmus quadrumanus JN700969-JN700974 Hydrozoa Trachylina Limnomedusae Cubaia aphrodite JN700942 Hydroidolina Aplanulata Ectopleura larynx JN700938 Hydra magnipapillata NC_011220-NC_011221 Hydra oligactis NC_010214 Hydra vulgaris BN001179-BN001180 Capitata Millepora platyphylla JN700943 Pennaria tiarella JN700950
Filifera III Clava multicornis JN700935 Filifera IV Nemopsis bachei JN700947 Leptothecata Laomedea flexuosa JN700945 Scyphozoa Coronatae Linuche unguiculata JN700939 Discomedusae Rhizostomeae Cassiopea andromeda JN700934 Cassiopea frondosa JN700936 Catostylus mosaicus JN700940 Rhizostoma pulmo JN700987 Semaeostomeae Aurelia aurita A NC_008446 Aurelia aurita B HQ694729 Chrysaora sp JN700941 Chrysaora quinquecirrha HQ694730 Cyanea capillata JN700937 Pelagia noctiluca JN700949 Staurozoa Stauromedusae Craterolophus convolvulus JN700975 & JN700976 Haliclystus sanjuanensis JN700944 Lucernaria janetae JN700946 Placozoa BZ10101 NC_008832 BZ243 NC_008834 BZ49 NC_008833 Trichoplax adhaerens NC_008151 Porifera Homoscleromorpha Corticium candelabrum NC_014872 Oscarella carmela NC_009090 Plakina monolopha NC_014884 Plakinastrella cf. onkodes NC_010217 Demospongiae G1=Keratosa Igernella notabilis NC_010216 Ircinia strobilina NC_013662 G2=Myxospongiae Aplysina fulva NC_010203 Chondrilla aff. nucula NC_010208 Halisarca dujardini NC_010212 G3=marine Haplosclerida Amphimedon compressa NC_010201 Callyspongia plicifera NC_010206 Xestospongia muta NC_010211 G4=Democlavia Agelas schmidti NC_010213 Axinella corrugata NC_006894 Cinachyrella kuekanthali NC_010198 Ephydatia muelleri NC_010202 Geodia neptuni NC_006990 Lubomirskia baicalensis NC_013760 Suberites domuncula NC_010496 Tethya actinia NC_006991 Topsentia ophiraphidites NC_010204
Additional Figure 2 – Cnidarian phylogeny of mitochondrial protein genes using the alternative AliMG alignment.
Phylogenetic analyses of cnidarian protein coding genes under the CATGTR model with
PhyloBayes for the AliMG alignments (3485 positions, 106 taxa) created using the combination MUSCLE+Gblocks. Support values correspond to the posterior probabilities for
166 the GTR(BI) and bootstraps for GTR(ML) analyses. Stars denote maximum support values. A dash denotes discrepancy between the results obtained by different models.
* Placozoa 0.99/1/100 Porifera * Zoantharia
* * Actiniaria 1/1/78 * Antipatharia 1/1/91 * H * Corallimorpharia 1/1/91 e
* * a * * * c
a 0.51/-/16 * l * * Scleractinia l
* a * *
* * 1/1/96 1/1/94 * Ceriantharia Helioporacea 0.99/-/12 Alcyonacea *
O 1/1/96 Pennatulacea
0.63/-/45 * c
* * o 0.67/-/-
0.47/-/- a * Alcyonacea
1/1/99 l 1/1/74 l
0.74/0.89/49 a 1/0.98/54 1/1/99 0.95/1/92 0.55/-/20 Coronatae * 0.87/-/43 Stauromedusae Staurozoa 0.83/1/58 0.99/1/99 0.37/0.62/65 Carybdeida * Cubozoa * Chirodropida * Limnomedusae H * * Filifera III Filifera IV y
* Leptothecata d r
0.99/-/- 1/1/95 Capitata o z 1/1/70 * o
* Aplanulata a
e a s u d e m o c s i D
* * * Semaeostomeae * * 0.85/1/89 * * 0.5 * Rhizostomeae
Additional Figure 3 – Cnidarian phylogeny of mitochondrial protein genes using the reduced alignments AliS and AliMG with 103 species.
Phylogenetic analyses of cnidarian protein coding genes under the GTR model and CAT approximation with RAxML for the AliS alignments created using CluatalW+SOAP, where the coronate Linuche unguiculata, the tube anemone Ceriantheopsis americanus and the blue octocoral Heliopora coerulea were removed. Support values correspond to the bootstraps for both AliS and AliMG alignments. Stars denote maximum support values. A dash denotes discrepancy between the two alignments.
* Porifera 97/86
* * 79/78 *
95/91 * *
97/90 * 84/88 * * 99/100 * Hexacorallia * *
99/100 * * * * *
91/96 73/97 99/100
37/- * * 77/67 * Octocorallia 98/99 33/- 99/100
95/97 51/74 84/88
* 43/40 Staurozoa 85/90 37/58 97/98 * Cubozoa *
* * * 91/88 Hydrozoa * 98/95
82/87 * 66/77 * *
* * 0.2 99/100
* * 86/87 Discomedusae * * 99/100
Additional Figure 4 – Composition of the amino acid alignment used in this study.
Principal component analysis of the amino acid composition per species for each of the alignments (AliS and AliMG) used in this study. Species have been color-coded per group corresponding to each of the main cnidarian clades (Coronatae, Cubozoa, Discomedusae,
Hexacorallia, Hydrozoa, Octocorallia, and Staurozoa), Porifera and Placozoa. The axes explain
33 and 23 per cent of the data for AliS and 33 and 22 percent for AliMG.
Additional figure 5 – The use of several statistical tests verifying the validity of some groups in cnidarians for the reduced alignments.
Probability values for the AU, KH and SH tests and BI values for several clades traditionally recognized in Cnidaria for the reduced alignments AliS and AliMG containing 103 species, where the coronate Linuche unguiculata, the tube anemone Ceriantheopsis americanus and the blue octocoral Heliopora coerulea were removed.
!"#$%&'() !"#-.%&'() $*+, -/01"2343.5"*160 !" #$ %$ !" #$ %$ !&'()*+,( -./0 -./1 -.02 -.23 -.44 -.0- %&5676(*898:;<676(898$5,'676( -.10 -.11 -.=/ -.=> -.== -.>0 %&5*?676(898:;<676(898%@(;'676( -.-1 -.-= -.1> -.-/ -.1/ -.=4 %&5*?676(898%@(;'676( -.-0 -.-/ -.11 -.-= -.-= -.=-
Additional figure 6 – Table of morphological characters mapped on the best tree.
Mapping of morphological characters under DELTRAN and ACCTRAN models differed only for characters (5) and (7). (1) symmetry: medusozoan taxa have been scored radial, but
Marques and Collins (2004) subdivided it into radial, biradial, or radial tetramerous;
Octocorallia, Zoantharia and Ceriantharia are scored bilateral (Won et al. 2001), while the remnant of Hexacorallia was scored radial (Marques and Collins 2004); bilateral symmetry is inferred the ancestral state for Cnidaria. (2) free-swimming medusoid stage: Hexa-,
Octocorallia and Staurozoa all lack a free-living adult form (Marques and Collins 2004); the medusoid stage is also lacking in some Hydrozoa species but Collins (2002) and Cartwright and Nawrocki (2010) have inferred that the ancestral Hydrozoa had a medusoid stage. (3)
172 velum: only present in Hydrozoa (Marques and Collins 2004). (4) strobilation: only described in Coronatae and Discomedusae (Marques and Collins 2004). (5) gastric filaments: we scored them as present in Cubozoa (Brigde et al. 1995), Staurozoa, Coronatae and Discomedusae
(Marques and Collins 2004), and absent in Hydrozoa (Brigde et al. 1995), although suggested to be present in some Aplanulata (Bouillon et al. 2004); we decided to opt for the most parsimonious scenario. (6) ephyrae: only described in Coronatae and Discomedusae (Marques and Collins 2004). (7) radial canals: described in Coronatae, Discomedusae, and some
Hydrozoa, absent in Staurozoa and unresolved in Cubozoa (Marques and Collins 2004); we choose two independent gains in Coronatae and Discomedusae as the most parsimonious scenario. (8) circular canals: present in Discomedusae and Hydrozoa, absent in the rest of
Medusozoa. Marques and Collins (2004) have further distinguished the level of development of these structures that are partial in Discomedusae and full in Hydrozoa. (9) quadrate symmetry of horizontal cross section: present only in Cubozoa and Staurozoa (Marques and
Collins 2004). (10) organization of the gastrodermal muscles of the polyp: organized in bunches of gastrodermic origin in all Hexacorallia but Ceriantharia, and inferred as the ancestral state for Cnidaria; organized in bunches of epidermic origin in all Medusozoa but
Hydrozoa. In Hydrozoa and Ceriantharia, gastrodermal muscle are not organized in bunches
(Marques and Collins 2004).
! " # $ % & ' ( ) !* Actiniaria 1 0 0 0 0 0 0 0 0 1 Antipatharia 1 0 0 0 0 0 0 0 0 1 Corallimorpharia 1 0 0 0 0 0 0 0 0 1 Scleractinia 1 0 0 0 0 0 0 0 0 1 Zoantharia 0 0 0 0 0 0 0 0 0 1 Ceriantharia 0 0 0 0 0 0 0 0 0 2 Alcyonacea 0 0 0 0 0 0 0 0 0 1 Helioporacea 0 0 0 0 0 0 0 0 0 1 Pennatulacea 0 0 0 0 0 0 0 0 0 1 Carybdeida 1 1 0 0 1 0 ? 0 1 0 Chirodropida 1 1 0 0 1 0 ? 0 1 0 Limnomedusae 1 1 1 0 0 0 1 1 0 2 Aplanulata 1 1 1 0 0 0 1 1 0 2 Capitata 1 1 1 0 0 0 1 1 0 2 Filifera III 1 1 1 0 0 0 1 1 0 2 Filifera IV 1 1 1 0 0 0 1 1 0 2 Leptothecata 1 1 1 0 0 0 1 1 0 2 Coronatae 1 1 0 1 1 1 1 0 0 0 Rhizostomeae 1 1 0 1 1 1 1 1 0 0 Semaeostomeae 1 1 0 1 1 1 1 1 0 0 Stauromedusae 1 0 0 0 1 0 0 0 1 0
First of all, I would like to thank Dennis V. Lavrov for having admitted me as his student, and who, despite all our ups and downs, restrained himself from throwing me out of his lab. That was not an easy task. I also thank my POS Committee members Anne Bronikowski, John Downing, Eric Henderson, Stephan Q. Schneider, and Jeanne Serb for their support. I would like to acknowledge the past and present members of the Lavrov Lab (Karri Haen, Xiujuan Wang, Romulo Segovia, and Walker Pett) for also coping with me all these years. I am particularly indebted to Andrea Bekic, Philip Lange, Selena Leonard, and Matthew Palen for their help in the lab and more. I would also like to mention some of my fellow graduate students Arun Sethuraman, Matthew Karnatz, Alvin Mercado Alejandrino, and many others, and tell them "Good luck!" I would like to thank the Wendel Lab, and particularly Corrinne Grover and Kara Grupp for their help in acquiring data and moral support during the hard periods. I also thank Dr. Jonathan Wendel and the EEOB Department for supporting for most of my time at ISU. I would also like to acknowledge the Porifera Tree of Life project (www.portol.org) and particularly Dr. Allen G. Collins, Dr. Robert W. Thacker and Dr. Cristina Diaz for their support in my work. I thank the Smithsonian Institution for having provided me with an opportunity to work at the National Museum of Natural History, and all the people from the LAB group, particularly Matthieu and Natalia. I also want to thank Hervé Philippe, Béatrice Roure and Henner Brinkmann for their help regarding phylogenetics and de- growth! I would not have been able to survive Ames for four years without the friendship of Zee, Rishi, Michael, Scott, Lando, the food-and-movie addicts (Paula, Diego, Tomoko, Kaya, Basak and Fatih, Giorgi, did I forget anyone?), the French collection (among other Boris and Jane, Estelle and Etienne, Jean-Pierre, Sophie, Charlène, Fabienne, Camille, Stéphanie), Casino Tehran members (Mostafa, the Alis, Fardad, the Mohammads, Dr Mina and family, Reza and Jami, Mirzad and Mina, Elham and Pedram, Omid, Hamed, and many more) and many more. Finally, this PhD work could not have been achieved without the help and support of my family (thanks bro!), Hannah and her family, and many friends back in France and other parts of the World who believe in me.