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Home , Echinidae, Echinoida, Echinozoa, Eleutherozoa, Loveniidae, Loxechinus albus

Mol Biol Rep DOI 10.1007/s11033-014-3847-5

Complete mitogenome of the edible : genetic structure and comparative genomics within

Graciela Cea • Juan Diego Gaita´n-Espitia • Leyla Ca´rdenas

Received: 7 May 2014 / Accepted: 25 November 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract The edible Chilean , Loxechinus , whereas the other included species of albus, is the only species of its and endemic to the and Pseudocentrotus. Southeastern Pacific. In this study, we reconstructed the mitochondrial genome of L. albus by combining Sanger Keywords Loxechinus albus Mitochondrial genome and pyrosequencing technologies. The mtDNA genome Genome architecture Echinodermata had a length of 15,737 bp and encoded the same 13 pro- tein-coding genes, 22 transfer RNA genes, and two ribo- somal RNA genes as other mtDNAs. The size of Introduction this mitogenome was similar to those of other Echinoder- mata species. Structural comparisons showed a highly Metazoan mitochondrial DNA (mtDNA) is typically a conserved structure, composition, and gene order within circular double-stranded molecule of approximately Echinoidea and Holothuroidea, and nearly identical gene 12–20 kb length that contains 13 protein-coding genes organization to that found in Asteroidea and Crinoidea, (PCGs) involved in oxidative phosphorylation (OXPHOS), with the majority of differences explained by the inversions 22 transfer RNA (tRNA) genes necessary to translate the of some tRNA genes. Phylogenetic reconstruction sup- PCGs, two ribosomal RNA genes (rRNA), and a major AT- ported the monophyly of Echinozoa and recovered the rich non-coding region usually denominated the mito- monophyletic relationship of Holothuroidea and Echinoi- chondrial control region [1, 2]. Although the gene content dea. Within Holothuroidea, Bayesian and maximum like- and organization are notably conserved in most animal lihood analyses recovered a sister-group relationship phyla [3], some differences can be found among taxa; in between Dendrochirotacea and Aspidochirotida. Similarly particular, tRNA genes switch position more frequently within Echinoidea, these analyses revealed that L. albus than do larger protein-coding and rRNA genes [2, 4, 5]. In was closely related to , both being this context, a wide range of comparative studies have used part of a sister group to and Echino- mitochondrial genomes (mitogenomes) as phylogenetic metridae. In addition, two major were found within markers to resolve deep evolutionary relationships [6]by Strongylocentrotidae. One of these clades comprised all of comparing entire mitogenome sequences [1, 7–10]orby the representative species and analyzing shared genome rearrangements (e.g., inversions and transpositions) at different taxonomic levels [3, 11, 12]. Electronic supplementary material The online version of this Thanks to rapid advances in next generation sequencing article (doi:10.1007/s11033-014-3847-5) contains supplementary material, which is available to authorized users. (NGS) techniques in recent years, the availability and number of entire mitogenome sequences for different tax- G. Cea J. D. Gaita´n-Espitia L. Ca´rdenas (&) onomic groups have considerably increased [10, 13]. To Facultad de Ciencias, Instituto de Ciencias Ambientales y date, a total of 38 mitogenomes of have been Evolutivas, Universidad Austral de Chile, Casilla 567, Valdivia, Chile described and are publicly available in the Organelle e-mail: [email protected] Genome Resources of NCBI (http://www.ncbi.nlm.nih. 123 Mol Biol Rep gov). Of these mtDNA sequences, four represent , was extracted from gonads of eleven adults using a com- eight ophiuroids, eight asteroids, five holothurians, and 13 mercial RiboPureTMKit (Ambion, Carlsbad, CA, USA). echinoids. Comparative genomics within the phylum RNA quality was analyzed by Agilent 2100 Bioanalyzer Echinodermata have identified a very different gene order RNA assays (Santa Clara, CA, USA) and evaluated by involving at least eight rearrangement events (two inver- calculating the ratio of the 28S and 18S rRNA intensity sions, three transpositions, and three tandem-duplication– peaks. Total RNA was then submitted to Macrogen, Seoul, random-loss events) [12]. This order places crinoids and Korea (http://www.macrogen.com) for cDNA library con- ophiuroids, with the presence of several unassigned struction, library normalization, and cDNA pyrosequencing sequences (UAS) and some pseudogenes, as the most in a 454-GS FLX Titanium System (454 Life Sciences, divergent groups [14]. Additionally, holothurians and Branford, CT, USA). echinoids possess the highest degree of conservation in mtDNA gene order, with few changes in gene structure and Mitogenome assembly and annotation strong similarities in the origin of replication [15]. In particular, the mitogenome architecture of the latter group The raw data was trimmed and assembled using CLC (sea urchins and sand dollars) has been characterized by a Genomics Workbench software (CLC Bio, Aarhus, Den- pattern of organization unique from that of other echino- mark), using the parameters described in [21]. Gene derms [12, 16]. annotation and mapping were performed using Blast2GO Within the class Echinoidea, the edible Chilean red sea software [22] according to the main categories of Gene urchin, Loxechinus albus, is the only species of its genus. Ontology (GO; molecular functions, biological processes This sea urchin is distributed throughout the Chilean coast, and cellular components) [23]. From this database, genes from Arica (18°S) to Tierra del Fuego (55°S) [17]. Loxe- were filtered by homology (Blastx algorithm v2.2.29) to chinus albus is one of the most economically important those of other echinoids (Table 1) using Geneious Pro 5.5.6 species in the littoral benthic systems of the southeastern software [24]. To complete the remaining tRNAs and to Pacific in South America [18]. Harvesting of L. albus verify the intergenic region we design PCR primers for represents the largest extraction volume among world walking amplification (Table S1) using traditional Sanger urchin fisheries [19]. However, overfishing of the red sea sequencing. Similar procedure was used to validate the urchin along Chilean coasts is driving the decline or sequence quality of some genes with ambiguous features depletion of natural populations of this biological resource (e.g. nad4) by using independent primers located in the [20]. Currently, no information exists on the population flanking regions of the target genes (Table S1). genetic background of L. albus or its functional genomics Several PCRs were set up for a total of 10 sea urchins. that could link the metabolic responses of this species (e.g., Each reaction (25 lL total volume) consisted of 2 lLof expression of genes involved in OXPHOS) to the great DNA template, 2.5 lL109 PCR buffer, 0.5 lL BSA, environmental variation experienced along the Chilean 1 lL MgCl2 (2 mM), 1.5 lL of each primer, 0.5 lLof coastal upwelling. In this study, we reconstruct the com- dNTP mix (2.5 mM), 0.6 lL of Taq DNA polymerase plete mitogenome of L. albus by combining Sanger and (Invitrogen, Carlsbad, CA, USA), and 15.4 lL sterile dis- 454 sequencing technologies. Furthermore, we character- tilled water. PCR was performed with the following ized general features such as genome length, nucleotide parameters: an initial cycle of denaturation at 95 °C for biases, and gene structure and compared them with those of 5 min; followed by 30 cycles at 95 °C for 5 min, 50 °C for other echinoderms. Finally, we established the taxonomic 1 min, and 72 °C for 1 min; and terminated by a final relationships of L. albus at the class and family level. This extension at 72 °C for 10 min. PCR products and sequen- work should be useful for studies on and con- ces were developed at Macrogen. servation genetics, as well as for those that integrate functional genomics with the physiology of this species Sequence analysis and genome annotation under environmental variation and climate change scenarios. Protein coding genes (PCGs), rRNA and noncoding sequences were identified by comparing alignments of homologous genes within complete mitogenomes of other Materials and methods echinoids (Table 1) using the BLAST tool implemented in Geneious Pro 5.5.6 [24]. The boundaries of both PCGs and RNA preparation, sequencing, and cDNA library rRNA genes were adjusted manually based on the locations of adjacent genes and the first start and stop codons in Samples of L. albus were collected in Los Molinos frame. All tRNA genes were located and folded into their (39°400S–73°120W), southern Chile. Total L. albus RNA proposed clover-leaf structures to confirm their secondary 123 Mol Biol Rep

Table 1 List of the Echinodermata species included in the present study Taxonomic position GenBank accession Genome Length (bp) Class Order Family Species

Echinoidea Loxechinus albus JX888466 15,737 Paracentrotus lividus J04815 15,696 Strongylocentrotidae Strongylocentrotus purpuratus X12631 15,650 Strongylocentrotus fragilis* KC898200 15,748 Strongylocentrotus intermedius* KC898198 15,718 Strongylocentrotus droebachiensis EU054306 15,710 Strongylocentrotus pallidus KC898197 15,552 Mesocentrotus nudus JX263663 15,709 Mesocentrotus franciscanus KJ526170 15,650 Pseudocentrotus depressus* KC898203 15,736 Hemicentrotus pulcherrimus* KC898202 15,721 crassispina KC479025 15,702 Evechinus chloroticus* PRJNA190637 – Arbacoiida Arbaciidae Arbacia lixula X80396 15,719 cordatum FN562581 15,767 Maretiidae Nacospatangus alta KC990834 15,763 Holothuroidea Aspidochirotida Holothuriidae Holothuria forskalii FN562582 15,841 Stichopodidae Stichopus horrens HQ000092 16,257 Parastichopus nigripunctatus AB525762 16,112 Apostichopus japonicus AB525760 16,106 Dendrochirotacea Cucumariidae Cucumaria miniata AY182376 17,538 Asteroidea Forcipulatida Asteriidae Pisaster ochraceus X55514 14,837 Ophiuroidea Ophiurida Ophiocomidae FN562577 17,383 * Partial sequences of the mitochondrial genome structures using tRNAscan-SE v.1.21 [25] and the gen- with default settings. Additionally, sequence regions that eralized mitochondrial tRNA settings. Addi- included mainly gaps were removed from the alignments tionally, tRNA genes were adjusted by visual inspection (Table S2). based on specific anticodons in regions between identified genes. Phylogenetic analyses

Alignment Nucleotide sequences for individual PCG alignments were concatenated before the phylogenetic analysis (Supple- PCGs of L. albus and other eleutherozoans (asterozoans mentary File 1). These sequences included some repre- and echinozoans) obtained from GenBank (Table 1) were sentatives of Echinoidea, Holothuroidea, Asteroidea, and translated into amino acid sequences using the echinoderm Ophiuroidea (Table 1). These last two groups were used as mitochondrial genetic code and aligned separately using outgroups in the phylogenetic reconstruction, because As- the TranslatorX multiple sequence alignment program [26]. terozoa is the sister group of Echinozoa within subphylum Alignments were done using the MAFFT platform of [16]. Nucleotide composition was evaluated TranslatorX with the L-INS-i option (accurate for align- using Geneious Pro 5.5.6 [24]. ment of B200 sequences) and default settings. The align- Best partition scheme (BPS) analysis for the concate- ments were back-translated into the corresponding nated PCG alignment (Table S3) was conducted with the nucleotide sequences. This alignment procedure helped PartitionFinder program [28] using a heuristic search avoid the destruction of codons and displacement of algorithm and the Bayesian information criterion (BIC). nucleotides and aimed to obtain a reliably homologous This allowed us to compare different partition schemes for region [26, 27]. Ambiguously-aligned sites were removed each codon position in each gene. This method is an using Gblocks v.0.19b implemented in TranslatorX [26] accurate strategy to account for variable evolutionary

123 Mol Biol Rep histories of different loci in mitochondrial phylogenomic Singleton sequences are not generally used in an EST analyses [29]. A total of 39 data blocks were defined, with project, and hence are not included in transcriptome ana- one data block for each codon position in each gene. lysis [39, 40]. In this study, we obtained a high number of Maximum likelihood (ML) inference was performed with mitosequences from the singleton database, with an aver- RAXML v.7.2.6 [30] using the RAxML-GUI graphical age of 1,648 sequences per gene (i.e. reads of the same interface [31], the GTRGAMMA model, and the rapid sequence without overlap), highlighting the usefulness of bootstrap option with 1,000 replicates. In addition, a the singleton database. It has been shown that the pyrose- Bayesian inference (BI) MCMC analysis was conducted quencing technology employed by 454 sequencing using MrBayes v.3.2 [32]. The rate parameter was allowed machines produces characteristic sequencing errors, mostly to vary. Parameter estimation was ‘‘unlinked’’ for the imprecise signals for longer homopolymers runs [41]. In shape of the gamma distribution used to model rate vari- order to deal with these problems we develop a stringent ation between sites, the substitution matrix, the proportion quality control using reads with high score of quality (i.e. of invariable sites, and the estimation of state frequencies. over 40 Q-score), and we used the condensation tool found Six Markov chains were used, and each chain was started in the NextGENe software v2.3.3 (Softgenetics, State from a random tree. The ‘‘temperature’’ parameter was set College, PA, USA). Additionally, we used Sanger to a default value of 0.2. Two simultaneous runs of sequencing as an alternative method to verify and complete 10,000,000 generations were conducted, and trees were detected gap in the mitogenome reconstruction. The sampled every 1,000 generations. To establish whether the method employed here to obtain the mitogenome of L. Markov chains had reached a steady state, we plotted the albus (using EST transcripts) has only been previously -ln likelihood scores of sampled trees against generation used a few times in other marine and terrestrial organisms time using Tracer v.1.5 [33]. Trees inferred prior to sta- (two ascidian species, Halocynthia roretzi and Ciona in- tionarity (i.e., lack of improvement in the likelihood score) testinalis [42]; two tiger salamanders, Ambystoma mex- were discarded as burn-in (first 10 % of the sampled trees), icanum and A. t. tigrinum [43]; two sea lice, Caligus and the remaining trees were used to construct a 50 % clemensi and C. rogercresseyi [44]; a marine gastropod, majority-rule consensus tree. Concholepas concholepas [21] ) and recently in the giant red sea urchin Mesocentrotus franciscanus [45].

Results and discussion General features of the mitogenome

Here we report the complete mitogenome sequence of the The complete nucleotide sequence of the L. albus mtDNA sea urchin Loxechinus albus, the sole representative of its was determined and deposited in the Organelle Genome genus and native to the southeastern Pacific. Traditionally, Resources of NCBI with accession number JX888466. long PCR has been used to sequence whole mitogenomes Overall, the mitogenome of L. albus was a circular mole- of metazoans. However, the results of this study support cule of 15,737 bp in length (Fig. 1b) and contained the previous reports suggesting that NGS is a viable method to same 37 genes found in other metazoans: 13 PCGs, 2 sequence organellar genomes because of its high coverage, rRNAs, and 22 tRNAs (Fig. 1a; Table 2). From these 37 high quality, and lower cost [34–37]. Furthermore, some mtDNA genes, only six were coded on the minus strand: authors have shown that the 454 pyrosequencing approach tRNA-Q, tRNA-A, tRNA-V, tRNA-D, tRNA-S (UGA), and is more reliable than traditional sequencing methods (e.g., nad6 (Fig. 1; Table 2). The total length of the L. albus Sanger) for highly AT-rich regions like those found in mitogenome was similar to those of other Echinodermata mitogenomes [36, 38]. mitogenomes (Table 1), and it had the same transcriptional From the transcriptome sequencing we obtained a total orientation [12, 16]. New rearrangements were not found in of 41,658 contigs (i.e., consensus sequences derived from L. albus, consistent with the highly conserved genome overlapping reads) with an average read length of 593,8 bp architecture of Echinoidea [12]. and 136,520 singletons (i.e., non-overlapping reads). In Overall, the mtDNA genome of L. albus included 20 total, we identified 506 contigs (1.2 % of total contigs) and intergenic spacers of 1–139 bp. In addition, a total of 37 24,675 singletons (18 % of total singletons) that were overlapping zones were found in this mitogenome, with the associated with mitogenome and covered 99 % of the mi- largest overlap having a length of 16 nucleotides and being togenome of L. albus. These reads covered information located between atp8 and atp6. The overall base compo- derived from 28 genes (cox1-3, cob, nad1-6, nad4L, atp6, sition of this mitogenome showed a high AT content, atp8, 16s rRNA, 12s rRNA, and 13 tRNAs). The missing similar to that of P. lividus but higher than in other echi- 1 % was mostly related to tRNAs. noids (Table S4).

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AB

Fig. 1 Comparative mitogenomics within Echinodermata. a Linear direction. b Representation of the mitochondrial complete genome of representation of the gene order and t/rRNA locations in five classes the red sea urchin Loxechinus albus. The 13-coding genes are in of echinoderms. Mitochondrial genomes scaled to 100 %. tRNAs are green, two rRNAs in red, and 22 tRNAs in blue. Black lines represent denoted by single-letter codes according to the amino acid they sequences obtained from EST contigs/singletons from 454 sequenc- represent: A, Ala; G, Gly; P, Pro; T, Thr; V, Val; S, Ser; R, Arg; L, ing. Orange ovals represent regions obtained from PCR ? Sanger Leu; F, Phe; N, Asn; K, Lys; D, Asp; E, Glu; H, His; Q, Gln; I, Ile; M, sequencing Met; Y, Tyr; C, Cys; W, Trp). Shaded boxes represent the reverse

Mitogenome organization, PCGs, tRNAs, and rRNAs ATG, whereas only the cob (TAA) stop codon is produced genes by polyadenylation in mRNA processing. The existence of non-canonical start codons (i.e., ATT, GTT and TTG) has The mtDNA genome of L. albus had identical genome been described in other echinoderms [15, 45, 47, 48]. On organization to that reported for other echinoids (Fig. 1a) the other hand, twenty-two tRNA genes were identified in [16]; it was similar to the mitogenome, the red sea urchin mitogenome through analysis of differing only in the transposition of the nad4 and 16S- sequence similarity and potential secondary structure rRNA genes [1, 15, 16]. Overall, the mitogenome of L. (Figure S1). The tRNAs were predicted to fold into the albus shared the typical conserved segment of all Echino- expected secondary clover-leaf structures with normal base dermata classes, which is located between cox1 and cob pairings and some atypical pairings. The tRNAs ranged in (Fig. 1a) [14, 46]. Additionally, the putative replication size from 65 (tRNA-Ser)to73(tRNA-Leu CUN, tRNA-Leu origin of L. albus was highly compact, with a length of UUR, tRNA-Phe, and tRNA-Met) nucleotides in length 136 bp, and located between the tRNA-Thr and tRNA-Pro (Table 2). Most were located in a cluster of 4.1 kb between genes (Fig. 1b). This feature is also shared with holothu- tRNA-Thr and Cox1 (Fig. 1a). This cluster is unique within roids (except Cucumaria miniata) (Fig. 1a). Echinodermata, with some rearrangements in Ophiuroidea, The L. albus mitogenome encodes the same 13 proteins Crinoidea, and Asteroidea (Fig. 1a) [14]. Lastly, rRNAs encoded by other animal mtDNAs. The start and stop showed similar characteristics to other echnoid and holo- codons for the 13 PCGs in the L. albus mitogenome are thuroid rRNAs, with both subunits (12s- and 16s-rRNA) shown in Table 2. With the exception of the nad4L (ATT), encoded on the major strand (Fig. 1a). The 12s-RNA gene aTP8 (GTG), and nad4 (TTG), all of the start codons were (897 bp) was located between tRNA-Phe and tRNA-Glu,

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Table 2 Mitochondrial genome content and general features of the red sea urchin Loxechinus albus Name Direction Length (bp) Min Max Start codon Stop codon Anti-codon AT % tRNA-Phe F Forward 73 1 73 35–37 12 s rRNA Forward 897 74 970 57.9 tRNA-Glu E Forward 69 966 1,034 996–998 tRNA-Thr T Forward 74 1,041 1,114 1,076–1,078 tRNA-Pro P Forward 70 1,254 1,323 1,285–1,287 tRNA-Gln Q Reverse 71 1,395 1,325 1,361–1,363 tRNA-Asn N Forward 72 1,402 1,473 1,434–1,436 tRNA-Leu (CUN) Forward 73 1,474 1,546 1,507–1,509 tRNA-Ala A Reverse 71 1,616 1,546 1,582–1,584 tRNA.Trp W Forward 69 1,617 1,685 1,647–1,649 tRNA-Cys C Forward 68 1,686 1,753 1,716–1,718 tRNA-Val V Reverse 70 1,822 1,753 1,789–1,791 tRNA-Met M Forward 73 1,837 1,909 1,870–1,872 tRNA-Asp D Reverse 70 1,980 1,911 1,947–1,949 tRNA-Tyr Y Forward 71 1,991 2,061 2,023–2,025 tRNA-Gly G Forward 68 2,067 2,134 2,096–2,098 tRNA-Leu (UUR) Forward 73 2,135 2,207 2,168–2,170 nad 1 Forward 972 2,210 3,181 ATG TAA 60.2 tRNA-Ile I Forward 72 3,183 3,254 3,215–3,217 nad 2 Forward 1,059 3,255 4,313 ATG TAA 63.3 16s rRNA Forward 1,537 4,316 5,852 63.8 cox 1 Forward 1,557 5,850 7,406 ATG TAA 57.9 tRNA-Arg R Forward 70 7,416 7,485 7,447–7,449 nad4L Forward 294 7,487 7,780 ATT TAA 65.3 cox 2 Forward 690 7,780 8,469 ATG TAA 59 tRNA-Lys K Forward 70 8,472 8,541 8,502-8,504 atp 8 Forward 165 8,542 8,706 GTG TAA 66.7 atp 6 Forward 699 8,691 9,389 ATG TAA 62.4 cox 3 Forward 786 9,390 10,175 ATG TAA 58 tRNA-Ser S (UGA) Reverse 70 10,245 10,176 10,218–10,220 nad 3 Forward 348 10,262 10,609 ATG TAA 61.6 nad 4 Forward 1,389 10,361 12,019 TTG TAG 60.4 tRNA-His H Forward 69 12,010 12,078 12,044–12,046 tRNA-Ser (AGN) Forward 66 12,081 12,146 12,177–12,179 nad 5 Forward 1,944 12,148 14,082 ATG TAA 61 nad 6 Reverse 489 14,083 14,571 ATG TAG 63.6 cob Forward 1,143 14,594 15,737 ATG TAA 59.7 whereas the 16s-RNA gene (1,537 bp) was located between topologies with similar branch lengths. Most clades and nad2 and cox1. subclades were well supported with [85 % posterior probabilities and bootstrap values (Fig. 2). BI and ML Phylogenetic relationships of L. albus analyses recovered a monophyletic group containing all within Echinodermata holothuroids and echinoids (i.e., Echinozoa). Within the Holothuroidea, Parastichopus nigripunctatus and Apos- The BPS for the concatenated alignment of the 13 PCGs tichopus japonicus grouped together in a sister-group indicated nine subsets of partitions (Table S3). This BPS relationship with Stichopus horrens (Fig. 2), consistent and established models of molecular evolution were used with the monophyly of the family Stichopodidae [49]. This for both BI and ML analyses, which produced identical along with Holothuria forskali formed the

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Ophiocomina nigra Ophiuroidea

Cucumaria miniata

1 Holothuria forskali 100 0.98 Holothuroidea 100 1 Parastichopus nigripunctatus 90 1 100 1 Apostichopus japonicus 91 1 Stichopus horrens 87

1 Nacospatangus alta Maretiidae 100 Loveniidae Arbacia lixula Arbaciidae Mesocentrotus nudus 1 1 100 67 1 Pseudocentrotus depressus 65 Mesocentrotus franciscanus Strongylocentrotus intermedius 1 1 1 100 Echinoidea 93 100 Strongylocentrotus droebachiensis Strongylocentrotidae 0.87 1 56 100 Strongylocentrotus pallidus 1 100 Strongylocentrotus fragilis 1 100 Strongylocentrotus purpuratus Hemicentrotus pulcherrimus 1 1 Heliocidaris crassispina 100 Echinometridae 100 Evechinus chloroticus

Paracentrotus lividus 1 Echinidae 100 Loxechinus albus

Pisaster ochraceus Asteroidea

0.3

Fig. 2 Maximum likelihood tree of the nucleotide sequences of all thirteen protein-coding genes in Echinozoa. The numbers on the nodes show the Bayesian posterior probabilities and maximum likelihood bootstrap percentages monophyletic order Aspidochirotida. The order Dendro- regular sea urchins based on partial sequences of the 12S- chirotacea, represented by the species Cucumaria miniata, rRNA and COI genes [50]. In addition, within the family was sister to Aspidochirotida, supporting the monophyletic Strongylocentrotidae, two major clades were found origin of the class Holothuroidea [16]. Furthermore within (Fig. 2). One comprised all of the representative species of Echinozoa, our analysis recovered a monophyletic clade of Strongylocentrotus and Hemicentrotus, while the other species of the families Loveniidae (E. cordatum) and included species of Mesocentrotus and Pseudocentrotus Maretiidae (N. alta) (i.e., order Spatangoida). This clade (Fig. 2). The molecular differentiation between these two was sister to the species representing the family Arbaciidae major clades was consistent with a previous morphological (A. lixula), and both were grouped with the order Echinoida analysis of the ultrastructure of primary spines [51] and (i.e., Echinidae, Strongylocentrotidae and Echinometridae). with phylogenetic inferences using mitochondrial genes This finding supports the monophyly of Echinoidea [45, 52, 53]. (Fig. 2)[16]. Previous attempts to reconstruct the evolutionary rela- Within Echinoida, our BI and ML analyses showed that tionships of echinoderms based on mitochondrial gene L. albus shares a most recent common ancestor with P. order revealed the monophyly of holothuroids and echi- lividus, both being part of a sister group to Strongylo- noids [12, 16]. This finding is mainly explained by the centrotidae and Echinometridae (Fig. 2). These findings paucity of rearrangements observed between these mitog- agree with previous studies on the molecular phylogeny of enomes (Fig. 1a). Nevertheless, at higher taxonomic levels

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