Comparative Studies On Mitochondrial Genomes Of Hoplonemerteans (Nemertea)

The phylum Nemertea (ribbon worms), containing about1275species, is a groupof worm-like Bilaterian animal. The most special structure of nemerteans is arhynchocoel containing a proboscis. Recent ultrastructural and molecular studiessupport Nemertea to be a member of Lophotrochozoa rather than Acoelomata. Thephylogenetic relationship of the phylum is still unsettled in parts, and conclusionsmay be dependent on different markers and analytical methods. Mitochondrialgenomes (mitogenomes) have been widely used as molecular markers forphylogenomic analyses, due to its fast evolution rate, lack of recombination andother advantages. So far, only ten complete or nearly complete mitochondrialgenomes are determined for nemerteans. In the present work, mitogenomes of nineMonostilifera nemerteans were sequenced with short and long PCR method andcompared with other previously published nemertean mitogenomes. Thephylogenetic relationships of nemerteans were ananlyzed using nucleotide sequencesas well as amino acid sequences. The main results are:1. The lengths of newly sequenced mitochondrial genomes of nine Hoplonemerteaspecies are: Prosadenoporus spectaculum,14655bp; Nipponnemertes punctatula,15354bp; Amphiporus formidabilis,14616bp; Gononemertes parasita,14742bp;Nemertopsis bullocki sensu Sun and Dong,1998,>12803bp; Emplectonema cf.gracile,14688bp; Tetrastemma olgarum,14580bp; Carcinonemertes sp.,14739bp;Ototyphlonemertes sp.,>11774bp. They are obviously shorter than those of knownHeteronemertea and Palaeonemertea species except for Nipponnemertes punctatula.The mitochondrial genomes of Hoplonemertea are compact and economic, withsmaller non-coding regions than that in Palaeonemertea and Heteronemerteamitogenomes. Like in other metazoans, the coding strand is AT rich, and two strands have significant nucleotide skewness. Many poly-T tract structures have been foundin these mitogenomes, which are not so frequent in Heteronemertea andPalaeonemertea mitogenomes. Thirteen protein-coding genes seem to have differentevolutionary speed, with cox1gene the most conservertive. The ratio ofasynonymous substitution rate and synonymous substitution rate (Ka/Ks) shows thatthe evolutionary rate of Hoplonemertea and Heteronemertea is faster thanPalaeonemertea. Transfer RNA genes usually can be folded into cloverleaf-likestructure, while the DHU-arm of trnS2is usually absent in most mitogenomes(except for that of Nemertopsis bullock sensu Sun and Dong,1998). Among codinggenes, the ribosomal RNA genes have the lowest evolution rate. The majornon-coding regions have faster evolutionary rate than the coding genes, withunconservative characteristics such as the tandemly repeat sequences in A.formidabilis and N. punctatula, the hairpin-like structures in A. formidabilis andCarcinonemertes sp. and so on. Gene arrangement is conservative withinHeteronemertea or Hoplonemertea mitogenomes, but it is variable amongHoplonemertea, Heteronemertea and Palaeonemertea mitogenomes. Comparison ofgene order among nemerteans and other Lophotrochozoa shows that the gene orderof Heteronemertea and Hoplonemertea is very close to that of Gastropod andPolyplacophora.Some differences are found between the parasitic/commensal mitogenomes andother free-living hoplonemertean mitogenomes. For example, in G. parasita themNCR is biased toward T and C (contrary to that in other hoplonemerteans) and therrnS gene has a unique58-bp insertion at5′end, the coding strand ofCarcinonemertes sp. has the lowest AT content and that of Nemertopsistetraclitophila is the highest, in N. tetraclitophila the nad3gene starts with the ATTcodon (ATG in other hoplonemerteans). However, we cannot conclude that thesedifferences are related to their special lifestyle, because similar variations may alsoexist among free-living nemerteans and available mitogenomic data of nemerteansare stilled limited.2. Phylogenetic analyses by maximum likelihood and Bayesian inference of18 nemerteans and32Lophotrochozoa mitochondrial genome sequences yieldeddifferent results. Overall, nemerteans have the closest relationship with Phoronida orPolyplacophora, and also close to the clade containing annelids, brachiopods andentoprocts, all of which belong to coelmate animals. All the results with highBootstrap values and posterior probabilities support the hyposesis thatHeteronemertea and Hoplonemertea are sister groups and Palaeonemertea is at thebasal position; Cratenemertidae is a taxon well separted from the othermonostiliferans which constitute the clade Distromatonemertea. Within the cladeDistromatonemertea, the position of other species is consistent with most previousstudies, but the position of Carcinonemertes sp. is variable amongDistromatonemertea inferred with nucleotide dataset or amino acid dataset. Moredata is still necessary to resolve detailed phylogenetic relationships of nemerteans.