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Among ~1300 species of world nemerteans, seven species in five genera of lineid heteronemerteans have been known to possess a branched proboscis. In this paper, we describe the eighth branched-proboscis species: Gorgonorhynchus citrinus sp. nov. from Okinawa, Japan. We also report Gorgonorhynchus cf. repens Dakin & Fordham, 1931 with uniformly orange body, as a new member for the Japanese nemertean fauna. We infer the phylogenetic relationships between these forms and other members of Lineidae McIntosh, 1874 for which partial sequences of the mitochondrial 16S rRNA and cytochrome c oxidase subunit I, and the nuclear 18S rRNA, 28S rRNA, and histone H3 genes are available in public databases, along with newly sequenced data of another branched-proboscis heteronemertean, Polydendrorhynchus zhanjiangensis (Yin & Zheng, 1984) from China. In the resulting tree, Gorgonorhychus Dakin & Fordham, 1931 was sister group to non-branched-proboscis Dushia Corrêa, 1963, whereas P. zhanjiangensis was sister group to likewise non-branched-proboscis Cerebratulus lacteus (Leidy, 1851). http://zoobank.org/urn:lsid:zoobank.org:pub:685992C5-F595-4C28-9178-256D945E595A

The genus Parahubrechtia Gibson and Sundberg, 1999 was first described within the family Hubrechtiidae (class Pilidiophora) and subsequently transferred to the family Callineridae (class Palaeonemertea). Here we describe two new species, Parahubrechtia rayi sp. nov. from the Sea of Japan (Russia) and P. peri sp. nov. from the South China Sea (China). A phylogenetic analysis based on partial sequences of five nuclear and mitochondrial gene regions, 18S rRNA, 28S rRNA, histone H3, 16S rRNA, and COI, has confirmed the monophyly of the genus Parahubrechtia, and indicated a close relationship to Callinera Bergendal, 1900, whose monophyly is not confirmed. Both genera belong to the family Tubulanidae, with its junior synonym being Callineridae. Three major subclades are distinguished within the Tubulanidae: subclade Tubulanus s. str., subclade Tubulanus punctatus, and subclade Parahubrechtia + Callinera. The further status of Parahubrechtia depends on whether the paraphyly of Callinera is confirmed or not and how the problem of paraphyly of the genus Tubulanus Renier, 1804 is resolved.

The transposon silencer piwi genes play important roles in germline determination and maintenance, gametogenesis, and stem-cell self-renewal, and the expression of certain piwi genes is indispensable for regeneration. Knowledge about piwi genes is needed for phylum Nemertea, which contains members (e.g., Lineus sanguineus) with formidable regeneration capacity. By searching the L. sanguineus genome, we identified six Argonaute genes including three ago (Ls-Ago2, Ls-Ago2a, and Ls-Ago2b) and three piwi (Ls-piwi1, Ls-piwi2, and Ls-piwi3) genes. In situ hybridization revealed that, in intact females, Ls-piwi2 and Ls-piwi3 were not expressed, while Ls-piwi1 was expressed in ovaries. During regeneration, Ls-piwi1 and Ls-pcna (proliferating cell nuclear antigen) had strong and similar expressions. The expression of Ls-piwi1 became indetectable while Ls-pcna continued to be expressed when the differentiation of new organs was finished. During anterior regeneration, expression signals of Ls-piwi2 and Ls-piwi3 were weak and only detected in the blastema stage. During posterior regeneration, no expression was observed for Ls-piwi2. To date, no direct evidence has been found for the existence of congenital stem cells in adult L. sanguineus. The &ldquo, pluripotent cells&rdquo, in regenerating tissues are likely to be dedifferentiated from other type(s) of cells.

The transposon silencer piwi genes play important roles in germline determination and maintenance, gametogenesis, and stem-cell self-renewal, and the expression of certain piwi genes is indispensable for regeneration. Knowledge about piwi genes is needed for phylum Nemertea, which contains members (e.g., Lineus sanguineus) with formidable regeneration capacity. By searching the L. sanguineus genome, we identified six Argonaute genes including three ago (Ls-Ago2, Ls-Ago2a, and Ls-Ago2b) and three piwi (Ls-piwi1, Ls-piwi2, and Ls-piwi3) genes. In situ hybridization revealed that, in intact females, Ls-piwi2 and Ls-piwi3 were not expressed, while Ls-piwi1 was expressed in ovaries. During regeneration, Ls-piwi1 and Ls-pcna (proliferating cell nuclear antigen) had strong and similar expressions. The expression of Ls-piwi1 became indetectable while Ls-pcna continued to be expressed when the differentiation of new organs was finished. During anterior regeneration, expression signals of Ls-piwi2 and Ls-piwi3 were weak and only detected in the blastema stage. During posterior regeneration, no expression was observed for Ls-piwi2. To date, no direct evidence has been found for the existence of congenital stem cells in adult L. sanguineus. The “pluripotent cells” in regenerating tissues are likely to be dedifferentiated from other type(s) of cells.

The brine shrimp Artemia exhibits two reproductive modes: 1) oviparity, producing diapause embryos; and 2) ovoviviparity, producing free-swimming nauplii. Previous studies have suggested the existence of a critical stage that determines the reproductive mode. Physicochemical factors, such as photoperiod, temperature, and salinity, have been suggested to irreversibly affect the reproductive mode of oocytes during this critical stage. In this study, experiments were carried out using a photoperiod and temperature-sensitive parthenogenetic Artemia clone where maternal Artemia were shifted bidirectionally between ovoviviparity (18 h L:6 h D, 27°C) and oviparity (6 h L:18 h D, 19°C) culture conditions. In the main experiment (Artemia shifted at six different stages including the post-larva II to adult II), the reproductive mode of first brood was converted when shifting was performed on post-larva II and III but was not converted when females were shifted after post-larva III. A supplementary experiment further revealed that the reproductive mode of first brood could be altered when shifting females at an “early phase of post-larva IV”, characterized by a developing ovisac reaching the middle of the third abdominal segment, ventral spines, and some oocytes growing larger than the others. In both experiments, reproductive modes of the second brood were significantly affected when the shifting was performed on post-larva IV. These results suggest that the critical stage for inducing oviparity and embryonic diapause is at the previtellogenic stage of oocytes, or at maternal “early phase of post-larva IV” for the first-brood offspring. During this stage, differential gene expression patterns of the two destined oocytes may be triggered by the token stimuli signals received by the oocytes.

The genus Artemia Leach, 1819 is a cosmopolitan halophilic crustacean, consisting of bisexual species and obligate parthenogenetic populations. Asia is rich in Artemia biodiversity. More than 530 Artemia sites have been recorded from this area and more than 20 species/subspecies/variety names have been used for them. There exist various problems in the nomenclature, identification, and phylogenetic status of Artemia native to Asia, which are discussed in this paper., This study was supported by the Fundamental Research Funds for the Central Universities (201762017), and Science and Technology Research Funds of Tibet Autonomous Region (XZ201703-GB-04)

Urmia Lake, the largest natural habitat of the brine shrimpArtemia urmiana, has progressively desiccated over the last two decades, resulting in a loss of 80% of its surface area and producing thousands of hectares of arid salty land. This ecological crisis has seriously affected the lake’s native biodiversity.Artemia urmianahas lost more than 90% of its population during the decade from 1994 (rainy period) to 2004 (drought period) due to salinity increasing to saturation levels (∼300 g/l). We studied the influence of this ecological crisis on the genetic diversity ofA. urmianain Urmia Lake, based on one cyst collections in 1994 and 2004. AMOVA analysis on ISSR data demonstrated a 21% genetic variation and there was a 5.5% reduction of polymorphic loci between samples. PCoA showed that 77.42% and 68.75% of specimens clustered separately in 1994 and 2004, respectively. Our analyses of four marker genes revealed different genetic diversity patterns with a decrease of diversity atITS1and an increase forNa+/K+ATPase. There was no notable difference in genetic variation detected forCOIand16Sgenes between the two periods. However, they represented distinctly different haplotypes.ITS1andCOIfollowed a population expansion model, whereasNa+/K+ATPaseand16Swere under demographic equilibrium without selective pressure in the 1994 samples. Neutrality tests confirmed the excess of rare historical and recent mutations present inCOIandITS1in both samples. It is evident that a short-term ecological disturbance has impacted the genetic diversity and structure ofA. urmiana.

Parthenogenetic Artemia from seven Chinese locations with different elevations and various ploidies are characterized by phenotypic and morphometric analyses. Our findings show that the studied populations exhibit dissimilar patterns of ovisac. Four phenotypic patterns of furca are qualified and one of them is shared among di-, tetra- and pentaploid Artemia. Results of discriminant analysis based on morphometric data reveal that tetra- and pentaploid populations are grouped together, but the Aqqikkol Lake population is clearly differentiated. Previous hypothesis/conclusion that polyploid Artemia are larger than diploids is only partly supported by the present results, which show that pentaploid and tetraploid populations are larger than the mostly diploid populations in terms of the total length, but the body size of the Aibi Lake triploids has not significant difference with the sympatric diploids and the mostly diploid Aqqikkol population that inhabit in very high altitude has the largest body size among all parthenogenetic populations. The founding confirms that body size of Artemia is following with Bergmann's rule.

11 pages, 4 figures, 4 tables, Heteronemerteans, such as Lineus ruber, L. viridis, Ramphogordius sanguineus, R. lacteus, Riseriellus occultus, and Micrura varicolor, share many similar external characters. Although several internal characters useful for distinguishing these nemertean species have been documented, their identification is based mostly on coloration, the shape of the head, and how they contract, which may not be always reliable. We sequenced the mitochondrial COI gene for 160 specimens recently collected from 27 locations around the world (provisionally identified as the above species, according to external characters and contraction patterns, with most of them as R. sanguineus). Based on these specimens, together with sequences of 16 specimens from GenBank, we conducted a DNA-based species delimitation/identification by means of statistical parsimony and phylogenetic analyses. Our results show that the analyzed specimens may contain nine species, which can be separated by large genetic gaps; heteronemerteans with an external appearance similar to R. sanguineus/Lineus ruber/L. viridis have high species diversity in European waters from where eight species can be discriminated. Our 42 individuals from Vancouver Island (Canada) are revealed to be R. sanguineus, which supports an earlier argument that nemerteans reported as L. ruber or L. viridis from the Pacific Northwest may refer to this species. We report R. sanguineus from Chile, southern China, and the species is also distributed on the Atlantic coast of South America (Argentina). In addition, present analyses reveal the occurrence of L. viridis in Qingdao, which is the first record of the species from Chinese waters, This study was supported by funding from the National Natural Science Foundation of China to SCS (31172046, 31471957) and by the Swedish Research Council to PS

The cysts of nine Chinese populations of parthenogenetic Artemia were studied by scanning electron microscope. In the 270 cysts examined, 15 different morphological patterns were recognized with most of them not recorded in previous studies and the "tubercled shell surface" being the most common pattern. Results also displayed high intrapopulation variability, with the maximum of 11 patterns (in 30 cysts) recorded from the Barkol population. No positive correlation between the diversity of cyst shell patterns and ploidy compositions was found. Principal components analysis suggests higher similarity among coastal populations than among inland populations, which may be attributed to the identity of physicochemical conditions among coastal salterns and dissimilarity among inland saline lakes. Microsc. Res. Tech. 77:1005-1014, 2014. V C 2014 Wiley Periodicals, Inc.

Strand, Malin ... et al.-- 2 pages, Nemertean classification has closely followed Stiasny‐Wijnhoff's scheme (1936) that was based on Schultze's (1851) division of the taxon into the two classes Anopla and Enopla. In August 2018, the 9th International Conference of Nemertean Biology took place in the Wadden Sea Station of the Alfred Wegener Institute in List auf Sylt, Germany. At this meeting, the community reached consensus to revise nemertean taxonomy at the class level, based on the compiled evidence from studies on nemertean systematics published in the last 15 years (Andrade et al., 2012, 2014 ; Thollesson & Norenburg, 2003). Previous classifications (e.g., Stiasny‐Wijnhoff, 1936) are not based on phylogenetic grounds, and the use of these names is therefore nowadays not wholly informative. With the purpose of facilitating the practical use of the nemertean taxonomy and also making nemertean taxonomy reflect a wealth of more recent information, we conclude that the ranks Anopla and Enopla should be eliminated with the following argumentation: “Enopla” has for long held no more information than the name “Hoplonemertea”. “Anopla” is paraphyletic and the name usually corresponds to the following traits: (a) not bearing stylet; and (b) mouth and proboscis having separate openings. This information is equally well presented in a system that also bears information on natural groups. The names “Anopla” and “Enopla” are hence dismissed and the following system of the Nemertea phylum is implemented: Class: Palaeonemertea Currently no Orders Class: Pilidiophora Order: Heteronemertea Class: Hoplonemertea Order: Monostilifera Order: Polystilifera The changes do not affect lower levels of taxonomy, but in accordance with Thollesson and Norenburg (2003), who first presented Pilidiophora, genus Hubrechtella is transferred from class Palaeonemertea to the class Pilidiophora

We compared the anatomy of the holotype of the palaeonemertean Cephalothrix simula ( Iwata, 1952 ) with that of the holotypes of Cephalothrix hongkongiensis Sundberg, Gibson and Olsson, 2003 and Cephalothrix fasciculus ( Iwata, 1952 ), as well as additional specimens from Fukue (type locality of C. simula) and Hiroshima, Japan. While there was no major morphological discordance between these specimens, we found discrepancies between the actual morphology and some statements in the original description of C. simula with respect to supposedly species-specific characters. Our observation indicates that these three species cannot be discriminated by the anatomical characters so far used to distinguish congeners. For objectivity of scientific names, topogenetypes of the mitochondrial cytochrome c oxidase subunit I (COI) sequences are designated for C. simula, C. hongkongiensis, and C. fasciculus. Analysis of COI sequence showed that the Hiroshima population can be identified as C. simula, which has been found in previous studies from Trieste, Italy, and also from both the Mediterranean and Atlantic coasts of the Iberian Peninsula, indicating an artificial introduction via (1) ballast water, (2) ship-fouling communities, or (3) the commercially cultured oyster Crassostrea gigas ( Thunberg, 1793 ) brought from Japan to France in 1970s. Cephalothrix simula is known to be toxic, as it contains large amounts of tetrodotoxin (TTX). We report here that the grass puffer Takifugu niphobles ( Jordan and Snyder, 1901 )-also known to contain TTX- consumes C. simula. We suggest that the puffer may be able to accumulate TTX by eating C. simula.

The first complete mitochondrial genome sequence for a nemertean, Cephalothrix simula, was determined by conventional and long PCR and sequencing with primer walking methods. This circular genome is 16,296 bp in size and encodes 37 genes (13 protein-coding genes, 2 ribosomal RNAs, and 22 transfer RNAs) typically found in metazoans. All genes are encoded on H-strand except two tRNAs (trnT and trnP). It differs from those reported for other metazoans, but some gene junctions are shared with those of other protostomes. Structure of the mitochondrial genome of C. simula is mostly concordant with the partial mitochondrial genome known for Cephalothrix rufifrons, but notable differences include three large indel events and transposition of 2 tRNAs. Nucleotide composition of the mitochondrial genome of C. simula is highly A+T biased. The compositional skew is strongly reflected in the codon-usage patterns and the amino acid compositions of the mitochondrial proteins. An AT-rich noncoding region with potential to form stem-loop structures may be involved in the initiation of replication or transcription. Gene adjacencies and phylogenetic analysis based on the 12 concatenated amino acid sequences (except atp8) of mitochondrial protein-coding genes show that the nemertean is close to the coelomate lophotrochozoans, rather than the acoelomate platyhelminths.

The taxonomic assignment of the Artemia from Qinghai-Xizang Plateau has been argued. In the present paper, the morphology of frontal knobs and gonopods of five Artemia populations from Qinghai-Xizang Plateau (Lagkor Co, Cam Co, Dagdong Co and Jibu Caka, Tibet; Jingyu Hu, Xinjiang) and three populations from North China (Badain Jaran and Yimeng, Inner Mongolia; Xiechi Lake, Shanxi) is described and compared with four non-Chinese species, the results of cross-breeding tests between the eight Chinese populations are documented and the taxonomic assignment of these Artemia populations is discussed. Morphological observations show that gonopods of the Chinese populations are significantly longer than those of the other species; distal gonopods of the Chinese populations possess spines that can be classified into three groups (posterior spines, exolateral spines and orthostichous spines). In the four non-Chinese species, however, orthostichous spines are absent (thus providing a morphological separation between A. urmiana and A. tibetiana) and the number of spines is much smaller than those of the Chinese populations. Discriminant analysis on numerical characters showed that three North China populations were divergent from four Tibetan populations, with Jingyu Hu population intermediate. Compared with the North China populations, the Tibetan populations possess a longer gonopod (longer gonopod, longer distal gonopod and longer finger-like tip), larger basal gonopod spine, larger percent of frontal knob spines that emerge by 2 spines from the same region and smaller percent of frontal knob spines that emerge by 4 and 5 spines from the same region. The Artemia from Jingyu Hu is morphometrically close to the Tibetan populations by possessing a larger basal gonopod spine, larger percent of frontal knob spines emerging by 2 spines from the same region and smaller percent of frontal knob spines emerging by 4 and 5 spines from the same region, but different from the latter by having a shorter gonopod (shorter gonopod, shorter distal gonopod and shorter finger-like tip), fewer frontal knob spines, more distal gonopod spines and more posterior spines. The results of cross-breeding tests showed that isolating barrier did not exist among the three North China populations or among the five Qinghai-Xizang Plateau populations, while they might exist between A. urmiana and the populations of Xiechi Lake (type locality of A. sinica) and Lagkor Co (type locality of A. tibetiana) and between the Qinghai-Xizang Plateau populations and A. sinica. The present results thus support the validity of A. tibetiana. Among the eight Chinese populations, the five populations from Qinghai-Tibet Plateau belong in A. tibetiana, whereas three populations from North China belong in A. sinica.

Of the 14 nominal species that are now or have ever been assigned to the genus Paranemertes Coe, 1901 , four have been reported to have stylets with a spirally fluted or braided appearance. Although differentiation in color patterns has been documented among species/populations, these nemerteans share similar external characters. Using the sequence datasets of mitochondrial cytochrome c oxidase subunit I (COI), 16S rRNA, and nuclear 28S rRNA genes of specimens from 14 localities of Canada, USA, Russia, Japan, and China, we analyzed the genetic differentiation and reconstructed the phylogenetic trees for these nemerteans. In conjunction with the external characters, we discuss their taxonomy and species delimitation. An analysis based on COI dataset showed high genetic variations among populations and even among worms from the same geographic area. The analyzed 111 individuals were assigned into seven networks by statistical parsimony analysis. The inter-network uncorrected p-distances ranged from 0.044 to 0.172 and the mean intra-network uncorrected p-distances varied from 0.001 to 0.005. With the exception of two networks that contain specimens from the East China Sea, all networks were well-supported by the results of Bayesian and neighbor-joining analyses on the COI data. Phylogenetic trees based on 16S rRNA and 28S rRNA datasets were basically similar to the COI trees, but specimens in some networks were merged into larger clades. Present molecular analyses support the validity of P. sanjuanensis and the synonymization of P. cylindracea with P. peregrina. Nemerteans previously recorded as P. peregrina may contain several species and sympatric speciation might have been occurred in this nemertean group.

Parthenogenetic Artemia from seven Chinese locations with different elevations and various ploidies are characterized by phenotypic and morphometric analyses. Our findings show that the studied populations exhibit dissimilar patterns of ovisac. Four phenotypic patterns of furca are qualified and one of them is shared among di-, tetra- and pentaploid Artemia. Results of discriminant analysis based on morphometric data reveal that tetra- and pentaploid populations are grouped together, but the Aqqikkol Lake population is clearly differentiated. Previous hypothesis/conclusion that polyploid Artemia are larger than diploids is only partly supported by the present results, which show that pentaploid and tetraploid populations are larger than the mostly diploid populations in terms of the total length, but the body size of the Aibi Lake triploids has not significant difference with the sympatric diploids and the mostly diploid Aqqikkol population that inhabit in very high altitude has the largest body size among all parthenogenetic populations. The founding confirms that body size of Artemia is following with Bergmann's rule.

The cysts of nine Chinese populations of parthenogenetic Artemia were studied by scanning electron microscope. In the 270 cysts examined, 15 different morphological patterns were recognized with most of them not recorded in previous studies and the "tubercled shell surface" being the most common pattern. Results also displayed high intrapopulation variability, with the maximum of 11 patterns (in 30 cysts) recorded from the Barkol population. No positive correlation between the diversity of cyst shell patterns and ploidy compositions was found. Principal components analysis suggests higher similarity among coastal populations than among inland populations, which may be attributed to the identity of physicochemical conditions among coastal salterns and dissimilarity among inland saline lakes.

The complete mitochondrial genome (mitogenome) of Tetrastemma olgarum is sequenced. It is 14,580 bp in length and contains 37 genes typical for metazoan mitogenomes. The gene order is identical to that of the previously published Hoplonemertea mitogenomes. All genes are encoded on the heavy strand except for trnT and trnP. The coding strand is AT-rich, accounting for 69.2% of overall nucleotide composition.

1Institute of Evolution & Marine Biodiversity, Ocean University of China, 5 Yushan Road, Qingdao 266003, China 2Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan 3A.V. Zhirmunsky Institute of Marine Biology, 17 Palchevsky St, 690059, Vladivostok, Russia 4Far Eastern Federal University 29 Oktyabrskaya St, 690600, Vladivostok, Russia 5Organizers of the 8th International Conference on Nemertean Biology; Guest Editors, Zoological Science

The phenomenon of codon usage bias is known to exist in many genomes and it is mainly determined by mutation and selection. To understand the patterns of codon usage in nemertean mitochondrial genomes, we use bioinformatic approaches to analyze the protein-coding sequences of eight nemertean species. Neutrality analysis did not find a significant correlation between GC12 and GC3. ENc-plot showed a few genes on or close to the expected curve, but the majority of points with low-ENc values are below it. ENc-plot suggested that mutational bias plays a major role in shaping codon usage. The Parity Rule 2 plot (PR2) analysis showed that GC and AT were not used proportionally and we propose that codons containing A or U at third position are used preferentially in nemertean species, regardless of whether corresponding tRNAs are encoded in the mitochondrial DNA. Context-dependent analysis indicated that the nucleotide at the second codon position slightly affects synonymous codon choices. These results suggested that mutational and selection forces are probably acting to codon usage bias in nemertean mitochondrial genomes.

We sequenced the complete mitochondrial genomes (mitogenomes) of three Hoplonemertea species, Amphiporus formidabilis, Prosadenoporus spectaculum and Nipponnemertes punctatula, which are 14,616, 14,655 and 15,354 bp in length, respectively. Each of the three circular mitogenomes consists of 37 typical genes and some non-coding regions. The nucleotide composition of the coding strand is biased toward T, almost a half of total nucleotides in these mitogenomes. There are many poly-T tracts across these mitogenomes, which exhibit T-number variation within different clones of protein-coding genes, mainly resulting from false PCR amplification. The major non-coding regions have tandem repeat motifs and hairpin-like structures that may be associated with the initiation of replication or transcription. Data published to date for nemerteans show that Palaeonemertea species usually bear the largest mitogenomes, while representatives in the more recently derived Distromatonemertea clade bear the smallest ones; and that the gene arrangement of mitogenomes seems to be variable within the phylum Nemertea, but stable within either of Heteronemertea and Hoplonemertea.

Background The mitochondrial genome is important for studying genome evolution as well as reconstructing the phylogeny of organisms. Complete mitochondrial genome sequences have been reported for more than 2200 metazoans, mainly vertebrates and arthropods. To date, from a total of about 1275 described nemertean species, only three complete and two partial mitochondrial DNA sequences from nemerteans have been published. Here, we report the entire mitochondrial genomes for two more nemertean species: Nectonemertes cf. mirabilis and Zygeupolia rubens. Results The sizes of the entire mitochondrial genomes are 15365 bp for N. cf. mirabilis and 15513 bp for Z. rubens. Each circular genome contains 37 genes and an AT-rich non-coding region, and overall nucleotide composition is AT-rich. In both species, there is significant strand asymmetry in the distribution of nucleotides, with the coding strand being richer in T than A and in G than C. The AT-rich non-coding regions of the two genomes have some repeat sequences and stem-loop structures, both of which may be associated with the initiation of replication or transcription. The 22 tRNAs show variable substitution patterns in nemerteans, with higher sequence conservation in genes located on the H strand. Gene arrangement of N. cf. mirabilis is identical to that of Paranemertes cf. peregrina, both of which are Hoplonemertea, while that of Z. rubens is the same as in Lineus viridis, both of which are Heteronemertea. Comparison of the gene arrangements and phylogenomic analysis based on concatenated nucleotide sequences of the 12 mitochondrial protein-coding genes revealed that species with closer relationships share more identical gene blocks. Conclusion The two new mitochondrial genomes share many features, including gene contents, with other known nemertean mitochondrial genomes. The tRNA families display a composite substitution pathway. Gene order comparison to the proposed ground pattern of Bilateria and some lophotrochozoans suggests that the nemertean ancestral mitochondrial gene order most closely resembles the heteronemertean type. Phylogenetic analysis proposes a sister-group relationship between Hetero- and Hoplonemertea, which supports one of two recent alternative hypotheses of nemertean phylogeny.

BACKGROUND: It has been suggested that statistical parsimony network analysis could be used to get an indication of species represented in a set of nucleotide data, and the approach has been used to discuss species boundaries in some taxa. METHODOLOGY/PRINCIPAL FINDINGS: Based on 635 base pairs of the mitochondrial protein-coding gene cytochrome c oxidase I (COI), we analyzed 152 nemertean specimens using statistical parsimony network analysis with the connection probability set to 95%. The analysis revealed 15 distinct networks together with seven singletons. Statistical parsimony yielded three networks supporting the species status of Cephalothrix rufifrons, C. major and C. spiralis as they currently have been delineated by morphological characters and geographical location. Many other networks contained haplotypes from nearby geographical locations. Cladistic structure by maximum likelihood analysis overall supported the network analysis, but indicated a false positive result where subnetworks should have been connected into one network/species. This probably is caused by undersampling of the intraspecific haplotype diversity. CONCLUSIONS/SIGNIFICANCE: Statistical parsimony network analysis provides a rapid and useful tool for detecting possible undescribed/cryptic species among cephalotrichid nemerteans based on COI gene. It should be combined with phylogenetic analysis to get indications of false positive results, i.e., subnetworks that would have been connected with more extensive haplotype sampling.

The mitochondrial genome sequences were determined for two species of nemerteans, Cephalothrix sp. (15,800 bp sequenced, near-complete) and Paranemertes cf. peregrina (14,558 bp, complete). As seen in most metazoans, the genomes encode 13 protein, 2 ribosomal RNA and 22 transfer RNA genes. The nucleotide composition is strongly biased toward A and T, as is typical for metazoan mtDNAs. There is also a significant strand skew in the distribution of these nucleotides, with the coding strand being richer in T than A and in G than C. All genes are transcribed in the same direction except for trnP and trnT, which is consistent with that reported for Cephalothrix hongkongiensis and Lineus viridis. Gene arrangement of Cephalothrix sp. is identical to that of C. hongkongiensis, while in P. cf. peregrina it is similar to L. viridis, but differs significantly from the three Cephalothrix species in the position of four protein-coding genes and seven tRNAs. Some protein-coding genes have 3′ end stem-loop structures, which may allow mRNA processing without flanking tRNAs. The major non-coding regions observed in the two genomes with potential to form stem-loop structures may be involved in the initiation of replication or transcription. The average Ka/Ks values, varying from 0.12 to 0.89, are markedly different among the 13 mitochondrial protein-coding genes, suggesting that there may exist different selective pressure among mitochondrial genes of nemerteans.

The complete mitochondrial genome of Iwatanemertes piperata (Nemertea: Heteronemertea) was determined. The genome, which contains 13 protein-coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes, is 16,382 bp in length and has a base composition of G (25.87%), A (21.53%), T (40.64%) and C (11.95%). The gene order is identical to other Heteronemertea mitogenomes published to date.