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Wholemounts of polyclad flatworms in the suborder Cotylea. (A) Prosthiostomum grande; (B) Prosthiostomum vulgaris; (C) Pseudoceros velutinus; (D) Pseudoceros nipponicus; (E) Pseudobiceros nigromarginatus; (F) Pseudoceros atropurpureus; (G) Pseudobiceros flavomarginatus; (H) Pseudobiceros hancockanus; (I) Thysanozoon brocchii; (J) Thysanozoon japonicum; (K) Cyclo porus japonicus
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The order Polycladida comprises a highly diverse and cosmopolitan group of marine turbellarian flatworms. Owing to the great morphological diversity and the absence of a molecular phylogeny, the classification of this group has always been controversial. Here we seek to add resolution by reporting the results of molecular phylogenetic analysis base...
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... n. The combination of previously published datasets (Bahia et al. 2017;Tsunashima et al. 2017;Dittmann et al. 2019;Litvaitis et al. 2019;Tsuyuki et al. 2020;Cuadrado et al. 2021;McNab et al. 2022) provides a new and updated phylogeny of the Polycladida, with special attention to the families Euryleptidae and Stylostomidae Dittmann, Cuadrado, Aguado, Noreña & Egger, 2019. ...
We describe Cycloporus pinkipus sp. n., a new polyclad flatworm species from the Adriatic coast of Croatia using live images, histological sections, and a molecular marker. It is the fifteenth described species of Cycloporus Lang, 1884 and the second described congener in the Mediterranean. The genus Cycloporus is characterised by a small oval body, tentacular bumps and the name-giving marginal pores. Cycloporus pinkipus sp. n. has a smooth dorsal surface, which is transparent creamy white with light brown to yellow spots, covered with prominent serial pink spots on the inner rim of the body margin. There is little variation of the genital organs between different species of the genus, therefore we recognise C. pinkipus sp. n. as a new species in particular due to its unique coloration, and a unique partial large nuclear ribosomal subunit (28S) sequence. In recent years the family Euryleptidae Stimpson, 1857, which also contains the genus Cycloporus, was discussed and revised in several molecular studies. In an updated molecular phylogeny of the Polycladida based on partial 18S and 28S rDNA marker genes, C. pinkipus sp. n. was recovered in a clade of many other Cycloporus species within Euryleptidae.
... Both species could be identified after flatworm photos, and advice on their morphology and molecular analyses was given by Dr. Tsuyuki (pers. comm.), who has been involved in various studies describing Pacific flatworm species, based on both morphology and molecular analyses (e.g., [55]). ...
... To analyse the flatworm specimens, the DNA was extracted in a similar manner as described above for the moon jellyfish. The Qiagen Rotor-Gene Q standard PCR mix was used, with the flatworm primers HRNT-F2 and HRNT-R2 [55] aimed at the marker 28S, and the primers HRpra2 and HRprb2-2 [55] aimed at the marker COI. For these two markers, the PCR programs that were used in a Rotor-Gene Q apparatus consisted of the following steps: for the marker 28S: 95 • C for 5 , 55× (95 • C for 2 ; 55 • C for 10 , 72 • C for 60 ), extension at 72 • C for 5 , and an HRM step going from 75 • C to 90 • C at a rate of 0.1 • C every 2 s; for the marker COI: 95 • C for 4 , 55× (95 • C for 60 ; 48 • C for 60 , 72 • C for 60 ), extension at 72 • C for 8 , and an HRM step going from 75 • C to 90 • C at a rate of 0.1 • C every 2 s. 1850) [54]. ...
... To analyse the flatworm specimens, the DNA was extracted in a similar manner as described above for the moon jellyfish. The Qiagen Rotor-Gene Q standard PCR mix was used, with the flatworm primers HRNT-F2 and HRNT-R2 [55] aimed at the marker 28S, and the primers HRpra2 and HRprb2-2 [55] aimed at the marker COI. For these two markers, the PCR programs that were used in a Rotor-Gene Q apparatus consisted of the following steps: for the marker 28S: 95 • C for 5 , 55× (95 • C for 2 ; 55 • C for 10 , 72 • C for 60 ), extension at 72 • C for 5 , and an HRM step going from 75 • C to 90 • C at a rate of 0.1 • C every 2 s; for the marker COI: 95 • C for 4 , 55× (95 • C for 60 ; 48 • C for 60 , 72 • C for 60 ), extension at 72 • C for 8 , and an HRM step going from 75 • C to 90 • C at a rate of 0.1 • C every 2 s. 1850) [54]. ...
Information on temporal and spatial trends with regard to the introduction of non-indigenous species (NIS) is often sparsely available. These trends may potentially help improve the design and focus of monitoring programs, give insights into new pathways and hotspots, and facilitate horizon scanning. We provide an overview of 215 marine and brackish water NIS recorded in The Netherlands. Temporal trends over the most recent three decades for taxonomic groups, species origin, introduction vectors, and water systems were analysed. We attempt to explain the observed patterns and discuss factors that hamper their explanation. A shift in the region of origin from Pacific to W Atlantic can potentially be linked to legislation prohibiting Pacific oyster imports, whereas a subsequent shift backwards cannot. Case studies illustrate that NIS may not be first detected in the water systems where they were originally introduced. Additionally, it is shown that changes in allegedly native species’ distribution or seasonal pattern should be linked to an introduced cryptic NIS instead. We also discuss the shortcomings of monitoring programs that were originally not focused on NIS, the importance of naturalists’ observations, and the added value of a more recent network that is focused on NIS detection in the coastal waters of The Netherlands.
... cotyleans [20,27,28,33], or as the sister group of Diposthidae, together forming the sister group of all remaining cotyleans [20,38] [20,27,28]) will be important to see if these species form part of clade 1 and whether clade 1 remains the sister to the rest of Cotylea. In our reconstruction, Pseudoceros Lang 1884 is the sister group of all other pseudocerotids, reflecting topologies of single-and few-gene trees [26][27][28]33]. The family Pseudocerotidae contains genera with single or duplicated male copulatory organs. ...
... Genera with duplicated male organs (e.g. Thysanozoon Grube, 1840, Pseudobiceros Faubel, 1984) were supported as a monophyletic cluster in the phylogenetic reconstruction of Litvaitis et al. [20], although genera with single and duplicated genital organs were intermingled in other studies [20,[26][27][28]33]. Here, Thysanozoon brocchi (duplicated male apparatus) is the sister group of 'Acanthozoon or Thysanozoon sp.', where Acanthozoon Collingwood, 1876 features a single male apparatus. ...
... All three studies used Euprosthiostomum mortenseni Marcus, 1948, and recovered the conflicting topologies with high support. Although our tree recovers a monophyletic Euryleptidae consistent with Tsunashima et al. [26], other recent phylogenies recovered paraphyletic Euryleptidae [20,27,28,33], which may be attributable to increased taxon coverage, i.e. inclusion of members of Stylostomidae Dittmann et al. [28]. ...
Platyhelminthes (flatworms) are a diverse invertebrate phylum useful for exploring life-history evolution. Within Platyhelminthes, only two clades develop through a larval stage: free-living polyclads and parasitic neodermatans. Neodermatan larvae are considered evolutionarily derived, whereas polyclad larvae are hypothesized to be ancestral due to ciliary band similarities among polyclad and other spiralian larvae. However, larval evolution has been challenging to investigate within polyclads due to low support for deeper phylogenetic relationships. To investigate polyclad life-history evolution, we generated transcriptomic data for 21 species of polyclads to build a well-supported phylogeny for the group. The resulting tree provides strong support for deeper nodes, and we recover a new monophyletic clade of early branching cotyleans. We then used ancestral state reconstructions to investigate ancestral modes of development within Polycladida and more broadly within flatworms. In polyclads, we were unable to reconstruct the ancestral state of deeper nodes with significant support because early branching clades show diverse modes of development. This suggests a complex history of larval evolution in polyclads that likely includes multiple losses and/or multiple gains. However, our ancestral state reconstruction across a previously published platyhelminth phylogeny supports a direct developing prorhynchid/polyclad ancestor, which suggests that a larval stage in the life cycle evolved along the polyclad stem lineage or within polyclads.
... [20,[25][26][27][28][29][30][31][32][33][34]. However, these studies used one or a few genes for their inferences, primarily 18S, 28S and COI. ...
... One novel finding resulting from our analysis is a new monophyletic clade (clade 1, Figure 2 In our reconstruction, Pseudoceros Lang, 1884 is the sister group of all other pseudocerotids, reflecting topologies of single-and few-gene trees [26][27][28]33]. The family Pseudocerotidae contains genera with single or duplicated male copulatory organs. ...
... The family Pseudocerotidae contains genera with single or duplicated male copulatory organs. Genera with duplicated male organs (e.g., Thysanozoon Grube, 1840, Pseudobiceros Faubel, 1984) were supported as a monophyletic cluster in the phylogenetic reconstruction of Litvaitis et al. [20], although genera with single and duplicated genital organs were intermingled in other studies [20,[26][27][28]33]. ...
Platyhelminthes (flatworms) are a diverse invertebrate phylum that are useful for exploring life history evolution. Within Platyhelminthes, only two clades develop through a larval stage: freeliving polyclads and parasitic neodermatans. Neodermatan larvae are considered evolutionarily derived, whereas polyclad larvae are hypothesized to be retained from the last common ancestor of Platyhelminthes – and Spiralia – due to ciliary band similarities among polyclad and other spiralian larvae. However, larval evolution has been challenging to investigate within polyclads due to low support for deeper phylogenetic relationships. To investigate polyclad life history evolution, we generated transcriptomic data for 21 species of polyclads to build a wellsupported phylogeny for the group. We then used ancestral state reconstruction to investigate ancestral modes of development (direct vs indirect) within Polycladida, and flatworms in general. The resulting tree provides strong support for deeper nodes and we recover a new monophyletic clade of early branching cotyleans. Early branching clades of acotyleans and cotyleans possess diverse modes of development, suggesting a complex history of larval evolution in polyclads that likely includes multiple losses and/or multiple gains. Our ancestral state reconstructions in previous platyhelminth phylogenies also suggest that larval similarities between flatworms and other phyla are most likely convergently evolved.
... Faubel (1983Faubel ( , 1984 placed more importance in the traits of the reproductive systems due to their complexity while Prudhoe (1985) proposed a classification based on the arrangement of the eyes and tentacles. Current molecular studies (Aguado et al. 2017;Bahia et al. 2017;Tsunashima et al. 2017;Litvaitis et al. 2019;Dittmann et al. 2019;Oya & Kajihara 2020) have recovered three clades containing the different acotylean families yet these do not completely correspond to the ones established by either of the traditional classifications based on morphology. Following the definitions proposed by Dittmann et al. (2019) and amended by Oya & Kajihara (2020), Acotylea is currently classified into three superfamilies: Discoceloidea, Leptoplanoidea and Stylochoidea. ...
Flatworms of the Order Polycladida are a group of free-living invertebrates found in a diversity of marine habitats, with over 800 species described worldwide. Marine flatworms are a conspicuous component of Australia’s marine fauna yet have received little attention. Less than 30 scientific articles have been published on Australian marine flatworms since 1855, of which only nine include species from southeastern Australia. Here, the biodiversity and distribution of species belonging to the Order Polycladida inhabiting intertidal rocky beaches in southeastern Australian waters were identified and analysed. Sampling was conducted at low tide along the coasts of New South Wales and Victoria. Collected samples were serially sectioned for comparative anatomical studies, and tissue was removed from each individual for molecular sequencing and analyses. Both nuclear and mitochondrial DNA sequences were obtained and used as an additional source of evidence for the description of new species as well as providing further insight into the phylogenetic relationships between them. A total of 20 species, six of which are new (e.g., Eulatocestus australis sp. nov.), and a new genus (Parabolia gen. nov.) have been described, as well as two new records for Australia (e.g., Stylochoplana clara Kato, 1937) have been identified increasing our knowledge of this important component of the Australian marine biota.
... The cotylean polyclad family Prosthiostomidae Lang, 1884 is characterized by i) an elongated body with a ventral sucker posterior to the female gonopore, ii) a plicate tubular pharynx, and iii) paired prostatic vesicles. Monophyly of this family has been supported in previous molecular phylogenetic studies based on partial sequences of the 28S rRNA gene alone (Bahia et al. 2017;Tsunashima et al. 2017;Litavaitis et al. 2019) or in combination with the 18S rRNA gene (Dittmann et al. 2019). Aguado et al. (2017) argued that Prosthiostomidae is not monophyletic, but this is probably due to the fast-evolving gene markers that they utilized (i.e., the mitochondrial 16S rRNA and cytochrome c oxidase subunit I (COI) genes). ...
... For a phylogenetic analysis, a concatenated dataset comprised of partial 28S rRNA and COI gene sequences was prepared. We employed the 28S rRNA gene as one of the two markers because it is the most commonly used for determining members of Polycladida (Bahia et al. 2017;Tsunashima et al. 2017;Dittmann et al. 2019;Litvaitis et al. 2019). In addition to the sequences determined for the four species from Misaki and for P. grande (see above), 24 other partial 28S rRNA gene sequences from 19 prosthiostomid species downloaded from GenBank were used in the analysis; multiple sequences derived from individuals representing geographically different local populations were used for Enchiridium periommatum Bock, 1913; Enchiridium sp. 2 of Litvaitis et al. (2019); and P. siphunculus (Table 1). ...
... In terms of the entire polyclad phylogeny, the analysis by Litvaitis et al. (2019) represented the densest sampling of Prosthiostomidae taxa before our study (cf. Aguado et al. 2017;Bahia et al. 2017;Tsunashima et al 2017;Dittmann et al. 2019). While a different taxonomic view was once proposed in terms of the generic affiliation (see Remarks for P. auratum), P. auratum was more closely related to P. siphunculus (type species of Prosthiostomum) than to Euprosthiostomum mortenseni in our tree (Fig. 7). ...
The present study provides morphological descriptions of four species of Prosthiostomum (Polycladida, Prosthiostomidae)—P. auratum Kato, 1937; P. hibana sp. n.; P. cf. ostreae Kato, 1937; and P. vulgare Kato, 1938—based on specimens collected among branching coralline algae and kelp holdfasts in Misaki, Japan. The new species P. hibana sp. n. is characterized by i) the dorsal surface of the body covered with numerous orange maculae, some of which coalesce together to form larger ones; ii) a pair of linear cerebral-eyespot clusters, each consisting of relatively few (7–9) cerebral eyespots; iii) 3–4 pairs of ventral eyespots embedded in parenchyma: iv) the inner wall of the male atrium deeply ruffled; v) the lumen of the seminal vesicle being narrow and elongated in shape; and vi) a large sucker situated in the center of the body. We remark on some orphological characters that were not mentioned in the original description of P. auratum. We infer the phylogenetic positions of these four species within Prosthiostomidae using the aximum-likelihood analysis based on partial 28S rRNA and COI gene sequences determined de ovo, in addition to those that are currently available in public databases. In the resulting tree, the four species—P. auratum, P. hibana sp. n., P. cf. ostreae, and P. vulgare—were nested in a clade that was composed of all the other Prosthiostomum species included in the analysis.
... Total genomic DNA was extracted from the excised tissue using standard phenolchloroform extraction with some modification [56]. The 28S rRNA genes were PCRamplified from the extracted DNA using the primers, HRNT-F2 (5 -AGTTC AAGAG TACGT GAAAC C-3 ) and HRNT-R2 (5 -AACAC CTTTT GTGGT ATCTG ATGA-3 ) [56]. ...
... Total genomic DNA was extracted from the excised tissue using standard phenolchloroform extraction with some modification [56]. The 28S rRNA genes were PCRamplified from the extracted DNA using the primers, HRNT-F2 (5 -AGTTC AAGAG TACGT GAAAC C-3 ) and HRNT-R2 (5 -AACAC CTTTT GTGGT ATCTG ATGA-3 ) [56]. The PCR reaction mixture contained 1 µL of DNA template, 2 µL of 10 × buffer for Ex Taq, 1.6 µL of dNTPs, 2.6 µL of 5 mM of each primer, 0.625 unit of Ex Taq (Takara Bio, Shiga, Japan), and H 2 O for a total volume of 20 µL. ...
Several polyclad flatworm species are known to contain high levels of tetrodotoxin (TTX), but currently TTX-bearing flatworms seem to be restricted to specific Planocera lineages belonging to the suborder Acotylea. During our ongoing study of flatworm toxins, high concentrations of TTXs were detected for the first time in the flatworm Prosthiostomum trilineatum, suborder Cotylea, from the coastal area of Hayama, Kanagawa, Japan. Toxin levels were investigated by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing that this species contains comparable concentrations of toxins as seen in planocerid flatworms such as Planocera multitentaculata. This finding indicated that there may be other species with significant levels of TTXs. The distribution of TTXs among other flatworm species is thus of great interest.
... Total genomic DNA was extracted from the excised tissue sample for each individual, following Tsunashima et al. (2017). Amplicons for a partial sequence of the 28S rRNA gene (approximately 1100 bp) were obtained by PCR using primers HRNT-F2 (5′-AGTTC AAGAG TACGT GAAAC C-3′) and HRNT-R2 (5′-AACAC CTTTT GTGGT ATCTG ATGA-3′). ...
Tetrodotoxin (TTX), also known as pufferfish toxin, causes a respiratory disorder by blocking neurotransmission, with voltage-gated sodium channel inhibition on muscle and nerve tissues. The toxin is widely distributed across vertebrates, invertebrates and bacteria. Therefore, it is generally thought that TTX in pufferfish accumulates via the food webs, beginning with marine bacteria as a primary producer. Polyclad flatworms in the genus Planocera are also known to be highly toxic, TTX-bearing organisms. Unlike the case of pufferfish, the source of TTX in these flatworms is unknown. In this study, taxonomical distribution patterns of TTX were investigated for acotylean flatworms from coastal waters using molecular phylogenetic analysis and high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). A maximum likelihood tree based on the 28S rRNA gene sequence showed that the flatworms belonged to several different lineages among the genera Planocera, Stylochus, Paraplanocera, Discocelis, Notocomplana, Notoplana, Callioplana and Peudostylochus. After LC-MS/MS analysis, the distribution of TTX was mapped onto the molecular phylogenetic tree. TTX-bearing flatworm species were seen to be restricted to specific Planocera lineages, suggesting that the TTX-bearing flatworm species have common genes for TTX-accumulating mechanisms.
... So far, no members of Zygantroides and Zygantroplana have been included in molecular phylogenetic studies (cf. Aguado et al. 2017;Bahia et al. 2017;Tsunashima et al. 2017;Dittmann et al. 2019;Litvaitis et al. 2019). ...
We describe a new species of ilyplanid polyclad, Zygantroides serpulidicola sp. nov., from Amakusa, Kumamoto, Japan. This is the third member of Zygantroides Faubel, 1983 and the first record of the genus from the Pacific Ocean. Zygantroides serpulidicola sp. nov. is characterized by i) the mouth opening near the common gonopore, ii) the sperm ducts separately entering a pear-shaped seminal vesicle, iii) an elongated Lang’s-vesicle duct, and iv) the horseshoe-shaped Lang’s vesicle located posterior to the common gonopore. We provide a partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene (712 bp) as a DNA barcode for the species. Our phylogenetic analyses based on concatenated sequences of the 16S, 18S, and 28S ribosomal DNA and COI indicate that Z. serpulidicola sp. nov. is nested in a clade comprised of Discocelidae and Ilyplanidae; the latter does not appear to be monophyletic.
... Taxonomic remarks. Thysanozoon brocchii Grube, 1840, the type species of the genus, was described in the Gulf of Naples, Italy, and is considered a cosmopolitan species (Brusa et al. 2009;Bulnes et al. 2011;Bahia et al. 2015), ranging from the Mediterranean to the Indian Ocean (Stummer-Traunfels 1895, Pitale & Apte 2017), Japan (Tsunashima et al. 2017), the Southwestern and Eastern Atlantic (Brusa et al. 2009;Bulnes et al. 2011;Noreña et al. 2014;Bahia et al. 2015), and the Caribbean Sea (Marcus & Marcus 1968). Thysanozoon estacahuitensis sp. ...
The flatworms of the suborder Cotylea live in diverse marine substrates such as rocks, seaweed and sand or in association with corals. In the Mexican Pacific, particularly on the coast of Oaxaca, knowledge about cotyleans is scarce, with only one record of the genus Pseudoceros. The aim of this work was to describe the cotylean species present in the area. A total of 48 specimens were collected, fixed, mounted in whole mounts or histological sagittal and frontal histological sections of the reproductive systems. These were examined for taxonomic identification, determining five new species of four genera: Boninia oaxaquensis sp. nov., Pericelis sigmeri sp. nov., Pericelis nazahui sp. nov., Pseudoceros bicuti sp. nov. and Thysanozoon estacahuitensis sp. nov. The present study establishes the first record of the genus Boninia and the family Boniniidae for the Tropical Eastern Pacific. It also confirms the presence of the genus Pseudoceros on the coast of Oaxaca, and break the disjunct distribution of this genus (from Gulf of California to Gulf of Tehuantepec), as well as the genus Pericelis (from Canada to Galapagos Islands) and Thysanozoon (from Chile to Gulf of California).
