Drosophila montium species group, 22 of which are presented here for the first time. The montium group is well-positioned for clade genomics. Within the montium clade, evolutionary distances are such that large numbers of sequences can be accurately aligned while also recovering strong signals of divergence; and the distance between the montium group and D. melanogaster is short enough so that orthologous sequence can be readily identified. All genomes were assembled from a single, small-insert library using MaSuRCA, before going through an extensive post-assembly pipeline. Estimated genome sizes within the montium group range from 155 Mb to 223 Mb (mean = 196 Mb). The absence of long-distance information during the assembly process resulted in fragmented assemblies, with the scaffold NG50s varying widely based on repeat content and sample heterozygosity . The total scaffold length for most assemblies is also shorter than the estimated genome size, typically by 5–15%. However, subsequent analysis showed that our assemblies are highly complete. Despite large differences in contiguity, all assemblies contain at least 96% of known single-copy Dipteran genes . Similarly, by aligning our assemblies to the D. melanogaster genome and remapping coordinates for a large set of transcriptional enhancers , we showed that each montium assembly contains orthologs for at least 91% of D. melanogaster enhancers. Importantly, the genic and enhancer contents of our assemblies are comparable to that of far more contiguous Drosophila assemblies. The alignment of our own D. serrata assembly to a previously published PacBio D. serrata assembly also showed that our longest scaffolds (up to 1 Mb) are free of large-scale misassemblies. Our genome assemblies are a valuable resource that can be used to further resolve the montium group phylogeny; study the evolution of protein-coding genes and cis-regulatory sequences; and determine the genetic basis of ecological and behavioral adaptations.Large groups of species with well-defined phylogenies are excellent systems for testing evolutionary hypotheses. In this paper, we describe the creation of a comparative genomic resource consisting of 23 genomes from the species-rich  Drosophila have included twelve species spanning the entire Drosophila lineage  species are closely related enough such that sequence similarity produces accurate alignments, but distantly related enough to recover a strong signal of sequence divergence. In this paper, we describe the creation of such a resource by assembling 23 genomes from the Drosophila montium species group, which is well-positioned for clade genomics  (montium group diverged from D. melanogaster roughly 28 million years ago (mya)  , but givhilidae) , 2018, ihilidae)  currentlhilidae) . The mony 19 mya . Members Oceania . More thmorphism , and coumorphism . Previoumontium genomes have already been assembled. The D. kikkawai genome .Fly lines for each All fly lines were maintained in small population vials. Prior to sequencing, some lines went through several generations of inbreeding. Other lines were not inbred, either due to difficulty maintaining the fly line, or time limitations.D. kikkawai genome  were explored using FastQC (v. 0.11.2)   and Stri 0.11.2) . SGA Prei.e., 101 bases instead of 100). This extra base was trimmed using BBDuk (BBMap v. 36.11) (Bushnell), with the option ftr = 99. Reads were adapter-trimmed for known Illumina adapters using BBDuk, with the options ktrim = r, k = 23, mink = 9, hdist = 1, minlength = 75, tpe = t, and tbo = t. The adapter-trimmed reads were then quality-trimmed to Q10 using BBDuk , with the options qtrim = rl, trimq = 10, and minlength = 51.Reads from some sequencing runs contained an extra base . Low levels of bacteria were also present in the D. burlai library. We utilized two different decontamination strategies.The D. pectinifera, we adopted a decontamination strategy similar to vs. average k-mer coverage plot. Scaffolds with 35 <= GC % <= 66 and 40.5 <= average k-mer coverage <= 68 were classified as candidate contaminant scaffolds. To avoid removing Drosophila scaffolds, candidate contaminant scaffolds were aligned to sequences in NCBI’s Nucleotide database  (For 5-dirty) , and the5-dirty) , potenti5-dirty)  using BL5-dirty) , with thD. burlai, D. jambulina, D. mayri, D. seguyi, and D. vulcana libraries appeared to be contaminated with highly abundant individual sequences, or groups of similar sequences. These sequences created spikes in the Per Sequence GC Content plots from FastQC , with the options bhist, lhist, and gcbins = auto. The output was then used to calculate the GC % of the reads, which are reported in montium genomes / samples represent a diversity of genome size estimates, repeat contents, heterozygosity levels, and sequencing error rates. Extensive tests were conducted to identify the assembler that performed the best across these diverse samples.Exploration of the data using SGA Preqc  showed tFRCbam) (We tested the following assemblers: ABySS . The resAll genomes were assembled using MaSuRCA (v. 3.2.2)  was set to the product of the estimated genome size and coverage.Our post-assembly pipeline started with assemblies in the MaSuRCA  were later filtered from the broken assemblies.REAPR - Recognition of Errors in Assemblies using Paired Reads (v. 1.0.18) , but varied if Sealer became stuck on a given k-mer size. After two rounds of gap closing, the D. triauraria assembly contained more than 2,000 single-N gaps. The remaining single-N gaps and associated flanking sequence were hard-masked with 300 Ns, and Sealer was run a second time using the above settings. This potentially extended the flanking sequence extracted by Sealer beyond the boundaries of the original tandem allele, thereby making it possible to find a connecting path in the graph. This decreased the number of single-N gaps below 2,000.Gaps in the assemblies were closed using a two-step process with adapter-trimmed and quality-trimmed reads. The first round of gap closing was performed using GapCloser (v. 1.12) (The assemblies were further improved using Pilon (v. 1.22) , with thAfter running Pilon , with thPilon  were randomly assigned to a single base, and scaffolds shorter than 1 kb in length were removed.Contaminants in the final assemblies were identified by NCBI’s Contaminant Screen. Most assemblies contained small numbers of scaffolds from bacterial or yeast species, which were removed. A total of four scaffolds across all assemblies also contained suspected adapter / primer sequences. These scaffolds were trimmed if the potential contaminant was located at the end of the scaffold, or split if it was located in the middle of the scaffold.10(repeat content) was calculated using the R (v. 3.4.1) (k = 41), as calculated by SGA Preqc (The correlation between the estimated genome size and the log. 3.4.1)  functionGA Preqc .k = 41), respectively. These values were calculated by SGA Preqc  and the percentage of the assembly present in scaffolds greater than or equal to 6.3 kb in length was calculated using the R  and the D. melanogaster reference genome   , with the sequence search engine RMBlast (v. 2.2.28) and Tandem Repeat Finder (TRF) (v. 4.04) (Each v. 4.04) , and thev. 4.04) , with thv. 4.04) . The LASD. melanogaster embryo. These fragments are approximately 2 kb in length. A total of 3,457 fragments were positive for enhancer activity and PCR-verified. D. melanogaster coordinates were remapped onto each montium assembly using liftOver  and melanogaster (query) sequences were aligned using BLASTn  to the previously published D. kikkawai (D. serrata (strain Fors4)  (van Rossum and de Boer 1991), and plots were created using Matplotlib (v. 1.5.1) .montium assemblies. Figure S2 contains additional dotplots.Table S1 reports a regression analysis for predictors of scaffold NG50. Table S2 reports a regression analysis for predictors of the percentage of the estimated genome size that was assembled. Figure S1 contains NG graphs showing the distribution of scaffold lengths for 23 Drosophila montium Species Group Genomes Project, NCBI BioProject Accession PRJNA554346. This record provides links to the assemblies, BioSamples, and sequencing data. The Whole Genome Shotgun projects have been deposited at DDBJ/ENA/GenBank under the accession numbers listed in Table 2. The versions described in this paper are versions XXXX01000000. Raw sequencing data were uploaded to the NCBI Sequence Read Archive (SRA). Besides removing reads that did not pass filtering, the FASTQ files were unprocessed.All assemblies and sequencing data are publicly available through the D. cf. bakoue, D. kanapiae, D. mayri, D. punjabiensis, D. tani, D. truncata, and D. vulcana were spread across two lanes of a flowcell. When the FASTQ files were uploaded to the NCBI SRA, the R1 and R2 files from both lanes were combined into individual R1 and R2 FASTQ files. If users wish to demultiplex reads by lane for these samples, lane information  is preserved in the sequence identifier line of the original FASTQ files.Sequencing libraries for et al. 2013–2015) annotation / summary tables, liftOver chain files from whole genome alignments, and BUSCO assessment tables were deposited in the Dryad repository: https://doi.org/10.6078/D1CH5R. Supplemental material available at figshare: https://doi.org/10.25387/g3.11301932.Repeat-masked assemblies, RepeatMasker , PCR-free, library to roughly 35x coverage for each species. The genomes were assembled using the Maryland Super Read Cabog Assembler (MaSuRCA), which combines de Bruijn graph and overlap-layout-consensus (OLC) approaches into a novel algorithm based on “super-reads”  Preqc (montium assembly. A) Preqc . The scamontium group range from 155.1 Mb to 223.4 Mb . These sizes are consistent with the previously assembled D. kikkawai (D. serrata (D. serrata assembly (strain 14028-0681.02) has an estimated genome size of 184.7 Mb. The relatively small difference between our genome size estimate and the total contig length of the previously published PacBio D. serrata assembly (strain Fors4) .Estimated genome sizes within the D. kanapiae assembly , to the highly fragmented D. triauraria assembly . The contiguity of the D. kanapiae assembly is somewhat surprising, given the use of a small-insert library, but is related to genome and sample characteristics described below. The average scaffold NG50 across all montium species is 73,813 bp .Scaffold NG50s vary widely, from the remarkably contiguous montium genomes were assembled using small-insert libraries (350 bp), they are especially sensitive to repeat content and heterozygosity. In the absence of long-distance information, in the form of mate-pair libraries or long-reads, large repeats form unresolvable structures in the graph. This results in fragmented assemblies that are missing many repeat copies . As expected, the scaffold NG50 is inversely proportional to repeat content and heterozygosity, with repeat content impacting assemblies nearly twice as much as heterozygosity. The scaffold NG50 is generally unaffected by sequencing depth, as most genomes reach the minimum coverage necessary to effectively assemble contigs. While the sample size is small, the regression results are reassuring in that for each variable, the direction and relative magnitude of change is consistent with general genome assembly predictions.Repeat content and heterozygosity vary widely across genomes / samples , which imontium assemblies reaches 85–95% of the estimated genome size. In Figure S1, this is where the curves intersect the x-axis. Given that our assemblies are missing many large repeat copies (see above), they should generally be shorter than the estimated genome size, with the magnitude of the difference proportional to the number, size, and divergence of repeat copies . As expected, the percentage of the estimated genome size that was assembled is inversely proportional to repeat content, but positively correlated with heterozygosity - with repeat content being the primary driver. Once again, the sample size is small for a regression analysis, and the heterozygosity results only reach statistical significance at an alpha level of 0.10, but the results are generally as expected.To investigate the combined effect of repeat content, heterozygosity, and coverage on the percentage of the estimated genome size that was assembled across all montium assemblies are fragmented, as evidenced by their modest scaffold NG50s. However, taken in isolation, the NG50s say little about the quality of the assemblies. Any single metric  can be a poor predictor of the quality / utility of an assembly. It is best to evaluate assemblies using a variety of methods, with an eye toward the downstream application , in practice, this effect is modest. Even for the most fragmented assemblies, roughly 80% of the assembly is present in at least gene-sized fragments.To study genes, a genome assembly should be present in at least gene-sized fragments  are single-copy genes present in more than 90% of surveyed species , to the large, repeat-rich, highly heterozygous, and fragmented D. triauraria . Strikingly, despite the wide range of contiguities, there is little variation in gene content: at least 96.1% of BUSCOs are complete (single-copy or duplicated) across all species. The D. kanapiae assembly exceeds 98%. For comparison, the previously assembled D. kikkawai and D. serrata genomes, which approach scaffold / contig N50s of 1 Mb, reach 98.1% and 96.2%, respectively , including nine of the ten BUSCOs that appear to be missing in the montium clade. Given that D. melanogaster was used to construct the Dipteran BUSCO set, and contributed orthologs for all 2,799 genes, no BUSCO should in theory be classified as missing in the reference assembly. However, high sequence divergence and complex gene structures can make BUSCOs difficult to identify in an assembly, making it appear as if fragmented or even complete genes are missing . The average percent identity is 75.1%, and the Expect value (E) for the vast majority of alignments is essentially zero (data not shown). Based on these results, it is clear that we can remap coordinates for thousands of D. melanogaster enhancers onto any montium assembly, and with a high level of confidence extract orthologous sequences.With the genomes aligned, we turned to looking for known enhancer sequences in the remapped . To dete genomes . Of noteD. serrata assembly (strain 14028-0681.02) to orthologous contigs in the previously published - and far more contiguous - PacBio D. serrata assembly (strain Fors4) , our scaffolds should generally align end-to-end within the longer PacBio contigs, with only relatively small insertions or deletions. Dotplots for pairwise alignments are shown in D. kikkawai assembly  from our e.g., collapsed repeats, small inversions, or tandem alleles) within these scaffolds. We used REAPR  (montium assembly contains orthologs for at least 91% of D. melanogaster enhancers. (This same approach can be used for any annotated feature in the D. melanogaster genome.) Importantly, the genic and enhancer contents of our assemblies are comparable to that of far more contiguous Drosophila assemblies. Finally, the alignment of our D. serrata assembly to a previously published PacBio D. serrata assembly  with mate-pair libraries or PacBio long-reads, they can easily generate vastly more contiguous assemblies that include most repeat copies.While all of our assemblies are complete enough to study genes and enhancers, if other researchers are interested in repeat structure, any montium group phylogeny; study the evolution of protein-coding genes and enhancers; and determine the genetic basis of ecological and behavioral adaptations.Going forward, our genome assemblies will be a valuable resource that can be used to further resolve the


Xenopus tropicalis is an excellent model organism for studies on vertebrate development and regeneration  and is also useful for the study of tumor formation . Spontaneously occurring neoplasia in amphibians have been reported, such as in X. laevis, Rana pipiens, and Andria japonicus . In X. laevis, one of 4,000 frogs , which were maintained in artificial outdoor ponds, displayed renal adenocarcinoma without other developmental disorders, indicating that the tumor formation was quite rare in X. laevis . In contrast to X. laevis, 3.66 % of R. pipiens in the wild heterogeneous populations had renal adenocarcinoma , which was experimentally induced by herpesvirus . Currently, there are no reports of spontaneous neoplasia in X. tropicalis, although recently developed genome editing technologies such as TALEN and CRISPR/Cas9 offer opportunities to induce neoplasia by simple disruption of tumor suppressor genes , which would allow modeling of human cancer in this species. The spontaneously occurring neoplasia in X. tropicalis could provide excellent opportunities to understand how the genetic background of this species influences the neoplasia phenotypes in combination with disruption experiments of tumor suppressor genes.X. tropicalis in Japan has successfully developed the four highly inbred wild-type strains, Nigerian A (NA), Nigerian H , Nigerian BH , and Ivory Coast (IC) . These strains are available for international research communities upon request. We found spontaneous neoplasia formation in the frog stocks at the NBRP . Histological analysis of X. tropicalis neoplasia using hematoxylin–eosin (HE) staining showed that melanocytes were enriched in the black stone-like nodules and that a layered structure was present  of the NBRP . Analysithe NBRP . Almost  nodules . Previou present . Two fro present . The aveNeoplasias were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4°C and were successively washed with PBS containing 0.1 % Tween 20. Fixed neoplasias were frozen in Optimum Cutting Temperature Compound, and cryosectioned with a thickness of 10 μm. Sections were stained with HE by a standard method. Each slide was photographed under a Zeiss Axio Vert. A microscope using an AxioCam MRc digital camera.X. tropicalis strains used in this experiment were maintained at the Amphibian Research Center, Hiroshima University. All treatment procedures for X. tropicalis were approved by Yamagata University Animal Research Committee (#310300). For HE staining, we used the following reagents: Mayer’s hematoxylin solution and 1% eosin Y solution , Tissue-Tek optimum cutting temperature compound .


Eruca sativa Mill. (Brassicaceae) is an important edible vegetable and a potential medicinal plant due to the antibacterial activity of its seed oil. Here, the complete chloroplast (cp) genome of E. sativa was de novo assembled with a combination of long PacBio reads and short Illumina reads. The E. sativa cp genome had a quadripartite structure that was 153,522 bp in size, consisting of one large single-copy region of 83,320 bp and one small single-copy region of 17,786 bp which were separated by two inverted repeat (IRa and IRb) regions of 26,208 bp. This complete cp genome harbored 113 unique genes: 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Forty-nine long repetitive sequences and 69 simple sequence repeats were identified in the E. sativa cp genome. A codon usage analysis of the E. sativa cp genome showed a bias toward codons ending in A/T. The E. sativa cp genome was similar in size, gene composition, and linearity of the structural region when compared with other Brassicaceae cp genomes. Moreover, the analysis of the synonymous (Ks) and non-synonymous (Ka) substitution rates demonstrated that protein-coding genes generally underwent purifying selection pressure, expect ycf1, ycf2, and rps12. A phylogenetic analysis determined that E. sativa is evolutionarily close to important Brassica species, indicating that it may be possible to transfer favorable E. sativa alleles into other Brassica species. Our results will be helpful to advance genetic improvement and breeding of E. sativa, and will provide valuable information for utilizing E. sativa as an important resource to improve other Brassica species. Eruca sativa Mill. is an annual or perennial species of Eruca (Brassicaceae), mainly distributed in Europe and Western Asia. E. sativa is believed to have originated from the Mediterranean region and has been widely used as an oil crop and edible vegetable [E. sativa is also a popular salad and spice in Middle Eastern and European countries [E. sativa has many medicinal and therapeutic properties, including antioxidant and antimicrobial activities [E. sativa is resistant to white rust, drought, and aphids [Brassica species. Therefore, many attempts have been made to use somatic fusion and sexual hybridization to transfer such desirable agronomic traits from E. sativa to other Brassica species [E. sativa and other species in Brassicaceae family will undoubtedly improve the ability to transfer desirable agronomic traits to related species.egetable . Due to ountries . Moreovetivities  as well tivities . Additiod aphids , traits  species . A phyloChloroplasts (cp) have an independent circular genome and play essential roles in photosynthesis, development, and the physiology of green plants , 8. Cp gE. sativa is not available.Many cp genomes have recently been decoded due to the advancements in next-generation sequencing technology, particularly third-generation sequencing technology which yields reads longer than 10 kb –21. ThusE. sativa was de novo assembled with a combination of long PacBio reads and short Illumina reads, and the features of this cp genome were fully elucidated. Next, 59 cp genomes of other species in the Brassicaceae family from GenBank were used to determine the genealogical relationships between E. sativa and other species. Our results will enable further genetic improvements and breeding of E. sativa and provide valuable information for utilizing E. sativa as a resource to improve of important Brassica species.In the present study, the complete cp genome of E. sativa seeds were provided by Professor Zaiyun Li (Huazhong Agriculture University), and cultivated in a glasshouse at Guizhou Normal University . A total of 5 g of fresh young E. sativa leaves of were collected to isolate cp DNA using the Plant DNA Extraction Mini Kit C  according to the manufacturer’s instructions. After determining the integrity of the DNA, 1 μg of DNA was fragmented, and a short-insert library (with the insertion of 450 bp) was constructed for Illumina sequencing (HiSeq X Ten), according to the manufacturer’s instructions . Then, 5 μg DNA was used to prepare the DNA libraries with insert sizes of 20 kb for PacBio sequencing, according to the manufacturer’s instructions . All the raw data, including short Illumina reads and long PacBio reads were submitted to the figshare (https://figshare.com) with the DOI: 10.6084/m9.figshare.13653515.Arabidopsis thaliana (NC_000932) cp genome using BLASR  [The 150 bp paired-end reads were produced by the Illumina sequencing platform. After removing the sequencing adapters and low-quality reads, the clean reads were obtained by Trimmomatic , accordiinement)   . The lowinement) . To corrinement) . Frame-sE. sativa cp genome with default settings [E. sativa circular gene map was drawn in OGDraw software version 1.2 [E. sativa is available in the public GenBank database (https://www.ncbi.nlm.nih.gov/) under the accession number of MT013255.The Dual Organellar Genome Annotator was used to annotate the settings . The stasion 1.2 . The welE. sativa cp genome, including the palindrome, reverse, forward, and complement types, were detected by the web-service REPuter (https://bibiserv.cebitec.uni-bielefeld) [https://webblast.ipk-gatersleben.de/misa/) [The long repeat sequences of the elefeld)  with thee/misa/)  with theE. sativa cp genome, the CodonW1.4.2 program [http://downloads.fyxm.net/CodonW-76666.html) was employed to calculate the synonymous codon usage of the protein-coding genes with default settings. The relative synonymous codon usage (RSCU) of all coding genes was also analyzed.To understand the translation dynamics of the  program  (http://http://genome.lbl.gov/vista/mvista/submit.shtml) [E. sativa cp genome and those of six related species. The related cp sequences were downloaded from the National Center for Biotechnology Information (NCBI), including B. rapa (NC_040849), B. oleracea (NC_O41167), B. juncea (NC_0282720), B. nigra (NC_030450), B. napus (NC_016734) and A. thaliana (NC_000932). IRscope (https://irscope.shinyapps.io/irapp/) was used to compare LSC/IRb/SSC/IRa junction regions among the seven selected cp genomes according to the annotated information.The mVISTA program (t.shtml)  was usedE. sativa and six related Brassicaceae species were employed to calculate non-synonymous (Ka) and synonymous (Ks) substitution rates. Pairwise alignments of these genes were carried out by with MAFFT, and the Ka/Ks value was determined with KaKs calculator (version 2.0) according to the default parameters.Pairwise comparisons of 79 protein-coding genes shared between E. sativa and related Brassicaceae species, 59 Brassicaceae species cp genomes  method with 1000 replicates using MEGA7 [To analyze the phylogenetic relationships between  genomes  were dowE. sativa cp genome . The complete E. sativa cp genome had a quadripartite structure comprised of 153,522 bp, including an SSC region of 17,786 bp and an LSC region of 83,320 bp, which were separated by a pair of inverted repeats (IRa and IRb) of 26,208 bp  possessed two introns , and the model species: A. thaliana. As shown in B. napus) to 154,478 bp  in length, and each component of the quadripartite cycle was comparable among the selected cp genomes. The overall GC content was also quite similar (36.29–36.39%) among these cp genomes. The gene content in these cp genomes was consistent, except that the ycf15 gene was missing in the A. thaliana cp genome. The ycf15 gene was also missing in the S. alba cp genome [rps19 gene in all seven selected species. Furthermore, the rps19 gene had an expansion of 113/114 bp in the IRb region in all selected cp genomes. The trnH-GUG gene and rpl2 gene resided at the LSC/IRa border, respectively, in all seven cp genomes, and the trnH gene was 2–30 bp from the border. Additionally, the boundary of IRb/SSC was located in the repetitive regions of the ycf1 and ndhF genes in all seven species, with only 1 bp of ycf1 and 36 bp of ndhF located in the SSC region. The IRa/SSC junction extended into the ycf1 gene in the other five species and the length of the ycf1 genes ranged from 1,027 bp to 1,030 bp in the IRa region, but the ycf1 gene was missing from the IRb/SSC regions in the E. sativa and B. juncea cp genomes. Similar results were observed in mustard species, such as S. alba [S. arvensis.To detect divergence between the p genome , indicatp genome . In this S. alba  and S. aE. sativa cp genome, we used mVISTA to compare the homology of the whole cp sequence among the seven selected cp genomes of Brassicaceae, using the E. sativa cp genome as a reference  and non-synonymous (Ka) nucleotide substitution patterns of genes are important indicators of gene evolution [E. sativa cp genome compared to six closely related species, including B. rapa, B. oleracea, B. juncea, B. nigra, B. napus and A. thaliana  gene, E. sativa is closely related to S. alba [Eruca had the closest relationship with Diplotaxis harra [Eruca is closely related to the genus Brassica. In this study, the cp genomes of 59 Brassicaceae species were used for the phylogenetic analysis based on 62 homologous CDs. The phylogenetic tree generated 58 branches with node values > 48%  regions of 26,208 bp separated by one LSC region of 83,320 bp and one SSC region of 17,786 bp. The cp genome harbored 112 unique genes, including 79 protein-coding genes, 29 tRNA genes, and four rRNA genes. The synonymous (Ks) and non-synonymous (Ks) substitution rate analysis showed that protein-coding genes generally underwent purifying selection pressure, except ycf1 and ycf2. A phylogenetic analysis revealed that E. sativa is closely related to agriculturally important Brassica species, and most closely related to B. juncea, indicating that it may be possible to transfer favorable E. sativa alleles into other Brassica species. These results are helpful to further genetic improvement and breeding of E. sativa, and also provide valuable information for understanding the evolutionary history of E. sativa.In the present study, we obtained the complete cp genome of S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Pseudomonas taiwanensis is a novel bacterium that uses shrimp shell waste as its sole sources of carbon and nitrogen. It is a versatile bacterium with potential for use in biological control, with activities including toxicity toward insects, fungi, and the rice pathogen Xanthomonas oryzae pv.oryzae (Xoo). In this study, the complete 5.08-Mb genome sequence of P. taiwanensis CMS was determined by a combination of NGS/Sanger sequencing and optical mapping. Comparison of optical maps of seven Pseudomonas species showed that P. taiwanensis is most closely related to P. putida KT 2400. We screened a total of 11,646 individual Tn5-transponson tagged strains to identify genes that are involved in the production and regulation of the iron-chelator pyoverdine in P. taiwanensis, which is a key anti-Xoo factor. Our results indicated that the two-component system (TCS) EnvZ/OmpR plays a positive regulatory role in the production of pyoverdine, whereas the sigma factor RpoS functions as a repressor. The knowledge of the molecular basis of the regulation of pyoverdine by P. taiwanensis provided herein will be useful for its development for use in biological control, including as an anti-Xoo agent.The Gram-negative bacterium  Pseudomonas comprises Gram-negative aerobic gammaproteobacteria which are capable of inhabiting a variety of ecological niches. Its members exhibit great metabolic diversity. Several species benefit plants or degrade environmental pollutants, and some species act as human and plant pathogens, such as P. aeruginosa PAO1 and P. syringae pv. tomato DC3000 [Pseudomonas species have been sequenced, including P. aeruginosa PAO1, P. syringae pv. tomato DC3000, insect pathogen P. entomophila, plant promoting growth P. fluorescens SBW25 and xenobiotic-compound-degrading bacterium P. knackmussii B13 [Pseudomonas species have been used to study potential virulence, regulatory and resistance genes. Genomic background may also provide better understanding of phylogenetic position, horizontal gene transfer, gene organization, and unique genes or islands among Pseudomonas species.The genus ssii B13 ,5,6. ThePseudomonas taiwanensis CMS, which is a novel Gram-negative bacterium and displays a board host range. It was isolated from soil in northern Taiwan, and uses shrimp shell waste as the sole carbon and nitrogen source [P. taiwanensis could produce high levels of extracellular chitinase, chitosanase and nattokinase [Plutella xylostella, Spodoptera exigua, and Trichoplusia ni [Drosophila melanogaster [Here, we focus on n source ,8. When tokinase ,10. It hnogaster .P. taiwanensis displayed strong antagonistic activity against the rice pathogen Xoo [Xoo is a major rice disease worldwide, which has been reported in Asia, northern Australia, Africa, the southern part of the United States and Latin America [Xoo agents will likely be essential in the future.In a previous study, we found that ogen Xoo . Rice ba America . Rice yi America . So far,P. taiwanensis has anti-Xoo activity by the secretion of iron-chelator pyoverdine and that T6SS was found to be involved in the secretion of pyoverdine in P. taiwanensis through an unknown mechanism [P. taiwanensis by high-throughput DNA sequencing (Roche 454 and Illumina Solexa). A combination of 454 and Illumina scaffolds were aligned onto an optical whole genome restriction map (WGRM). The de novo assembly contigs from next-generation sequencing were aligned to form large scaffolds and understand their orientation based on an optical WGRM that has been used in many projects [P. taiwanensis, we screened the Tn5 transposon tag library for P. taiwanensis mutants with reduced or increased anti-Xoo activity. The flanking sequences of Tn5 transposon insertion sites were determined and blasted against the genomic sequences of P. taiwanensis (NCBI accession no. CP011858), which allowed us to efficiently identified the genes that are mutated by Tn5 transposon. Taken together, combining data mining of the P. taiwanensis draft genome and mutant toxicity data, we were able to precisely identify virulence factors and pathogenic pathways involved in the antagonistic activity against rice pathogen Xoo.Previously, we showed that echanism . In thisprojects ,14. TranP. taiwanensis genome was sequenced and generated via a de novo assembly from 454 (Roche) long reads and paired-end Illumina short reads. Using Solexa paired-end sequencing, the total output produces 3389 million paired-end total read length with an average length of 79 bases after adaptor trimming, giving approximately 666-fold coverage of the genome. In addition, the 454 sequencing produces total output of 248 million read length with an average length of 382 bases after adaptor trimming, giving approximately 48.7-fold coverage of the genome. The contigs from Solexa and 454 were organized by high-resolution restriction map  from a single strain DNA of P. taiwanensis  with 62.6% GC content and annotated to encode 4477 proteins  and terminus (ter)  contains a specific DnaA-binding site which is located near the dnaA gene [P. taiwanensis, the GC skew shows a symmetrical structure of oriC located opposite the terminus position  did not hit any sequence against the COG database  functional classifications using the COGs database . In P. tbase see . The larbase see . The amis putida ,23. Thisbacteria . The larnfection . In addients see .P. taiwanensis was performed by optical restriction mapping based on the unweighted-pair group method with arithmetic averages (UPGMA) [P. taiwanensis was compared to those of other Pseudomonas species that were derived from public genomic sequences with in silico BamHI digestion  analysis  . The whoigestion A. The whanalysis B. The phtion map A and multion map B, and th studied ,27. The P. taiwanensis to P. putida KT2400 and P. entomophila L48, the genome sizes among these three species are different. In comparison to P. putida KT2400 and P. entomophila L48, the P. taiwanensis genome has a smaller size that contains fewer open reading frames (4477), tRNA (69), and rRNA (16)  . HoweverP. taiwanensis (Y-axis) and P. putida KT2400 (X-axis) or P. entomophila L48 (X-axis). MUMmer software revealed an X-shape alignment pattern in the dot plot in which a conserved region formed a straight line and a scattered distribution indicates less homology between two species. P. taiwanensis displayed less homology of large genomic regions when compared to P. putida KT2400 and P. entomophila L48  was used to detect the flanking sequence of the transposon. The TAIL-PCR products were sequenced and subjected to BLAST search against the CDS of P. taiwanensis (NCBI accession no. CP011858) and the Pseudomonas database.In our prior studies, we found that ogen Xoo . Severalcterized . To gaintivities . To dete5-inserted mutants were annotated by the COG database. These genes were found to be involved in metabolism, information storage and processing, cellular processes and signaling  and have no toxicity against Xoo. The pvdL gene encodes non-ribosomal peptide synthetase (NRPS) which is involved in the biosynthesis of pyoverdine chromophore [5-inserted mutants, we collected genes that have been shown to synthesize and regulate iron-chelator pyoverdine  under iron-limited conditions  compared to wild type  was used in this study [P. taiwanensis against Xoo was conducted on 1/2 trypticase soy (TSB) agar plates  at 28 °C. P. taiwanensis was grown in 100 mL of iron-limited liquid medium  in a 500 mL flask at 28 °C and 200 rpm with shaking for 24 h. Xoo was cultured in 1/2 TSB medium at 28 °C for 1 day. Xoo was mixed with melted 1/2 TSB agar medium before being poured into an empty plate for competition assay. For bioassay, bacteria culture broth (109 CFU/mL) or filtered (0.22 µm) supernatant was injected (50 µL) into the hole in the Xoo-mixed 1/2 TSB agar plate until the inhibition zones had been characterized. The bacteria pellets were washed three times with PBS at 4 °C and resuspended in PBS. Cell density and cell viability were determined using optical density at OD600 and by counting CFU/mL.is study . AntagonP. taiwanensis was grown overnight in LB broth. The Qiagen RNA/DNA mini kit  was used for extraction of genomic DNA. DNA purity and concentration were determined by a nanodrop spectrophotometer and agarose DNA electrophoresis.P. taiwanensis BCRC 17,751 was sequenced using the Roche 454 GS FLS and Illumina GAII performed at the High Throughput Genomics Core Facility of the Biodiversity Research Center in Academic Sinica. The DNA reads of Illumina were used to generate 120 bp pair-end read lengths, and the Roche 454  was used to generate 300–400 bp read lengths. The paired-end raw reads of Illumina were trimmed to remove linkers and adaptors and further assembled by CLC Genomic Workbench (4.0.2). Trimming of adaptors and primers of Roche 454 lone reads was performed by Newbler (2.3). The contigs of Illumina and Roche 454 were oriented and assembled based on the whole genome optical BamHI-restriction map of P. taiwanensis  to construct one circular genome (P. taiwanensis was submitted to NCBI Genbank (NCBI accession no. CP011858).The genomic DNA of r genome . AssemblP. taiwanensis open reading frames (ORFs) were predicted and annotated using the NCBI prokaryotic genome annotation pipeline [P. taiwanensis is deposited in NCBI (NCBI accession no. CP011858). The prophage elements were identified via PHAge Search Tool (PHAST) [The pipeline . Annotat (PHAST) .P. taiwanensis compared with in silico BamHI-restriction map data of other Pseudomonas species using the unweighted pair group method with arithmetic mean (UPGMA). The whole genome-wide phylogenetic tree was generated in MapSolverTM from optical restriction map data. The MLST phylogenetic tree analysis of Pseudomonas species and other biocontrol agents was built by means of the seven housekeeping genes amino acid sequence, rpoD, gyrB, acnB, cts, gap, pgi, and pfk. A neighbor-joining tree based on the amino acid sequences of seven concatenated housekeeping genes was built by using MEGA 4.0 with 1000 bootstrap replicates.Phylogenetic trees were constructed based on an optical map and multi-locus sequence typing (MLST). A whole genome-wide phylogenetic tree was built based on the whole genome optical BamHI-restriction map data of Whole-genome DNA alignments were generated in two ways. First, the MUMmer program was used for pair-wise alignment of two entire bacterial genomes . Second,P. taiwanensis, P putida KT2400, and P. entomophila L48 were compared to each other using BLASTP with E-value cut-off of 10−5. The Markov cluster (MCL) algorithm was used to identify specific gene families by clustering the BLASTP results, which can be separated into different groups based on homology of proteins, with an inflation parameter of 2.0 [All predicted protein sequences of r of 2.0 .dnaA and GC skew [The position of the genomic replication origin was determined based on initiator gene  GC skew . GC skew5 insertion mutants using an EZ-Tn5 transposon mutagenesis kit <KAN-2> (Epicentre). To determine the Tn5 transposon insertion site, thermal asymmetric interlaced PCR (TAIL-PCR) was used to detect the sequences flanking the transposon. The tail-PCR products were sequenced and subjected to BLAST search against the P. taiwanensis database and Pseudomonas database. To screen the Tn5 mutant library, we utilized a P. taiwanensis mutagenesis library to incubate with Xoo to find pathogenicity-related genes.We made Tn600) of cell culture. Cell density was detected at 600 nm in a microplate spectrophotometer  after washing twice in PBS.Pyoverdine was measured from culture supernatants using fluorescence spectrometer  detection at excitation wavelength 405 nm and emission wavelength 460 nm ,60. Pyovm/z 1044) on the surface of the agar plate, as reported in a previous study [P. taiwanensis. The iron-limited agar samples with bacterial colonies were excised and placed on glass slides, and then covered with a thin layer of universal MALDI matrix (Sigma-Aldrich). Samples were detected in positive reflectron ion mode, and screened at 200 μm laser intervals with the acquisition mass range set at 1000–1500 Da. The standard peptide calibration mixture  and universal MALDI matrix were used to calibrate and test the MALDI-TOF mass spectrometer. The IMS data were analyzed using Fleximaging 3.0 software . The intensity of the molecules is presented as gradient colors in the figures.MALDI-IMS allows us to detect pyoverdine .TnP. taiwanensis as a biocontrol bacterium (Xoo activity of P. taiwanensis involves many genes that play distinct roles according to their characteristics. For induction of toxicity against Xoo, P. taiwanensis requires iron-limited conditions which activate iron-chelator pyoverdine synthesis genes and a specific EnvZ/OmpR two-component signal transduction pathway. In contrast, we found that the sigma factor RpoS negatively affects pyoverdine production. In a number of Gram-negative bacteria, EnvZ/OmpR is known to regulate osmotic stress response. However, this is the first study to show that EnvZ/OmpR plays a major role in regulating a network of downstream genes under iron-limited stress after mid-stationary phase growth of P. taiwanensis. EnvZ/OmpR signal transduction can be activated under iron-limited stress. In contrast, in envZ mutant of P. taiwanensis, many virulence factors show a lower level of expression in nutrient-rich conditions. The finding that the extracellular level of pyoverdine is affected Tn5-transposon inserted mutant of envZ in an iron-limited environment offers us an opportunity to examine how EnvZ/OmpR senses the iron signal to trigger signaling steps to regulate downstream genes. In the future, we will perform transcriptomic analyses of the wild type and Tn5-transposon tagged envZ mutant in iron-rich and iron-limited environments to address the role of iron in EnvZ/OmpR transduction and pyoverdine production. The results may provide information to improve the biocontrol capability of P. taiwanensis.In this work, we have provided a global view of the molecular basis of acterium . Antagon


Tritonia tetraquetra  and Tritonia exsulans Bergh, 1894 is confirmed. T. tetraquetra distributes across the large geographic and bathymetric distances in the North-Eastern (NE) and North-Western (NW) Pacific. In turn, at NE Pacific coasts the separate species T. exsulans is commonly occured. Thus, it reveals a misidentification of T. tetraquetra and T. exsulans species in neuroscience applications. Presence of more hidden lineages within NW Pacific T. tetraquetra is suggested. The long lasting confusion over identity of the species from the genera Tritonia and Tochuina is resolved using molecular and morphological data. We also disprove a common indication about “edible T. tetraquetra” at the Kuril Islands. It is shown that Tochuina possesses specialized tritoniid features and also some characters of “arminacean nudibranchs”, such as Doridoxa and Heterodoris. Diagnoses for the families Doridoxidae and Heterodorididae are provided. Taxonomy of the genus Doridoxa is clarified and molecular data for the genus Heterodoris presented for the first time. A taxonomic synopsis for the family Tritoniidae is provided. A new genus among tritoniid taxa is proposed. Importance of the ontogeny-based taxonomy is highlighted. The cases when apomorphic characters considerably modified in a crown group due to the paedomorphosis are revealed. Tracing of the character evolution is presented for secondary gills–a key external feature of the family Tritoniidae and traditional dendronotacean nudibranchs.Nudibranch molluscs of the family Tritoniidae are widely used neuroscience model systems for understand the behavioural and genetic bases of learning and memory. However species identity and genus-level taxonomic assignment of the tritoniids remain contested. Herein we present a taxonomic review of the family Tritoniidae using integration of molecular phylogenetic analysis, morphological and biogeographical data. For the first time the identity of the model species  Tritonia and Tochuina have been no less useful model systems for studies neural basis of behaviour than Aplysia ” . H. aurantium was described , remarkably, in the same publication of Pallas  because nudibranch T. tetraquetra has the same colour and after taken out of water was similar to ascidian H. aurantium. It is also possible that a subsequent confusion arose after Steller applied the indigenous name for ascidian or nudibranch in his diaries. Ascidian H. aurantium is still used for food in the neighbouring Japanese Islands and available on the modern Japanese fish markets ” (: p. 326) Pallas  is recorded in a comprehensive Ainu dictionary of the animal names [Halocynthia roretzi . Importantly, in XIX century the chordate ascidians still were incorrectly assigned to “molluscs”. That explain the indication of “molluscous or soft-bodied animals” for both togoi and totsui , and not inedible nudibranch T. tetraquetra. We therefore disprove the common indication [Tritonia tetraquetra”. As a taxonomically important implication, the name tochui was incorrectly applied by Odhner  is extiraquetra . All thedication , 23, 42 Tritonia. However, some genera, e.g. Tritoniopsis Eliot, 1905 is continued to use currently. Instead, other taxa, e.g. Tochuina are sometimes omitted [Marionia Vayssière, 1877 is different from other tritoniid taxa because of presence of stomach plates, but otherwise contains numerous species with disparate morphology, and molecular phylogenetic data show within Marionia at least two distinct clades [Tritonia has been especially controversial. Several disparate morphological groups supported by molecular data currently falls within single genus Tritonia. Thus, an assessment of the taxonomic placement of the model species of the North Pacific tritoniids is impossible without a broad-scope phylogenetic framework for the family Tritoniidae. Otherwise it is not possible clearly indicate for what reason the model species Tritonia tetraquetra was assigned to the genus Tochuina for a long time and why Tochuina has some intriguing external and internal similarities to the non-tritoniid species Doridoxa Bergh, 1899 and Heterodoris Verrill et Emerton, 1882  is a sister to Tritoniopsis. The genus Tritoniopsis in turn is morphologically more similar to the type species of the genus Tritonia but phylogenetically is more close to the genera Marianina, Tochuina and Tritoniella  into a single genus must be used. The argumentation on the “presence of intermediate forms” [Onchimira that links cryptobranch and phanerobranch dorid nudibranchs [Marianina and Tritoniopsis demonstrating the profoundly different morphology at adults stages  and Tochuina gigantea (previously known as “Tochuina tetraquetra”) well illustrates the need to use this approach. The misidentification between these two distantly related tritoniid clades, Tochuina and Tritonia has persisted for more than a century. In the present study using combination of morphological data from the type specimen and recent molecular data we finally evidently demonstrate the significant differences between Tochuina and Tritonia in a broad phylogenetic framework , but much finer encompasses the molecular and morphological data. Also, the genus Tochuina shows external similarity to the distantly related non-tritoniids Doridoxa and Heterodoris , though largely was not involved the neurobiological works, but closely related to the important model species, T. tetraquetra and T. exsulans Type species. Diagnosis. Body narrow. Dorsolateral appendages distinct, branched, relatively few in number. Notal edge indistinct. Anterior corners of notum absent. Rhinophoral sheaths closed, without lateral opening and appendage. Oral veil not bilobed with few long processes. Anal opening usually placed towards anterior part of lateral side. Jaws oval. Masticatory edge with conical elements. Central radular teeth tricuspid. Moderate number of rows of lateral teeth, about 20–30 per half row. Stomach plates absent. Seminal receptacle relatively large, oval with long stalk and without large bag-like base. Copulative organ conical, without distinct folds. Usually small forms with body length no more than ca. 30 mm. Sources of the morphological data used in the diagnosis are indicated in the Duvaucelia lineata , Duvaucelia manicata , Duvaucelia nilsodhneri  comb. nov., Duvaucelia plebeia , Duvaucelia striata ,? Duvaucelia taliartensis  comb. nov.Species composition. Duvaucelia in the restricted sense is resurrected here (see list of included species above). For a species Tritonia taliartensis molecular or internal morphological data are not available from the original description [Duvaucelia.Remarks. This is a maximal supported clade in the present phylogenetic analysis  that concription , but genMarianina Pruvot-Fol, 1931Genus Aranucus Odhner, 1936= Marianina rosea  Type species. Diagnosis. Body very narrow. Dorsolateral appendages distinct, bifid, cerata-like, few in number. Notal edge indistinct. Anterior corners of notum absent. Rhinophoral sheaths closed, without lateral opening and with distinct fleshy lateral appendage. Oral veil with two long processes pointed forward and two shorter processes directed laterally. Anal opening placed towards anterior part of lateral side. Jaws oval. Masticatory edge with small granule-like denticles. Central radular teeth with strong triangular cusp and distinct lateral denticles. Small number of rows of lateral teeth . Stomach plates absent. Seminal receptacle large, oval with short stalk and without large bag-like base. Copulative organ elongated, with a rounded tip, without distinct folds. Small forms with body length no more than 15 mm. Sources of the morphological data used in the diagnosis are indicated in the Marianina khaleesi  comb.nov., Marianina rosea .Species composition.? Marianina with bifid but not branched dorsolateral appendages is phylogenetically sister to the genus Tritoniopsis . The juvenile appearance of some set of characters and simultaneous phylogenetic placement inside of a group with generally complex morphology is a robust evidence for the paedomorphosis [Marianina and phylogenetic relation to a complex group is characteristic for paedomorphic taxa and commonly resulting in proposals for higher taxonomic groupings. Two genera were proposed two times for the same paedomorphic tritoniid species: Marianina Pruvot-Fol, 1930 [Aranucus Odhner, 1936 [Marianina. Intriguingly, there is a species, currently assigned to the genus Tritonia, T. khaleesi Silva, Azevedo et Matthews-Cascon, 2014 which shows a remarkable combination of small size (up to 12 mm), a gracile external shape with just two long and two short processes of the oral veil, the radula with central teeth bearing strong triangular cusp with lateral denticles and a few lateral teeth (max 5)  also shows partly similar to Marianina rosea and “T.” khaleesi few elongate processes of oral veil and a lateral lobe at rhinophoral sheaths. However radula of “Tritonia”pickensi has common for tritoniids tricuspid central teeth, whereas central teeth of “T.” khaleesi is very similar to Marianina. While there is a possibility that T. khaleesi is independently from Marianina acquired paedomorphic radular morphology, taking into consideration several significant similarities in different characters, it is more probable that T. khaleesi is more closely related to M. rosea. We therefore transferred here “T.” khaleesi into the genus Marianina  comb. nov.) with a reservation. When the molecular data for M. khaleesi will be available, it will be possible to further conclude either it is a sister species to M. rosea, or a separate new genus within some other tritoniid lineage. In any scenario, compare to M. rosea, M. khaleesi demonstrates remnants of the branching pattern of the dorsolateral appendages, which is common to the majority of tritoniids. This is very consistent with the results of the molecular analysis shows that simplified paedomorphic Marianina is sister to the externally complex Tritoniopsis  Type species. Diagnosis. Body broad to moderate. Dorsolateral appendages distinct, branched, from few to numerous in number. Notal edge commonly remains as narrow folds between of dorsolateral appendages. Anterior corners of notum absent or indistinct. Rhinophoral sheaths closed, without lateral opening and appendage. Oral veil bilobed or not, with numerous long processes. Anal opening usually placed towards middle or posterior part of lateral side. Jaws oval. Masticatory edge with elements of various degree and numbers. Central radular teeth tricuspid. From 15 to more than 150 lateral teeth per half row in various species. Stomach plates present. Seminal receptacle very large with long stalk and without bag-like large base. Copulative organ conical or bulbous, without distinct folds. Usually large forms with body length more than 30 mm (up to 200 mm), but several species apparently are small-sized. Sources of the morphological data used in the diagnosis are indicated in the M. abrahamorum Silva, Herrero-Barrencua, Pola et Cervera, 2019, M. albotuberculata , M. arborescens Bergh, 1890, M. babai Odhner, 1936, M. bathycarolinensis Smith et Gosliner, 2005, M. blainvillea , M. cabindae White, 1955, M. chloanthes Bergh, 1902, M. cucullata , M. cyanobranchiata , M. dakini , M. distincta , M. echinomuriceae Jensen, 1994, M. elongoviridis Smith et Gosliner, 2007, M. elongoreticulata Smith et Gosliner, 2007, M. fulvicola Avila, Kelman, Kashman, et Benayahu, 1999, M. gemmii Almón, Pérez et Caballer, 2018, M. ghanensis Edmunds et Carmona, 2017, M. hawaiiensis , M. kinoi Angulo-Campillo et Bertsch, 2013, M. levis Eliot, 1904, M. limceana Silva, de Meirelles et Matthews-Cascon, 2013, M. olivacea Baba, 1937, M. ramosa Eliot 1904, M. rubra , M. pellucida Eliot, 1904, M. platyctenea , M. pusa Er. Marcus et Ev. Marcus, 1968, M. pustulosa Odhner, 1936, M. semperi Jensen, 1994, Marionia tedi Ev. Marcus, 1983, M. vanira Ev. Marcus et Er. Marcus, 1966, M. viridescens Eliot 1904.Species composition. Marionia in spite of the external similarity to the genus Tritonia possesses such a very distinctive feature as the solid chitinous stomach plates. However this large genus currently is united only by this single character. Intriguingly, stomach plates which are very similar to Marionia also present in a distantly related dendronotoidean family Tethydidae [Marionia [Marionia” genus. The available names such as Marioniopsis and Paratritonia, after clarification to which clades they actually belong can be used for further genus-level delineation among “Marionia”. For example, the so far monotypic genus Paratritonia possesses small-sized species with few dorsolateral appendages but with a large number of the radular lateral rows , new geMarionia invariably forms a separate clade from other tritoniids [Marionia with related genera Marioniopsis and Paratritonia (and more genera which have to be separated) should be placed in a separate family. In this connection, there is another considerable issue with the genus Marionia that affects all tritoniids. The majority of the available phylogenetic analyses [Marionia renders the family Tritoniidae paraphyletic regarding Dendronotoidea. A revision of the large Marionia clade is out of scope of the present paper and pending a separate study.Importantly, according to all available data itoniids . Therefoanalyses , 72, 92 Paratritonia Baba, 1949Genus Paratritonia lutea Baba, 1949.Type species. Diagnosis. Body narrow. Dorsolateral appendages distinct, branched, few in number. Notal edge indistinct. Anterior corners of notum absent. Rhinophoral sheaths closed, without lateral opening and appendage. Oral veil not distinctly bilobed with few long processes. Anal opening usually placed towards middle or posterior part of lateral side. Jaws oval. Masticatory edge with teeth-like elements of various degree and numbers. Central radular teeth unicuspid with additional denticles. Numerous rows of lateral teeth . Stomach plates present. Anterior and posterior portions of digestive gland fused. Reproductive organs unknown. Small forms with body length up to 30 mm. Source of the morphological data used in the diagnosis are indicated in the Species composition. Only type species so far was included .Marionia.Remarks. See under genus Tochuina Odhner, 1963Genus Tritonia gigantea Bergh, 1904 Type species. Diagnosis. Body broad. Dorsolateral appendages indistinct, branched, numerous. Notal edge distinct, well-defined. Anterior corners of notum present, distinct. Rhinophoral sheaths partly open laterally; lateral appendage absent. Oral veil not bilobed without processes or with small tubercles. Anal opening placed towards middle or posterior part of lateral side. Jaws square. Masticatory edge smooth or possibly with indistinct elements. Central radular teeth unicuspid. Additional fine denticles often present on central teeth. Numerous rows of lateral teeth, more than 50 (up to 200) per half row. Stomach plates absent. Seminal receptacle relatively large with long stalk and without bag-like base. Copulative organ conical, without distinct folds. Large forms with body length more than 30 mm and include very large form up to 300 mm. Sources of the morphological data used in the diagnosis are indicated in the Tochuina gigantea , Tochuina nigritigris  comb. nov., Tochuina nigromaculata  comb. nov.Species composition. T. gigantea  [Heterodoris robusta Verrill et Emerton 1882 . As results, we robustly reveal that a recently described species which was assigned to “Tritonia”, T. nigritigris from NE Pacific [Tochuina  to the species that was described considerably earlier by Roginskaya in 1984 [Tritonia nigromaculata also from the deep sea of the North Pacific .Remarks. The incompletely closed rhinophoral sheaths and the well defined anterior notal corners of tea Figs  represene, 1892)   per half row. Stomach plates absent. Seminal receptacle small or larger, oval with long stalk and a bag-like large base. Copulative organ massive, cylindrical or conical, with variously expressed folds. Usually large to moderate in size forms with body length more than 30 mm (including very large species up to 300 mm). Sources of the morphological data used in the diagnosis are indicated in the Tritonia antarctica Pfeffer in Martens et Pfeffer, 1886, T. australis , T. bollandi Smith et Gosliner, 2003, T. challengeriana Bergh, 1884,? T. coralliumrubri Doneddu, Sacco et Trainito, 2014, T. dantarti Ballesteros et Avila, 2006, T. episcopalis Bouchet, 1977, T. exsulans Bergh, 1894, T. festiva , T. griegi Odhner, 1922, T. hombergii Cuvier, 1803, T. incerta Bergh, 1904, T. indecora Bergh, 1907, T. ingolfiana , T. newfoundlandica Valdés, Murillo, McCarthy et Yedinak, 2017, T. odhneri Er. Marcus, 1959, T. olivacea Bergh, 1905, T. pallescens Eliot, 1906, T. pallida Stimpson, 1855, T. primorjensis Minichev, 1971, T. tetraquetra , T. psoloides Aurivillius, 1887, T. vorax .Species composition. Tritonia to several predominantly large forms with a numerous processes on the bilobed oral veil and a large number of the lateral teeth. In our present analysis this morphological group forms a clade that closely related to the type species T. hombergii and including these model species T. tetraquetra and T. exsulans  is inconsistent with the morphological data on the type and closely related to it Tritonia species. Particularly, “T.” flemingi is a very small species, with non-bilobed oral veil with only few processes, and indistinct dorsolateral appendages [T.” flemingi partly approaches the genera Duvaucelia and Tritonicula gen. nov., but because of significant geographic gap it may represents an independent lineage of small-sized tritoniids. Therefore “T.” flemingi does not include here into the genus Tritonia pending molecular phylogenetic data to propose a separate genus for this species. For several other species, including for instance a deep-sea T. griegi Odhner, 1922, the molecular data are also still unavailable, and inclusion of these taxa into the genus Tritonia needs to be confirmed. Also not all included species are consistent morphologically with the type species T. hombergi from the northern Atlantic and related North Pacific T. tetraquetra and T. exsulans. For instance, Valdés et al. [T. newfoundlandica with T. nigritigris. However, according to the present analysis the deep-sea species from NE Pacific T. nigritigris belongs to the genus Tochuina, which is distantly related to the genus Tritonia , some of them will be needed to resurrect in a future. In addition, the present Tritonia species composition is inconsistent biogeographically. While the type species T. hombergii from the northern Atlantic, and the North Pacific species such as T. tetraquetra, T. exsulans and T. festiva are closely related with a maximal support  [Remarks. Based on the present molecular phylogenetic analysis  we restrans Figs –6. The gays high . More lipendages , 96. By  Powell, 37 is incTritonia . The Noral teeth , which a support , another species . Tritonieview in ). Howeven, 1837)  is an avGenus Tritonicula gen. nov.Urn:lsid:zoobank.org:act: 7E6448CD-78DA-4A65-8141-0A9EB40D0D0C.Tritonia hamnerorum Gosliner et Ghiselin, 1987Type species. Tritoni- and a diminutive Latin suffix–cula in reference to a “little Tritonia” because the new genus contains so far only small, gracile species.Etymology. From common family stem Diagnosis. Body usually narrow. Dorsolateral appendages distinct, branched, relatively few in number. Notal edge commonly indistinct. Anterior corners of notum absent. Rhinophoral sheaths closed, without lateral opening, in some species with elongate lateral appendage. Oral veil not bilobed with few long processes. Anal opening usually placed towards middle lateral side, or partly moves to dorsal side. Jaws oval. Masticatory edge with delicate tubercle-like elements or more distinct conical plates. Central radular teeth tricuspid. Additional fine denticles may present irregularly on central teeth. Few rows of lateral teeth, so far reported no more than 11 per half row. Stomach plates absent. Seminal receptacle relatively large with long stalk and without large bag-like base. Copulative organ conical or rounded, without distinct folds. Small forms with body length no more than 20 mm. Sources of the morphological data used in the diagnosis are indicated in the Tritonicula bayeri Ev.  comb. nov., Tritonicula hamnerorum  comb. nov., Tritonicula myrakeenae  comb. nov., Tritonicula pickensi Ev.  comb.nov.,? Tritonicula wellsi  comb. nov.Species composition. Tritonia s. str. and Duvaucelia s.str. This group contains generally smaller-sized species (with body length no more than 20 mm) such as “T”. bayeri Ev. Marcus et Er. Marcus, 1967, “T.” hamnerorum Gosliner et Ghiselin, 1987, “T.” myrakeenae Bertsch et Osuna, 1986 and “T.” pickensi Ev. Marcus et Er. Marcus, 1967 [Tritonicula gen. nov. from both generally large-bodied Tritonia and small-sized Duvaucelia. The genus Tritonia possesses in adult specimens bilobed oral veil with numerous processess and more than 50 lateral teeth per row. Small-sized Duvaucelia commonly has larger number of lateral teeth than Tritonicula gen. nov. , though in few species this number can be smaller. Also species of the genus Duvaucelia, compare to Tritonicula gen. nov. does not have lateral extension on the rhinophoral sheaths. In addition, the new genus is consistent biogeographically. All included species inhabit warm waters of the American Atlantic and the Caribbean, or neighbour tropical Pacific waters. Tritonia wellsi was originally described from North Carolina (and further founded in Brazil) and placed in the subgenus Tritonidoxa [Tritonidoxa demonstrates completely different characters including absence of dorsolateral appendages [T.” wellsi are consistent with Tritonicula gen. nov. We include this species into new genus with reservation because of absence of molecular data. A species “T.” khaleesi (for which there are no available molecular data) which is included here to the genus Marianina (see above) can be also potentially related to Tritonicula gen. nov. According to our analysis it is likely that Duvaucelia and Tritonicula gen. nov. acquired small body size and smaller number of the lateral teeth independently (T. tetraquetra and T. exsulans), but also at the genus level, because otherwise such convergent evolutionary events will be considerably masked.Remarks. In the present analysis we confirm a separate and morphologically distinct clade  from botus, 1967 , 78, 99.tonidoxa . Howeverpendages . Small sendently . This isTritonidoxa Bergh, 1907Genus Tritonidoxa capensis Bergh, 1907Type species. Diagnosis. Body narrow. Dorsolateral appendages absent. Notal edge distinct. Anterior corners of notum indistinct. Rhinophoral sheaths closed, without opening laterally and lateral appendage. Oral veil not bilobed with few processes. Anal opening placed towards posterior part of lateral side. Jaws oval. Masticatory edge with conical elements. Central radular teeth tricuspid. Large number of rows of lateral teeth . Stomach plates absent. Seminal receptacle relatively large. Copulative organ conical, without folds. Body length about 30 mm. Source of the morphological data used in the diagnosis are indicated in the T. capensis [Species composition. Only type species, capensis .Tritonidoxa [Remarks. There are more species that could potentially be included in tonidoxa , 78, 101Tritoniella Eliot, 1907Genus Tritoniella sinuata Eliot, 1907Type species. Diagnosis. Body narrow to moderately broad. Dorsolateral appendages absent or indistinct, non-branched. Notal edge distinct. Anterior corners of notum indistinct. Rhinophoral sheaths closed, without lateral opening, but expanded laterally. Oral veil not bilobed or indistinctly bilobed, without processes. Anal opening placed towards middle part of lateral side. Jaws square. Masticatory edge with fine tubercles. Central radular teeth in adults unicuspid or tricuspid. Additional distinct cusps may present on central teeth. Numerous rows of lateral teeth, commonly more than 50 per half row. Stomach plates absent. Seminal receptacle relatively large with long stalk and without large bag-like base. Copulative organ conical, with distinct circular fold or bulge. Large forms up to 80 mm. Source of the morphological data used in the diagnosis are indicated in the Tritoniella belli Eliot, 1907 .Species composition. Tritoniella well distinguishes from the majority of tritoniids by absence of distinctly branched dorsolateral appendages. Phylogenetically it is related to the genus Tochuina and important for the present study. Here it is shown that the Antarctic Tritoniella forms a sister clade to the North Pacific genus Tochuina . Stomach plates absent. Seminal receptacle relatively large, oval with long stalk and without large bag-like base. Copulative organ conical, without distinct folds. Small to medium forms with body length from ca. 20 to 50 mm. Sources of the morphological data used in the diagnosis are indicated in the T. brucei Eliot, 1905, T. cincta , T. elegans , T. frydis Er. Marcus et Ev. Marcus, 1970.Species composition. Tritoniopsis, T. brucei Eliot, 1905 comes from temperate southern Atlantic (Gough Island). Other currently assigned to the genus Tritoniopsis species come from tropical and subtropical waters. The molecular data are not available for T. brucei and phylogenetic placement of the genus Tritoniopsis is based on other included species   and a second poorly known South African species D. benthalis Barnard, 1963 [Composition. The genus rd, 1963 .Doridoxa and family Doridoxidae until recently was among the most enigmatic and poorly known nudibranch taxa  [D. ingolfiana Bergh, 1899 syn. nov.). The external and internal morphology of the described from the Barents Sea D. walteri [D. ingolfiana from the waters of the northern Atlantic [D. ingolfiana (including “D. inglofiana var.”) the presence of both smooth and denticulate central teeth is indicated [D. walteri from the type locality region (Barents Sea off the East Spitzbergen) well agreed with the original description of D. walteri in Krause [D. ingolfiana, D. walteri has both smooth and denticulate central teeth   Diagnosis. Body moderately broad. Dorsolateral appendages non-branched or absent. Notal edge distinct, well-defined. Anterior corners of notum present, distinct. Rhinophoral sheaths partly open laterally; lateral appendage absent. Rhinophores perfoliate. Oral veil not bilobed without processes. Anal opening placed towards posterior part of lateral side. Jaws oval. Masticatory edge smooth or possibly with indistinct elements. Central radular teeth unicuspid with a narrow, weak cusp. Additional fine denticles may present on central teeth. Numerous lateral teeth, more than 50 (up to 200 and more) per half row. Stomach plates absent. Digestive gland with several external lobes. Seminal receptacle with long stalk and without bag-like base. Copulative organ conical or cylindrical, without distinct fold. Medium sized forms with body length up to ca. 35 mm. The sources of the morphological data are in , 112–115Heterodoris Verrill et Emerton, 1882  contains two confirmed species: H. robusta Verrill et Emerton 1882 and H. anthipodes Willan, 1981.Composition. The genus Heterodoris and the family Heterodorididae show intriguing similarity to the family Doridoxidae  respectively. The external similarity between the families Doridoxidae and Heterodorididae, as well as similarity to the tritoniid genus Tochuina are likely due to the plesiomorphic similarity of the rhinophoral sheaths and oral veil patterns (see details below). Internally families Doridoxidae and Heterodorididae are different in presence of an entire  Tochuina . HoweverTochuina  the moleTochuina . The famTochuina . The famTochuina . TogetheTochuina . The famTochuina . Thus, ibranches ) digestibranches  in Heterreported  and thisAtthila Bergh, 1899 and the family Atthilidae, introduced by Bergh [Heterodoris Verrill et Emerton, 1882 and the family Heterodorididae Verrill et Emerton 1882 respectively. It was already proposed [Atthila without a study of additional materials [Atthila and Heterodoris show the same incompletely closed rhinophoral sheaths integrated to the oral veil [Atthila as a “two-lobed edge of rhinophoral cavity” [Atthila and Heterodoris also come from the same geographic region of the western part of the North Atlantic and similar depth range (800–1500 meters) [H. robusta from more southern localities in the north-eastern Atlantic [Heterodoris worldwide needs in a further investigation, including clarification of the species status “Heterodoris ingolfiana” [Heterodoris robusta from the type locality region of both Heterodoris and Atthila (the northern Atlantic coast of North America) were investigated. It was discovered that both denticulated and non-denticulated central teeth may occur in the same exemplar   differ from Tochuina . These taxa belong to different families, which acquired specialized characters in course of the phylogeny  was placed with a lower support in a basal position to the majority of the non-dorid nudibranch families. The present analysis places Doridoxidae and Heterodorididae as a sister group to Arminidae  in course of the process of peramorphosis  is nested together with Doridoxa and Heterodoris clades . In true dorids the oral veil is instead enclosed by the frontal notal edge [Doridomorpha does not show any distinct oral veil around the mouth under the putative “anterior notal edge” [Doridomorpha is anterior part of the modified and fused oral veil. In dorids the frontal edge instead is the true notum and separate oral veil is placed under the frontal notum [In a previous analysis  Doridoxarminidae . This isrminidae . This sporphosis   together with Doridomorphidae and Arminidae come as a sister clade to tritoniids  at the edge of the notum  is sister to the North Pacific genus Tochuina (Marionia (with distinct branched appendages) either basal to all tritoniids [Tochuina) and short cerata-like structures, sometimes partly branched (as in the modern Lomanotus) (Marianina (body length up to 15 mm)  is entirely lacking any gills [Doridoxa and Heterodoris serve as a respiratory structure. The oxygen-rich environment of cold waters [Tochuina (up to 300 mm in length) possesses small branched appendages and distinct notal edges at the same time , but lacking a distinct notal edge and have large branched appendages  does not possess distinct branched appendages, and hence can respire using only ample notum and short unbranched/weakly branched notal processes. Therefore if in the oxygen-rich cold water environment a reducing of body size is occurred, special respiratory structures can become unnecessary. Doridoxa and Heterodoris two times smaller than Tritoniella (up to 30–40 mm in length), and either completely devoid of any special respiratory structures or possesses only weak appendages ..Doridoxany gills . A majord waters  can promd waters , 134. Hoame time . The modges Figs . Insteadpendages . Therefoerodoris  may reprTritonia tetraquetra  and characterizes externally by commonly orange colour without white lines . Tritonia exsulans instead has a restricted geographic range and so far reliably recorded from California to British Columbia in shallow waters, in a range circa 5–100 m. Described from deeper waters (more than 500 m) off Alaska and California Tritonia diomedea Bergh, 1894 shares same morphological characters with molecularly confirmed T. tetraquetra Click here for additional data file.


J. Sport Heal. Sci. 9, 103–104. doi: 10.1016/j.jshs.2020.02.001. It should be Chen, P., Mao, L., Nassis, G. P., Harmer, P., Ainsworth, B. E., and Li, F. (2020). Coronavirus disease (COVID-19): The need to maintain regular physical activity while taking precautions. J. Sport Health. Sci. 9, 103–104. doi: 10.1016/j.jshs.2020.02.001.In the original article, the reference for Chen et al.  was incoCan Training Status Protect Against COVID-19 Infection?. Paragraph five should read:In the original article . Some studies have reported reductions in the lymphocyte proliferative response (Papa et al., Can an Acute Exercise Session Increase the Risks for Covid-19 Infection?In addition, the citation has now been inserted in the section Paragraph twoSeveral studies have investigated the effects of a single exercise session on immune function by measuring acute changes in various parameters (Flynn et al., Paragraph threeNieman et al.  had partParagraph fiveThe different results across these studies may be due to the variability in the immune system responses to an acute exercise session. Although a number of studies have observed an immunosuppressive response after prolonged and intense exercise bouts (Steensberg et al., In the original article, there was an error. Incorrect references were cited.Can Training Status Protect Against Covid-19 Infection?, paragraph three:A correction has been made to the section On the other hand, other studies have shown no protective effects of exercise training programs on URTI incidences (Nieman et al., The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.