Solanum melongena L.) is an economically and nutritionally important fruit crop of the Solanaceae family, which was domesticated in India and southern China. However, the genome regions subjected to selective sweeps in eggplant remain unknown. In the present study, we performed comparative transcriptome analysis of cultivated and wild eggplant species with emphasis on the selection pattern during domestication. In total, 44,073 (S. sisymbriifolium) to 58,677 (S. melongena cultivar S58) unigenes were generated for the six eggplant accessions with total lengths of 36.6-46 Mb. The orthologous genes were assessed using the ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitutions to characterize selective patterns during eggplant domestication. We identified 19 genes under positive selection across the phylogeny that were classified into four groups. The gene (OG12205) under positive selection was possibly associated with fruit-related traits in eggplant, which may have resulted from human manipulation. Eight positive selected genes were potentially involved in stress tolerance or disease resistance, suggesting that environmental changes and biotic stresses were important selective pressures in eggplant domestication. Taken together, our results shed light on the effects of artificial and natural selection on the transcriptomes of eggplant and its wild relatives. Identification of the selected genes will facilitate the understanding of genetic architecture of domesticated-related traits and provide resources for resistant breeding in eggplant.Eggplant ( Studies on several organisms suggest that very few genetic loci contributed to the rapid phenotypic divergence relative to domestication [Over human history, wild plants and animals were selected and adapted for cultivation and commercial use, a process known as domestication. Darwin proposed that domestication and adaptation were in parallel in the wild and are powerful in generating phenotypic diversity . Selectitication –6.With the improvements of sequencing technologies and increasing availability of plant genomes, the consequences of domestication could be studied at a transcriptional or even whole-genome level. Several domestication-associated genes were characterized in some crops such as rice, maize, tomato, and pepper –10. A reSolanum melongena L., 2n = 2x = 24) is an economically important vegetable crop and is widely grown in America, Europe, and Asia, with 51.3 million tons of agricultural production in 2016 (http://faostat.fao.org). The eggplant belongs to the large family Solanaceae, which compromises >3000 species with diverse genetic and phenotypic variation including tomato, pepper, and potato. Eggplants exhibit a wide biodiversity of local landraces/wild relatives with huge variation respect to growth habit, pathogen resistance, and fruit size and color. The subgenus Leptostemonum (“spiny solanums”) comprises approximately 500 species, including three closely related cultivated eggplant species: S. melongena , S. macrocarpon , and S. aethiopicum , which are all of Old World origin and grown as food crops [S. incanum L. in India and S. undatum L. in southern China). Domesticated eggplant exhibits substantial phenotypic differences from its wild progenitors, reflecting rapid and pronounced evolutionary changes. Identification of genetic changes underlying these phenotypic differences will give insight into the genetic architecture of complex traits, the response to selection, and provide resources for eggplant improvement.Brinjal eggplant  [S. aethiopicum, S. integrifolium, and S. sisymbriifolium). Evidence for significant changes in both gene expression levels and sequences was found which might have occurred as a result of selective sweeps and specifically identified a subset of genes that were likely targets of selection during domestication.The availability of the eggplant genome (hinkuro)  provideshinkuro) –17. HoweS. sisymbriifolium, S. aethiopicum, and S. integrifolium) and three domesticated eggplant cultivars from S. melongena  , and it is cultivated in Thailand. The seeds of S. sisymbriifolium, S. aethiopicum, and S. integrifolium were obtained from Xishuangbanna Botanical Garden . S. aethiopicum is mostly grown in tropical Africa [S. aethiopicum has four classifications ; S. integrifolium (S. aethiopicum gr. Aculeatum) is one of them and mainly used as rootstocks (mansfeld.ipk-gatersleben.de). S. aethiopicum and S. integrifolium belong to the section Oliganthes which forms a sister group to S. melongena within the Old World clade [S. sisymbriifolium belongs to the Sisymbriifolium clade, distant from the Old World clade [Six different eggplants were used in the present study, three wild species (and LYQ) . Two eggand LYQ) . The seel Africa ; howeverld clade , whereasld clade .μmol photons m−2 s−1). When the plantlets had three true leaves, the leaf tissue was collected from the seedlings 14 days postgermination. The leaf tissue for each species was collected from three individual plants then mixed for RNA extraction. Total RNA of each sample was extracted using an RNAprep Pure Plant Kit  according to the manufacturer's protocol.Seeds (10 per species) from the six eggplants were germinated in soil and in the dark for 7 days. Plants were then transferred into growth chambers (28°C/22°C day/night temperatures and 14 h photoperiod with 300 http://www.novogene.cn). mRNA was purified from 3 μg total RNA using poly-T oligo-attached magnetic beads. Sequencing libraries were generated using Illumina TruSeq™ RNA Sample Preparation Kit  following the manufacturer's recommendations. In order to select cDNA fragments of preferentially 200 bp in length, the library fragments were purified with the AMPure XP system , then sequenced on an Illumina Hiseq 2000 platform, and 90 bp paired-end reads were generated. Clean reads were obtained by removing reads containing adapter and poly-N and low-quality reads from raw data. All the downstream analyses were based on clean data with high quality. Transcriptome assembly was accomplished based on the left files and right files using Trinity [Illumina sequencing was performed at Novogene Bioinformatics Technology Co. Ltd., Beijing, China (http://www.genome.jp/kegg/). We used KOBAS [To annotate the unigenes of the eggplant transcriptomes, each of the unigenes from all six eggplants was searched against different public databases, including KEGG Orthology database, Gene Ontology (GO) database, NCBI nonredundant protein sequences (NR) database, protein family (PFAM) database, Swiss-Prot protein database, NCBI nucleotide sequence (NT) database, and Eukaryotic Orthologous Group (KOG) database. GO enrichment analysis of genes was implemented by the GOseq R package-based Wallenius noncentral hypergeometric distribution , which ced KOBAS  softwarehttp://eggplant.kazusa.or.jp/). The index of the reference genome was built using Bowtie v2.0.6, and paired-end clean reads were aligned to the reference genome using TopHat v2.0.9. We selected TopHat as the mapping tool for TopHat can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other nonsplice mapping tools.Reference genome and gene model annotation files were directly downloaded from the eggplant genome database (The Cufflinks v2.1.1 Reference Annotation-Based Transcript (RABT) assembly method was used to construct and identify both known and novel transcripts from TopHat alignment results. Picard-tools v1.96 and SAMtools v0.1.18 were used to sort, mark duplicated reads, and reorder the bam alignment results of each sample.Gene expression levels were estimated by RSEM  for eachμg total RNA by HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper)  in 10 μL of reaction mixture. The quantitative PCRs were performed according to the manufacturer's instructions using the DyNAmo™ Flash SYBR Green qPCR Kit  in an ABI Prism StepOnePlus real-time thermal cycler . The amplification was performed as follows: 10 min at 94°C followed by 40 cycles of 94°C for 20 s and 60°C for 60 s. A melting curve was generated to ensure product uniformity. Gene expression was evaluated by the 2_△△Ct method [RNA-seq data for 16 different genes was validated by real-time quantitative PCR (qPCR). The primers of selected genes were designed using Primer Priemer 5 software  and synthesized by Sangon. The primer pair SmActin-F and SmActin-R was used to amplify an eggplant actin fragment as a control for normalizing the starting amounts of cDNA. The sequences of primers used in this study are listed in Supplementary t method . The expKa/Ks ratio represents the ratio of the number of nonsynonymous substitutions per nonsynonymous site (Ka) to the number of synonymous substitutions per synonymous site (Ks), which could be used as an indicator of selective pressure acting on protein-coding genes. In order to estimate the Ka/Ks ratio, the bidirectional best hit (BBH) method [Ka/Ks calculation was performed with the PAML package using default settings [Ka/Ks ratio significantly >1 are usually considered to be evolving under positive selection, whereas Ka/Ksratio < 1 indicates purifying selection on the gene loci, and Ka/Ks ratio close to 1 indicates neutral mutation.In genetic analysis, the ) method  was usedsettings . CompariS. melongena domestication, the leaf transcriptomes of three S. melongena cultivars, HZHQ (MEL_HZHQ), LYQ (MEL_LYQ), and S58 (MEL_S58), and distant relatives, S. aethiopicum (AET_SG), S. integrifolium (INT_2006), and S. sisymbriifolium (SIS_2007), were analyzed.Molecular analyses have been conducted to evaluate genetic diversity among wild and domesticated eggplant species using various approaches such as RAPD, SSR, and sequence analysis –32. Howede novo assembly of high-quality clean read sequences into transcript reads, ranging from 73,161 transcripts for S. sisymbriifolium to 101,387 for S. melongena S58. Only the longest transcripts were defined as final unigenes, yielding 44,073 (S. sisymbriifolium) to 58,677 (S. melongena S58) unigenes of at least 200 bp in length for all species, with an average length of 408-850 bp and a total length of 36.6-46 Mb , with 381 million Illumina reads (89.45%) passing the quality filtering. The average Phred quality score was at least 95% for 99% base accuracy for filtered reads, indicating the high quality of filtered reads . The Tri.6-46 Mb . N50 bei.6-46 Mb . The dec's study .S. sisymbriifolium had the highest ratio of UTR and CDS per unigene  of all unigenes was predicted by searching against the NCBI nonredundant (NR) and Swiss-Prot protein databases using ESTScan (3.0.3) or BLAST. The upstream and downstream sequences of the CDS in each unigene were considered as the 5′ and 3′ UTR sequences. 22%-31% unigenes contained the 5′ or 3′ UTR sequence, and 77%-83% CDS sequence among all sample was found from unigenes . S58 con unigene . We annog et al. , indicatS. melongena var. Nakate-Shinkuro) using the TopHat software [S. melongena cultivars mapped ~88% of the published eggplant genome reads, whereas S. aethiopicum and S. sisymbriifolium mapped 71% and 27% respectively (S. sisymbriifolium contained the most count of the novel transcripts (1257 genes), followed by S. aethiopicum (579 genes), S. integrifolium (459 genes), and S. melongena cultivars .Clean reads for all samples were aligned to the eggplant reference genome , S. macrocarpon L. (section Melongena), and S. aethiopicum L. (section Oliganthes). The origin of the eggplant lineage is most likely started in Africa; however, the relationships among African species and their Asian relatives are not well understood [S. melongena (HZHQ), S. aethiopicum, S. integrifolium, and S. sisymbriifolium. The published reference eggplant genome (S. melongena Nakate-Shinkuro) was also included in the analysis. Orthologous genes among the six species were determined using predicted protein sequences and the OrthoMCL algorithm (OrthoMCL Software-v2.0.3) [e < 0.00001) was more than 80% of the size of the genes being compared. Overall, 2,695 single-copy genes and 1,793 multicopy genes were common to all five species analyzed  , 35. Genanalyzed . Interesectively ; the numS. sisymbriifolium, such as locomotion in biological process and items within a cellular component, which contain more specific genes and specific GO items  [Solanum lycopersicum) [Capsicum annuum) [S. aethiopicum and S. integrifolium, are in a sister clade; this is somehow supporting the classification that S. integrifolium is one of the four groups in S. aethiopicum, whereas the wild species S. sisymbriifolium was most distant from the domesticated species. This category result is consistent with previous reports [Bayesian inference methods were then used to construct a phylogenetic tree using the single-copy orthologous gene sequences with the reference genomes of eggplant  , potato berosum) , tomato ersicum) , and pep annuum) . The res reports , 19. Mor reports  and Song reports  showed tS. sisymbriifolium (SIS_2007) gene expression branch length (S. aethiopicum (AET_SG) and S. integrifolium (INT_2006) were in the same cluster  between RNA-seq data and qPCR data. The qPCR expression quantities were basically consistent with their transcript abundance changes identified by RNA-seq, which confirms the reliability of RNA-seq data.To validate the RNA-seq data, qPCR of 16 randomly selected genes was performed. As shown in S. melongena domestication, the leaf transcriptome of the six eggplant accessions was analyzed. The ratio of Ka/Ks by random substitution throughout the coding gene within specific loci can be used to estimate selective pressure; a gene with Ka/Ks > 1 indicates that this gene is subjected to positive selection, whereas Ka/Ks < 1 indicates purifying selection, and Ka/Ks score close to 1 indicates neutral mutation [Ka/Ks in all species, we identified 19 unigenes (listed in P < 0.05) evidence of evolution under positive selection across the phylogeny. Many of these genes have been annotated in tomato and potato and characterized in details in rice and Arabidopsis. The result supports the hypothesis that relatively few mutations have gone through strong selective sweeps during domestication.The genetic basis of domestication-related traits has been studied in several organisms, including important crops such as maize, millet, rice, and cucumber , 44–46. mutation . From coOG06215, OG08277, and OG12205 are likely related to the hormone response. OG06215 is characterized as a Domon superfamily which is auxin-induced protein in root cultures. OG08277 is a ribosomal protein, with annotated functions of controlling vacuole trafficking and developmental programs through the regulation of lipid metabolism in Arabidopsis. Ribosome proteins serve as translational regulators of auxin response, and lipid metabolism modulates auxin-mediated tissue differentiation [OG12205 was identified as an Aux/IAA family gene; its orthologous genes in tomato interact with ARF (auxin response factor) proteins and involved in the regulation of quality parameters over tomato fruit development and play diverse roles in flower and fruit development [The 19 genes subjected to positive selection could be divided into four groups: hormone response, development and response to disease and abiotic stress, oxidation-reduction pathway, and development. Orthologous genes ntiation . Auxin pntiation . In the elopment –53.WRKY, E3 ubiquitin ligase, and DnaJ homolog subfamily proteins [Arabidopsis thaliana more susceptible to pathogenic bacteria and fungi. PPR proteins also involved in chloroplast, mitochondrial and seed development, and abiotic stress response in rice and maize [Pseudomonas solanacearum in transgenic tobacco [OG0372 functions as pentatricopeptide repeat-containing protein; one gene (OG10870) belongs to the DnaJ homolog subfamily. A series of positive genes were annotated as hydrothermal carbonisation (HTC) in leaves or roots and involved in the oxidation-reduction pathway, such as OG06179, OG06849, and OG05048. OG09458 belongs to ubiquinone oxidoreductase, also functions in HTC in the leaf. OG05094 is annotated as 2-aminoethanethiol dioxygenase-like protein. In addition, there were three development-related genes found to have been subjected to positive selection, a thiamin pyrophosphokinase (TPK) encoding gene (OG07167) and two Ca2+-dependent phospholipid-binding protein encoding genes (OG06647 and OG05019).Many gene/protein families are reported to be associated with stress tolerance and disease resistance, such as PPR (pentatricopeptide repeat) protein, proteins –59. Parkproteins  found thnd maize , 54, 61. tobacco . We idenOG07232 and OG07554 were E3 ubiquitin ligases which respond to drought stress in maize [OG09141 and OG08464 that are positively selected. OG06038 and OG03732 were annotated as Calvin cycle protein and early nodulin 16 precursor, respectively, which also involved in plant response to stresses and rapid environmental changes [in maize  and invoin maize . We iden changes . HoweverS. melongena species  and the N50 values  are both higher than that for the recent transcriptomic study on eggplant [S. aethiopicum and S. integrifolium from section Oliganthes were included in the sister clade, close to the cultivated eggplants, whereas the wild species S. sisymbriifolium in the Sisymbriifolium clade was in the most distant clade from the domesticated species. Functional analysis of species-specific genes indicate that eggplant has gone through complex molecular and metabolic changes during domestication, evolving its own gene pool with increased new genes as well as rapid adaptation to different environmental conditions. Moreover, relatively few mutations (19 positively selected genes) have gone through strong selective sweeps during eggplant domestication. Our results provide insights into the selection patterns of the transcriptomes of wild and cultivated eggplants and the understanding of complex domesticated-related traits.Previous studies have shown that domestication-related traits are often controlled by few genetic loci. However, the selective sweeps in eggplants remain unknown. We identified 44,073-58,677 unigenes for six wild and cultivated eggplants; the average number of unigenes for the three eggplant . The phy


Introduction: Medication, herbal and dietary supplement induced lupus and liver injuries have been well documented in the literature. We report two cases of skyfruit seed induced autoimmune phenomena and hepatic injury. Swietenia macrophylla seeds, also known as skyfruits or mahogany seeds in Southeast Asia have been used as a remedy for diabetes, hyperlipidemia and shown to have antibiotic properties by its effect on the innate immune system. Due to its perceived benefits, it is being used as part of traditional medication in Southeast Asia for various ailments.Case description: A 62 year-old Indian male with a history of diabetes mellitus, hypertension, hyperlipidaemia, coronary artery disease and non-alcoholic fatty liver disease presented to the gastroenterology clinic for transaminitis. He had been on stable doses of medications for the above including atorvastatin 10mg ON for the last one year. He presented with polyarthralgia with tenosynovitis and severe restriction of motion of his right shoulder, left wrist and dorsum of right hand at onset with minimal response to a short course of prednisolone. His symptoms improved but were still present until review in the rheumatology clinic six weeks later. Of note he had taken skyfruit seeds six weeks ago. Systemic review was negative for rashes, oral ulcers, sicca symptoms, Raynaud's, hematuria, frothy urine or abdominal pain. He reported a significant unintentional weight loss of 14kg in the preceding 12 months. There was no family history of autoimmune conditions. Physical examination showed synovitis of the right metacarpophalangeal and proximal interphalangeal joints. Full blood count, renal function and thyroid function tests were normal. His liver function test showed transaminitis with alanine transaminase (ALT) of 699 U/L , aspartate aminotransferase (AST) of 645 U/L and gamma-glutamyl transferase (GGT) of 125 U/L. C-reactive protein (CRP) was 75.7 mg/L. Anti-nuclear antibody (ANA) screen was positive; anti-ENA (extractable nuclear antigen) profile and rheumatoid factor were negative. Anti-mitochondrial M2, anti- liver cytosol 1, anti- liver kidney microsomal and anti- soluble liver antibodies were negative. Hepatitis B surface antigen and antibodies against hepatitis A, C and E were non-reactive. He realized that the onset of symptoms - arthritis, weight loss and transminitis correlated with his intake of skyfruit seeds and stopped consuming them. The inflammatory arthritis had completely resolved and CRP and liver enzymes had markedly improved six weeks later. Within a year, his liver function tests had normalised and he had no recurrence of his arthritis.A 59 year-old Chinese female, previously well, presented with right upper quadrant discomfort for two weeks. Liver function tests were deranged with a total bilirubin 1.51 umol/L, ALT 859 U/L, AST 491 U/L and GGT 98 U/L. Hepatitis B, hepatitis C and human immunodeficiency virus (HIV) antibodies were non-reactive. A CT (Computed tomography scan) of the abdomen and pelvis showed fatty liver, and mesenteric panniculitis with prominent mesenteric nodules. She had noted a 6kg weight loss over six weeks and loss of appetite. Oesophagogastroduodenoscopy and colonoscopy showed chronic atrophic gastritis without dysplasia or malignancy. She had started taking five sky fruit seeds daily for duration of one month prior to presentation. Systemic review was negative for features of connective tissue diseases. Her physical examination was normal except for dry skin over hands and feet related to frequent ablutions. Her transaminitis resolved one month after stopping consumption of the seeds. Laboratory testing for autoimmune conditions showed a positive ANA 1:320, negative anti-double stranded deoxyribonucleic acid (DNA), anti-ENA profile and anti-neutrophil cytoplasmic antibody (ANCA). She had an elevated aldolase and creatinine kinase (CK) at 30.8 U/L and 463 U/L respectively. These normalised two weeks after stopping her recently initiated exercise regimen. Further work-up showed normal full blood count, renal function, thyroid function tests, erythrocyte sedimentation rate, needle electromyography of proximal and distal limbs and high resolution CT thorax.Discussion: To our knowledge, these are the first reports of Swietenia macrophylla seeds’ association with liver injury and induction of autoimmune phenomena such as inflammatory arthritis, mesenteric panniculitis and positive ANA. Our patients did well on prompt discontinuation of the skyfruit seed intake. These reports emphasize the importance of being aware of the regional trend in herbal supplement intake as there has been an increase in advertising by local markets about the benefits of skyfruit seeds. This may lead to serious health sequelae if not recognized promptly and discontinued.Key Learning Points: Consumption of Swietenia macrophylla seeds can lead to hepatitis and autoimmune phenomena.Disclosure: J. Leong: None. W. Soe: None. A. Vasudevan: None. E. Teo: None. K. Fock: None.


Gecarcinus lateralis YOs across molt was utilized in this study to curate the list of GPCRs and their expression in order to better assess which GPCRs are involved in the molt process.G-protein coupled receptors (GPCRs) are ancient, ubiquitous, constitute the largest family of transducing cell surface proteins, and are integral to cell communication via an array of ligands/neuropeptides. Molt inhibiting hormone (MIH) is a key neuropeptide that controls growth and reproduction in crustaceans by regulating the molt cycle. It inhibits ecdysone biosynthesis by a pair of endocrine glands  through binding a yet uncharacterized GPCR, which triggers a signalling cascade, leading to inhibition of the ecdysis sequence. When MIH release stops, ecdysone is synthesized and released to the hemolymph. A peak in ecdysone titer is followed by a molting event. A transcriptome of the blackback land crab G. lateralis putative GPCRs were obtained by screening the YO transcriptome against the Pfam database. Phylogenetic analysis classified 49 as class A (Rhodopsin-like receptor), 35 as class B (Secretin receptor), and 9 as class C (metabotropic glutamate). Further phylogenetic analysis of class A GPCRs identified neuropeptide GPCRs, including those for Allatostatin A, Allatostatin B, Bursicon, CCHamide, FMRFamide, Proctolin, Corazonin, Relaxin, and the biogenic amine Serotonin. Three GPCRs clustered with recently identified putative CHH receptors (CHHRs), and differential expression over the molt cycle suggests that they are associated with ecdysteroidogenesis regulation. Two putative Corazonin receptors showed much higher expression in the YOs compared with all other GPCRs, suggesting an important role in molt regulation.Ninety-nine Molting requires an orchestrated regulation of YO ecdysteroid synthesis by multiple neuropeptides. In this study, we curated a comprehensive list of GPCRs expressed in the YO and followed their expression across the molt cycle. Three putative CHH receptors were identified and could include an MIH receptor whose activation negatively regulates molting. Orthologs of receptors that were found to be involved in molt regulation in insects were also identified, including LGR3 and Corazonin receptor, the latter of which was expressed at much higher level than all other receptors, suggesting a key role in YO regulation.The online version of this article (10.1186/s12864-018-5363-9) contains supplementary material, which is available to authorized users. Crustaceans are a diverse subphylum of arthropods comprising of close to 67,000 species, classified into six classes, which together with their derived insects, comprise the pancrustacea superphylum clade . MalacosIn crustaceans, like other arthropods, locomotion is facilitated by a rigid exoskeleton. Chitin, the second most abundant carbohydrate in nature following cellulose, is the major component of the exoskeleton, providing a scaffold for cuticular proteins that form links between the chitin fibers, as well as stabilize deposition of minerals that harden the cuticle. The skeletal muscles attach internally to the exoskeleton and contract to enable limb movements . In someThe molt cycle involves well-defined stages including intermolt, premolt, ecdysis, and postmolt. The longest stage is intermolt, when the animal accumulates organic compounds and stores the energy required for reproduction and molting. In premolt, the epidermal layer enlarges and starts to separate from the old exoskeleton. It then synthesizes the outer layers of the new exoskeleton, while degrading the inner layers of the old exoskeleton. Ecdysis then takes place, by which the animal emerges from its old exoskeleton. The postmolt stage begins by the rapid uptake of water and minerals  and harden the new exoskeleton to complete the molt cycle. During this stage, the animals start to consume food . MoltingIn decapod crustaceans, ecdysone is secreted from the YOs into the hemolymph and is carried to target tissues, where it is hydroxylated into the active molt hormone: 20-hydroxyecdysone (20HE) –16. 20HEGecarcinus lateralis was published in 1952 by Bliss and Welsh [et al. performed binding assays using radio-labeled MIH with YO membranes collected from blue crab juveniles in intermolt stage and hepatopancreas membrane of mature vitellogenic females. MIH was found to bind to both YO and hepatopancreas membranes, but with far higher affinity to the YO, suggesting that the main binding site of MIH is the YO membrane [Our model organism is arguably the most well studied decapod crustacean in the context of molting. One of the earliest scientific studies on nd Welsh , describnd Welsh , 11, as nd Welsh –30 and tnd Welsh –34 have nd Welsh , 35 for membrane .Caenorhabditis elegans [Drosophila melanogaster [Bombyx mori, using in vitro binding assays with ITP peptides and 30 GPCRs [B. mori as references for phylogenetic study, Veenstra identified one contig (Pc-GPCRA9) as an ITP-like receptor based on clustering with BNGR-A24 and three receptors as putative ITP receptors  in the red swamp crayfish Procambarus clarkii, based on clustering with BNGR-A34 [Sagmariasus verreauxi has also proposed two putative ITP receptors based on phylogenetic alignment to BNGR-A34 and Pc-GPCRA53 [G. lateralis YO transcriptome across the molt cycle.G-protein coupled receptors (GPCRs) are ancient, ubiquitous, constitute the largest gene family of transducing cell surface proteins, and are integral to cell communication –39. All  elegans , while tnogaster , adding 30 GPCRs . Using tBNGR-A34 . A recen-GPCRA53 . These s-GPCRA53 , 51, makP. clarkii and found several neuropeptide receptors, including those for Ast C, LGR, PDF, DH31, and DH44 [et al. computationally identified 86 GPCRs in the eastern spiny lobster S. verreauxi, including important neuropeptide receptors [G. lateralis YO. Of these, 71 were annotated either by phylogenetic or domain analysis, which include Ast receptor, Crz receptor, CHHamide receptor, and FMRFamide receptor. These outcomes are comparable to previous studies in Decapoda [GPCRs play a central role in cell signaling as receptors for several transmitters, mediators, hormones, and neuropeptides. In crustaceans, most of the neuropeptides with known receptors act through GPCRs. Although many dozens of neuropeptides have been identified in hundreds of crustacean species over the last decade , 52, 53,and DH44 . In 2016eceptors . In thisDecapoda , 49. N-gIn silico transcriptomic analysis identified 299 sequences with seven TM   in five It is noteworthy that one GPCR (A37) showed specific expression at the postmolt stage, and one putative corazonin receptor (A6) was not expressed in the intermolt and early premolt stages, but was expressed in the mid and late premolt stages with higher expression in the postmolt stage. All three predicted CHHRs  were expressed throughout the molt cycle except in the postmolt stage. GPCR families known to be involved with molting in arthropods and those that show differential expression in the present study are discussed in further detail below.Manduca sexta [Allatostatins (ASTs) are pleiotropic neuropeptides that function as inhibitors of juvenile hormone (JH) production. JH is synthesized in the corpora allata in insects, while its crustacean active analogous compound  is synthesized in the mandibular organ . JH/MF mca sexta . It is aca sexta –62.Drosophila insulin-like peptides (DILPs) [G. lateralis Ast A receptor (AstA_R) contains three N-glycosylation motifs at the N-terminus and two N-glycosylation motifs at the C-terminus, while P. clakii AstA_R has three N-glycosylation motifs at the N-terminus and one at the C-terminus  and  (DILPs) . One putnus Fig. a. In G.lD. melanogaster, Ast B  blocks the receptivity of copulated females and increases food uptake after copulation [Platynereis larvae [. One putative Ast B receptor  was identified, which clustered with AstB_R identified in P. clarkii ), where AstB_R expression increases towards the molt and persists in the postmolt, when the animal is not feeding, suggesting a different role for Ast B in crustaceans. Another plausible explanation is that in this study we focused on the expression of AstB_R in the YOs, the crustacean analog of the prothoracic gland (PG) in insects, while the AstB_R temporal expression pattern in insects was examined in whole animals.Ast B is known for its myoinhibitory role and is therefore also referred to as myoinhibitory peptide (MIP) . In D. mpulation . Ast B is larvae . One putkii Fig. b. Gl-GPCkii Fig. . This exM. sexta [D. melanogaster [Tribolium castaneum [P. clarkii [S. vereauxi [M. sexta, Helicoverpa zea and Aedes aegypti [Ast C or PISCF-type Ast was originally described as an unknown neuropeptide in M. sexta . Its ananogaster , Triboliastaneum , and lat clarkii  and S. vvereauxi . Ast C i aegypti . It also aegypti . One putPeriplaneta americana [M. sexta [Corazonin (Crz) was initially discovered as a strong cardioaccelerator in the American cockroach mericana  and latemericana . It is americana –76. Crz mericana , while imericana . In addiM. sexta , servingM. sexta . AlthougM. sexta .et al. (2017) who report that, in C. maenas, Crz_R1  is higA6 Fig.. b. Crz inD. melanogaster, CCHamide-2 is mainly located in endocrine cells in the gut, where the cells sense the quality of food and signal for the transport of CCHamide-2 to the brain, where it binds to a CCHamide-2 receptor and alters feeding behavior [CCHamide is an invertebrate neuropeptide that was initially designated as ‘synthetic peptide, CCM’ . Roller behavior .P. clarkii CCHamide receptor in the phylogenetic analysis , which were clustered with sis Fig. a. CCH_R2sis Fig. . The rapsis Fig. . The SEMCarcinus maenas, where it accelerates heart contraction [Homarus americanus and Cancer productus [Macrobrachium nipponense, CCAP is among six key neuropeptides found to be involved in reproduction regulation [P. americana, D. melanogaster, Baculum extradentatum, and Locusta migratoria [M. sexta [D. melanogater [P. clarkii CCAP_R in the phylogenetic tree  is produced in the pericardial organ of the shore crab traction . It was roductus , 86. CCAroductus . In Macrgulation . Althouggratoria . CCAP alM. sexta  and reguanogater . One putree Fig. . RPKM exree Fig. .et al. identified two putative GPCRs, BNGR-A2 and A34, as ITP receptors, and A24 as an ITP-like receptor (member of CHHR family) in the silk moth B. mori. This led to the identification of CHH-like family receptors in crustaceans. Veenstra identified three CHH-like receptors clustered with BNGR-34 in P. clarkii, and two putative CHH-like receptors have also been found in S. verreauxi [MIH is a neuropeptide that controls molting in decapod crustaceans. The identification of its receptor has been the focus of crustacean endocrinological research for decades. In 2014, Nagai erreauxi .et al. conducted MIH binding assays in the blue swimmer crab Callinectes sapidus [Three putative GPCRs  were identified as CHH-like receptors, as these sequences clustered into the CHHR clade Fig. a. Furthe sapidus . This st sapidus . Based oM. sexta, two alternatively spliced ETHRs (ETHR-A and ETHR-B) are encoded by ethr gene and expressed in discrete central neurons [D. melanogater, the eth gene encodes two neuropeptides designated ETH1 and ETH2. Injection of ETH1 into pharate pupae strongly induces proecdysis within 1-3 minutes, followed by ecdysis, while injection ETH2 has no effect [P. clarkii ETH_R1. ETHR was expressed at a similar level of RPKM through the molt cycle except the postmolt stage, where no expression was detected  . Many is cloning . More FL cloning , 101. Th cloning –104. FMR cloning , 106. Thnogaster  through nogaster , 108.G. lateralis putative FMRFamide receptors clustered with P. clarkii and S. verreauxi FMRFamide receptors . Motif analysis of the S. serreauxi and G. lateralis FMRFamide receptors showed high similarity between FMRF_R1 and FMRFamide receptor of S. serreauxi. In particular, there were three N-glycosylation motifs at the N-terminus and one N-glycosylation motif at the seven TM domain. The only difference was at the C-terminus where two additional N-glycosylation motifs were predicted in G. lateralis  is activated by GPA2/GPB5 neuropeptide, a heterodimer formed by GPA2 and GPB5 [C. maenas, Bursicon is co-localized with CCAP, both being released from neurons in the CNS. Bursicon plays a key role in cuticle hardening during post molt [Penaeus monodon [Leucine-rich repeats-containing GPCRs (LGRs) belong to the rhodopsin-like GPCR family. LGRs have, in addition to the GPCR-conserved 7-TM domain, multiple repeats of leucine-rich regions (LRRs) and low density lipoprotein (LDL) motifs for hormone binding. LGRs are classified into three types  based on the number of LRRs, number of LDL motifs, and the structure of the hinge region . Type A and GPB5 , and LGRand GPB5 . Bursicoost molt , 113. Bu monodon . The num monodon .D. melanogaster, LGR3 is activated by Dilp8, a member of the insulin-like neuopeptide family [. The identification of LGR3 in G. lateralis YOs suggests a similar function for this receptor in delaying the molt by damaged or lost limbs [pro) (11), that delays molting by lowering hemolymph ecdysteroid titer (109). Given that several insulin-like peptides were recently identified in decapods [pro functions as the Dilp8 ortholog.Five putative LGRs were predicted in the phylogenetic analysis. Gl_GPCRA4 and Gl_GPCRA19 clustered with the Bursicon receptor (type B LGR) and Gl_GPCRA4b clustered with the GP2/GP5 receptor (type A LGR). Another LGR identified (Gl-GPCRA14) clustered with GRL 101-like. Like other GRL 101-like receptors, Gl-GPCRA14 consists of 11 LDLs and 7 LRRs in its ectodomain, which defines it as type C2-like LGR Fig. d. Gl-GPCe family . LGR3 ace family , 117. The family . The idest limbs , 120. Audecapods , 122, itNilaparvata lugens, Rhodnius prolixus [S. verreauxi [G. lateralis TRHR clustered with P. clarkii and S. verreauxi TRHRs.In mammals, thyrotropin-releasing hormone (TRH) is a hypothalamic releasing factor that is synthesized mainly in the hypothalamus. Upon release TRH stimulates the release of thyroid-stimulating hormone and prolactin by the pituitary. TRH is also produced in peripheral tissues and the nervous system . TRHs haprolixus ) and a cerreauxi  by phyloP. clarkii [Macrobrachium malcolmsonii [Drosophila PG [Biogenic amines are neuroactive molecules involved in synaptic transmission in the nervous system . This gr clarkii  and Macrolmsonii , but it olmsonii . Autocriphila PG .Three putative receptors clustered in the biogenic amine clade. Gl_GPCRA30 and Gl_GPCRA32 clustered with serotonin receptor 1 and serotonin receptor 2, respectively. Gl_GPCRA34 clustered with the octapamine receptor clade. One adenosine (Gl_GPCRA33) and four prostaglandin (Gl-GPCRA35-A38) receptors were also identified  [P. clarkii HPR1 in the phylogenetic analysis  . Three psis Fig. . It is yD. melanogaster [C. maenas and its function in rhythmic coordination was established [D. melanogaster [Diuretic hormones (DH) regulate water balance in arthropods . There anogaster . DH31 waablished . CT peptnogaster . One putB. mori, PDF binds to the BNGR-B2 receptor and stimulates the ecdysone biosynthesis in the PGs [D. melanogaster (CG13758), including those in P. clarkii and M. rosenbergii [Pigment dispersing factor (PDF) is a neuropeptide produced by the XO in crustaceans, and is found across inverterates. It has a variety of functions, including pigment dispersal in chromatophore cells  and regu the PGs . Decapodenbergii , 142. ThG. lateralis YOs transcriptome clustered with P. clarkii PTHR in the phylogenetic tree  belongs to the parathyroid hormone family, which includes PTH, PTH-related peptide (PTHrP), and tuberoinfundibular peptide (PTH2). In vertebrates, the PTH family regulates the calcium titer in serum, affecting most organs . One putree Fig. .G. lateralis YO transcriptome  , sweet and amino acid taste, pheromone, and odorant receptors in fish, as well as several orphan receptors [D. melanogaster mGlu and boss receptors, respectively  and marginal E-value (higher than 1.0E-7). With the rapid expanding availability of transcriptomes and genomes of decapod species, the spatial-temporal expression pattern and genomic context of these receptors is expected to be elucidated.Several GPCRs that are conserved across arthropods were not identified in the G. lateralis . Using this approach, we identified contigs encoding 99 GPCRs in the YO transcriptome of . Using tlis Fig. ; 27 . The identity of the MIH receptor has remained elusive, but it is assumed that it is a GPCR, the activation of which initates the triggering phase. Three contigs, designated CHH_1 (A9), CHH_2 (A10), and CHH_3 (A12), were identified as receptors for the CHH neuropeptide family, which includes MIH, CHH, and ILP , and therefore are candidates for the MIH receptor , early premolt (EP - stage D0), mid premolt (MP - stage D1), late premolt (LP - stage D2) and postmolt (PM), were collected and sequenced in triplicates . The transcriptomic data was screened for the longest open reading frames (ORFs) using transdecoder (version 5.0), generating a fasta file with the amino acid sequences. The amino acid sequences were scanned against the PfamA database (version 27.0) using hidden Markov models (HMMs) to identify the seven TM families. Sequences with seven TM profile were extracted , secretin and adhesion (class B), metabotropic glutamate (class C) based on Pfam. The references list were created using annotated sequences from previous studies [Arthropoda organism filter, and protein sequences blasted against the FlyBase database. The reference list was entered into TMMHMM [The transcriptome as well as FASTQ of us study . In brieus study . Animals studies , 156, ano TMMHMM  using deThe Reads Per Kilobase of transcript, per Million mapped reads (RPKM) were obtained by mapping sequence reads against YOs database using the RNA-seq tool of CLC Genomics Workbench  with default parameters. The RPKM was calculated as follows:P < 0.05). To avoid false positive result, the P values were then corected using false discovery rate (FDR < 0.05).The RPKM values of proteins relating to molt cycle were then imported into CLC for statistical analysis to determine whether there were significant differences in RPKM between different molt stages. The Empirical analysis of DGE was performed to compare RPKM between molt stages with the probability distribution less than 5% . The sequence alignment file was used to generate a phylogenetic tree with CLC Genomics Workbench . The lists of GPCRs used for phylogeny are given in Additional file To further annotate the G. lateralis males. A competitive ELISA assay was performed to confirm that the animals were in intermolt (stage C4) by measuring hemolymph ecdysteroid titer . The samples were stored in RNA later at -20 °C until extraction. Total RNA was extracted from 10 tissues comprising claw muscle (CM), eyestalk ganglia (ESG), gill (G), heart (H), hindgut (HG), hepatopancreas (HP), midgut (MG), testis (T), thoracic ganglia (TG), and Y-organs (YOs). Trizol® Reagent (Invitrogen), was used to extract total RNA according to manufacturer’s instruction and quantified using a ND-2000 . The extractions were stored at -80 °C for RT-PCR experiment. Primers were designed using Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/) and synthesized by Sigma–Aldrich company Additional file 2:Table S2. Statistical analysis of defined GPCRs using empirical analysis of DGE and false discovery rate method. (XLSX 80 kb)


MUSK gene. One variant, c.79+2T>G, is a known pathogenic variant, and the other, c.2165T>C (p.V722A), is a novel missense variant, predicted to be pathogenic by in silico analysis. The two variants were proven to be in trans. This case expands the clinical and molecular spectrum of MuSK congenital myasthenic syndromes.A 30-year-old woman with congenital vocal cord paralysis presented for evaluation of fatigable proximal upper limb weakness and difficulty maintaining the neck erect. Neurologic examination showed bilateral asymmetric eyelid ptosis, mild weakness (MRC 4/5), and atrophy of neck extensors and shoulder girdle muscles, whereas lower limb muscle strength was normal. Repetitive nerve stimulation revealed decremental responses in orbicularis oculis and trapezius. Needle electromyography demonstrated myopathic changes in proximal and paraspinal muscles. Acetylcholine receptor and muscle skeletal receptor tyrosine kinase (MuSK) antibodies, creatine kinase (CK), and lactate were negative or normal. Next-generation sequencing detected two heterozygous variants in the  Congenital myasthenic syndromes (CMS) are a heterogeneous group of hereditary neuromuscular disorders due to defects of the neuromuscular transmission. CMSs are characterized by fatigable muscle weakness, ptosis, and ophthalmoparesis and often manifest in infancy or childhood . HoweverMUSK gene. One variant, c.79+2T>G, affects a donor splice site in intron 1 and is expected to result in absent or disrupted protein product. This variant was previously reported in two MuSK CMS patients with CVCP (trans (C.79+2T>G from mother and c.2165T>C from father). Albuterol 4 mg orally resulted in normal strength of the neck flexors, neck extensors, biceps brachii, and triceps bilaterally.The patient is a 30-year-old woman who was born at term to non-consanguineous parents. Immediately after uncomplicated delivery, she developed respiratory distress and required intubation. She received a diagnosis of bilateral CVCP. She was discharged home with a tracheostomy without ventilator support or oxygen. She had no episodes of apnea. She underwent bilateral arytenoidectomy was successfully decannulated at age 3. In the mean time, she achieved normal motor milestones and kept up with her peers in gymnastics. She had, however, some exertional dyspnea and stridor. Mild eyelid ptosis was noted in childhood. During pregnancy, 15 months before presenting to our institution, she noted fatigable weakness of proximal upper limb and neck extensors muscles as well as shoulder girdle muscle atrophy. She had no diplopia, dysphagia, lower limb weakness, or exacerbation of the respiratory symptoms. She had no myalgia. Her 11-month-old daughter is well and has met motor development on time. Her 33-year-old brother and parents, of English and German descent, are asymptomatic. Neurological examination showed bilateral asymmetric eyelid ptosis , slight MUSK variant. The novel p.V722A variant is located in the tyrosine kinase region of MuSK and very close to a previously reported pathogenic variant (p.A727V)  . Owen etweakness . Howeverweakness . Dok7 CMweakness , 2, had weakness , 12, 13.weakness , 15. DokOur patient, similarly to most MuSK CMS patients , respondWritten informed consent was obtained from the participant for the publication of this case report and all identifiable data and images.MP and MM performed data collection, drafting of final manuscript, approval, and critical review of final form. J-LS performed data collection, approval, and critical review of final form.Neurology Genetics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.MM receives compensation to serve as associate editor of


Petroperla mickjaggeri gen. nov. sp. nov. and Lapisperla keithrichardsi gen. nov. sp. nov.—are placed in a new family Petroperlidae within the stemline of Systellognatha. The first Cretaceous larval specimen of Acroneuriinae, Electroneuria ronwoodi gen. nov. sp. nov., is also described along with another four new species that are placed within the Acroneuriinae genus LargusoperlaLargusoperla charliewattsi sp. nov., Largusoperla brianjonesi sp. nov., Largusoperla micktaylori sp. nov., and Largusoperla billwymani sp. nov. Additional specimens of Acroneuriinae are described without formal assignment to new species due to insufficient preservation. Implications for stonefly phylogeny and palaeobiogeography are discussed.This contribution describes seven new species of fossil stoneflies from Cretaceous Burmese amber, all of which are dedicated to present and past members of the Rolling Stones. Two species— Stoneflies (Plecoptera) is a small insect order with approximately 3,700 extant and 250 fossil species currently described . Extant Plecoptera is considered either as sister group to all other Neoptera or as one of the basal clades within Polyneoptera . PhylogeMegaleuctra), and Perloidea as monophyletic. Recently, The first molecular phylogeny was published by Gulou carpenteriStoneflies are relatively rare in the fossil record, which might be related to their delicate bodies and their preferred lotic habitat preventing fossilization. Stoneflies date back to the Carboniferous, with the alleged stem-Plecoptera LargusoperlaPinguisoperla Chen, 2018, including a single species Pinguisoperla yangzhouensis.The best-preserved fossil specimens are found in amber, allowing the observation of even minute morphological details. However, amber inclusions of stoneflies are in general very rare. Only one species attributed to the Perlidae subfamily Acroneuriinae  was descLargusoperla and describe the first larval specimen of stoneflies in Burmese amber. More importantly, we report two new genera within a new family of fossil stoneflies, which probably represent stem group representatives of Systellognatha. We discuss their systematic affinities and evaluate possible implications for Plecoptera phylogeny. We also provide novel insights into several aspects of the palaeocology and palaeobiogeography of stoneflies.In this study, we add four new species of All pieces of fossil-bearing Burmese amber originate from the type locality Hukawng Valley, Kachin State, Myanmar. The exact outcrop among the various amber mines in this valley is unknown, because the specimens were acquired from traders. A review of these amber deposits and relative geological history is available in The material was studied under a Leica M205 C  and Olympus SZX7  stereomicroscope. Leica Z16 APO Macroscope with Leica Application Suite Version 3.1.8 and Helicon Focus Pro was used to obtain stacked photographs with extended depth of field. Photographs were sharpened and adjusted in contrast and tonality in Adobe Photoshop™ version CS6 .High-resolution μ-CT scanning with a Bruker Skyscan 1272 tomograph was performed on specimen SMNS BU-79, but yielded no sufficient contrast to produce any useful tomographic image.Abbreviations for wing veins used throughout the text follow All type specimens examined are housed in the State Museum of Natural History, Stuttgart, Germany (SMNS) and catalogue numbers are specified for individual species below.When discussing the affinities of individual taxa, we refer to the phylogenetic system of Plecoptera proposed by http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:486E9A01-EF59-41D7-B001-4AD6D7FBB11F. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central, and CLOCKSS.The electronic version of this article in portable document format will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix Burmese amber is one of the oldest resins with insect inclusions, and stoneflies are one of the oldest pterygote lineages. What lies closer at hand than to link fossil stoneflies in ancient stones with the Rolling Stones and to name the new species after the members of the oldest and greatest Rock ‘n’ Roll Band in the world. The discerning reader will notice that the new family and genera are named after ‘the Stones,’ and all present and former members of the Rolling Stones are honoured with their own species.Class Insecta Linnaeus, 1758Subclass Pterygota Lang, 1888Order Plecoptera Burmeister, 1839Suborder Arctoperlaria Zwick, 1973Infraorder Systellognatha Zwick, 2000Petroperlidae, fam. nov.urn:lsid:zoobank.org:act:3F9EB209-A5DD-49C9-A5D6-7952F05F94A4Type genus.Petroperla gen. nov.Diagnosis. Glossae and paraglossae of approximately same size (plesiomorphy of Systellognatha), rather stout labial palps (plesiomorphy of Systellognatha), short first tarsomere  (apomorphy of Systellognatha); euplantulae present on tarsi (plesiomorphy of Systellognatha); setose arolium (apomorphy of Systellognatha); forewing with numerous crossveins in costal field (plesiomorphy of Systellognatha); vein RA almost reaching wing apex (apomorphy of Petroperlidae), proximal origin of vein RP  (plesiomorphy of Systellognatha).Petroperla gen. nov.urn:lsid:zoobank.org:act:68557BD3-F4E1-43BB-A10B-68D796971769Type species.Petroperla mickjaggeri, gen. et sp. nov.Diagnosis. By monotypy, as for the type species.Etymology. The first part of the compound noun refers to the Rolling Stones and is derived from Latin ‘petra,’ meaning ‘stone,’ the second part ‘perla,’ refers to the stonefly genus Perla.Petroperla mickjaggeri sp. nov. . Individual segments covered with short hair-like setae. Antennal segments in middle third of antenna approximately 2.2× longer than wide. Maxillary palps five-segmented, two basal palpomeres short, three distal palpomeres longer, of approximately same length. Labium with glossae and paraglossae of approximately same size, labial palps rather stout, three-segmented .Head. Not preserved.Thorax. Prothorax approximately quadrangular . Abdominal segments of Lapisperla keithrichardsi sp. nov. are equipped with posterolateral projections (no projections in Petroperla mickjaggeri sp. nov.), and meso- and metathorax of Lapisperla keithrichardsi sp. nov. ventrolaterally with gill remnants (apparently absent in Petroperla mickjaggeri sp. nov.). We consider these differences pronounced enough to justify a placement of these species in two separate genera within Petroperlidae.Order Plecoptera Burmeister, 1839Suborder Arctoperlaria Zwick, 1973Infraorder Systellognatha Zwick, 2000Superfamily Perloidea Latreille, 1802Family Perlidae Latreille, 1802Subfamily Acroneuriinae Klapálek, 1914Electroneuria, gen. nov.urn:lsid:zoobank.org:act:3A4CA23C-2D23-4F8E-9492-93C490AE736CType species.Electroneuria ronwoodi, gen. et sp. nov.Diagnosis. By monotypy, as for the type species.Etymology. Latin ‘electrum’ refers to both amber and electric guitars, the suffix ‘–neuria’ to the stonefly subfamily Acroneuriinae.Electroneuria ronwoodi sp. nov. . Length of antennal segments in the middle third of antenna approximately equal to segment width. Mouthparts of predaceous type. Mandibles with only apical part of right mandible recognizable, with four rounded teeth. Left maxilla well visible . Individual segments covered with short hair-like setae. Antennal segments in the middle third of antenna length approximately 2.5× longer than wide.Maxillary palps slender, five elongated palpomeres covered with short hair-like setae . Labial Thorax. Prothorax approximately quadrangular. Colour brown with pale median longitudinal line.Forewings : length Hind wings . Another diagnostic character  is not very pronounced in Largusoperla charliewattsi sp. nov. and is probably variable within the genus.Within Acroneuriinae, Pinguisoperla Chen, 2018, the second Perlidae genus described from Burmese amber, the subfamilial attribution is uncertain . Individual segments covered with short hair-like setae. Antennal segments in the middle third of antenna length approximately 2.5× longer than wide.Maxillary palps slender, with five elongated palpomeres covered with short hair-like setae  and 10E.Thorax. Prothorax trapezoidal, distinctly wider anteriorly. Surface of pronotum with numerous short hair-like setae. Colour brown with pale longitudinal median band, poorly visible due to state of preservation.Forewings  and 10B:Hind wings : length Thoracic sterna  with appLegs slender, covered with short hair-like setae. Several longer and thicker spine-like setae occasionally on tibia. Two thickened tibial spurs and several setae apically on tibia  and 9E. Abdomen. Colour brown, ventral side paler. Individual segments not distinctly enlarged posteromedially. Elevated, knob-shaped and circular hammer near posterior margin of sternum IX . Slightly moniliform in shape; segments covered with long hair-like setae .Affinities.Largusoperla billwymani sp. nov. share all the diagnostic characters of the Perlidae subfamily Acroneuriinae and the genus Largusoperla, as discussed above.Largusoperla billwymani sp. nov. clearly differs from Largusoperla charliewattsi sp. nov. based on the general shape of body and wings (more slender with narrower wings in Largusoperla billwymani sp. nov.) and pronotum shape . Largusoperla billwymani sp. nov. also exhibits larger paraprocts in comparison with L. charliewattsi sp. nov. and a different shape of hammer .Largusoperla billwymani sp. nov. are similar to Largusoperla flata, but slightly differ in shape, lateral contour being concave in Largusoperla flata . Largusoperla difformitatem can be distinguished from Largusoperla billwymani sp. nov. by having a smaller, transversally elongated hammer and also highly divergent paraprocts.Another two species Largusoperla micktaylori sp. nov. . Individual segments covered with short hair-like setae. Antennal segments in the middle third of antenna length approximately 2.2× longer than wide.Maxillary palps slender, with five elongated palpomeres covered with short hair-like setae  and 13C.Thorax. Pronotum trapezoidal, anteriorly slightly wider. Colour brown with central longitudinal pale band.Forewings  and 14C:Hind wings  and 14E:Thoracic sterna  with appLegs slender, covered with short hair-like setae. Two thickened tibial spurs apically on tibia. Very long hair-like setae subapically on tarsomere 3, approximately as long as claw length. Tarsi with first two tarsomeres short  and apical tarsomere long, approximately 1.5× longer than first two combined. Arolium present, slightly wider than width of apical tarsomere. Euplantulae prominent, present on tarsomeres 2 and 3.Abdomen. Individual segments not distinctly enlarged posterolaterally. Cerci short, 2.2–2.3 mm in length (approximately 0.2× body length), covered with short setae . Relative size of glossae and paraglossae is apparent, with paraglossae distinctly larger  together with overall high similarity in the arrangement of tarsi, mouthparts, wing venation and simultaneous occurrence with Largusoperla males, we include Largusoperla micktaylori sp. nov. into Largusoperla. From Pinguisoperla Chen, 2018, another Perlidae genus described from the Burmese amber, Largusoperla micktaylori sp. nov. clearly differs by the absence of an enlarged, plump basal segment of the cercus, which constitutes the main diagnostic character of Pinguisoperla.y larger  and 13C Largusoperla micktaylori sp. nov. with three pronounced extensions on posterior margin is rather unusual and unique among known stoneflies and represents a crucial diagnostic character of Largusoperla micktaylori sp. nov. Since the shape of the subgenital plate is exactly the same in both specimens and since they also share other morphological characteristics , we consider these specimens to represent a single new species.The subgenital plate in Largusoperla micktaylori sp. nov. differ from each other in the wing venation details such as number of crossveins and RP branches . Subgenital plate bilobed with wide rounded notch in middle of posterior margin .Largusoperla sp.  is assignable to Acroneuriinae, but without visible genitalia and generally too poorly preserved to allow generic attribution.Discriminating species characters for males of Acroneuriinae are the mainly detailed shape of hammer and paraprocts . For femThe absence of crossveins in the distal half of (ScP+) RA–RP area allows the placement of Petroperlidae in the suborder Arctoperlaria .Petroperla gen. nov. and Lapisperla gen. nov. . This character has been proposed as diagnostic for Systellognatha by Moreover, in the forewing of al field  is stronPetroperla mickjaggeri sp. nov. to Perloidea, the latter characterized by apomorphic shape of labium with glossae much shorter than paraglossae . It is more parsimonious to assume that it is either an autapomorphy of Petroperlidae, or even a synapomorphy of Petroperlidae and Kargaloperla.Within Systellognatha, a labium with glossae only slightly smaller than paraglossae and with rather stout labial palps  and 2A eaglossae . Therefolineages , which alineages  and 4A. G. carpenteri, and according to G. carpenteri to Plecoptera. It is also present in Permian Palaeoperla exacta Sharov, 1961  and Perlopsis filicornis Martynov, 1940 (Perlopseidae) and can be observed in Euholognatha as well, e.g. recent Leuctridae or fossil Perlariopseidae  might be related to Petroperlidae, and Palaeoperlidae might not constitute a monophyletic group.Relationships with several fossil groups considered as stem-Systellognatha  cannot bPredaceous mouthparts with long and slender palps are diagnostic for larvae of the superfamily Perloidea within the arctoperlariid group Systellognatha .Electroneuria ronwoodi sp. nov. rather resembles Perlidae and Chloroperlidae, since the ‘rounded shape of larval wing-pads whose edges meet medially, without leaving a separate notal contour’ is mentioned as a possible synapomorphy of these two families within Perloidea by Of the three families of Perloidea , the wing pad arrangement of Electroneuria ronwoodi sp. nov. can be excluded from Chloroperlidae, as larvae of this family exhibit a slender body with usually an oval pronotum and cerci distinctly shorter than the abdomen (see examples in Electroneuria ronwoodi sp. nov. is relatively robust bearing long cerci. Another apomorphic character of Chloroperlidae (except for subfamily Paraperlinae) is a thin, asymmetrically inserted, terminal maxillary palpomere  in the shape of thoracic segments with clearly visible notal contour between wingpads  occipital spinule row complete medially, (2) fringe of long thin setae laterally on pronotum , (3) long hair-like setae on surface of wing pads and abdominal terga, (4) posterior margin of abdominal terga with numerous very long thin setae, and (5) long setae on cerci absent.Based on the combination of characters as presented above, we attribute e tribes , but froPerla are known from Eocene Baltic amber , we might assume that the stonefly community in the Burmese amber forest streams was dominated by representatives of a limited number of higher taxa. This might be caused by the nature of the original habitat, which has been assumed to be a tropical Araucaria forest , a taxon, which today is restricted to the Nearctic realm. This points to an oriental origin of Anacroneuriini, thus confirming the respective hypothesis of


Chrysolophus, golden pheasant (Chrysolophus pictus) and Lady Amherst's pheasant (Chrysolophus amherstiae), exhibit brilliant colors in their plumage, but with extreme phenotypic differences, making these two species great models to investigate plumage coloration mechanisms in birds.As one of the most recognizable characteristics in birds, plumage color has a high impact on understanding the evolution and mechanisms of coloration. Feather and skin are ideal tissues to explore the genomics and complexity of color patterns in vertebrates. Two species of the genus Chrysolophus and golden pheasant against different backgrounds, we found that four melanogenesis biosynthesis genes and some lipid-related genes might be candidate genomic factors for the evolution of melanin and carotenoid pigmentation, respectively. In addition, a study among 47 birds showed some candidate genes related to carotenoid coloration in a broad range of birds. The transcriptome data further reveal important regulators of the two colorations, particularly one splicing transcript of the microphthalmia-associated transcription factor gene for pheomelanin synthesis.We sequenced and assembled a genome of golden pheasant with high coverage and annotated 15,552 protein-coding genes. The genome of Lady Amherst's pheasant is sequenced with low coverage. Based on the feather pigment identification, a series of genomic and transcriptomic comparisons were conducted to investigate the complex features of plumage coloration. By identifying the lineage-specific sequence variations in Analysis of the golden pheasant and its sister pheasant genomes, as well as comparison with other avian genomes, are helpful to reveal the underlying regulation of their plumage coloration. The present study provides important genomic information and insights for further studies of avian plumage evolution and diversity. Gypaetus barbatus, and turacin in Tauraco macrorhynchus [The plumage colors of birds serve functions in crypsis, social signaling, and mate choice . Due to rhynchus . In addirhynchus .dN/dS across birds [Feather complex coloration is likely coordinated through multiple genes that regulate diverse mechanisms. The melanogenesis biosynthetic pathway has been elucidated , 8, and ss birds , 16. HowChrysolophus pictus) at the genome and transcriptome levels, together with its sister species, the Lady Amherst's pheasant (Chrysolophus amherstiae). These species are two important organisms for studies of plumage coloration because of their phenotypic differences and close relationship. These two species can even crossbreed to produce fertile offspring under human feeding conditions. In adult male golden pheasant, both the crest and rump feathers are golden-yellow in color, the belly and upper tail coverts are dark red, the nape feathers are light orange with two black stripes, the mantle is iridescent green, and the tail is black spotted with cinnamon , cell development , and kidney development , respectively  , and 89.ectively . FunctioThe phylogenetic placement is a critical background for many comparative genomic analyses. To assess the phylogenetic position of the golden pheasant in Galliformes, a phylogenetic tree was constructed with five other sequenced Galliformes  was duplicated to three copies in the golden pheasant genome . However, the expression of CYP2J19 in the orange nape of golden pheasant was significantly higher than other colored feathers. It could be suggested that CYP2J19 might influence coloration at the transcriptional level in Chrysolophus. Additionally, CYP2D6 had the maximum allelic polymorphism among the CYP family in human [CYP2D gene. The expanded CYP2D6 genes in golden pheasant may function in metabolism or biotransformation of some foreign chemicals and could be a candidate for carotenoid deposition in its feathers.Sequence divergences and/or gene duplications have been proposed as important mechanisms in the course of evolution . Identifome Fig. , whereasome Fig. . The CYPotenoids , and twocanaries , 31. Comin human  and was in human , indicatChrysolophus species possessed darker eumelanic and brighter pheomelanic colors in their integument plumage, particularly the most impressive bright red and yellow feathers in male individuals , endothelin receptor B (EDNRB), KIT proto-oncogene tyrosine-protein kinase (KIT), and agouti signaling protein (ASIP) , which could prevent the formation of the “Kelch repeat type 1” domain (PF01344) based on the InterProScan annotation [ASIP gene, a single nucleotide is inserted after the initiation codon at exon 2A, which may impact 50% of ASIP isoforms by disabling this initiation codon or causing a frameshift, resulting in a premature transcription termination at the 13th cordon  with its ligands, alpha-melanocyte-stimulating hormone (α-MSH) and ASIP. ASIP can competitively antagonize α-MSH to bind MC1R, and ATRN is an obligatory accessory receptor for ASIP that enhances ASIP-Mc1R binding [Chrysolophus.We identified the lineage-specific varied genes in 48 birds . Four meIP) Fig. . ATRN hanotation   that are translated from exon-1B and exon-1M , which is the main apolipoprotein of chylomicrons and low-density lipoproteins (LDL). The biological functions of lipids include the storage and transportation of fat-soluble vitamins, including carotenoids. The transportation of carotenes requires LDL, and the transportation of xanthophylls requires high-density lipoprotein (HDL) [The two other sequenced Galliformes, chicken and turkey, also do not accumulate carotenoids in feathers. It is likely that golden pheasant acquired this new ability. Thus, the variations after its speciation from the ancestral species, but conserved in nonfeather-carotenoid birds, may contain the clues related to the new phenotype of feather carotenoids. In the 48 published avian genomes , four biin (HDL) . The evoP < 0.001; Zyxin (ZYX), is present at cell-cell contact sites and shuttles to the nucleus, where it affects cell fate and growth [ZYX participates in an interaction network with the gamma subfamily of peroxisome proliferator-activated receptor (PPAR-gamma) [in vivo and enter and transport in the bloodstream, where they are incorporated with lipoproteins, such as HDL and lLDL [In addition, the five feather-carotenoid birds are from five different clades , indicating that these birds may have independently acquired this ability. To detect whether some genes experience potential convergent variations in feather-carotenoid birds, we separated the birds into two groups, feather-carotenoid and nonfeather-carotenoid, and then performed a whole orthologous gene-wide association study between the two groups. We identified 48 genes containing genotypes (at the amino acid level) that might be associated with the accumulation of carotenoids in feathers  , which iR-gamma) , 53. Theand lLDL   and Lady Amherst's pheasant (non-carotenoid contained) feathers were enriched in the PPAR signaling pathway Fig. , which mage Fig. . In addiage Fig. . APOA1 ile bonds . A nonsele bonds , 57, prile bonds , chickenle bonds , and golle bonds . Based oion Fig. .P value = 1.23E-61; P value = 1.99E-16, Pearson test; Beta-keratins are major components of plumage, and evolution of the β-keratin multigene family may contribute to the novel characteristics of feathers , 61. In Chrysolophus. For melanin pigmentation, by identifying the lineage-specific variations in Chrysolophus, four melanogenesis genes might be associated with the evolution of eumelanin/pheomelanin regulation in the two pheasants. Additionally, the RNA sequencing (RNA-seq) data showed that the alternative splicing of ASIP and MITF were consistent with pigment composition in red and yellow feathers of Chrysolophus, particularly the MITF-1M transcript. For carotenoid pigmentation, we first identified genes that recently varied in golden pheasant but were conserved in the other 40 nonfeather-carotenoid birds, and the results indicated that the evolution of lipid-related genes might be highly related to carotenoid consumption in golden pheasant. Second, by a whole orthologous gene-wide association study between the sequenced feather-carotenoid and nonfeather-carotenoid birds, we identified 48 candidate genes that contain some lipid-related genes directly or indirectly, which may be associated with the carotenoid deposition in a broad range of avian plumage. In addition, the DEGs between the two pheasants were also enriched in some lipid-related pathways. It could be proposed that extraordinarily complex plumage patterns are not only encoded by the genome but also produced by the mechanisms underlying multilayered plumage coloring  [The genomic DNA from blood samples of a male golden pheasant was sequenced on the Illumina Hiseq 2000 platform. A series of paired-end sequencing libraries with insert sizes of 170 bp, 500 bp, 800 bp, 2 kb, 5 kb, 10 kb, and 20 kb were constructed, sequenced, and assembled using SOAPdenovo  . Contigshttp://www.bx.psu.edu/miller_lab/dist/README.lastz-1.02.00/README.lastz- 1.02.00a.html). More details about the methods are described in a study of the Chinese rhesus macaque genome [Taking advantage of the close evolutionary relationship between golden pheasant and turkey, the turkey genome was used as a reference and linked the assembled genome of golden pheasant to construct pseudochromosomes. The genome of golden pheasant was aligned to the genome of turkey using LASTZ  and mapped onto the golden pheasant genome using TblastN [RRID:SCR_015054) [Homology-based and RNA-seq combined data were used to annotate coding genes of golden pheasant. For the homology-based prediction, protein sequences of  TblastN . Next, h_015054)  to definRRID:SCR_014597) [The RNA-seq data are a good supplement for gene annotation because most of the homology alignments have no intact ORFs. Almost 100 G RNA-seq data from 25 samples were used and assembled into transcripts as follows. The reads were mapped to the golden pheasant genome using Tophat (version 2.0.8) . Cufflin_014597)  was used_014597) .RRID:SCR_005829) [Gene functions were assigned according to the best match of the alignment to the public databases, including Swiss-Prot, KEGG, and National Center for Biotechnology Information (NCBI) NR protein databases. Gene Ontology was annotated by Basic Local Alignment Search Tool (BLAST)2GO based on the alignment with the NCBI NR database. The motifs and domains in protein sequences were annotated using InterProScan   by searcde novo approaches. For the homology-based prediction, RepeatProteinMask and RepeatMasker  [http://www.girinst.org/repbase/) [de novo approach, RepeatModeler  and LTR-FINDER  [de novo repeat library, and then RepeatMasker was used to find TEs in the genome using the de novo repeat library. For the comparative analysis, the TEs of chicken, turkey, and zebra finch were annotated using the same pipeline to avoid the influence of different releases of the Repbase database or a different prediction pipeline.Tandem repeat searching was carried out using Tandem Repeats Finder . TEs in _012954)  against epbase/)  were use_015247)  were useA total of 22 libraries of different organizations or different color feathers from golden pheasants and Lady Amherst's pheasants  were constructed by using the Illumina TruSeq RNA sample preparation kit according to manufacturer's instructions. The libraries (insertion size ∼200 bp) were sequenced 90 bp at each end by using the Illumina Hiseq 2000 platform. We achieved 48∼83 million reads per library . RNA reaRRID:SCR_011812) for the protein sequences and then guided to align the corresponding coding sequences (CDS). A total of 996,755 4D synonymous sites were obtained and used in the phylogenomic construction. The phylogenome was constructed by using RAxML  (version 8.1.19) [The Treefam pipeline  was used 8.1.19)  with the 8.1.19) , was use 8.1.19) , chickenRRID:SCR_014932) package (version 4) [P values were computed by using the Chi-square statistics adjusted by the false discovery rate method to enable multiple testing, and the cutoff was used as 0.01. Further, the selected positive sites were retained by the homology prediction and RNA transcripts to avoid false-positive results from the assembly error or different splicing transcripts.For the 6,538 one-to-one orthologous paired genes (from the TreeFam pipeline as above described) in the eight avian species, the selected positive genes in golden pheasant were investigated. The protein sequences of orthologues were aligned by using the Muscle  softwarersion 4) . P valueP value ≤ 0.01.For the multicopy families, using the gene family results and timed-tree generated in the above as inputs, we studied the expansion and contraction of gene families using the Computational Analysis of Gene Family Evolution (version 2.1) , which iRRID:SCR_010602) [A total of 46.65 Gb paired-end data (read length 100 bp) of the Lady Amherst's pheasant were sequenced from a library with an insert size of 500 bp, and 44.88 Gb high-quality data were generated. All short reads were aligned twice to the golden pheasant genome using SOAP2 (version 2.22) . The fir_010602) , which uBy using the assembly of golden pheasant as a reference, we obtained the putative gene sequences of Lady Amherst's pheasant by changing the assembly of golden pheasant at the homozygous SNP and indel detections. The sites with map depth <5 or >120 are replaced by “N.”In the studies of 48 birds as background , 16, theChrysolophus. To avoid false-positive results from different splicing transcripts, only the results supported by both the homology prediction and RNA transcripts were retained.After identifying the orthologous gene pairs, we used five other Galliformes  and 11 other birds  from 11 different clades according to the phylogenetic analysis of 48 birds as background. The orthologous protein sequences of golden pheasant, Lady Amherst's pheasant (putative gene sequences as above described), and the 16 birds were aligned using Muscle. The alignments were compared site by site. We selected the gene containing the site that was same in the 16 birds but specific in Chrysolophus species, lineage-specific varied genes in golden pheasant as well as conserved in Lady Amherst's pheasant and the other 39 non-carotenoid birds were selected. A multisequences alignment was conducted by using MUSCLE to select the genes containing pheasant-specific sites, which is common in Lady Amherst's and the other 39 birds. Finally, 258 recent varied genes were annotated to KEGG pathways and scored by the following methods: if pathway A has total number of N(a) genes in golden pheasant and there are number n(a) genes in the 258 recent varied genes, then the score of pathway A is S(A) + = n(a)/N(a), and if pathway B has number of O(ab) genes shared with pathway A and has total number of N(b) genes in golden pheasant, then there are number of e(b) genes in pathway B, except the members shared with pathway A, and the S(A) + = e(b)/N(b)*O(ab)/N(a).Previous investigations have displayed the avian species that did or did not express carotenoid in their feathers . These rP value < 0.001.Otherwise, we referred to the population resequencing analysis strategy, such as that in Hilma Holm's research , and divTo avoid bias from different prediction methods applied in different bird genomes, we downloaded protein sequences of keratin genes of chicken from NCBI and then mapped against golden pheasant and 52 other avian genomes by using the same pipeline. Homology-based gene prediction was obtained by using the gene prediction pipeline mentioned above, except the threshold alignment rate was greater than 50%. Domain annotation was performed by using InterProScan, and only the results with domain of IPR003461 (avian keratin), IPR002957 (Type I keratin), or IPR003054 (Type II keratin) were retained. The correlation between the copy number of β-keratin and the assembly quality (contig N50) refers to the studies of the 48 birds (part of “Correlation between average substitution rates and number of species within different avian orders” and “color Discriminability”) . The subBoth melanin and carotenoid pigments in feathers were examined in two ways: spectrum and chromatogram. Raman spectroscopy was carried out using a Labram HR1800 spectrometer , referring to the strategy of Galvan  and ThomChrysolophus pictus genome assembly has been deposited under the accession number SAMN02980944 (BioProject PRJNA257945). Raw Illumina sequencing reads of the C. pictus reference genome have been deposited at NCBI in the SRA under accession number SRA753467. The C. pictus RNA-seq reads have been deposited at NCBI in the SRA under accession number SRA743586. The C. amherstiae RNA-seq reads have been deposited at NCBI in the SRA under accession number SRA743973. Supporting data, including assembly and annotation files, have been deposited in the GigaScience database GigaDB [The genome assembly is available via GenBank. The e GigaDB .Additional file 1: This doc file contains the supplementary figures: S1–S20.Additional file 2: This xls file contains the supplementary tables: S1-S24.Additional file 3: This doc file contains supplementary notes of pigments identification, animal sampling and transcriptome analysis.The authors declare that they have no competing financial interests.α-MSH: alpha-melanocyte-stimulating hormone; 4D: 4-fold degenerate; ASIP: agouti signaling protein; ATRN: attractin; BLAST: Basic Local Alignment Search Tool; CDS: corresponding coding sequence; CMC: ; DEG: differentially expressed gene; EDNRB: endothelin receptor B; GO: ; HDL: high-density lipoprotein; HPLC: high-performance liquid chromatography; HSP: high-scoring segment pair; indels: insertions and deletions; KEGG: Kyoto Encyclopedia of Genes and Genomes; KIT: KIT proto-oncogene tyrosine-protein kinase; LDL: low-density lipoprotein; MAPK: mitogen-activated protein kinase; MC1R: melanocortin-1 receptor; MITF: ; MULE: ; Mya: million years ago; NCBI: National Center for Biotechnology Information; ORF: open reading frame; OS: orthologous set; PPAR-gamma: peroxisome proliferator-activated receptor; RE: repetitive element; RNA-seq: RNG.P.L., J.Y., and C.Z. conceived the study. J.Y., G.Q.G., Y.C.Z., J.G., and X.K.Y. prepared the samples. M.X., J.M.M., Y.L.Y., H.M.C., and C.Z. performed genome sequencing, assembly, and annotation. G.P.L. and C.Z. supervised genome sequencing, assembly, and annotation. M.X., G.Q.G., Y.L.Y., J.M.M., X.Q.Z., X.M.X., and J.Y.X. performed genome analyses. G.Q.G., M.X., C.L.B., and G.H.S. carried out the transcriptome analyses. G.Q.G., Z.Y.W., and Y.H. carried out carotenoids and eu-/pheomelanins analysis. R.B.W., G.P.Z., and C.M.C. discussed the data. All authors contributed to data interpretation. G.P.L., G.Q.G., and M.X. wrote the paper with significant contributions from Y.C.Z., C.L.B., J.Y., C.M.C., and C.Z.This study was approved by the Institutional Animal Care and Use Committee of the Inner Mongolia University.This research was partly funded by the State Key Development Program for Basic Research of China, 973 Program (2012CB22306), the Open Project of Key Development Program for Basic Research of Inner Mongolia Autonomous Region, National Natural Science Foundation of China (31660301), Natural Science Foundation of Inner Mongolia (2013ZD06), and State Key Laboratory of Agricultural Genomics (2011DQ782025).GIGA-D-18-00007_.pdfClick here for additional data file.GIGA-D-18-00007_Revision_1.pdfClick here for additional data file.GIGA-D-18-00007_Revision_2.pdfClick here for additional data file.Response_to_Reviewer_Comments_Report_.pdfClick here for additional data file.Response_to_Reviewer_Comments_Report_Revision_1.pdfClick here for additional data file.Reviewer_1_Report_ -- Christopher Emerling2/12/2018 ReviewedClick here for additional data file.Reviewer_1_Report_Revision_1 -- Christopher Emerling7/8/2018 ReviewedClick here for additional data file.Reviewer_2_Report_ -- Matthew Greenwold4/1/2018 ReviewedClick here for additional data file.Reviewer_2_Report_Revision_1 -- Matthew Greenwold7/18/2018 ReviewedClick here for additional data file.Supplemental FilesClick here for additional data file.


Radopholus similis, commonly known as the burrowing nematode, is an important pest of myriad crops and ornamentals including banana (Musa spp.) and Citrus spp. In order to characterize the potential role of putative effectors encoded by R. similis genes we compared predicted proteins from a draft R. similis genome with other plant-parasitic nematodes in order to define the suite of excreted/secreted proteins that enable it to function as a parasite and to ascertain the phylogenetic position of R. similis in the Tylenchida order. Identification and analysis of candidate genes encoding for key plant cell-wall degrading enzymes including GH5 cellulases, PL3 pectate lyases and GH28 polygalactouranase revealed a pattern of occurrence similar to other PPNs, although with closest phylogenetic associations to the sedentary cyst nematodes. We also observed the absence of a suite of effectors essential for feeding site formation in the cyst nematodes. Clustering of various orthologous genes shared by R. similis with other nematodes showed higher overlap with the cyst nematodes than with the root-knot or other migratory endoparasitic nematodes. The data presented here support the hypothesis that R. similis is evolutionarily closer to the cyst nematodes, however, differences in the effector repertoire delineate ancient divergence of parasitism, probably as a consequence of niche specialization. These similarities and differences further underscore distinct evolutionary relationships during the evolution of parasitism in this group of nematodes. Meloidogyne spp., and the cyst nematodes belonging to the Heterodera and Globodera spp. Of these, genome and transcriptome sequences of several cyst nematodes, such as the soybean cyst nematode H. glycines [G. pallida, G. ellingtonae and G. rostochiensis. [M. hapla [M. incognita, M. javanica, M. arenaria [M. graminicola [M. floridensis [M. enterolobii [R. reniformis has also been released in the public domain [Global crop loss caused by plant-parasitic nematodes approaches ~$100 billion annually [glycines ,3 and thhiensis. –6 exist M. hapla , M. incoarenaria ,9, M. grridensis  and M. eerolobii  have beeerolobii . Also, mc domain .R. reniformis the burrowing nematode Radopholus similis is a migratory endoparasite. Radopholus is a member of the Pratylenchidae family, which have not been shown to form specialized feeding sites. Although the currently known host range of R. similis extends to more than 365 plant species [R. similis is an economically significant parasite of Musa spp. worldwide [Citrus spp., coconut, palm, coffee, sugarcane, avocado, black pepper, foliage ornamentals of the family  and ginger (Zingiberacieae). Additionally, bananas and plantains (Musa sp.) are consumed by ~400 million people worldwide and the export industry for these crops serves as a crucial mode of income for people living in many countries, including Colombia, Ecuador, Costa Rica and the Philippines [Unlike the sedentary endoparasitic RKN and cyst nematodes and the semi-endoparasitic,  species , R. simiorldwide , and alslippines .R. similis was first identified in 1893, in the banana growing areas in Fiji islands [R. similis populations from multiple regions worldwide indicated that R. similis radiation is a relatively recent event, occurring within the past 200–300 years [R. similis thrives in the tropical and the sub-tropical regions around the world including east Africa, south and central America, parts of Asia, Australia and multiple regions in Europe. In the United States, R. similis is found in Florida, Texas and the islands of Hawaii & Puerto Rico [R. similis is a sexually dimorphic PPN. Males and females can be classified by the unique sexual dimorphism that exists in their cephalic region, and this trait also supports the hypothesis that R. similis is closely related to the cyst nematodes, belonging to the ‘Hoplolaimidae’ family [R. similis males are generally 500–600 μm long and possess a raised lip, a feature not shared by the females [R. similis females are generally 550–800 μm long ending with a unique hyalinated tail region. R. similis reproduces as a facultative hermaphrodite, although out crossing occurs preferentially when males are present [00 years –21. R. srto Rico . R. simi’ family ,24. R. s females . Males a present .R. similis is completed in 18–20 days at a temperature range of 25–30°C, however populations in Europe seem to undergo reproduction even at lower temperatures [R. similis juveniles and females are parasitic, with the juveniles feeding outside the root system occasionally. The intercellular migration and feeding by these vermiform nematodes cause significant cell damage, which leads to the characteristic reddish-brown lesions associated with these nematodes. Additionally, damage caused by the tunneling and feeding activities, leaves the root system wounded and susceptible to opportunistic bacteria or fungi that may enter the plant through the wounded site.The life cycle of eratures . Eggs arR. similis infection seen in a banana plant is the characteristic necrotic lesions on the roots, that results in weakened root systems and reduced plant growth due to poor uptake of nutrients and water. This ultimately leads to toppling of banana plants especially during heavy rains and storm conditions and is a major concern among growers in the banana growing areas. Currently a variety of practices such as hot water and/or nematicidal treatment of the corms and suckers of banana plants, guying and propping of fallen banana plants [Musa spp. such as the Yangambi Km5 and Pisang Jari Buaya(AAA) have been very successful in reducing nematode populations [R. similis populations infecting Musa spp. in Uganda, which has been attributed to pathogenicity variability between R. similis populations [R. similis on bananas such as the “mutualistic endophyte Fusarium oxysporum strain 162” and the “antagonistic bacteria Bacillus firmus” have shown efficacy under controlled conditions; however, the protection they offer is generally limited to a single growing season[The major underground symptoms of an a plants  to hold ulations  in majorulations . Howeverulations . Furtherng season,29. ManaR. similis may provide insights into the variety of mechanisms adopted by this pathogen to maintain a plant-parasitic lifestyle. In order to characterize the potential role of putative PPN effectors, including plant cell-wall degrading enzymes, encoded by R. similis genes we compared predicted proteins from our draft R. similis genome [R. similis in the Tylenchida order. Small Subunit ribosomal DNA (SSU rDNA) studies have placed R. similis as a sister taxon of the endoparasitic cyst nematodes [G. rostochiensis, G. pallida, H. glycines, the root-knot nematodes M. hapla, M. incognita, the migratory endoparasite D. destructor and the free-living nematode C. elegans. We were able to corroborate the SSU rDNA phylogenetic studies that R. similis is indeed a sister-taxon to the cyst nematodes. In this article, we present key parasitism/effector features of R. similis and its phylogenetic position in comparison with several nematodes exhibiting different lifestyles.The genome sequence of s genome  with othematodes ,31,32. UR. similis effectors and parasitism genes have been reported including β-1,4 endoglucanase [R. similis predicted protein set possesses all the known parasitism PPN proteins , (CLE)—like peptides present in the cyst nematodes such as G. tabacum, G. rostochiensis and G. virginiae, which has been proven to be a required component to achieve development of a syncytium in host tissues [R. similis transcriptome analysis as well [R. similis not forming elaborate feeding sites in roots. However, effectors associated with host defense induction such as venom allergen- like effector proteins (VAPs) and suppression such as calreticulin (3) were present in R. similis [M. incognita and the migratory PPN, B. xylophilus, where it has been shown to be involved in host defense suppression and reproduction respectively [R. similis , which is a key enzyme involved in modification of the host salicylic acid pathway [R. similis , glycosyl transferase (GT), carbohydrate esterase (CE), polysaccharide lyase (PL) and auxillary activity (AA). GHs are enzymes that accelerate the degradation of glycosidic bond between carbohydrates and/or non-carbohydrates. GHs are found across a wide gamut of organisms including bacteria, plants, fungi and nematodes [R. similis belong to multiple families, notably, GH5 (cellulase), and GH28   have carbohydrate binding modules (CBM) such as CBM2 appended. Cellulases from the root-knot nematode M. incognita and the cyst nematode, G. pallida also possess an appended CBM2 domain. In the sugar beet cyst nematode, H. schachtii, a secreted protein with a CBM domain, was demonstrated to directly interact with an Arabidopsis pectin methylesterase, thereby implicating it in facilitating nematode parasitism [CAZymes comprise of enzyme families that are involved in the degradation, synthesis and/or remodeling of carbohydrates and their conjugates. CAZymes were searched in the CAZy database, dbCAN database and the peptide pattern recognition (PPR) library to look for homologues of proteins encoding putative CAZymes in the predicted protein set of ematodes . Within rasitism .R. similis encodes ten putative cellulase genes belonging to the GH5 family, subfamily 2 Phylogenetic analyses demonstrated that the R. similis GH5 proteins are distributed in four distinct clades, of which three are clustered and each is closely related to the cyst nematodes belonging to the Heterodera and Globodera genus  and found the predicted localization likelihood patterns to be extracellular  of cellulases in most PPNs [ra genus . This reematodes . Moreoven et al.  have supn et al. ,51. Thisn et al. ,52 that oderidae”,32,52. Ibacteria . Additiobacteria . DeepLoccellular . No cellost PPNs ,55.R. similis. Polygalactouranase is required for the breakdown of galactouronic acid, which is an essential monomer that makes up pectin. GH28 polygalactouranase has also been found in the transcriptome of R. similis by Huang et al. [Ralstonia solanacearum. Intriguingly, R. solanacearum is a xylem colonising pathogen and R. similis has been reported to block xylem vessels which might imply that these functions might have been a characterisic of niche specialization. Furthermore, within plant-parasitic nematodes, GH28 genes acquired from bacteria have been found in root-knot nematode M. hapla, M. incognita and the family Pratylenchidae. Notably, polygalactouranase from RKNs as well as the false root-knot nematode, Nacobbus aberrans also demonstrates high sequence similarity with the R. solanacearum GH28 protein [R. reniformis also possesses GH28s, but the GH28 in R. reniformis appears to be non-functional. Because GH28 does appear to be functional in R. similis, this finding supports the hypothesis of early gain in the last common ancestor of root knot and cyst, and subsequent loss in the cyst nematodes [A single gene coding for a putative polygalactouranase, belonging to the GH28 family has also been found in g et al. . A BLAST protein ,31,56. SR. similis. These genes belong to five families, namely PL1, PL3, PL5, PL9 and PL22. R. similis possesses four putative PL3 (pectate lyase) proteins, fewer than other PPNs in clade 12 such as M. hapla (20), M. incognita (33), H. glycines (15), G. pallida (8) and the clade 10 PPN B. xylophilus (15)  have been predicted in R. similis with other nematodes such as plant-parasitic, human-parasitic and free-living. R. similis forms a total of 8542 orthologous clusters and 5115 singletons. All the species combined, shared a total of 3690 orthologous clusters (R. similis shares higher number of orthologous clusters with the cyst nematode G. rostochiensis (4034), compared to the root-knot nematode M. hapla (3911), the free-living nematode C. elegans (3726), the migratory endoparasitic nematode D. destructor (3785) and the human parasitic nematode B. malayi (3723).Plant-parasitic nematodes occur in clade 1 (Triplonchida), clade 2 (Dorylaimida), clade 10 (Aphelencoididae) and clade 12 (Tylenchida) of the 12 clades comprising the phylum Nematoda. In a phylogenetic tree presented by Holterman et al., 2017 , disparaclusters . We obseR. similis, G. pallida, G. rostochiensis, H. glycines, M. incognita M. hapla, D. destructor and C. elegans was performed to gain insight into the phylogenetic relationships shared by R. similis. The resulting species tree corroborates the existing hypothesis, that, R. similis is indeed a sister taxon to the cyst nematodes  and thereby share the latest common ancestor with the cyst nematodes than with the root-knot nematodes, M. hapla and M. incognita or the migratory endoparasite D. destructor  values less than 1 were extracted using a custom perl script.Gene prediction was performed using the MAKER v.2.31.8 pipeline  in threeR. similis protein sequences were blasted against CAZy database [R. similis proteome . The E value used was 1e-5. dbCAN2 was utilized to perform this job[database   blastp analysis of relevant genes was done against the NCBI nr database (nematoda) with an E- value of 1e-5. A blastp analysis was also performed on wormbase parasite with an e-value of 1e-5. The relevant hits were extracted, following which the sequences were aligned using MUSCLE   has been deposited in NCBI under the accession number SJFO00000000 and BioProject PRJNA522283.S1 File(DOCX)Click here for additional data file.S1 FigR. similis proteins are indicated in red. Genbank accession numbers are shown adjacent to each nematode. The numbers on the branches indicate bootstrap support value. 1000 bootstrap replications were performed. Scale indicates number of substitutions per site.(TIF)Click here for additional data file.S2 FigR. similis gpd gene is indicated in red. The numbers on the branches indicate bootstrap support value. 1000 bootstrap replications were performed. Scale indicates number of substitutions per site.(TIF)Click here for additional data file.S3 FigR. similis predicted cellulase protein sequence and (B) H. glycines cellulase protein sequence (acc: AAC15707.1). Numbers on branches indicate localization likelihood in different compartments. Ideal localization path is highlighted in red.(A) (TIF)Click here for additional data file.