ITS, matK, rbcL, trnH-psbA), and sequencing. Sequencing data were assembled using Bioedit software, and the BLAST algorithm was used on the NCBI database for species identification at the genus level. Molecular barcoding data based on genetic similarity identification was in full coherence with taxonomic classification based on morphological data. The combination of ITS and matK exhibited a greater capacity to identify a species among the Greek salep samples. Out of the 53 samples examined, 52.9% were classified as Dactylorhiza spp. and 33.3% as Anacamptis spp., whereas only 6 samples were identified as Orchis spp. (11.8%). Given that a superior-quality salep beverage comes from tubers of the latter, the number of samples classified as such in northwestern Greece is unexpectedly low. A database of 53 original reference sequences from wild-growing samples of Greek origin was generated, providing a valuable resource for the identification of other salep samples from different regions. The DNA barcoding results unveiled that salep samples from northwestern Greece are related to nine members of four different genera of Orchidaceae. All species are nationally protected and covered by the CITES convention, while many of these orchids are included in the EU Directive 92/43/EEC appendix as “Other Important Species”. Thus, expedited coordinated management actions are needed to ensure their survival in the future. Molecular DNA barcoding combined with botanical taxonomy can be used for the identification and conservation of collected Greek orchids used for salep production as well as in the regulation of fair salep trade. A modified CTAB protocol was used for DNA extraction, amplification of barcoding regions ( Consequay 2023) ,6,7. Gerard’s Herbal in 1633 [The commercial product commonly called ‘salep’  is the most famous folk preparation comprising orchids . For tho in 1633 . To date in 1633 ,20,21. I in 1633 ; however in 1633 ,10,15,19 in 1633 . Anacamptis coriophora (L.) R.M. Bateman, Pridgeon & M.W. Chase, Anacamptis morio (L.) R.M. Bateman, Pridgeon & M.W. Chase, Anacamptis papilionacea (L.) R.M. Bateman, Pridgeon & M.W. Chase, Orchis anthropophora (L.) All., and Orchis italica Poir. [Anacamptis pyramidalis (L.) Rich., Dactylorhiza sambucina (L.) Soó, Dactylorhiza saccifera (Brongn.) Soó, Orchis militaris L., Orchis provincialis Balb. ex Lam. & DC., and Orchis simia Lam. [Orchis mascula [Orchis mascula salep has presumed lung anticancer activity, strong antioxidant activity, and beneficial effects on hyperlipidemia and hypertension [Currently, the most popular form of salep is a hot beverage that is consumed to soothe the throat and ease stomach aches and intestinal cramps and as a remedy for colds and coughing . Severalca Poir. , or Anacmia Lam. . One waymia Lam. . Among d mascula . Apart frtension . Undoubtrtension .Orchis, Anacamptis, and Ophrys and also Dactylorhiza, Himantoglossum, Neotinea, Platanthera, and Serapias are reported as common among these studies [Given the ethnic importance of salep and the concomitant concerns for orchid conservation ,10,15,19 studies .ITS), and chloroplast DNA, such as rbcL, maturase K (matK), psbA-trnH, and the transfer ribonucleic acid leucine (trnL), are broadly used as markers for the molecular identification of plant species [psaB, trnL-F, and nuclear Xdh [ITS and matK markers can successfully identify members of the large genus Dendrobium in the family Orchidaceae [nrITS, trnL-F, and matK for barcoding DNA extracted from 150 collected tubers of Iranian orchids used to produce salep [Orchis, Anacamptis, and Dactylorhiza. Specifically, nrITS and the trnL-F spacer proved to be easier to amplify and sequence than the matK, providing a better-discriminating ability that eventually led to the recognition of the species that are most threatened by overharvesting in different regions of Iran [The above-mentioned studies coupled with the complex taxonomy of Orchidaceae ,26,27,28 species . Howeverlear Xdh ,26,27. Ilear Xdh ,33,34. Pce salep , reveali of Iran . Thus thGenomics technologies and approaches are of vital importance to maintain and conserve biodiversity as they can provide reliable results, even if DNA is too degraded and difficult to sequence using next-generation tools . HoweverThe scope of this study was first to check whether widely used DNA barcoding markers already used in other Orchidaceae studies can be used for the identification of collected wild-growing orchids used for salep by the Sarakatsani ethnic Greek population subgroup in northwestern Greece . The secITS and matK were successfully amplified, sequenced, and used for the identification of almost all samples, while three barcoding regions  were successfully used for only four samples. Although in most samples the trnH-psbA region was amplified and sequenced, the results were in some cases divergent from the results of the other two barcoding regions that were totally in agreement. Divergent results are accompanied by low BLAST similarities with our samples. A lack of information or taxonomic misclassifications in public DNA databases could lead to divergent results and misidentifications. In only a few samples, trnH-psbA was used instead of one of the other two regions for molecular identification. No dependence of the sequencing success rate on the origin of the sample (fresh or dried tissue) was observed. The combination of ITS and matK demonstrated a greater capacity to identify a species among the Greek salep samples, in agreement with previous studies concerning the family Orchidaceae [Orchis sp. due to out-flowering appearance  or O. quadripunctata Cirillo ex Ten. . In some other cases, the molecular barcoding data suggested the identification as Dactylorhiza maculata (L.) Soó or Dactylorhiza sambucina (L.) Soó for the specimen GR-1-BBGK-18,6097-26, and D. maculata for the specimens GR-1-BBGK-19,6097-27 and GR-1-BBGK-16,6097-29 , and the specimen GR-1-BBGK-18,6097-3 as Anacamptis sp.  Rich., all other samples (n = 16) were found to be Anacamptis morio (L.) R.M. Bateman, Pridgeon & M.W. Chase with molecular barcoding data; the latter belong to subsp. caucasica (K.Koch) H. Kretzschmar, Eccarius & H. Dietr. as determined using the taxonomic identification of the collected samples  P.F. Hunt & Summerh. subsp. pythagorae (Gölz & H.R. Reinhard) H.A. Pedersen, P.J. Cribb & Rolf Kühn  Kreutz; sample GR-1-BBGK-21,146) and Dactylorhiza sambucina  were only identified as Dactylorhiza sp. with molecular barcoding data due to contradictive matching results in the NCBI database. The taxonomically identified sample GR-1-BBGK-18,6097-24 as D. sambucina was matched as either D. sambucina or D. incarnata (L.) Soó with molecular barcoding data; however, it is highly possible that this sample may be D. sambucina due to ITS barcoding accuracy (Dactylorhiza maculata (L.) Soó subsp. saccifera (Brongn.) Diklic (synonym of D. saccifera (Brongn.) Soó subsp. Saccifera) was only identified at the species level with molecular barcoding data (n = 6 samples); a possible molecular match with D. maculata subsp. fuchsii (Druce) Soó for the specimen GR-1-BBGK-21,119  [D. sambucina or Dactylorhiza viridis (L.) R.M.Bateman, Pridgeon & M.W.Chase (synonym Coeloglossum viride (L.) Hartm.) was rejected  Schltr. subsp. rumelicum (H.Baumann & R.Lorenz) Niketic & Djordjevic  was only identified at the genus level with molecular barcoding data (H. caprinum (M.Bieb.) Spreng. (synonym H. affine (Boiss.) Schltr.); however, the latter is not reported as currently present in the Greek territory  [The taxonomically identified accuracy . The taxK-21,119  was disray 2023) . In anotrejected  due to ting data  due to pay 2023) . In geneDactylorhiza (52.9%). Seventeen samples were classified as Anacamptis (33.3%)   . In manys Orchis . Surpris (11.8%) .Orchis spp., the number of samples classified as such in the sample set from northwestern Greece is unexpectedly low. According to the BLAST results, the Greek samples probably belong to Orchis pallens or Orchis quadripunctata. A closer inspection of the ITS barcoding region nucleotide alignment analysis indicates that the Greek samples are highly similar to O. pallens, and thus are different from other Orchis spp.  [Along this line and with the aim to protect orchid resources, previously published recommendations have suggested the need for long-term monitoring of wild-growing salep orchid populations in Greece and elsewhere and effective modeling regarding the response of different salep species to different harvesting pressures . Other ray 2023)  for in say 2023) . With onay 2023) , and manPlant samples of Greek orchids used for salep were collected from mountainous areas in northwestern Greece (mainly) during 2018–2022 . Information regarding the localities and habitats where salep orchids naturally grow in the wild was obtained through informal conversations with (a) local residents of mountain areas in northwestern Greece who collect bulbs for personal use and (b) shepherds of the Sarakatsani ethnic Greek subpopulation group who traditionally graze their flocks at high altitudes and are aware of the local flora . These pAll collections were performed between May and early July each year. When not possible to collect living plant individuals, dried inflorescences were collected . The wilhttps://powo.science.kew.org, accessed on 1 May 2023) [https://portal.cybertaxonomy.org/flora-greece/intro, accessed on 1 May 2023) [To allow comparisons across geographical scales and with other studies, all the species and subspecies are hereby cited with their currently accepted names according to the POWO (Plants of the World Online) database (ay 2023)  and not ay 2023) . The latTotal genomic DNA was extracted using a modified CTAB protocol . The extInternal Transcribed Spacer (ITS) [trnH-psbA intergenic spacer region [maturase k (matK) [ribulose-bisphosphate carboxylase gene (rbcL) [Four barcoding regions were tested, namely, er (ITS) , trnH-psr region , maturask (matK)  and ribue (rbcL) . The useDiuris sulphurea R.Br., Goodyera schlechtendaliana Rchb. f., and Caladenia reticulata Fitzg. were used herein as outgroups based on previous references from the literature  and taxonomic classification of orchids collected from different sites in northern Greece showed that 53% represent members of the genus Dactylorhiza, 33.3% are members of the genus Anacamptis, and 11.8% are members of the genus Orchis. Considering the reasons that sometimes create boundaries in DNA barcoding, reliable projects require holistic approaches given that biological and technical complications/issues are inevitably insurmountable. The use of such straightforward DNA barcoding protocols for salep orchids may assist in tracking, monitoring, and regulating the trade of wild-harvested products  and can also facilitate the conservation of natural populations of Greek salep orchids. The investigation herein has shown that DNA barcoding markers widely used in different orchid-related studies can also be used for the identification of wild orchids collected for salep by the Sarakatsani ethnic Greek population subgroup in northwestern Greece. The combined molecular barcoding (Although in situ conservation of these orchid plants is ensured in various sites of the Natura 2000 network in Greece, there are still many wild-growing salep orchid populations outside the protected zones that suffer extensive over-collection directly from the wild. Therefore, long-term monitoring of wild-growing salep orchid populations is needed in Greece, which should be combined with ecological modeling regarding the response of different salep species to varied harvesting pressures. In addition, collection bans should be enforced in severely affected areas and in cases of illegal collections and exports from the country, while close monitoring of species diversity in extant salep trade should be coupled with informed controls at domestic customs offices. Training on sustainable collection practices and facilitation for the establishment of small-scale pilot cultivations of salep orchids are already in place in Greece, and these should be extended to foreign stakeholders with the aim to alleviate future collection pressure on wild-growing populations of these protected phytogenetic resources.


Pseudomonas aeruginosa is a significant pathogen identified with healthcare-associated infections. The present study evaluates the role of biofilm and efflux pump activities in influencing high-level resistance in virulent P. aeruginosa strains in clinical infection. Phenotypic resistance in biotyped Pseudomonas aeruginosa (n = 147) from diagnosed disease conditions was classified based on multiple antibiotic resistance (MAR) indices and analysed with logistic regression for risk factors. Efflux pump activity, biofilm formation, and virulence factors were analysed for optimal association in Pseudomonas infection using receiver operation characteristics (ROC). Age-specificity (OR [CI] = 0.986 [0.946–1.027]), gender (OR [CI] = 1.44 [0.211–9.827]) and infection sources (OR [CI] = 0.860 [0.438–1.688]) were risk variables for multidrug resistance (MDR)-P. aeruginosa infection (p < 0.05). Biofilm formers caused 48.2% and 18.5% otorrhea and wound infections  respectively and more than 30% multidrug resistance (MDR) strains demonstrated high-level efflux pump activity , protease , lipase , and hemolysin . Resistance relatedness of more than 80% and 60% to cell wall biosynthesis inhibitors  and, DNA translational and transcriptional inhibitors  were observed (p < 0.05). Strong efflux correlation  with MDR strains, with high predictive performances in efflux pump activity (ROC-AUC 0.78), biofilm formation (ROC-AUC 0.520), and virulence hierarchical-clustering. Combine activities of the expressed efflux pump and biofilm formation in MDR-P. aeruginosa pose risk to clinical management and infection control. Pseudomonas aeruginosa is one of the significant pathogens identified with increasing healthcare-associated infections (HAIs) [Pseudomonas infection to prevent growing resistance. Antibiotic selective pressures facilitate intrinsic resistance mechanisms to the drug of choice, making therapeutic applications difficult, expensive, and exacerbating morbidity [s (HAIs) ,2. It iss (HAIs) ,4,5. Froorbidity .P. aeruginosa biofilm functionally creates favourable medium that prevents accessibility of antibiotics to infection sites and promotes cell-surface interactions for tissue degradation. Biofilm formation in P. aeruginosa infection further enhances in vivo colonization, adaptation and persistence, through aggregation of exopolysaccharides (EPS) and biomolecules  induced by poly-N-acetylglucosamine [P. aeruginosa [P. aeruginosa resistance and increase virulence factor productions.cosamine . The actruginosa . MexAB-Oruginosa ,10. PersP. aeruginosa intracellular survival and cytosol lipid degradation leading to severe tissue pathology [P. aeruginosa isolates are associated with biofilm and efflux pump activities [P. aeruginosa and possible implications on antibiotic stewardship in Nigerian hospital settings. The association of biofilm formation, efflux pump activities, and virulence proteins from P. aeruginosa strains resulting in high-level multi-drug resistance in clinical infection is yet to be defined. The study aims to evaluate the functional role of biofilm-forming capacity and efflux pump activities of virulent MDR-P. aeruginosa isolates from clinical infections and association of these factors with pathogen tissue tropism.Increase efflux activity and virulence factors usually intensify disease morbidity, with activation of hemolysin that cause tissue damage, dysfunctional cellular immune responses, and red cell destruction ,12. Invoathology . Severaltivities ,19,20,21P. aeruginosa strains collected over the 6-month period, 27 were multi-drug resistance (MDR), showing significant resistance of more than 80% to cell wall biosynthesis inhibitors  and over 60% resistance to DNA translational and transcriptional inhibitors ;  with the highest prevalence rate among children ages 0–12 years (47.6%) and adult ages 24–50 (25.7%). Higher rate was recorded among female (20.8%) compared to male . Considerable high risk for otorrhea (37.0%), wounds (21.4%) and bloodstream (18.2%) infections were noted , but no MDR P. aeruginosa strain was isolated from other infection sites .The univariate analysis of the strain collections from the subjects presenting various systemic infections is shown in -P. aeruginosa strains (p = 0.001) were biofilm producers causing otorrhea and wound infections respectively. Strains found in otorrhea infection expressed significant efflux pump activity , protease , lipase , and hemolysin  compared to strains from other infections  with the MDR strain mostly from the ear infection, was recorded. Higher proportion of MDR strains overproducing efflux and biofilm clustered together (cluster a) compared to less number recorded in cluster b (P. aeruginosa. High biofilm ROC-AUC 0.78 (95% CI: 0.580–0.915) highlights a stronger predictive performance compare to efflux ROC-AUC 0.520 (95% CI: 0.321–0.715), showing a significant phenotypic adaptive resistance mechanism  with the degree of biofilm, hemolysin and lipase production , facilitate strain colonization and high infection rate [P. aeruginosa among female patients presents a different Pseudomonas infection phenomenon attributed to hormonal changes during the menstrual cycle, influencing behavioral and physiological changes that alter the immune responses [Pseudomonas infection [Pseudomonas wound infections are exacerbated by expired or ineffective topical antibiotics, contaminated or poorly sterilized dressing gauze. These further pose a significant challenge to clinical management, causing prolonged hospital stay and increasing morbidity [In the current study, age–specificity is a considerable risk factor for MDR ion rate ,23. The ion rate . Self-presponses . Increasnfection . Though nfection , poor clorbidity . P. aeruginosa protease degrades basement membranes and extracellular matrix components, it cleaves the laminin, fibronectin, proteoglycans, and various collagens, causing direct tissue invasion with extensive ulceration leading to the down-regulation of cascade path of humoral immune responses [P. aeruginosa lipase (an extracellular lipolytic enzymes), interacts with inflammatory mediators released from human platelets, neutrophilic and basophilic granulocytes, and monocytes to enhance massive tissue damage and bloodstream infection [Furthermore, the produced biofilm matrix plays structural function of inhibiting neutrophil elastase  from the host to enable persistence infectivity and virulence . The abiesponses . During nfection . Hemolysnfection . These vnfection .P. aeruginosa obtained from wounds, urinary tract and ear infections reveal active circulation and re-dissemination of strains with potential to exchange resistant genetic elements , thereby facilitating horizontal gene transfer. This results to relatively high recombination that could give rise to multi-clonal MDR population [Pseudomonas aeruginosa infection control could suffer major setbacks due to high-level resistance to cell wall biosynthesis inhibitors  which are most prescribed antibiotics. The recorded antibiotic resistance of more than 60% to translational and transcriptional inhibitors poses a serious challenge to clinical management with a remarkable capacity to develop an extend-spectrum resistance to other classes of antibiotics. Low outer membrane permeability, high efflux activity and producted inactivating enzymes are possible intrinsic or acquired resistance gained through horizontal gene transfer or mutational changes [P. aeruginosa adaptive resistance mechanisms, acting as a diffusion barrier to limit antibiotic access to the bacterial cells making the strains capable of surviving antibiotic attacks [Clustering of highly related MDR pulation . Dissemipulation . Hospita changes , limitin attacks . HospitaP. aeruginosa susceptibility corroborate the previous report of increasing MDR in similar clinical settings in northern and southern Nigerian with more prevalence rate of hospital-acquired Pseudomonas infection [Very low clinical MDR nfection ,19,20,33P. aeruginosa OM carries several substrate-specific porins that limit the intake of certain compounds and nutrients, further enhance expression of MexAB-OprM efflux, which provides resistance to varieties of antibiotics  [P. aeruginosa to intensify infection while maintaining high-level resistance. The interplay of virulence factor activity and biofilm formation demonstrates a dominant influence that increases infection burden, therapeutic failure, morbidity and poor quality of life [P. aeruginosa-host adaptation in severe infection. Further genomic studies would be needed to ascertain the pathways that facilitate the MDR-P. aeruginosa pathology. Clustering efflux pump and biofilm-producing P. aeruginosa obtained from different infection sources with related multi-antibiotic resistance indices reveals a remarkable MDR strains relatedness that could trigger an hospital and community-acquired P. aeruginosa infection which may be difficult to treat.Over-expression of a well-developed efflux system embedded across the outer membrane (OM) permeability barrier continuously enables active expulsion of antibacterial compounds, improving the cell persistence and multiplication during infection . Reportsobiocin) . The abuobiocin) . The wea of life . The obsP. aeruginosa infection pathology and important gene targets to be considered for new drug discovery. Comparative analysis of hierarchical clustering of MDR strains characterized with a combination of efflux genes, virulence factors, and biofilm further suggests a major impending public health challenge that calls for routine surveillance, monitoring, and definitive strategic interventions. The study provides a clue to the emerging clusters of virulent MDR P. aeruginosa that could be regarded as high risk for public health.The predictive performance of the biofilm matrix indicates phenotypic adaptations and protective capacity provided by the matrix for cellular efflux pump and persistent induction of modulators  that contribute to persistent resistance . FunctioP. aeruginosa isolates from diagnosed disease conditions were obtained from clinical samples of patients attending two major healthcare facilities; Federal Medical Centre, Abeokuta and General Hospital, Ota; in southwest Nigeria between September 2020 and March 2021 with ethical approval from Covenant Health Research Committees, Covenant University, Ota, Nigeria (CHREC/055/2020). All the isolates were preserved in semi-solid Brain Heart Infusion (BHI)  supplemented with glycerol and re-characterized for confirmation following standard biochemical methods previously described [One hundred and forty-seven escribed .Pseudomonas infections was determined using Kirby-Bauer disc diffusion in accordance with CLSI recommendations and guidelines [P. aeruginosa (MDR P. aeruginosa) according to Magiorakos et al. [The susceptibility profile of commonly prescribed antibiotics for idelines . Overnigidelines ,40. Isols et al. . Multipls et al. .v/v) crystal violet solution was added and further incubated at room temperature for 1 h. Following two washing to remove the stain, 200 µL glacial acetic was added to dissolve the attached stain. The optical density (OD) was measured using UV Microplate reader [The formation of biofilm was assayed in a microtitre plate as described by Mathur et al. . Brieflye reader .P. aeruginosa strains were selected for EP activity and biofilm detection. The EP activity was assayed with the Ethidium Bromide (EtBr) which is a substrate of efflux pumps using agar cartwheel method described by Ugwuanyi, et al. [MDR , et al. . AdjusteHemolysin production was demonstrated as described by Edberg et al. . OvernigmexA, mexB and OprM), which belong to the RND pump, was assayed with PCR [P. aeruginosa ATCC 27853 was used as positive control and nuclease-free water as negative control. The PCR assays were performed in Bio-Rad MJ thermal cycler  with initiation of 95 °C for 5 min; 30 cycles of denaturation at 95 °C for 60 s, annealing  according to the manufacturer’s instruction and evaluated for quality and purity with Nanospectrophotometer. Encoded efflux pump genes  using SPSS v22. Level of virulence factors production from MDR P. aeruginosa were expressed as percentages and further analysed with unpaired t-tests  to determine the differences between the variables and p-values less than 0.05 were considered statistically significant. The significance of the resistance rate of the selected translational and transcriptional inhibitors was determined with ANOVA in GraphPad Prism 8.0. Median resistance of MDR P. aeruginosa obtained from various clinical sample sources was evaluated with ANOVA. The correlation matrix of phenotypic antibiotic resistance with biofilm formation, efflux activity, protease, lipase, and hemolysin productions was calculated using the Pearson method. The possible association of these variables was illustrated in graphical presentations. To predict the intensity of antibiotic resistance of MDR P. aeruginosa with biofilm and efflux pump activity, receiver operating characteristics (ROC) curves were calculated, taking the significance of optimal predictions given by the area under the curve (AUC) [Among the patients, risk factors for ve (AUC) , using tP. aeruginosa population. Observed association and hierarchical clustering of virulent MDR strains reveal high capacity for effective adaptability, survival, and infectivity. Periodic community-wide intervention, monitoring of local drug prescriptions, and appropriate clinical management of the Pseudomonas infections are needed to mitigate the impending spread of virulent MDR P. aeruginosa.The activities of the produced virulence proteins, efflux pump and biofilm, contribute significantly to high antibiotic resistance and persistent chronic infection with possible transmission and exchange of genetic materials among the MDR


Sargassum horneri, a prevalent species of brown algae found along the coast of the northwest Pacific Ocean, holds significant importance as a valuable source of bioactive compounds. However, its rapid growth can lead to the formation of a destructive “golden tide”, causing severe damage to the local economy and coastal ecosystems. In this study, we carried out de novo whole-genome sequencing of S. horneri using next-generation sequencing to unravel the genetic information of this alga. By utilizing a reference-guided de novo assembly pipeline with a closely related species, we successfully established a final assembled genome with a total length of 385 Mb. Repetitive sequences made up approximately 30.6% of this genome. Among the identified putative genes, around 87.03% showed homology with entries in the NCBI non-redundant protein database, with Ectocarpus siliculosus being the most closely related species for approximately one-third of these genes. One gene encoding an alkaline phosphatase family protein was found to exhibit positive selection, which could give a clue for the formation of S. horneri golden tides. Additionally, we characterized putative genes involved in fucoidan biosynthesis metabolism, a significant pathway in S. horneri. This study represents the first genome-wide characterization of a S. horneri species, providing crucial insights for future investigations, such as ecological genomic analyses. Sargassum horneri is a major brown macroalga commonly found along the northwest coast of the Pacific Ocean [S. horneri thrives at depths ranging from 3 to 15 m. It plays a crucial role in carbon cycling by effectively sequestering substantial amounts of CO2, thus holding significant importance in marine ecosystems [S. horneri as a valuable source of bioactive compounds, particularly sulfated polysaccharides, which exhibit a wide range of functions, including antiviral and anti-inflammatory activities [Sargassum has shown significant efficacy in reducing Helicobacter pylori infection without inducing drug resistance [S. horneri as a source of bioactive compounds makes it a subject of great interest for further research and exploration. Understanding and harnessing the properties of S. horneri could have implications for environmental conservation and the development of novel pharmaceuticals.ic Ocean . Since iic Ocean . Typicaltivities ,5. For isistance . The potS. horneri can also pose significant challenges to coastal ecosystems and local economies. The phenomenon of drifting S. horneri, driven by heightened light energy compared to its benthic state, can result in rapid growth and the formation of what is commonly known as a “golden tide” [S. horneri golden tide hazard caused an economic loss of about CNY 0.5 billion due to damage to local seaweed aquaculture [S. horneri [It is important to note that en tide” ,8. This aculture . In rece horneri . Nitroge horneri . Phospho horneri . HoweverEctocarpus siliculosus [Saccharina japonica [Nemacystus decipiens [Cladosiphon okamuranus [Tribonema minus [Macrocystis pyrifera [Sargassum fusiforme [Undaria pinnatifida [S. horneri in public databases. However, the genome sequence of S. fusiforme has been successfully decoded, making it the first Sargassum genus genome to be assembled using a combination of PacBio and Ilumina reads. With a total length of approximately 394.4 MB and an N50 value of around 142.1 KB, the assembled genome of S. fusiforme is highly complete, with over 90% of the BUSCOs detected at the protein level. As such, it provides an invaluable reference for guiding the genome sequencing process of S. horneri. Incorporating comparative information during the assembly process can significantly enhance the quality of the reconstructed genome. Brown algae, which consists of approximately 2000 species and are categorized into 16 orders, are a prominent group of multicellular organisms that can be found extensively in marine environments . There aiculosus , Saccharjaponica , Nemacysamuranus , Tribonema minus , Macrocypyrifera , Sargassnatifida ,22. ThesS. horneri and generate molecular resources for studying the “golden tide” seaweed. To achieve this, we conducted de novo whole-genome sequencing of a strain of S. horneri using the Illumina HiSeq X-ten platform. The genome assembly was performed using a reference-guided assembly pipeline, followed by further analysis, including the characterization of genome features, identification of repetitive sequences, gene prediction, and comparative analysis. Additionally, we discovered and examined genes responsible for the regulation of fucoidan biosynthesis enzymes. This research aimed to characterize the genome of S. horneri strain was collected from the sea surface in the Wenzhou Dongtou District of Zhejiang Province, China. It underwent cleaning and disinfection using ddH2O, followed by freezing and storage in liquid nitrogen for genomic DNA extraction. Genomic DNA extraction was carried out using a plant genomic DNA extraction kit obtained from Annoroad Gene Technology in Beijing, China. Subsequently, the extracted DNA was quantified and assessed for quality using a NanoDrop 2000 microspectrophotometer, a Qubit fluorometer, and 1% agarose gel electrophoresis in same company.The Cardamine amara genome [S. fusiforme using the bowtie2 V2.3.5.1 with the default parameters. Blocks with continuous read coverage were obtained and nearby blocks were combined to form a superblock, which was then subjected to ABYSSS for de novo assembly. The scaffolds were subjected to redundancy removal and error correction by aligning the trimmed paired-end reads. Uncovered regions of the contigs were eliminated, and the remaining contigs were split. Contigs with a length shorter than 200 bp were discarded, and scaffolds shorter than 1 kb were also removed from further analysis. Finally, contigs were aligned to the Nt database using an E-value threshold of 1 × 10−5, and the top 10 matches to eukaryotes were filtered out. To assess the accuracy of the genome assembly, the clean reads were realigned to the assembly using bowtie2. A paired-end (PE) library was created following the Illumina standard protocol, with insert sizes of 350 base pairs (bps). The raw sequence data, consisting of reads with a length of 150 bp, were generated using an Illumina HiSeq X-ten platform. The entire set of sequencing reads was deposited in the Short Read Archive (SRA) database under the accession number PRJNA756794. After removing the adapters, the fastp program was employed to filter out reads containing more than 10% of N nucleotides or more than 20% of low-quality bases . The resa genome . First, S. fusiforme [To analyze the distribution of repetitive sequences within the assembled genome, we consolidated the genome into a singular sequence. This was achieved by inserting 100 “N” characters between each pair of linked contigs. Afterwards, Repeat-Modeler was utilized to construct a de novo repeat library for usiforme . This prusiforme , RepeatSusiforme , and TRFusiforme . SubsequS. horneri (PRJDB4109) were obtained from NCBI and aligned to the repeat-masked assembly using TopHat2 V2.1.1 [Genome annotation was conducted using the BRAKER1 pipeline, which combines the strengths of GeneMark-ET and AUGUSTUS . To begi2 V2.1.1 . The res2 V2.1.1 . AUGUSTU2 V2.1.1 . To asseS. horneri genes onto the KEGG metabolic pathways. Genes from other plant species, such as Arabidopsis thaliana, Chlamydomonas reinhardtii, Monoraphidium neglectum, Auxenochlorella protothecoides, Ostreococcus lucimarinus, Ostreococcus tauri, Micromonas commoda, Micromonas pusilla, Cyanidioschyzon merolae, Galdieria sulphuraria, and Chondrus crispus (carragheen), were included in the analysis. Functional annotations for the putative genes were performed by comparing their sequences with public databases, including the NCBI non-redundant protein database (Nr), Swiss-Prot, and Pfam. Gene Ontology (GO) terms and COG were annotated using eggNOG-mapper V2 using the default parameters . In addiSargassum genus, we embarked on an extensive study to identify and characterize the specific genes associated with the biosynthesis and metabolism of fucoidan. The synthesis of fucoidan is likely catalyzed by six enzymes, including GDP-mannose 4,6-dehydratase (GM46D), GDP-l-fucose synthetase (GFS), L-fucokinase (FK), GDP-fucose pyrophosphorylase (GFPP), fucosyltransferase (FT), and sulfotransferase (ST). Genes encoding these enzymes were manually confirmed based on the Blast search results against the Nr database.Given the significance of fucoidan in the S. horneri dataset, the analysis included eight other brown algae species: E. siliculosus, S. japonica, M. pyrifera, N. decipiens, C. okamuranus, T. minus, S. fusiforme, and U. pinnatifida. For S. japonica, transcriptome sequencing data from different tissue sites (SRA accession numbers: SRX5192067- SRX5192070) were used, while RNA-seq datasets for M. pyrifera from different genders  were utilized in the BRAKER1 pipeline. The data for the other species were directly download from the official website. In each organism, CD-HIT was employed to eliminate redundant sequences with a similarity of 90% or higher. Following this, the non-redundant protein sequences underwent an all-against-all Blastp analysis, utilizing an E-value threshold of 1 × 10−5. By employing OrthoMCL and examining the intersections of orthologs across the seven brown algae, a total of 14,819 groups were identified, as illustrated in Ortholog analysis was performed using OrthoMCL V2.0.9 and protein datasets obtained through the BRAKER1 pipeline. In addition to the S. horneri and seven other brown algae species: E. siliculosus, S. japonica, S. fusiforme, M. pyrifera, N. decipiens, C. okamuranus, and U. pinnatifida [S. horneri and S. fusiforme were designated as the target species, whereas S. fusiforme and E. siliculosus served as the anchor and reference species, respectively. Genes were considered positively selected if the branch-wide test yielded false discovery rates (FDRs) of less than 0.05. JPred4 was employed for secondary structure prediction [The PosiGene pipeline was used to search the genome for positively selected genes in natifida . In thisediction .S. horneri, a total of 229,605,305 paired-end raw reads were obtained using a library with insert lengths of 350 bp on a Hiseq X-ten platform. After pre-processing the raw reads, over 200 million paired-end reads were retained. Based on k-mer analysis, the haploid genome length of S. horneri was predicted to be ~413.6 MB by GenomeScope, with an estimated sequence coverage of approximately 73× and a genome repeat length of 227 Mb, and a heterozygosity of ~1.23%. Using a reference-guided de novo assembly pipeline, a draft genome with a total length of 385 Mb was assembled after filtering out candidate contaminants. About 70% of the clean reads could be realigned to the assembled genome using bowtie2.To investigate the genomic background of Ectocarpus sp. was the species with the best hit, with 16,337 genes identified.RepeatMasker identified a total length of approximately 118 Mb for repetitive sequences, accounting for approximately 30.6% of the draft genome size. After masking these repeats, de novo gene prediction was carried out using the BRAKER1 pipeline, resulting in the detection of 58,211 putative genes based on public RNA-seq data. The gene completeness was assessed, revealing that approximately 75.6% of the BUSCOs were detected at the protein level. Among the putative genes, 87.03% had hits in Nr, 52.67% had hits in Swiss-Prot, 57.96% had hits in Pfam, and 16.08% had hits in GO, as shown in Using eggNOG-mapper, a large number of genes (75.49%) was assigned to COG functional classifications . The larS. horneri  lower than 0.05 (FDR < 0.05)  . Eightee < 0.05) .S. horneri. This approach has yielded a valuable resource that will facilitate future investigations into algal genetics and genome evolution. Sargassum is a large genus of brown seaweed that includes over 300 species. It exhibits both dioecious and monoecious traits, making it a valuable model for studying algal evolution. The genome size of S. horneri was estimated to be 385 Mb, which is larger than the genome size of the Sargassum genus (196~319 Mb) previously estimated using static microspectrophotometry [Sargassum genus. Compared to two other well-known brown algae, the estimated genome size of S. horneri was larger than that of E. siliculosus and smaller than that of S. japonica. The percentage of repetitive sequences in S. horneri (30.6%) was slightly larger than that of E. siliculosus (22.7%) [S. japonica (39%) [S. horneri was much lower than that of S. fusiforme (60.7%), despite the two species having similar genome sizes (394.4 Mb). This difference in repetitive sequence content could be attributed to the inherent limitations of short-read assembly methods, which generally provide less comprehensive information on repetitive elements compared to long-read assembly methods. While a reference-based assembly can be a valuable approach for generating a high-quality genome assembly, it is important to acknowledge that errors or gaps present in the reference genome can potentially propagate to the assembled genome. Additionally, reference-based assembly runs the risk of introducing bias and overlooking crucial genomic regions that are specific to the organism under investigation. In this study, we employed the reference-based assembly method to conduct a comprehensive whole-genome survey analysis for otometry . This su (22.7%)  and S. jca (39%) , althougS. fusiforme [E. siliculosus [S. japonica [Ectocarpus sp., indicating that most of these putative genes have been properly assigned. The relatively high heterozygous ratio (1.23%) can have an impact on the N50 value during the assembly of NGS reads, indicating that additional efforts, such as implementing third generation sequence technology, would be necessary to improve the quality of the genome assembly. A total of 58,211 protein-encoding genes were predicted in the assembled genome, surpassing the reported numbers for usiforme , E. siliiculosus , and S. japonica . It is aS. japonica was found to be the best match for GM46D and GFPP, while E. siliculosus was the best match for GFS, FK, and FT, supporting the hypothesis that there are distinct phylogenetic sources of genes involved in the polysaccharide biosynthesis in brown algae [E. siliculosus with that of the Sargassum genus, it was observed that GT65 was not present in either species. This absence of GT65 suggests the potential existence of novel fucosyltransferases or alternative enzymes in Sargassum that play a role in fucoidan biosynthesis [Fucoidan biosynthesis is a complex metabolic pathway involved in the production of fucoidan, a sulfated polysaccharide found in brown algae. Fucoidan is known for its diverse biological activities, including antioxidant, anticancer, anticoagulant, and immunomodulatory properties. Understanding the genes and enzymes involved in fucoidan biosynthesis is crucial for elucidating the molecular mechanisms underlying its production and biological functions. Our study focused on genes associated with fucoidan biosynthesis and compared them with known genes from other brown algae species. In the fucoidan biosynthesis pathway, wn algae ,45. DiffSargassum provides strong support for the utilization of the S. fusiforme reference-guided assembly pipeline in the genome assembly of S. horneri. The unicellular filamentous yellow-green algae T. minus, belonging to the class Xanthophyceae, are widely distributed in both freshwater and saltwater ecosystems. The genome used in this study was initially isolated from wastewater treatment ponds in San Luis Obispo, CA. It was subsequently excluded from the subsequent evolutionary analysis. In the analysis of gene family evolution, a gene encoding an alkaline phosphatase family protein was found that exhibited positive selection in S. horneri. Alkaline phosphatase plays an important role in utilizing dissolved organic phosphorus compounds by catalyzing the decomposition or mineralization of organic phosphorus into biologically active phosphorus in algae in subtropical coastal water [S. horneri achieve population dominance, providing pivotal molecular materials for the study of the S. horneri golden tide.The purpose of concatenating the sequences is to combine the information from multiple protein sequences, thereby increasing the accuracy and reliability of the phylogenetic analysis. By combining multiple sequences into concatenated sequences, it allows for a more comprehensive examination of the evolutionary relationships between species and provides more information to infer their genetic relationships. After concatenation, the phylogenetic tree was constructed using RAxML V8.2.3 and the PROTGAMMALG model. RAxML is a commonly used software for phylogenetic tree construction, which utilizes the maximum likelihood method to infer the tree’s topology and branch lengths. The PROTGAMMALG model is a widely used protein evolution model that considers substitution rates and types among different protein sequences to accurately estimate the phylogenetic tree. The high degree of similarity between the genomes of these two species within the genus al water . Therefo


Xylocarpus. Two of the three species of the genus Xylocarpus are true mangroves (X. granatum and X. moluccensis), and one is a non-mangrove (X. rumphii). In order to resolve the phylogenetic relationship between the mangrove and non-mangrove species, we sequenced chloroplast genomes of these Xylocarpus species along with two non-mangrove species of the Meliaceae family (Carapa guianensis and Swietenia macrophylla) and compared the genome features and variations across the five species.The mahogany family, Meliaceae, contains 58 genera with only one mangrove genus: the rpl22 gene region only occurred in Xylocarpus, while the repetitive sequences in accD were found in X. moluccensis and X. rumphii. The TrnH-GUG and rpl32 gene regions and four non-coding gene regions showed high variabilities between X. granatum and the two non-mangrove species (S. macrophylla and C. guianensis). In addition, among the Xylocarpus species, only two genes (accD and clpP) showed positive selection. Carapa guianensis and S. macrophylla owned unique RNA editing sites. The above genes played an important role in acclimation to different stress factors like heat, low temperature, high UV light, and high salinity. Phylogenetic analysis with 22 species in the order Sapindales supported previous studies, which revealed that the non-mangrove species X. rumphii is closer to X. moluccensis than X. granatum. Overall, our results provided important insights into the variation of genetic structure and adaptation mechanism at interspecific (three Xylocarpus species) and intergeneric (mangrove and non-mangrove genera) levels.The five Meliaceae species shared 130 genes  with identical direction and order, with a few variations in genes and intergenic spacers. The repetitive sequences identified in  They arXylocarpus, which has three species with accepted names (the name currently accepted as the one which should be used in preference to refer to a species): X. granatum J.Koenig, X. moluccensis (Lam.) M.Roem., and X. rumphii (Kostel.) Mabb. (https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:26860-1#children). Among these three species, X. granatum  is the most widespread species in East Africa, tropical Australia, Southeast Asia, and the Pacific Islands  is distributed from the East-Africa to Philippines, Australia and the Pacific Islands , and possession of specialized morphological features  and physiological mechanism for salt exclusion and/or excretion  revealed that four non-mangrove species of the family Meliaceae, namely Carapa guianensis, Swietenia macrophylla, Swietenia mahagoni, and Khaya nasica, formed a cluster in a clade with Xylocarpus (Xylocarpus species (X. moluccensis). The phylogenetic pattern became different when all three Xylocarpus species and five other cpDNA loci  were considered in another study  and two non-mangrove species from the same family. We did not use nuclear markers , as these markers were found to be not sufficiently informative (like 26S) and contain saturated substitutions at family level (like ITS) for Sapindales  and for X. moluccensis in CNSA (CNP0000567)  , we charXylocarpus species, C. guianensis and S. macrophylla were collected from Singapore  and China, respectively, after confirming their taxonomic identities  as a reference. Some unannotated genes were further manually adjusted by Geneious R version 10.1.3 . To validate the accuracy of assembling, we designed four pairs of primers in four junctions between the large single-copy region (LSC hereafter), single small copy region (SSC hereafter), and two inverted repeats (IRA and IRB hereafter) (MH348115 (X. granatum), MH330688 (X. moluccensis), MH330687 (X. rumphii), MH396436 (C. guianensis), and MH348156 (S. macrophylla).Raw reads of the five Meliaceae species were filtered to remove the low-quality bases, and the clean reads were assembled to chloroplast genome in NOVOPlasty . The chlreafter) . PCR ampreafter) . Finallyreafter) . The annThe repeat sequences in the chloroplast genomes of the five study species were identified in the online program REPuter . Palindrhttps://github.com/tseemann/snippy) to analyze the SNPs and INDEL among five study species. The divergence among the species was visualized using the mVISTA viewer and the genome-wide sequence variation was computed using X. granatum as a reference. Variation percentages in coding regions and non-coding regions were calculated. By using the PAMLX version 1.3.1 (ω = dN/dS)   with the= dN/dS) . For eachttp://codonw.sourceforge.net). We selected coding genes of at least 300 bp to avoid the codon bias index associated with short sequences. In addition, the repeat sequences were removed to avoid repetitive computation. A total of 85 CDS of every species were used in PREP suit (https://bio.tools/prep_suite) to predict possible RNA edit sites in PREP-cp (for chloroplast genes). The cutoff value was selected as 0.8 to ensure the accuracy of the prediction . In this study, we collected sample from Sungei Buloh Wetland Reserve, Singapore, which was planted from Cooperative Republic of Guyana on 28 December 1938. We found 12 SNPs or INDEL between the earlier accession and our sample . The other two species of the genus Meliaceae, namely S. macrophylla and C. guianensis, had 37.95% and 37.91% GC content, respectively. A total of 130 functional genes were found in each of the study species: 85 protein-coding genes (CDS), 37 tRNA and 8 rRNA  included two introns. The rps12 was a trans-spliced gene and had two directions with 5′ exon in LSC region and 3′ end exon in IR region. In addition, rps19 and ndhD genes contained unusual start codons—GTG and ACG, respectively. 58 genes  were related to self-replication . The functions of some genes could not be deciphered   .X. granatum possessed highest number of repetitive sequences (n = 24) among the five species studied here. X. moluccensis and X. rumphii contained equal number of reverse repeats (n = 3); however, quantities of forward and reverse types were different between the two species. We found 11 forward repeats and four palindromic repeats in X. moluccensis, and nine forward repeats and five palindromic repeats in X. rumphii. The two related species of this family, S. macrophylla and C. guianensis, had almost identical repeats distribution. Comparing all repeat type sequences, the figure for palindromic appeared to be almost stable among these five species. However, forward repeats ranged from 10 in C. guianensis to 18 in X. granatum, and reverse repeats ranged from 1  to 3 (X. moluccensis and X. rumphii). The distributions of three type of repeats were different in each region: approximately half of forward repeats located in the CDS region, while 27% and 16% of the repeats were distributed in the spacer region and intron region, respectively. In palindromic repeats, 44% repeats were situated in the intron region, while 30% and 26% repeats were identified in the spacer region and CDS region, respectively. No repeats were discovered in spacer region of reverse repeats, while 56% and 44% repeats were found in intron region and CDS region, respectively  were located in the rpl22 gene region, while in X. moluccensis and X. rumphii, the number of repeats in the rpl22 gene region were 2 and 1, respectively. No repeat was found in the rpl22 gene region for S. macrophylla and C. guianensis. At ycf2 gene, one repeat was founded for the three Xylocarpus species, while four and three repeats were observed in S. macrophylla and C. guianensis, respectively. We found variable number of repeats (three to four) in the ycf3 intron range among these five Meliaceae species. At accD gene, two reverse repeats were found in X. moluccensis and X. rumphii, and these repeats were not identified in other species. Majority of these repeats were found in the CDS of the genome. Maximum number of repeats was observed in X. granatum (14), followed by X. moluccensis (nine), X. rumphii (seven), S. macrophylla (seven) and C. guianensis (five). Palindromic repeats were discovered in CDS, gene intron (intron) and intergenic spacer (spacer) regions, while reverse repeats were only found at CDS and intron regions (In  regions .X. moluccensis and S. macrophylla to 97 in C. guianensis (X. granatum). Most of these SSRs were located in the intergenic spacer region. We found 68 SSRs (72.3%), 64 SSRs (69.6%), 62 SSRs (66.0%), 64 SSRs (69.6%), 72 SSRs (74.2%) in the intergenic spacer regions of X. granatum, X. moluccensis, X. rumphii, S. macrophylla and C. guianensis, respectively  were identified across the five study species. The number of SSRs varied from 92 in ianensis . In all ianensis , Fig. S5ectively . It is iAilanthus altissima of the family Simaroubaceae (GenBank accession number: MG799542). We observed highly conserved IR boundary regions with a little variation in length of the boundary genes  was shown with a red line in X. granatum and C. guianensis in trnH-GUG, 3.49% between X. granatum and X. moluccensis in rpl22, 3.09% between X. granatum and C. guianensis, and 3.14% between X. granatum and S. macrophylla in rpl32. On the other hand, four non-coding regions showed more than 4% variability: 4.24% between X. granatum and S. macrophylla in trnK-UUU—rps16, 7.27% between X. granatum and C. guianensis in atpA - atpF, 4.28% between X. granatum and S. macrophylla in psbB - psbT, and 4.35% between X. granatum and C. guianensis in ccsA-ndhD  showed positive selection between the three Xylocarpus species  . AnalysiXylocarpus showed an identity prediction, implying that they were highly conserved  (The comparative estimates of RNA prediction editing sites of the five study species were found to be similar to each other. All three species in genus onserved . Compare genomes . Out of n genes) .Xylocarpus species were clustered together, X. moluccensis and X. rumphii clustered as one clade, while X. granatum was sister to this clade  and Bayesian inference (BI) methods. Each of the 22 species from six families of the order Sapindales clustered within their own families and each clade was highly supported by both phylogenetic analysis methods. Majority of the nodes in the ML tree were well supported with bootstrap value higher than 95%, except the most recent common ancestor (MRCA) of Burseraceae, namely Anacardiaceae and Sapindaceae. In BI tree, each node was supported with posterior probability of 1. In both phylogenetic trees, three is clade . The Xylresearch . In addiXylocarpus species, including the rare non-mangrove species X. rumphii and two true mangrove species. In addition, the study also reported the genome sequences of two closely related non-mangrove species of Xylocarpus, among which S. macrophylla is considered as a vulnerable species . Our study, therefore, adds novel information to the whole genome sequences of the order Sapindales and family Meliaceae.This is the first study which reported the whole chloroplast genome sequences of three Analysis of the interspecific variation in chloroplast genome sequences revealed that the five Meliaceae species showed similarity in GC contents and gene features. All genes shared identical direction and order, with only a few variations in intergenic spacer sequences. The chloroplast genomes in Meliaceae were highly conserved and no obvious contraction or expansion were found in IR borders.Xylocarpus from the other two species and between the three Xylocarpus species themselves. Our study identified presence of three repetitive sequences across five Meliaceae species. Based on the similar proportion of repeats among the three species of Xylocarpus, high similarity of chloroplast genomes among these species became evident.While the GC contents and gene features showed the conservatism of chloroplast genomes in Meliaceae, the distribution pattern of repeat sequences explained the divergence of X. granatum was found to have the highest number of repeats in rpl22, while only two repeats and one repeat were found in X. moluccensis and X. rumphii, respectively. No repeats were observed in C. guianensis and S. macrophylla. It has been reported that rpl22 gene was more likely to be transferred to nuclear genome from chloroplast genome  between 29 species from the mangrove ecosystem of the Bhitarkanika sanctuary , X. granatum had larger niche width (2.15) than X. moluccensis (1.15). The existence of more repetitive sequences in rpl 22, therefore, might explain the wide distribution of X. granatum, compared to the other two Xylocarpus species, and might also account for the divergence between X. granatum and other four Meliaceae species (two Xylocarpus species and two non-mangrove species) studied here.However, t genome , which cacc D in X. moluccensis and X. rumphii. The gene acc D is associated with leaf form and photosynthetic efficiency, since presence of acc D in plastids promotes gene expression in light-harvesting organs (X. moluccensis and X. rumphii may cause similar leaf appearances in these two species (Xylocarpus clade and other two relative clades) with the relationship between X. moluccensis and X. rumphii being closer than X. granatum.Finally, we observed similar repeat sequences in g organs . Similar species . Overall species . The phyIn addition to providing insights into the divergence pattern of the five species of Meliaceae, the repeat sequences, especially the simple sequence repeats (SSRs), provided powerful genetic resources for investigating population diversity. In this study, we identified 467 SSR loci, most of which showed polymorphisms among the five study species. These loci, therefore, could act as molecular markers for investigating population genetics and biogeography in Meliaceae family. The advantage of using cpDNA SSR marker is that it allows for the detection of population genetic structure and gene flow at the maternal level, which may be informative for understanding the demographic history and evolutionary relationships of these species. Moreover, in terms of feasibility, the use of cpDNA SSR markers, or cpDNA markers as a whole, is relatively straightforward and cost-effective, as it requires amplification and sequencing of a single locus. However, using only one marker may not provide a complete picture of the population genetics of a species or group, as different markers may capture different aspects of genetic diversity and evolutionary history.X. granatum and other four Meliaceae species (two Xylocarpus species and two non-mangrove species) studied here. Two out of three coding genes, namely rpl22 and rpl32, are associated with the large subunit of ribosome. Previous studies have identified that ribosomal proteins of the large subunit 22 and 23 are essential under heterotrophic conditions   .e.g., In coding sequences of the five study species, the usage frequencies of the most A/U-ending amino acids were higher than that of the G/C-ending amino acids. In addition, all the putative editing sites were C-to-U transitions, and majority of these sites were found to be situated in the second positions of these codons. These findings provide evidence of certain biasness in codon editing of chloroplast genomes. Our codon usage frequency results are consistent with previous studies, which showed the similar amino acids distribution among coding sequences of Sapindales members e.g., .ndhB and ndhD genes was found to be maximum in all five Meliaceae species, which might be because they participate in multiple biological processes, and their expression and regulatory activity were stronger than others. In coding sequences of the five Meliaceae species, the most abundant amino acid was Leu, a plant growth promoter having photosynthetic and regulating effect. In addition, differences in editing sites were found between the three Xylocarpus species and the two non-mangrove species (C. guianensis and S. macrophylla), which might contribute to the divergence between the mangrove and non-mangrove species of Meliaceae.Most editing sites variation may convert the original amino acids to hydrophobic amino acids, which may be conducive to the formation of protein stable structure and provided a certain basis for the plants in adapting to the environment. The number of editing sites of X. moluccensis to the non-mangrove species X. rumphii, rather than another mangrove species X. granatum. This finding is in line with the previous phylogenetic pattern based on specific cpDNA and nDNA loci sequences  revealed identical topological structure. Interestingly, the phylogenetic tree showed closer relationship of the mangrove species equences . One poslocarpus . Accordi habitat . HoweverXylocarpus species (two mangroves and one non-mangrove species) and two related non-mangrove species of Meliaceae and supported the previous observations. Our study also highlighted the importance of repetitive sequences and genes under positive selection in stress response and adaptation of the three Xylocarpus species, especially that of the X. granatum, in fluctuating environmental conditions. Finally, the polymorphic simple sequence repeats and gene regions identified in this study can act as genetic markers in future molecular taxonomy research for other members of the Meliaceae family.To the best of our knowledge, this is the first study that characterized complete chloroplast genomes for five Meliaceae species and identified the intraspecific variations between genomes of two mangrove and three non-mangrove species. Based on the distribution pattern of repetitive sequences, variability in coding and non-coding regions, differences in editing sites in coding sequences and phylogenetic analysis, our study identified the divergence pattern between the three 10.7717/peerj.15527/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15527/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.15527/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.15527/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.15527/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.15527/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.15527/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.15527/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.15527/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.15527/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.15527/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.15527/supp-12Supplemental Information 12Click here for additional data file.10.7717/peerj.15527/supp-13Supplemental Information 13Click here for additional data file.10.7717/peerj.15527/supp-14Supplemental Information 14Click here for additional data file.10.7717/peerj.15527/supp-15Supplemental Information 15Click here for additional data file.


Sarcocystis are characterized by a mandatory two-host, prey–predator life cycle. Several Sarcocystis species are known to form macroscopic or microscopic sarcocysts in the muscle tissues of domestic sheep and goats. It has been long considered that Sarcocystis species parasitizing farm animals are specific to intermediate hosts. However, some studies have recently reported the unexpected detection of Sarcocystis species in animals that are not considered to be their canonical hosts. In the current investigation, muscle samples of sheep and goats from Lithuania were molecularly tested for species previously described in such hosts and for other non-canonical Sarcocystis spp. Based on DNA sequence analysis, along with canonical Sarcocystis species present in their respective hosts, non-canonical  species, such as S. capracanis and S. morae, were detected in sheep, while S. arieticanis and S. tenella were found in goats. Possible explanations of the obtained results are discussed.Protozoan parasites of the genus Sarcocystis spp. in farm animals. Therefore, the current work aimed at molecularly testing samples of sheep and goats reared in Lithuania to identify Sarcocystis species described in other intermediate hosts but suspected to be non-canonical parasites to these small ruminants. For this purpose, muscle samples from 47 domestic sheep and nine goats were examined. Sarcocystis species were identified using direct and nested PCR targeting cox1 and sequencing of positive amplified products. Along with the detection of the canonical Sarcocystis spp. in their respective intermediate hosts, the DNA of S. capracanis and S. morae was detected in sheep, although these species were previously thought to be specific to goats and deer, respectively. In addition, DNA from S. arieticanis and S. tenella was found in goats, even though these two species were believed to be sheep-specific. Notably, under light microscopy, only sarcocysts of S. capracanis specific to goats were observed. Thus, future research on the life cycle and host-specificity of Sarcocystis spp. examined is warranted.Contradictory data is available on the intermediate host specificity of  Sarcocystis have a mandatory two-host life cycle that is usually rooted in an ecological prey–predator relationship. Typically, herbivores such as sheep (Ovis aries) and goats (Capra hircus), act as intermediate hosts, while carnivores, such as Felidae and Canidae, are definitive hosts. Specific intermediate hosts can become infected by ingesting food or water contaminated with Sarcocystis oocysts or sporocysts. Depending on the Sarcocystis spp., either microscopic or macroscopic tissue cysts (sarcocysts) will develop and allocate  in the striated muscle of the intermediate host. When meat containing mature sarcocysts is consumed by a predator or scavenger, the parasite undergoes sexual reproduction and forms isosporoid oocysts in the intestines of the definitive host [Parasites of the genus ive host . Sarcocystis spp. infection in sheep is available worldwide [Sarcocystis species, S. arieticanis, S. tenella, S. gigantea, and S. medusiformis, use sheep as intermediate hosts [S. arieticanis and S. tenella form microscopic sarcocysts in the muscles of sheep and use canids as definitive hosts [S. gigantea and S. medusiformis form macroscopic sarcocysts in the muscles of sheep and use felids as definitive hosts [S. mihoensis and S. microps, proposed to have dogs as the definitive host, have only been identified in two reports from Japan and China, respectively, and available molecular and biological details are scarce [Sarcocystis infection is non-lethal to the ruminant intermediate host; nonetheless, the economic impact on profits associated with reduced production and carcass condemnation should be taken into consideration [Sarcocystis spp. infecting domestic goats are available. Three different Sarcocystis species, S. capracanis, S. hircicanis, and S. moulei, are proposed to use goats as intermediate hosts. Two species, S. capracanis and S. hircicanis, form microscopic sarcocysts and use canids as definitive hosts, while S. moulei constitutes macroscopic sarcocysts and uses felids as definitive hosts . Howevein goats . To date18S rRNA molecular marker has limited discriminatory power when investigating closely related Sarcocystis spp. infecting ungulates as intermediate hosts. In contrast, cox1 was successfully used to identify Sarcocystis species present in sheep and goats [Sarcocystis genus. Recent studies indicate that the nd goats ,13,14. ASarcocystis spp. found in farm animals are strictly specific to the intermediate host [Sarcocystis species have recently been increasingly reported in non-specific hosts. For instance, goat-specific S. capracanis has been reported in domestic sheep [S. moulei that also utilizes goats as an intermediate host has been reported both in domestic sheep [Bubalus bubalis) [S. tenella has been detected in wild goats (Capra aegagrus) [For a long time, it has been widely assumed that ate host . Howeveric sheep , while Sic sheep ,17 and wbubalis) . Moreoveegagrus) ,19. Neveegagrus) . Bos taurus), horses , pigs (Sus scrofa), and sheep were previously examined for the presence of Sarcocystis spp. by means of morphological [cox1 sequence analysis revealed the occurrence of S. arieticanis and S. tenella [Sarcocystis spp. in goats bred in the country. In Lithuania, among farm animals, cattle  for detailed morphological and molecular analysis of Sarcocystis spp. The transported muscle samples were stored frozen (at −20 °C) until further analysis. The general information about sheep and goats is known to the company’s veterinarian, and the farmers’ consents have been provided to the company. None of the animals were intentionally euthanized for the purpose of this research, and all sample collection procedures adhered to accepted animal welfare guidelines. Between 2019 and 2021, diaphragm, oesophagus, and heart samples were collected from 47 healthy domestic sheep following the slaughter process. Furthermore, three diaphragms, five hearts, and eight oesophagi were collected from healthy goats. The entire organs were obtained to examine the presence of Sarcocystis-like structures. Subsequently, a morphological analysis of the microscopic sarcocysts was conducted using freshly squashed muscle samples. A microscopical examination was performed under a Nikon ECLIPSE 8oi light microscope . Sarcocysts excised from fresh muscle samples were preserved individually in separate tubes containing 96% of ethyl alcohol at −20 °C for the molecular examination.The collected tissue samples from goats were visually checked for macroscopic n = 2) was carried out with the GeneJET Genomic DNA Purification Kit  in accordance with the manufacturer’s recommendations.DNA extraction of sarcocysts  and a 3500 Genetic Analyzer , following the manufacturer’s specifications. The resulting cox1 sequences of Sarcocystis spp. were compared with the most similar ones available in NCBI GenBank using the Nucleotide BLAST function .Sequencing of the selected PCR samples was performed using a Big-DyeSarcocystis spp., muscle samples from 47 sheep (n = 141) and nine goats (n = 16) were subjected to identification of Sarcocystis spp. by combined acid-pepsin digestion and PCR techniques [Toxoplasma gondii from animal tissues [Aiming for sensitive detection of chniques . Artific tissues . Samples tissues . S. arieticanis, S. gigantea, S. medusiformis, S. mihoensis, and S. tenella) and goats  and to investigate their degree of unspecificity, conventional and nested PCR (nPCR) assays were employed (see below). In addition, given the previous reports of the wide host range of S. morae [cox1 data in the GenBank regarding S. moulei when the primers were designed, the 28S rRNA gene was chosen as the molecular target for investigating this particular species of Sarcocystis. All digested samples were subjected to the extraction of the genomic DNA using a PureLink Microbiome DNA Purification Kit . For the specific detection of the canonical species that are present in domestic sheep . For the phylogenetic analysis, sequences from this work were aligned with those gathered from the GenBank database by using the MUSCLE algorithm available in the MEGA7 software [S. truncata from wild ungulates.The software . The finsoftware  and the software . For theSarcocystis-like sarcocysts were found in a single oesophagus specimen. One type of microcyst similar to those of S. capracanis was observed under a light microscope. Sarcocysts were elongated and spindle-shaped, measuring 500–1310 × 65–150 µm  in size (n = 20) in length (n = 60) in size  and genetically characterized within cox1, two species, S. arieticanis and S. tenella, were identified in sheep tissues, and S. capracanis was confirmed in the muscles of goats by both conventional and nPCR  was obtained for the grouping of the newly identified Sarcocystis sp. OaLT1 variant with S. heydorni of cattle. However, the phylogenetic analysis showed close relationships of Sarcocystis sp. OaLT1 and Sarcocystis spp. parasitizing sheep (S. tenella), goat (S. capracanis), cattle (S. heydorni) [Alces alces)  [Capreolus capreolus) (S. gracilis) [Based on a short fragment of ectively . The exaeydorni) , moose   and roe racilis) .S. arieticanis and S. tenella in the diaphragm, heart, and oesophagus of sheep ranged from 83.0% to 100% and from 10.6% to 59.6% by nPCR and direct PCR, respectively. At the same time, the occurrence of S. capracanis and S. morae in three types of sheep muscles was in the range of 10.6–42.6% and 10.6–31.9%, respectively  goats tested. In addition, S. arieticanis was detected in a single goat (11.15%), and S. tenella was found in four (44.4%) individuals .The present study revealed the first identification of ividuals . Both spgle goat 1.15%, anSarcocystis spp. in goats have been mostly carried out in Asia and Africa. In Kunming (China), 174 of 225 (77.3%) goats were confirmed to be infected with Sarcocystis spp. using cox1 region. The prevailing S. capracanis was detected in 74.6% of goats, and S. hircicanis was found in 33.3% of individuals [Sarcocystis in Saudi Arabia was lower , as only S. capracanis was distinguished by cox1 [S. capracanis was identified in Malaysian goats by PCR targeting 18S rRNA, revealing a level of infection of around 90.5% [Sarcocystis spp. are difficult to be drawn.Studies on ividuals . Meanwhi by cox1 . Only S.r 42.9%; /84 goatsnd 90.5% ,40,41,42Sarcocystis species may not be as strict as previously believed [S. hjorti, a parasite known to infect cervids [S. hjorti sequences, suggesting that it is likely an undescribed Sarcocystis sp. rather than S. hjorti [S. gigantea, a species that typically parasitizes sheep [18S rRNA sequences exhibited high similarities to S. medusiformis and S. moulei, they consistently displayed distinguishable differences from Sarcocystis species identified in cattle and horses . There is also an investigation showing that S. cruzi, which typically infects cattle, was detected based on 18S rRNA in another bovid species, the wood bison (Bison bison athabascae) [18S rRNA with additional investigation on other genetic regions, such as the cox1 gene, as it possesses significant variability and serves as the most accurate method to differentiate taxonomically close Sarcocystis species [As commented above, an important number of recent studies suggest that the intermediate host-specificity of several believed . The relbelieved ,43 is in cervids , was foues sheep , infecties sheep  or horsees sheep . While tabascae) . Regardl species .S. tenella and S. capracanis can parasitize both sheep and goats [S. tenella has been detected in chamois (Rupicapra rupicapra tatrica) [S. tenella and S. capracanis have been identified in Barbary sheep (Ammotragus lervia) [Ovis or Capra genera, thus raising questions about Sarcocystis species-specificity for their intermediate host. Some existing reports mentioned that nd goats ,49. Furttatrica) , and S.  lervia) . HoweverSarcocystis spp. has been detected in heart, oesophagus, and diaphragm samples from sheep and goats  the agarose gel of all negative controls remained clean; (ii) not all samples were positive for atypical/unexpected species; (iii) the genetic identity of all positive samples for atypical species was confirmed by the Sanger sequencing method showing multiple single peaks in each chromatogram; (iv) the obtained sequences of non-host-specific  samples . Real-ti samples . In sum,S. capracanis in excised sarcocysts from goats constitutes the first report of Sarcocystis spp. infection in such a host in Lithuania. Species-specific PCR tests allowed the detection of two non-typical Sarcocystis species in sheep, S. capracanis and S. morae, in the muscles of sheep, and two non-typical goat Sarcocystis species, S. arieticanis and S. tenella, in the muscles of goats. Additional molecular data showed the presence of a new Sarcocystis genetic variant (Sarcocystis sp. OaLT1) with 78.6–83.8% similarity to the closest Sarcocystis species cycling in ungulates as intermediate hosts in the muscle samples of two sheep.Microscopical and molecular identification of Evidence of a lower intermediate host-specificity identified here warrants the need for further investigations, including additional effort on direct examination for individual sarcocysts and, when possible, experimental infections to demonstrate such cross-infections.