Genlisea a unique lobster pot trapping mechanism supplements nutrition in nutrient-poor habitats. A wide spectrum of microbes frequently occurs in Genlisea's leaf-derived traps without clear relevance for Genlisea carnivory. We sequenced the metatranscriptomes of subterrestrial traps vs. the aerial chlorophyll-containing leaves of G. nigrocaulis and of G. hispidula. Ribosomal RNA assignment revealed soil-borne microbial diversity in Genlisea traps, with 92 genera of 19 phyla present in more than one sample. Microbes from 16 of these phyla including proteobacteria, green algae, amoebozoa, fungi, ciliates and metazoans, contributed additionally short-lived mRNA to the metatranscriptome. Furthermore, transcripts of 438 members of hydrolases , mainly resembling those of metazoans, ciliates and green algae, were found. Compared to aerial leaves, Genlisea traps displayed a transcriptional up-regulation of endogenous NADH oxidases generating reactive oxygen species as well as of acid phosphatases for prey digestion. A leaf-vs.-trap transcriptome comparison reflects that carnivory provides inorganic P- and different forms of N-compounds  and implies the need to protect trap cells against oxidative stress. The analysis elucidates a complex food web inside the Genlisea traps, and suggests ecological relationships between this plant genus and its entrapped microbiome.In the carnivorous plant genus  Carnivory, including trapping and subsequent digestion of prey, has evolved several times in plants. About 800 species from five angiosperm orders  , Merzig (carnivorsandmore.de) and Nüdlingen ] were cultivated in the greenhouse of the IPK Gatersleben, Germany. Plants were grown in pots with a mixture of peat and sand. The soil was kept wet by rain water, containing small organisms living naturally inside. Leaves and traps of both species were collected in summer season 2010 (SS) and winter season 2011 (WS) after thorough cleaning with 2 l of running cold distilled water. Total RNA samples were isolated using RNeasy Kit (Qiagen) with DNaseI treatment. The sample quality was controlled on a 2100 Bioanalyzer (Agilent). Illumina RNA-TruSeq libraries were prepared from 1 μg RNA of each sample without mRNA enrichment or rRNA depletion. Illumina Hiseq2000 paired-end sequencing  resulted in at least 31 million reads per library (Table www.ebi.ac.uk/ena/).RNA-Seq reads from total RNA libraries were trimmed for sequence quality using the standard pipeline  of the CLC Genomics Workbench v5.5.1 . Using the RNA-seq module of the CLC Genomics Workbench, trimmed and high quality reads from each dataset were mapped to the non-redundant and truncated version of the ribosomal RNA SILVA reference sequences [LSURef_115 and SSURef_NR99_115, . NCBI Taxonomy IDs of assigned genera were extracted by the Tax Identifier tool (http://www.ncbi.nlm.nih.gov/Taxonomy/TaxIdentifier/tax_identifier.cgi) and used for drawing a phylogenetic tree by the phyloT tree generator (http://phylot.biobyte.de) and iTOL graphical editor (http://itol.embl.de/).Relative abundance  and reoccurrence of each assigned genus were categorized as Bacteria, SAR protozoans , green algae (Chlorophyta), metazoan or other eukaryote groups. For each category, a relative abundance cutoff of 0.1% and at least appearance within two samples was applied at genus level for each library. Trap enrichment was calculated as the fold change in abundance of each phylotype between trap sample and its corresponding leaf sample. For every phylotype, a paired www.metagenassist.ca) for mapping bacterial phenotypic information. Several phenotype categories including oxygen requirement, energy source, metabolism and habitat may have multiple phenotypic traits associated with a given taxon. A paired t-test was used to examine differences in species richness and intra-group similarity between different attributes such as organs, species and seasons.The same taxonomy assignment pipeline was applied for 18 published metatranscriptome Illumina sequencing datasets of creek, soil, feces, marine sediment, marine water body and lake habitats Table . A totalG. nigrocaulis library were separately de novo assembled by the CLC Genomics Workbench 5.5.1 with automatic bubble and word sizes and minimal 200 bp contig length. Contigs longer than 500 bp were sequentially filtered out of G. nigrocaulis high and low confidence transcripts , SILVA LSURef_115 and SSURef_NR99_115 sequences by using ublast (1E-09) of the Usearch software  as reference. Transcript amounts (in reads per kilobase of exon per million reads) were calculated for every gene and quantile-normalized. Log2 ratios were used to measure relative changes in expression level between each pair of trap and its corresponding leaf sample. Genes were considered expressed if they have (1) more than one unique mapped read and (2) have more than five total mapped reads. Absolute values of the corresponding log2 ratios higher than 2 and the p-value of a paired t-test (trap vs. leaf) lower than 0.05 are conditions for selecting differentially expressed genes.The 31,710 microbe transcript contigs  served as a reference for read mapping using the RNA-seq module of the CLC Genomics Workbench v5.5.1 with standard mapping parameters (minimum length 0.9 and minimum similarity 0.8) for all 8 Genlisea mRNA-seq datasets  and further annotated with default filtering parameters . Generic GO-slim categories were used to provide a summary of GO annotation results. Enzyme code class assignment was exploited to define the list of hydrolases. Species information and bit score of blastx from the best blast hit result of every transcript were exported and taxonomically summarized by LCA algorithm from MEGAN software with a minimum score 50. Phyla which have been detected by ribosomal RNA assignment were used as main categories. Best hits from Chordata species were referred to as the Metazoa group. Enrichment analysis using the Fishers's Exact Test with Multiple Testing Correction of standard false discovery rate (FDR) was carried out in Blast2Go for enriched GO categories with a p-value cutoff of 0.05.By using Blast2GO  were more diverse than in G. hispidula (39–73 genera) traps, regardless of seasonal sampling. Overall we found in Genlisea trap samples 184 out of total 220 uniquely detected genera having at least 0.1% relative abundance of either bacteria, SAR protists , green algae (Chlorophyta), metazoa, or other eukaryotic microbes . Of 220 detected genera, 33.2% were found only in a single trap sample and only 6.8% were in all trap samples regardless of season and Genlisea species tested. The green algae Carteria and the fungus Entophlyctis were prevalent in only one sample, while eight other dominant genera appeared in more than one trap sample.Deep sequencing has been shown to be a suitable approach for large-scale comparisons of microbial communities  in traps in comparison with corresponding leaves Figure . These aG. hispidula traps is less diverse than that of the dimorphic G. nigrocaulis traps, although under our cultivation conditions the microfauna composition was likely homogeneous . Within this purple non-sulfur bacterial family, the chemoheterotrophs include the facultative anaerobic genera Skermanella, Telmatospirillum and the strictly aerobic and microoxic genera Magnetospirillum are mainly found in Genlisea samples. (ii) In Proteobacteria phylum, the acetic acid bacterium Asaia and several genera in plant growth-promoting Rhizobiales are highly enriched in G. hispidula traps and Genlisea leave samples. The abundant Asaia genus  has recently recognized as bacterial symbionts of various insects . The opposite is found in G. hispidula traps with 7.2 and 18.7% bacterial reads, respectively  A To provide an additional level of functional understanding of the bacterial active-microbiome of Genlisea traps (trap-enriched set), available phenotype information of identified genera from the METAGENassist database . The main fraction of non-plant transcripts ranging from 1500 to 8000 bp  was analyzed. A total of 10,518 transcripts had significant BLAST hits (E ≤ 1.0E-3) in the NCBI protein reference database , and 1298 transcripts could be further assigned with an enzyme code. The top five KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of this microbial metatranscriptome include purine metabolism , prokaryotic carbon fixation pathways , pyruvate metabolism , thiamine metabolism  and the tricarboxylic acid (TCA) cycle . In total, only 15 transcripts  with assigned enzyme codes had a significant best hit from bacterial species, although the proportion of bacterial transcripts is in the same range as those from algae, amoebes, or ciliates. In total, 408 bacterial transcripts having E-value less than 1E-3 and a bit score higher than 50, originated from 300 bacterial species of 214 genera, belonging mainly to the most abundant bacteria phyla Proteobacteria (151 transcripts), Cyanobacteria (76 transcripts) and Firmicutes (75 transcripts).Of the 10,518 microbe transcripts, 6140 transcripts could be annotated , Volvox carteri f. nagariensis , Chlamydomonas reinhardtii , Acanthamoeba castellanii str. Neff  and Dictyostelium purpureum . Given the limited availability of genomic data for unicellular Eukarya, it is more likely that transcripts could have been from soil-borne related species. Among the whole microbiome, T. thermophila which is a voracious predator of bacteria  were accumulated. Enrichment of transcripts involved in transmembrane transport (FDR 2.7E-3) and other substance transport mechanisms  were observed in C. reinhardtii, while V. carteri produced transcripts enriched for generation of precursor metabolites and energy (FDR 2.5E-3) and for stress response (FDR 0.042). No statistically significant enrichment was found comparing transcripts of Acanthamoeba castellanii str. Neff, Dictyostelium purpureum, or of all bacteria species with the whole microbial transcriptome.The enzyme code distribution of the microbial transcriptome showed 33.7% transcripts encoding hydrolases Tables , S2. MaiG. nigrocaulis genome as reference, the Genlisea rhizophyll trancriptomes were characterized by RNA sequencing analysis in comparison to the corresponding leaf transcriptome. Samples of G. nigrocaulis and G. hispidula from two different seasons were included (Table p < 0.05) in G. nigrocaulis. Hence, 6.4% of all 17,113 G. nigrocaulis genes, corresponding to 8.5% of genes transcribed either in leaves or traps, were differentially expressed. Of the 1098 differentially expressed genes (DEGs), 69 showed an at least two-fold accumulation or reduction of transcripts .Using the annotated ed Table . RelativG. nigrocaulis traps and leaves indicates a switch from photosynthesis and chloroplast activities in leaves toward respiratory and mitochondrial activities in traps  and two NADH oxidases , which contribute to generate ATP via the respiratory pathway, were up-regulated. Interestingly, NADH oxidases are probably used to generate superoxide and further reactive oxygen species for prey digestion in Genlisea traps  in Utricularia traps are consistently high, even in species growing in highly oligotropic waters with low prey-capture rates , nitrate transporter (Gnig_g12092), amino acid transporter (Gnig_g1022), and oligopeptide transporter (Gnig_g2102) transcripts. Moreover, three transcription factors , likely involved in cellular nitrogen metabolism were the most up-regulated genes in Genlisea traps. Likely, Genlisea plants absorb N-nutrients via carnivory. For P-nutrient demand of Genlisea plants, there are four up-regulated genes, of which proteins are predicted to have acid phosphatase activity (EC:3.1.3.2/0), including Gnig_g15303, Gnig_g1090, Gnig_g9666 and Gnig_g2820. Although six (inorganic) phosphate (co)transporters  were expressed in Genlisea traps, these genes do not show a significant differential expression , green algae, microbial fungi and a large range of minute metazoans. Ribosomal RNA profiling indicates a highly dynamic structure of the trap bacterial community, reflecting their ecological importance mainly as prey of the one-way food web inside Genlisea traps. The enrichment in facultatively anaerobic bacteria suggests an occasionally interrupted anoxia environment in Genlisea digestive chambers. A high amount of superoxide and other reactive oxygen species is likely generated in Genlisea traps for killing prey and stimulates different oxidative stress responses in trap cells.T. thermophila is a voracious bacterial predator, while green algae, such as C. reinhardtii, seem to stay as commensals or inquilines inside Genlisea traps. A variety of mites, nematodes, rotifers and annelids are similarly entrapped and ingest in turn protozoans until they perish and their corpses serve themselves as nutrient. Further studies using microcosm experiments with less complex microbial community may be interesting to understand contributions of each microbe to the carnivory.The opportunistic feeding behavior, to catch and utilize various prey, provides Genlisea plants alternative N- and P- macronutrient sources from microbes. The abundance of bacteria involved in nitrogen cycling  indicates their importance for the gain of N- nutrients. In addition, various transporters for different N- forms such as ammonium, nitrate, amino acids and oligopeptides together with transcription factors involved in cellular nitrogen metabolism are highly up-regulated in Genlisea rhizophylls. Except for acidic phosphatases, only a limited range of Genlisea hydrolases were found up-regulated in the traps, suggesting that Genlisea plants rely on digestive enzymatic systems from microbes. Indeed, various hydrolases were identified from entrapped metazoan microbes, Alveolata protists, green algae and amoeboid protozoa. Among them, the cilliate HC and GV conceived and designed the study. HC and GV performed the experiments and analyzed the data. HC and GV wrote the paper with contributions from IS. AP, TS, US, and IS contributed reagents/ materials/ analysis tools. All authors read and approved the final manuscript.The 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.


Agrobacterium-mediated transformation is a commonly used method for plant genetic engineering. However, the limitations of Agrobacterium host-plant interactions and the complexity of plant tissue culture often make the production of transgenic plants difficult. Transformation efficiency in many legume species, including soybean and the common bean, has been reported to be quite low. To improve the transformation procedure in legumes, we screened for chemicals that increase the transformation efficiency of Lotus japonicus, a model legume species. A Chemical library was screened and chemicals that increase in transient transformation efficiency of L. japonicus accession, Miyakojima MG-20 were identified. The transient transformation efficiency was quantified by reporter activity in which an intron-containing reporter gene produces the GUS protein only when the T-DNA is expressed in the plant nuclei. We identified a phenolic compound, chloroxynil, which increased the genetic transformation of L. japonicus by Agrobacterium tumefaciens strain EHA105. Characterization of the mode of chloroxynil action indicated that it enhanced Agrobacterium-mediated transformation through the activation of the Agrobacterium vir gene expression, similar to acetosyringone, a phenolic compound known to improve Agrobacterium-mediated transformation efficiency. Transient transformation efficiency of L. japonicus with 5 μM chloroxynil was 60- and 6- fold higher than that of the control and acetosyringone treatment, respectively. In addition, transgenic L. japonicus lines were successfully generated by 5 μM chloroxynil treatment.Furthermore, we show that chloroxynil improves L. japonicus transformation by Agrobacterium strain GV3101 and rice transformation. Our results demonstrate that chloroxynil significantly improves Agrobacterium tumefaciens-mediated transformation efficiency of various agriculturally important crops. Genetically modified (GM) crops are commonly used for improving agricultural production and environmental protection. The first GM crop, FLAVR SAVR tomato, was commercially introduced in 1994 , where GAgrobacterium-mediated transformation, direct gene transfer into protoplasts and particle bombardment. Agrobacterium-mediated transformation is the most commonly used method because of its higher transformation frequency, integration of long DNA segments and stable expression of the transgene due to the low copy number of T-DNA insertions . Bot. BotAgroArabidopsis thaliana[L. japonicus. Hypocotyl explants suffered serious damage with 50 μM CX treatment , have been known to induce rapid cell death in  thaliana. Thus, treatment , and thereatment . No signreatment . Based oAgrobacterium vir genes are induced by CX treatment, vir gene expression level was quantified using real-time qPCR. The VirB1 gene was activated by CX treatment in a dose-dependent manner   . As a renil (IX) . Both 0.nil (IX) . The frenil (IX) . The Agrreatment .Agrobacterium-mediated transformation. A previous study reported that phenolic compounds and their analogues induce vir genes in Agrobacterium[VirB1 gene expression levels in 5 μM CX treated explants were lower than that those treated with 100 μM AS Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.


Background: The Atlantic Forest is globally one of the priority ecoregions for biodiversity conservation. In Argentina, it is represented by the Paranense Forest, which covers a vast area of Misiones Province between the Paraná and Uruguay rivers. The Uruguay River is a global hotspot of freshwater gastropod diversity, here mainly represented by Tateidae (genus Potamolithus) and to a lesser extent Chilinidae. The family Chilinidae  includes 21 species currently recorded in Argentina, and three species in the Uruguay River. The species of Chilinidae occur in quite different types of habitats, but generally in clean oxygenated water recording variable temperature ranges. Highly oxygenated freshwater environments  are the most vulnerable continental environments. We provide here novel information on three new species of Chilinidae from environments containing waterfalls and rapids in the Uruguay River malacological province of Argentina.Materials and Methods: The specimens were collected in 2010. We analyzed shell, radula, and nervous and reproductive systems, and determined the molecular genetics. The genetic distance was calculated for two mitochondrial markers (cytochrome c oxidase subunit I–COI- and cytochrome b -Cyt b-) for these three new species and the species recorded from the Misionerean, Uruguay River and Lower Paraná-Río de la Plata malacological provinces. In addition, the COI data were analyzed phylogenetically by the neighbor-joining and Bayesian inference techniques.Results: The species described here are different in terms of shell, radula and nervous and reproductive systems, mostly based on the sculpture of the penis sheath. Phylogenetic analyses grouped the three new species with those present in the Lower Paraná-Río de la Plata and Uruguay River malacological provinces.Discussion: Phylogenetic analyses confirm the separation between the Uruguay River and the Misionerean malacological provinces in northeast Argentina. These new endemic species from the Uruguay River add further support to the suggestion that this river is a diversity hotspot of freshwater gastropods . These endemic species from environments with rapids and waterfalls should be taken into account by government agencies before the construction of dams that modify those ecologic niches in the Uruguay River. The changes that result from damming rivers with waterfalls and rapids have caused the extinction of species—for example, those of the gastropod genus Aylacostoma (e.g.Aylacostoma from rapids in the High Paraná River (Argentina-Paraguay), based on materials collected in 2007. In 2011, however, the locations were flooded during the last stage of filling the Yacyretá Reservoir.Highly oxygenated freshwater environments  are the most vulnerable continental environments globally, supporting highly specific faunas (including gastropods) with narrow habitat requirements. Accordingly, many native snail populations are declining in numbers as a consequence of the continuous degradation and destruction of their natural ecosystems from unabated human activity . In partacostoma . Despiteoma e.g.. Vogler Potamolithus, with 12 endemic species), and to a lesser extent by Chilinidae (three endemic species) and Ampullariidae (endemic genus Felipponea with three species). The streams of Misiones Province contain waterfalls and rapids that have been poorly studied by malacologists. In these environments several endemic freshwater gastropod entities have been recorded—e.g., the genera Acrorbis (Planorbidae), inhabiting only waterfall environments (Felipponea spp. (Ampullariidae), recorded in the rapids of the Uruguay River and its tributaries , inhabiting the waterfalls of Iguazú National Park (Argentina and Brazil)  and Sineancylus rosanae  (Planorbidae), with the last being present in the rapids of the upper Iguazú River (Argentina and Brazil)  , Chilina rosanae  (Planorb Brazil) .Chilina genus found in Argentina, 15 are endemic and nine of this 21 are vulnerable (http://www.iucnredlist.org) lists only one species as “vulnerable”  from Chile), seven as “data-deficient,” and four as “least concern.” In the Del Plata basin  six species of Chilinidae have been recorded. Three are found in the Lower Paraná-Río de la Plata and the Uruguay River malacological provinces: Chilina fluminea , Chilina rushii Pilsbry, 1896 and Chilina gallardoi Castellanos & Gaillard, 1981   is one of the oldest families of freshwater gastropods . Of the lnerable . Vulneralnerable . Of thosrd, 1981 . The oth-Brazil) .Chilina nicolasi, Chilina santiagoi and Chilina luciae from rapids and waterfalls of the Uruguay River malacological province. Phylogenetic analyses were used to confirm the segregation of the three species and of the species in the different freshwater malacological provinces of Argentina.In this study we describe and provide information on the anatomy and molecular genetics of three news species: camera lucida. Figures were drawn only for characters that showed specific differences. The terminology used for the anatomical descriptions follows Chilina megastoma studied by C. iguazuensis described by C. fluminea fluminea and C. fluminea parva Martens, 1868 studied by C. fluminea fluminea studied by C. rushii and C. gallardoi studied by C. lilloi Ovando & Gutiérrez Gregoric, 2012, C. portillensis Hidalgo, 1880 and C. tucumanensis Castellanos & Miquel, 1980 all studied by C. mendozana Strobel, 1874, C. parchappii , C. cuyana Gutiérrez Gregoric, Ciocco & Rumi, 2014 and C. sanjuanina Gutiérrez Gregoric, Ciocco & Rumi, 2014 all studied by The specimens were collected in the Misiones Province  and deposited in the Malacological Collection at the Museo de La Plata, Buenos Aires Province, Argentina (MLP-Ma). Additional material in MLP-Ma was also studied. Adult specimens were first relaxed in menthol for 12 h, then immersed in hot water (70 °C), and finally stored in 96% (v/v) aqueous ethanol or fixed in modified Raillet-Henry (R-H) solution for freshwater animals—93% (v/v) distilled water, 2% (v/v) glacial acetic acid, 5% (v/v) formaldehyde, and 6 g sodium chloride per liter. Six shell measurements were taken: total length (TL), length of the last whorl (LWL), aperture length (AL), total width (TW), aperture width (AW), and aperture projection (AP) following The radulae were separated from the buccal mass and cleaned following the method of 3 samples from the foot of recently collected specimens (2010) using commercial kits . A partial sequence of the genes encoding the mitochondrial cytochrome c oxidase subunit I (COI) and cytochrome b (Cyt b) were amplified by the polymerase chain reaction (PCR) with the universal primers of C. megastoma, C. iguazuensis and C. fluminea  were obtained from the Barcode of Life Database (BOLD). The sequence alignment was performed with the Clustal X 2.0.12 software  can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:3140E36D-B1F5-4C1B-9F3C-0081CDE88B00. 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 (PDF) will represent a published work according to the International Commission on Zoological Nomenclature , and henFamily Chilinidae Dall, 1870ChilinaGray, 1828Genus Auricula (Chilina) fluctuosaType species: Diagnosis: Species in the genus and family have an oval (oblong to ventricose) shell with an expanded last whorl. Nervous system with partial detorsion. Roof of the mantle cavity pigmented with kidney occupying almost entire length. Kidney inner wall with numerous transverse trabeculae of irregular contour. Rectum on right side of mantle cavity, anus near pneumostome. Incomplete division of male and female ducts; common duct opens to hermaphrodite duct, with irregular contours on both sides. Proximal portion of uterus with glandular walls. Calcareous granules in vaginal lumen and secondary bursa copulatrix or accessory seminal receptacle present. Penial terminal portion with cuticularized teeth-like structures.Remarks: The Chilinidae includes only the genus Chilina with 36 species, 21 of which are found in Argentina , coll. D.E. Gutiérrez Gregoric, V. Núñez & R.E. Vogler, 23 March 2010.Holotype: MLP-Ma 13412-2 ; paratypes: MLP-Ma 13412 same data ; MLP-Ma 14134 same data .Etymology: Dedicated to the first son, Nicolás, of the first author of this paper.Diagnosis: Shell thick, oval, two columellar teeth (upper underdeveloped); radula with first lateral tooth with saw-like external side of mesocone; penis sheath twice the length of the prepuce; penis sheath inner sculpture with triangular regular pustules.Description:Shell (Shell  and 2B. Reproductive System (c. 8% the length of bursa copulatrix duct). Vagina cylindrical, longer than wide, folded over free oviduct and entering female atrium. (ii) Male reproductive system. Prostate gland extending to lower half of uterus and consisting of variable size and with cylindrical acini. Vas deferens coiled twice, overlapping vagina. At level of penis complex, vas deferens bent back on itself. Penis sheath muscular, twice the length of the prepuce, with slight convexity on right side. Penis sheath inner sculpture with triangular pustules over entire surface. Penis elongated (as long as the penis sheath), robust, with outer surface crossed by transverse lamellae, triangular in cross section. Prepuce cylindrical, thin, with constriction marked by oblique lines arranged in a V making connection with penis sheath.e System . (i) FemRadula  was deposited in GenBank under the number KT830419.Remarks: Of the Chilinidae species for which characters of the radula have been described so far, C. nicolasi is the only one with the first and second lateral tooth of the outer edge of the mesocone (tricuspid) or second inner cusp (tetracuspid) serrated. The radulae of C. gallardoi and C. nicolasi have a similar number of rows and teeth per row, but the outermost lateral teeth in C. nicolasi can have up to seven cusps, while those of C. gallardoi have only five (C. iguazuensis has more rows (57–65 vs 52–59) and teeth per half-row (43–63 vs 40–41) than C. nicolasi ; and in C. fluminea and C. lilloi the central tooth is tricuspid . The AL/LWL ratio in C. gallardoi is lower than in C. nicolasi  along the whorls (as does C. rushii), a character absent in C. nicolasi .nly five . The radnicolasi . Only thricuspid . The sheer 100%; . In addiLSID urn:lsid:zoobank.org:act:4238E0F8-4452-4818-A1F2-7C1A5D8FFC4E –4H and 6Type locality and type material: Horacio Foerster Falls, Misiones Province, Argentina , coll. D. E. Gutiérrez Gregoric, V. Núñez & R.E. Vogler, 24 March 2010.Holotype: MLP-Ma 14135 ; paratypes: MLP-Ma 13417 same data ; MLP-Ma 14136 same data .Other material examined: MLP-Ma 14137 Horacio Foerster Falls, Misiones Province, Argentina , coll. C. Galliari, May 2009 (four dry shells); MLP-Ma 14138 Moconá Falls, Misiones Province, Argentina , coll. C. Galliari, May 2009 ; MLP-Ma 14139 Moconá Falls, Misiones Province, Argentina , coll. A. Rumi, S.M. Martín & I. César, October 20, 2011 ; MLP-Ma 14140 Yerba Falls, Paraíso Stream, El Soberbio, Misiones Province, Argentina , no collector and date .Etymology: Dedicated to the second son, Santiago, of the first author of this paper.Diagnosis: Shell small, thin, aperture projection 50% of TW; radula with asymmetrical bicuspid central tooth; penis sheath inner sculpture with regular conical pustules and longitudinal folds.Description:Shell (Shell  and 2D. Reproductive System (c. 18% the length of bursa copulatrix duct), comprising of a long duct and expanded at the distal end. (ii) Male reproductive system. Muscular penis sheath, nearly twice as long as prepuce. Penis sheath inner sculpture with pustules of conical aspect and longitudinal folds. Penis slightly longer than penis sheath, robust, with outer surface cut by transverse lamellae, triangular in cross-section. Prepuce inner sculpture with numerous smooth, very tight longitudinal folds.e System . (i) FemRadula  were deposited in GenBank under the numbers KT820416 and KT820424 respectively.Remarks: The spire is not preserved in all specimens. This loss occurs in several species of Chilinidae, especially in those that inhabit fast-running water such as C. iguazuensis , strong rather than weak bands, higher AL/LWL, AP/TW and TW/LWL ratios , and has one columellar tooth whereas there are two in C. nicolasi. Chilina santiagoi differs from C. nicolasi in the length of the secondary bursa copulatrix (18% of bursa copulatrix duct length vs 8%), and internal sculpture of the penis sheath . Regarding the radula, C. santiagoi has fewer rows of teeth (average 44 vs 55 in C. nicolasi) and fewer teeth per half row (average 32 vs 40); the developed cusp of the central tooth (in both the tooth is bicuspid) is the right cusp (left in C. nicolasi) and the cusp has only one serrated edge (both in C. nicolasi), and the outermost lateral teeth have five cusps (up to seven in C. nicolasi).azuensis . Chilina 17.3 mm . Chilina ganglia , but thiLSID urn:lsid:zoobank.org:act:FE46F318-BA47-4D5B-8AD2-266C63EB87A4 –4K and 7Type locality and type material: Pesiguero stream, Misiones Province, Argentina , coll. D. E. Gutiérrez Gregoric, March 21, 2010.Holotype: MLP-Ma 14141 ; Paratypes: MLP-Ma 13413 same data ; MLP-Ma 14142 same data .Etymology: Dedicated to the daughter, Lucía, of the first author of this paper.Diagnosis: Shell thick, aperture projection 33% of TW; radula, central tooth bicuspid and with saw-like external side, first lateral tetracuspid; prepuce 37% of length of penis sheath; penis sheath inner sculpture with two regions, one with polygonal pustules and the other with longitudinal zigzag folds.Description:Shell (Shell  and 2F. Reproductive System (e System . (i) FemRadula  were deposited in GenBank under the numbers KT820420 and KT820425 respectively.Remarks:Chilina luciae, like C. gallardoi and C. rushii, was recorded in the rapids of a stream that flows into the Uruguay River. Chilina luciae differs from both those species by not having a shell keel. Chilina luciae has two strong columellar teeth as in C. gallardoi, C. fluminea and C. rushii; while C. nicolasi also has two columellar teeth, but weak ones. The AL/LWL ratio in C. luciae is lower than in C. nicolasi and C. santiagoi (83 vs 90 and 95% respectively), but higher than in C. gallardoi (78%)  and all lower than in C. santiagoi (50 and 80% respectively). Chilina luciae differs from C. nicolasi and C. santiagoi in the length of the secondary bursa copulatrix (11% of bursa copulatrix duct length vs 8 and 18% respectively), and internal sculpture of the penis sheath  . The AP/ of them . The firfluminea .C. nicolasi, 1; C. luciae, 1; C. fluminea, 1; C. gallardoi, 1) and 15 sequences of 655 bp for COI  were obtained  and endemic species (15)  . These nies (15) . The famFrom an anatomical viewpoint, the new species exhibit differences in shell, radula and reproductive system characteristic, especially in the sculpture of the penis sheath. In species recently described and re-described by The interspecific genetic distances found in the present study for COI were 1.2% or greater, and the intraspecific distances lower than 0.5%. Studies in Lymnaeidae  have suggested a similar interspecific genetic distance for COI among neotropical species . For lane.g. C. megastoma and C. iguazuensis. Nevertheless, the species from the Río de la Plata River (C. fluminea and C. rushii) are more closely associated with those of the Uruguay River. The species in the Cuyo malacological province (C. mendozana and C. sanjuanina) are distinct from those from the Del Plata basin. Likewise, species of Aylacostoma (Thiaridae) and Acrorbis (Planorbidae) in the Misioneran malacological province have not been recorded in the Uruguay River malacological province , and Chilina santiagoi in the Uruguay River. In addition, C. nicolasi and C. luciae have been added to the species recorded in rapids along rivers in Misiones Province, which include C. iguazuensis, Sineancylus rosanae, Felipponea spp. and Aylacostoma spp. A hydroelectric dam is going to be built in the area where C. nicolasi were collected. This hydroelectric dam will raise the level the Uruguay River, causing the disappearance of the environment inhabited by the species. These endemic species should be taken into account by government agencies before the construction of dams that modify these types of environments in the Uruguay River.With the examples described here, the number of endemic species known in waterfall environments increases. Thus, species living in Misiones Province are


Staphylococcus aureus causes many types of infections, ranging from self-resolving skin infections to severe or fatal pneumonia. Human innate immune cells, called polymorphonuclear leukocytes (PMNs or neutrophils), are essential for defense against S. aureus infections. Neutrophils are the most prominent cell type of the innate immune system and are capable of producing non-specific antimicrobial molecules that are effective at eliminating bacteria. Although significant progress has been made over the past few decades, our knowledge of S. aureus-host innate immune system interactions is incomplete. Most notably, S. aureus has the capacity to produce numerous molecules that are directed to protect the bacterium from neutrophils. Here we review in brief the role played by neutrophils in defense against S. aureus infection, and correspondingly, highlight selected S. aureus molecules that target key neutrophil functions. Staphylococcus aureus is adept at circumventing destruction by the human innate immune system, and the bacterium imposes a significant disease burden on at-risk and otherwise healthy populations worldwide [S. aureus commonly resides as a quiescent commensal within the human nares, and, as such, is a frequent cause of opportunistic infections [S. aureus has remained a leading cause of healthcare and community associated infections in the United States over the past 60 years. The organism causes a broad array of diseases and syndromes, such as endocarditis, toxic-shock syndrome, bacteremia, soft-tissue infections, surgical-site infections, and pneumonia [S. aureus increasingly undermines treatment regimens already hampered by a substantial lack of novel therapeutics [S. aureus to develop antibiotic resistance and cause significant morbidity, underscores the importance of developing new therapeutic approaches for treatment of infections.orldwide . S. aurefections . Indeed,neumonia ,4,5,6. Iapeutics ,8. TakenS. aureus infections, and this characteristic is attributed largely to the role played by neutrophils in host defense. Indeed, individuals with neutrophil defects are highly susceptible to severe and life-threatening S. aureus infections [S. aureus to circumvent elimination by PMNs.Polymorphonuclear leukocytes (PMNs or neutrophils) are the most prominent cellular component of the host innate immune response to bacterial and fungal infections. The vast majority of healthy individuals are seemingly protected against severe fections . This re9 cells/kg body weight every day in a healthy human adult [Neutrophils are the primary cellular constituent of the human innate immune system and are essential for host defense against invading microbial pathogens. PMNs originate and mature in the bone marrow, and are subsequently released into the peripheral vasculature. High numbers of neutrophils are maintained in circulation to facilitate rapid recruitment to infected tissue. Intravascular circulating neutrophils comprise approximately half of the entire granulocyte population, whereas marginated neutrophils maturing in the bone marrow or resident in organ systems make up the remaining half ,11. Matuan adult ,11. Circn-formyl peptides [Circulating neutrophils are recruited to infection sites through chemotactic signals produced by host cells and shed and/or secreted bacterial molecules. The ability of neutrophils to rapidly respond to invading microorganisms is a key facet of innate immunity. Neutrophils are highly sensitive to chemotactic signals that signify infection and promote migration. Host-derived chemotactic factors, such as interleukin 8, GRO-alpha, granulocyte chemotactic protein 2, and complement component C5a are potent proinflammatory mediators that are used to recruit additional PMNs to areas of infection ,13,14. Fpeptides ,18,19,20peptides ,22,23.Neutrophil recruitment from the bloodstream to infected tissue is governed by a series of complex signal transduction cascades that ultimately control the dynamics of PMN interaction with vascular cells and regulate chemotaxis ,13. The Neutrophils play a primary role in host defense and remove pathogens through a process known as phagocytosis. Phagocytosis is initiated by recognition and binding of bacteria, and is followed by internalization. Recognition of microbial pathogens is determined by pattern recognition receptors (PRRs) and opsonin receptors. PRRs bind microbe-associated molecules known as pathogen-associated molecular patterns (PAMPs), and PRRs are an essential component of the host-immune surveillance. The composition of PRRs includes a broad array of receptors, such as membrane-bound toll-like receptors (TLRs), that identify highly conserved bacterial surface and secreted molecules. PRR ligation induces intracellular signal transduction cascades that trigger enhanced phagocytosis and cytokine production, albeit pathogen recognition by PRRs alone is generally insufficient to stimulate phagocytosis ,26,27. T2−. The superoxide anion is readily converted to hydrogen peroxide by superoxide dismutase. Notably, hydrogen peroxide and other secondary oxygen derivatives such hydroxyl radical, chloramines and hypochlorous acid (HOCl), which is generated by the myeloperoxidase-halide system, contribute to PMN microbicidal activity [Neutrophils use both oxygen-dependent and oxygen-independent processes to kill invading microbial pathogens . Phagocyactivity ,34. Neutactivity ,36,37. Aactivity ,36,38. CThe rapid influx of large numbers of neutrophils to sites of infection is a hallmark of the acute inflammatory response. The combination of ROS, degradative enzymes, and cationic peptides create a potent microbicidal environment confined within neutrophil phagosomal membranes. Inasmuch as these molecules do not discriminate between host and microbe targets, the potential of host-tissue damage is high should the neutrophil undergo inappropriate cell lysis . To mitiS. aureus is capable of producing numerous freely secreted and surface-bound molecules that have potential to alter and/or limit the functional capacity of neutrophils. In vitro, these molecules have been shown to inhibit neutrophil recruitment, bacterial binding and phagocytosis, and killing by ROS and antimicrobial peptides (AMPs)   is a freely secreted protein that inhibits neutrophil and monocyte chemotaxis [S. aureus enterotoxin A or P (SEA or SEP)—is located on a mobile genetic element known as a β-hemolysin-converting bacteriophage [S. aureus strains tested to date [et al. reported that S. aureus extracellular adherence protein (Eap) and two homologs, EapH1 and EapH2, inhibit neutrophil elastase, proteinase-3, and cathepsin G [S. aureus immune evasion molecules, such as SCIN and CHIPS, and this process is blocked by the Eap molecules [Chemotaxis inhibiting protein of emotaxis ,44. CHIPriophage . SAK binriophage , and SEAriophage ,48,49. C to date . More rehepsin G . These nolecules . The extet al. found that Ssl7 blocked neutrophil recruitment in a mouse model of inflammation [in vitro [There is also a family of staphylococcal-like proteins (Ssls) that has been reported to function as immune evasion molecules. For example, Ssl7 binds host IgA and complement component C5, inhibiting generation of C5a, phagocytosis, and production of phagocyte reactive oxygen species ,53. In aammation . This sain vitro . In subsin vitro .S. aureus produces at least five molecules that inhibit serum complement convertases—host enzyme complexes important for a fully functional complement system. The activity of host C3 convertase is essential for the opsonization of microbes with complement components such as C3b and iC3b, which promote phagocytosis by complement receptors. Moreover, activation of the complement system ultimately generates C5a, a neutrophil chemoattractant. Staphylococcal complement inhibitor (SCIN) and homologues (SCIN–B and SCIN–C), extracellular fibrinogen-binding protein (Efb), and extracellular complement-binding protein (Ecb), bind and inhibit the C3 convertase (SCIN proteins), or alternatively, C3b–containing convertases and C5 convertases (Efb and Ecb) [S. aureus molecules with the host complement system inhibits leukocyte recruitment and neutrophil phagocytosis of S. aureus in vitro and ex vivo in human blood [S. aureus in a mouse infection model [et al. found that SCIN-B, SCIN-C, Efb and Ecb are located within a segment of the S. aureus genome that also encodes alpha-hemolysin (Hla), Ssl12, Ssl13, Ssl14, FPR-like 1 inhibitory protein (FLIPr), and a FLIPr homologue (FLIPr-like) [S. aureus evasion of host defense, this segment of the genome was named immune evasion cluster 2 (IEC-2) [and Ecb) ,57. The an blood ,59. Althet al. recently demonstrated that staphopain A (ScpA), a secreted S. aureus cysteine protease, inhibits in vitro neutrophil chemotaxis mediated by CXCR2 [Laarman by CXCR2 . ScpA clby CXCR2 . The redS. aureus molecules have been shown to inhibit neutrophil phagocytosis in vitro or in whole blood. For instance, S. aureus protein A (SpA) and the second binding protein of immunoglobulin (Sbi) associate with the Fc-region of immunoglobulin and can inhibit phagocytosis in vitro [S. aureus capsule polysaccharide [S. aureus phagocytosis in vitro. S. aureus strains lacking at least two of these molecules—e.g., those lacking Spa and ClfA—are ingested to a greater extent by neutrophils than those lacking only one of these molecules [in vitro uptake by neutrophils completely. The ability of these molecules to block uptake is also probably dependent on assay conditions. The vast majority of the studies of S. aureus phagocytosis by neutrophils have been performed with cells in suspension. The extent to which phagocytosis assays with neutrophils in suspension approximate the interaction of bacteria with neutrophils in the bloodstream is unclear. It has long been known that neutrophils are largely responsible for clearance of S. aureus from the bloodstream in animal infection models [S. aureus by adherent human neutrophils—which are representative of phagocytes at sites of infection—is highly efficient and seemingly unimpeded by anything produced by the microbe [S. aureus by neutrophils was noted by Rogers and Tompsett many years ago, and they also found that phagocytosis of S. aureus was significantly greater than that of Klebsiella pneumoniae, group A streptococcus, or Streptococcus pneumoniae [S. aureus “antiphagocytic” molecules in the pathogenesis of human infections remains unclear. The problem with S. aureus and human infections is not inefficient phagocytosis . Ratherhe topic ,73,75,76he topic ,75,77.S. aureus has the capacity to produce molecules that can moderate the cytotoxic effects of ROS and AMPs. Survival of S. aureus inside the phagosome is directly linked to the ability to mitigate the effects of host–derived ROS and AMPs. Superoxide dismutases (SodA and SodM), catalase (KatA), alkyl hydroperoxide reductase (AhpCF), and staphyloxanthin (encoded by crtOPQMN), have been shown to protect S. aureus from ROS [S. aureus also mitigates the effects of ROS by means of proteins and enzymes involved in metal ion homeostasis . Althouphylaxis . Most ev. aureus ,84,85.dlt operon of S. aureus (dltABCD) encodes a cell envelope modification system that attaches d-alanine to cell wall teichoic acids [d-alanine into teichoic acids decreases the negative charge on the bacterial surface and limits susceptibility of the microbe to cationic AMPs, including human neutrophil alpha-defensins [et al. used a mouse infection model to demonstrate that the dlt operon is important for S. aureus virulence [S. aureus transfers l–lysine to phosphatidylglycerol to produce lysylphosphatidylglycerol, which contributes to neutralization of the negative charge of the bacterial cell surface [S. aureus strains with mprF genetically inactivated are significantly more susceptible to killing by human neutrophils, a phenomenon linked to increased binding to host AMPs [S. aureusmprF mutants also have a decreased virulence phenotype in mouse infection models, which further underscores the importance of the resistance to AMPs in S. aureus pathogenesis [i.e., the AMPs no longer interact with the charge-neutral membrane of the microbe. There are several other notable molecules and immune evasion systems utilized by S. aureus to promote survival after phagocytosis by neutrophils. For example, lysozyme is a prominent antimicrobial agent present in neutrophil granules. It functions as an antibacterial agent by hydrolyzing beta-glyosidic linkages in cell wall peptidoglycan. Notably, S. aureus is completely resistant to lysozyme. This a characteristic conferred by O-acetylation of peptidoglycan, a process catalyzed by a protein known as OatA [The ic acids . Incorpoefensins ,87. Coll surface . S. aureost AMPs . S. aureogenesis . In gene as OatA .S. aureus is capable of producing an extensive arsenal of cytolytic toxins. Several of these molecules, including alpha-type phenol soluble modulins (PSMα) [S. aureus infections is an interesting topic, but outside of the scope of this review. Here we focus our brief discussion on the contribution of molecules that contribute to lysis of neutrophils after phagocytosis.s (PSMα) , Panton-s (PSMα) ,92,93, ls (PSMα) ,95, and s (PSMα) , cause oS. aureus within granulocytes can be trafficked to tissues and promote low-grade persistent infection [S. aureus causes lysis of neutrophils after phagocytosis [et al. reported that prominent community-associated MRSA strains such as USA300 and USA400 have high capacity to cause rapid lysis of human neutrophils after phagocytosis [et al. indicated that S. aureus—in this case USA300—is contained within the phagosome to the point of neutrophil lysis [S. aureus is consistent with programmed necrosis. Indeed, Greenlee-Wacker et al. then demonstrated that neutrophils undergo programmed necrosis following phagocytosis of S. aureus [S. aureus infections, although direct evidence is still lacking and more work is needed.Rogers and colleagues discovered that nfection ,74. Thisnfection . Rogers ocytosis . Voyich ocytosis . Recent ocytosis ,97. Howeil lysis . The obsil lysis . Rather,. aureus . Such cyS. aureus has capacity to produce many molecules directed to inhibit neutrophil functions such as recruitment and phagocytosis. Despite this repertoire of immune evasion molecules, severe invasive S. aureus disease is infrequent in otherwise healthy individuals who lack risk factors for infection. In addition, the localized presence of neutrophils is a hallmark of S. aureus infections and neutrophils readily phagocytose S. aureus. These observations bring into question the role of the S. aureus immune evasion molecules in human disease . Finally, the possible utility of an opsonophagocytic vaccine for S. aureus is confounded by two key features of S. aureus-neutrophil interactions: (1) phagocytosis is already efficient in the absence of a vaccine; and (2) neutrophils can undergo rapid lysis after phagocytosis of S. aureus. Despite the noted caveats, research in these areas must move forward. Advances in basic research that continue to elucidate the complex interactions between S. aureus and the human host will likely offer new inroads into developing more efficacious therapeutic treatments and perhaps, a preventative vaccine.To summarize,


Glycine max, while paired segments were rarely identified in Lotus japonicas, Medicago truncatula and Cajanus cajan. However, by comparing interspecies microsynteny, we determined that the great majority of Hsf-containing segments in Lotus japonicas, Medicago truncatula and Cajanus cajan show extensive conservation with the duplicated regions of Glycine max. These segments formed 17 groups of orthologous segments. These results suggest that these regions shared ancient genome duplication with Hsf genes in Glycine max, but more than half of the copies of these genes were lost. On the other hand, the Glycine max Hsf gene family retained approximately 75% and 84% of duplicated genes produced from the ancient genome duplication and recent Glycine-specific genome duplication, respectively. Continuous purifying selection has played a key role in the maintenance of Hsf genes in Glycine max. Expression analysis of the Hsf genes in Lotus japonicus revealed their putative involvement in multiple tissue-/developmental stages and responses to various abiotic stimuli. This study traces the evolution of Hsf genes in legume species and demonstrates that the rates of gene gain and loss are far from equilibrium in different species.Whole-genome duplication events (polyploidy events) and gene loss events have played important roles in the evolution of legumes. Here we show that the vast majority of Hsf gene duplications resulted from whole genome duplication events rather than tandem duplication, and significant differences in gene retention exist between species. By searching for intraspecies gene colinearity (microsynteny) and dating the age distributions of duplicated genes, we found that genome duplications accounted for 42 of 46 Hsf-containing segments in  Glycine up to 13 Mya Whole-genome duplication, or polyploidy, is a common phenomenon in the evolution of plants and is particularly widespread in angiosperms Arabidopsis genome, three whole-genome duplications that have occurred in the past 350 million years have brought about a greater than 90% increase in the number of transcription factor, signal transduction and developmental genes A gene family is a group of similar genes resulting from wholesale or partial gene duplication; the size of a gene family reflects the number of duplicated genes  in each species Drosophila, Caenorhabditis elegans, yeasts and animals Arabidopsis, rice and maize, respectively. On the basis of sequence divergence, three Hsf classes  and several subgroups are currently recognized Here, we examine the evolution of the heat shock transcription factor (Hsf) family in legume species. Hsfs serve as the terminal components of signal transduction and are the central regulators of the expression of heat shock proteins and other heat shock-induced genes that confer thermotolerance to all eukaryotes Lotus japonicus (birdsfoot trefoil), Medicago truncatula (barrel medic), Cicer arietinum (chickpea), Glycine max (soybean), Cajanus cajan (pigeonpea) and Phaseolus vulgaris (common bean). The aim of this investigation was to determine which genes were derived from genome duplication, subsequently giving rise to paralogs, which genes descended from speciation events, giving rise to orthologs and which genes have undergone gene loss. In addition to examining the phylogeny of the Hsf family, we performed a comprehensive examination of the legume genome structure anchored by Hsf genes. Furthermore, we searched for gene microsynteny within and between the genomes of the legumes to investigate the evolutionary history of the Hsf regions. Our data show that extensive synteny remains in the homeologous regions within/between legume species. In addition, most Hsf regions can be traced to ancient papilionoid-specific whole-genome duplication or recent Glycine-specific whole-genome duplication. However, different rates of gene loss in the Hsf family have occurred along separate lineages of legume. Our results may help facilitate the extrapolation of Hsf gene function from one lineage to another.In this study, we analyzed the Hsf gene families from six papilionoid legume species for which substantial information about genomes or transcriptomes was available, namely L. japonicus (version 2.5) from the L. japonicus Genome Sequencing Project (http://www.kazusa.or.jp/lotus/), M. truncatula (version 3.5) from the M. truncatula Genome Sequencing Project (http://www.medicagohapmap.org/?genome), G. max (version 1.01) from the Soybean Genome Sequencing Project (http://www.phytozome.net/soybean.php), C. cajan (version 1.0) from the International Crops Research Institute for the Semi-Arid Tropics (http://www.icrisat.org/gt-bt/iipg/Genome_Manuscript.html), P. vulgaris (version 0.9) from the US Department of Energy Joint Genome Institute (http://www.phytozome.net/commonbean.php), P. patens (version 1.6) from the Joint Genome Institute (http://www.phytozome.net/physcomitrella), S. moellendorffii (version 1.0 filtered model 3) from the Joint Genome Institute (http://genome.jgi-psf.org/Selmo1/Selmo1.download.ftp.html) and C. reinhardtii (version 4.3) from the Joint Genome Institute (http://www.phytozome.net/chlamy.php). Although information about the whole genome of C. arietinum is not currently available, the C. arietinum transcriptome has been sequenced using next-generation sequence technology http://www.nipgr.res.in/ctdb.html). The downloaded nucleotide and protein sequences of each species were in turn used to build local databases using DNATOOLS software. Published Hsf protein sequences C. arietinum transcriptome, a TBLASTN search was performed, and the identified full-length cDNAs were translated in the correct frame. Only hits returning E-values of less than 0.001 were considered for further analysis. This step was crucial for finding as many similar sequences as possible. Moreover, on the basis of BLASTN search results in the genome databases using the predicted cDNA sequences of Hsf genes, information was obtained about the chromosome locations of these genes. Redundant sequences with different identification numbers and the same chromosome locus were eliminated from the data set. To confirm the presence of both Hsf-type DBD domain and HR-A/B regions in the sequences obtained, the predicted protein sequences of Hsf genes were analyzed in the Pfam HMM database and the SMART tool The most recent versions of genome, protein and cDNA sequences of each species were downloaded from the respective genome sequence sites as follows: In addition to the DBD and HR-A/B domains, many Hsfs also contain an NLS and an NES domain, and most plant class A Hsfs contain one or several AHA motifs. To identify the NLS domain in the Hsfs, the program PredictNLS Saccharomyces cerevisiae (ScHsf1), and the Hsfs of C. reinhardtii, S. moellendorffii and P. patens, were used as the outgroup. In order to analyze the classes and subgroups of the legume Hsf families, 25 maize Hsfs (ZmHsfs) Arabidopsis Hsfs (AtHsfs) Multiple sequence alignments using ClustalX (version 1.83) L. japonicus, M. truncatula, G. max and C. cajan. Tandem duplication is characterized by multiple gene family members occurring within either the same or neighboring genomic regions. Tandem duplicated genes were defined as genes in any gene pair, T1 and T2, that (1) belong to the same gene family, (2) are located within 100 kb each other, and (3) are separated by zero, one or fewer, five or fewer, or 10 or fewer nonhomologous (not in the same gene family as T1 and T2) spacer genes et al. et al. E-value <10−10) with a protein-coding gene neighboring another anchor point. When four or more such gene pairs with syntenic relationships were detected, the two regions were considered to have originated from a large-scale duplication event.To categorize the expansion of the Hsf gene families, the physical locations of all members of this family were examined in L. japonicus, M. truncatula, G. max and C. cajan were set as the anchor points according to their physical locations. The protein-coding sequences assigned to the flanking regions of each Hsf gene in one species were aligned with those in the other species by pairwise comparisons. A syntenic block is defined as the region in which three or more conserved homologs  were located within a 100 kb region between genomes The analysis of microsynteny across species was based on comparisons of the specific regions containing Hsf genes. Similarly, the Hsf genes of The duplicated gene pairs within each duplicated block were used to calculate Ks and to analyze Ka/Ks ratios. Protein sequences of the gene pairs were aligned using MUSCLE −9 substitutions per site per year. The divergence time (T) was calculated as T = Ks/(2×6.1×10−9) 10−6 Mya When dating large-scale duplication events, Ks can be used as the proxy for time. For each pair of duplicated regions, the mean Ks of the flanking conserved genes were calculated, and these values were then translated into divergence time in millions of years assuming a rate of 6.1×10Codeml program is available under PAML  V. 4.7 software L. japonicus acquired with the Lotus 52 K Affy chip were obtained from the Lotus Transcript Profiling Resource L. japonicus Genome Sequencing Project were used to query the corresponding probe set IDs in the GeneChip. The log transformed expression values for the retrieved probe sets were then used to perform cluster analysis by Cluster The data for evaluating Hsf gene expression in various tissues of L. japonicus plants (Miyakojima MG-20) were grown in a greenhouse at 25±2°C with a 14/10 h (light/dark) photoperiod. Four-week-old seedlings were prepared for abiotic stress treatments. For temperature treatments, the uniform-sized seedlings were transferred to the temperature-controlled growth chambers, which were maintained at 42±1°C for heat stress and at 4±1°C for cold stress. For oxidative stress, seedling leaves were sprayed with 10 mM H2O2 solution. After each treatment, the leaves of the seedlings were harvested at 0, 1 h and were immediately frozen in liquid nitrogen and kept at −80°C pending the extraction of RNA.β-tubulin was used as an endogenous control. The relative expression of each gene was calculated as the ΔΔCT value in comparison to unstressed samples . These experiments were independently replicated at least three times for each sample.Total RNA was extracted from the collected samples using Trizol reagent , followed by Dnase I digestion to remove residual genomic DNA contamination. The quality and quantity of the total RNA was measured by electrophoresis on 1% (w/v) agarose gels and examined with a NanoDrop ND-1000 UV-Vis spectrophotometer . For each sample, the first strand cDNA was reverse transcribed from 1 µg total RNA using the QuantiTect Rev. Transcription Kit  according to the manufacturer's instructions. Quantitative real-time PCR was conducted on an ABI PRISM 7300 real-time PCR system . All the gene-specific primer sequences for quantitative real-time PCR were designed by Primer Express Version 3.0 software  and are listed in L. japonicus, M. truncatula, G. max, C. cajan and P. vulgaris as well as transcriptome data for C. arietinum. A total of 11, 19 and 13 Hsfs were identified in the cool season legumes L. japonicus, M. truncatula and C. arietinum, respectively, while 46, 22 and 29 Hsfs were identified in the tropical season legumes G. max, C. cajan and P. vulgaris, respectively  and the Millettioid clade . To determine the number of full-length Hsf proteins in the six legumes, BLAST and HMM searches were performed against the annotated genomes of ectively . To obtaectively . Sequencectively . These dCombining the six legume protein sequences, we constructed a phylogenetic tree using neighbor-joining analysis . The legG. max was present compared with that from the other species. On the other hand, in each clade, the members of different species may have evolved from a common ancestral gene by divergence of the lineage. Therefore, the 18 defined clades provide a framework for inferring parologs and orthologs of Hsfs. The phylogenetic relationships based on the ML tree were largely consistent with these results , we considered these gene pairs to be conserved and defined these two regions as derived from a large-scale duplication event. We also defined a flexible set as a set of genes in which the flanking regions of an Hsf pair contained two or three conserved genes to avoid the possibility that pairs of Hsf genes resided within more divergent blocks.To investigate this possibility, we searched for gene similarity in the Hsfs flanking regions. If four or more of the 15 up- and downstream genes flanking two Hsf genes achieved a best non-self match using BLASTP  was found to have involved large-scale duplication. However, it should be noted that approximately 45% of the Hsf family could not be assigned to any chromosome. In M. truncatula, genes flanking both pairs, MtHsf-03/MtHsf-16 and MtHsf-11/MtHsf-19, were found to be conserved. In addition, two gene pairs (MtHsf-04/MtHsf-05 and MtHsf-08/MtHsf-09) were located near each other on chromosomes 2 and 4, and thus most likely resulted from tandem duplication. Moreover, we found that the DNA sequences for MtHsf-08 and MtHsf-09, as well as their four flanking genes (within an approximately 40-kb region) were completely identical to each other. In G. max, no tandem duplication was identified, but 42 out of 46 Hsf genes (approximately 91.3%) were arranged into duplicated chromosomal regions. These 42 genes were classified into 16 duplication groups; each group had two to four members that had conservation between their flanking genes  were judged according to the flexible set.We identified three conserved genes flanking the pair ng genes . In the G. max were divided into two groups (except for GmHsf-18 and GmHsf-38) based on the Ks values of paralogs flanking the Hsf pair  occur at a constant rate over time, the conserved flanking protein-coding genes were used to estimate the dates of the large-scale duplication events Hsf pair . The Ks Hsf pair , correspG. max Hsf duplicated network, when two duplicated genes from recent duplication could not be found simultaneously, we reasoned that a possible ancient gene loss event occurred. As shown in G. max lineage, ancient genome duplication should have produced at least 32 Hsf genes. However, eight of these lines lacked both copies, which would have been obtained from recent genome duplication, suggesting that approximately 25% of the ancient duplicates were lost over millions of years. Moreover, taking ancient gene losses into account, the number of G. max Hsf genes derived from recent genome duplication should be 50. Among these, eight pairs lost one copy of the gene, indicating that only about 16% of recent duplicates were lost. On the contrary, in L. japonicas, M. truncatula and C. cajan, only a few Hsf-containing segments could be matched in duplicated pairs. What is the origin of the remaining Hsf genes in these species?In the L. japonicus, M. truncatula, G. max and C. cajan  were present in the 17 orthologous groups of segments. A representative synteny diagram for three of these groups is shown in L. japonicas-M. truncatula-G. max-C. cajan microsynteny also allowed us to verify the 18 ancient gene lineages inferred from the phylogenetic analysis. These results demonstrate that there is a one-to-one correspondence between syntenic orthologous groups and ancient gene lineages, except for the ancient gene of clade 18 (class C Hsfs).To identify the evolutionary origins and orthologous relationships within the Hsf genes of legumes, Hsf family members were used as anchor genes to study the molecular history of the chromosomal regions in which they reside. Using a stepwise gene-by-gene reciprocal comparison of the regions hosting the Hsf genes, we observed strongly conserved microsynteny among these regions across C. cajan . After tC. cajan  A–Q. We L. japonicus, M. truncatula, G. max and C. cajan. Synteny quality was calculated as twice the number of matches divided by the total number of genes in both segments; this process discounts gene amplification but counts conservation of genes between species M. truncatula and G. max syntenic regions. The G. max and C. cajan comparison exhibited the highest conservation, 70.91%. These results support the orthology of the segment groups used in this study.Each orthologous group of segments includes between four and 24 orthologous groups of genes  with representation in at least two species. These groups are shown connected by black lines in L. japonicus, M. truncatula and C. cajan) and networks of duplicated regions in G. max, each anchored by the Hsf gene. Within an orthologous group, segments of different legume species are thought to have shared the ancient legume whole-genome duplication that occurred outside of the papilionoid lineage. In many groups, only one region of L. japonicus, M. truncatula and C. cajan was comparable to homoeologous regions of G. max, suggesting that one member of the Hsf gene pair produced from ancient genome duplication was lost in their ancestral lineages. For example, the MtHsf-17/LjHsf-07/CcHsf-09 anchored regions showed microsynteny with two G. max duplicated regions containing GmHsf-13/GmHsf-15 . The four G. max segments arose from two rounds of whole-genome duplication. Moreover, the Hsf orthologs were usually found in the syntenic regions of the three legume species, and there were no counterparts in one species of L. japonicus, M. truncatula or C. cajan, indicating that two Hsf copies produced from ancient genome duplication were lost in its ancestral lineage. We also uncovered four cases in which the regions containing Hsf orthologs was syntenic between only two legume species. MtHsf-12 were conserved with those of G. max (GmHsf-01/GmHsf-20), while the orthologs of MtHsf-12/GmHsf-01/GmHsf-20 were missing in L. japonicus and C. cajan  were lost in the L. japonicas Hsf family. Moreover, there were 11 orthologous groups with the single orthologous region in M. truncatula and four groups without sharing microsynteny with Hsf anchored regions in M. truncatula. This suggests that 19 out of 34 ancient duplicated genes (approximately 56%) were lost in the M. truncatula Hsf family. In C. cajan, all 12 mapped Hsf genes were found to possess conserved microsynteny among the species investigated, and Hsf orthologs were found in nine groups located in the single syntenic region compared with other legume species, but 10 other Hsfs in this species could not be localized on the genome, and their regions were not used for comparison. Therefore, the number of duplicated Hsfs that remain in C. cajan is uncertain.Because the 17 orthologous groups of Hsf-containing segments indicate that there are at least 17 Hsf genes in this ancestor, after ancient whole-genome duplication, 34 Hsf genes should have been produced in their progenitor. In nine orthologous groups, only one Hsf-containing region of G. max has been expanded by two genome duplications. To better understand the evolutionary constraints acting on this gene family, we measured the Ka/Ks ratios for 35 unambiguous pairs of Hsf paralogs  in the network of duplicated regions of G. max. The resulting pairwise comparison data showed that all the paralog pairs have Ka/Ks ratios <1  was higher than that of the early-duplicated Hsfs (0.25), but there was no significant difference between these ratios . Moreover, the variance of the Ka/Ks ratios for the recent-duplicated Hsfs (0.013) was not significantly different from that of the early-duplicated Hsfs . This indicates that the younger and older proteins in the Hsf family are under similarly stable evolutionary constraints, which supports the notion that this family is essential for the regulation of cellular processes in G. max.Almost the entire Hsf family of atios <1 , suggestG. max. Interestingly, all Ka/Ks values for 322 pairs of duplicated non-Hsf genes were lower than 1  between the duplicated regions containing Hsfs in r than 1 , clearlyG. max. As expected from the basic Ka/Ks analysis, sliding window analysis clearly showed that numerous sites/regions are under moderate to strong negative selection  were highly accumulated in both underground (nodule and root) and aerial  parts, but the expression level was higher in the underground parts  were expressed preferentially in stem and flower, and LjHsf-02 showed higher expression than LjHsf-10 , the genes predominantly expressed in leaves  in at least one of the stress conditions examined (LjHsf-04 and LjHsf-11) were significantly up-regulated by all three stresses. Three  were expressed at remarkably high levels in response to both heat and H2O2 stress. LjHsf-08 was induced by heat stress but was suppressed by H2O2 stress. LjHsf-07 was significantly down-regulated upon exposure to heat and H2O2 stresses. A few genes were primarily responsive to one stress treatment. LjHsf-05 and LjHsf-10 responded specifically to heat stress, while LjHsf-06 was distinctively up-regulated under H2O2 stress. In contrast, LjHsf-03 showed minor fluctuations during all three stresses. It is worth noting that five genes  were strongly heat-inducible in our experiments, suggesting that they could have important roles in the heat shock regulatory network.Quantitative real-time PCR analysis was then performed to evaluate the response of the examined . Among tL. japonicus, M. truncatula and C. arietinum, respectively, and 46, 22 and 29 Hsfs in the tropical season legumes G. max, C. cajan and P. vulgaris, respectively. Before reconstructing the gene gain/loss history of legume Hsf families, it is necessary to trace the Hsf genes in different legume genomes back to a common ancestor. Phylogenetic trees are quite informative for inferring the number of Hsfs in the most recent common ancestor of the six legume species analyzed in this study G. max are present in at least an extra copy compared with the other species, and the extra copy is very close to its potential paralog. These results are consistent with the well-documented fact that G. max has undergone an additional WGD not shared with by the other three species G. max vs. the other legumes, which indicates that differential gene loss events may have occurred in different species. Furthermore, all of the clades were confirmed by the following interspecies microsynteny analysis (except for the shared clade representing class C Hsfs), indicating most duplicates of class C Hsfs have been lost in legumes. When we compared the number of ancestral genes with those in the extant legumes species, it appeared as though the Hsf family had contracted in most of the cool season legumes and expanded in the tropical season legumes. For example, compared with the number of ancestral genes, the number of Hsf genes was nearly halved in L. japonicus but increased approximately 2.5-fold in G. max. These results most likely reflect the complex evolutionary history of the Hsf family in legume species.In this study, we identified 11, 19 and 13 Hsfs in the cool season legumes G. max, most Hsf genes were assigned to the duplicated segments, and these segments could be divided into two groups based on Ks values. The members of one group were supported with an average Ks value of 0.701, which corresponds to the second peak in L. japonicus, M. truncatula and C. cajan, a few Hsf-containing segments were matched in pairs. The Ks values for these pairs were included in MtHsf-03/MtHsf-16 (0.80) and MtHsf-11/MtHsf-19 (0.77). All of these Ks values from G. max and other legumes probably represent the same polyploidy that occurred in all members of Papilionoideae subfamily approximately 59 Mya. Thus, the rate of synonymous substitution in M. truncatula appears to be greater than that in G. max. In a previous study of Rxp regions, higher rates of synonymous substitution were also detected in M. truncatula compared with G. maxIn G. max is known to have undergone a second whole-genome duplication approximately 13 Mya; this duplication did not occur in L. japonicus, M. truncatula or other legume lineages. Two whole-genome duplications in the ancestor of G. max lead to the expectation of a maximum of four homoeologs in G. max genome G. max and formed two pairs of paralogs, accordingly. In some cases, these four homoeologous Hsf regions were retained. For instance, GmHsf-12, GmHsf-17, GmHsf-32 and GmHsf-36 are located in four homoeologous regions, respectively. More conserved flanking protein-coding genes were identified between the two paralogous Hsf-containing segments derived from the recent duplication event than those from the ancient duplication  and the two Hsf regions of M. truncatula (MtHsf-03/MtHsf-16) were found to have conserved microsynteny to the four Hsf regions of G. max Click here for additional data file.Figure S2HR-A/B region alignment of Hsf proteins from L. japonicus, M. truncatula, C. arietinum, G. max, C. cajan, P. vulgaris and three lower plants.(PDF)Click here for additional data file.Figure S3NJ phylogeny of A. thaliana, O. sativa, Z. mays, L. japonicus, M. truncatula, C. arietinum, G. max, C. cajan and P. vulgaris Hsf proteins.(PDF)Click here for additional data file.Figure S4ML phylogeny of L. japonicus, M. truncatula, C. arietinum, G. max, C. cajan and P. vulgaris Hsf proteins.(PDF)Click here for additional data file.Figure S5The full syntenic maps of chromosome regions containing Hsf genes across L. japonicus, M. truncatula, G. max and C. cajan genes.(PDF)Click here for additional data file.Figure S6Sliding window analysis employed to estimate selective pressures on 35 pairs of duplicated Hsf genes in G. max.(PDF)Click here for additional data file.Table S1Primers used in quantitative real-time PCR.(PDF)Click here for additional data file.Table S2Information about Hsfs in L. japonicus, M. truncatula, C. arietinum, G. max, C. cajan, P. vulgaris, S. moellendorffii, P. patens and C. reinhardtii.(PDF)Click here for additional data file.Table S3Domain and motif survey of legume and three lower plant Hsfs.(PDF)Click here for additional data file.Table S4Likelihood ratio tests and parameter estimations for the six site models based on the coding sequences of G. max Hsf genes.(PDF)Click here for additional data file.


Gracilaria vermiculophylla, a widespread non-native species in coastal estuaries of the Northern hemisphere. Forty-two loci were screened for amplification and polymorphism. Nine of these loci were polymorphic across four populations of the extant range with two to eleven alleles observed. Mean observed and expected heterozygosities ranged from 0.265 to 0.527 and 0.317 to 0.387, respectively. Overall, these markers will aid in the study of the invasive history of this seaweed and further studies on the population dynamics of this important haploid–diploid primary producer.Microsatellite loci are popular molecular markers due to their resolution in distinguishing individual genotypes. However, they have rarely been used to explore the population dynamics in species with biphasic life cycles in which both haploid and diploid stages develop into independent, functional organisms. We developed microsatellite loci for the haploid–diploid red seaweed  In the last decade, genetic approaches to answering evolutionary and ecological questions have become less expensive and more easily applied to non-model species . MicrosaWhile theory predicts that selection should favor either diploidy or haploidy , Hughes Asparagopsis spp.; Lygodium spp.; Campylopus introflexus; Understanding the consequences of biphasic life cycles and land- or seascape features on population structure is particularly relevant in light of the increasing frequency of biological introductions. There are numerous examples of widespread, and putatively invasive species, that have free-living haploid and diploid stages, including macroalgae  Papenfuss is native to the northwest Pacific and, in the last 30–40 years, has spread throughout high to medium salinity estuaries of the eastern North Pacific  from a single individual collected from Charleston, South Carolina, USA. For library preparation, DNA was extracted using CTAB . Samples were electrophoresed on an ABI 3130xL genetic analyzer equipped with 36 cm capillaries (Applied Biosystems). Alleles were scored manually using GENEMAPPER ver. 4 (Applied Biosystems) and allele sizes were binned with TANDEM ver. 1.08 software  in which approximately 1 cm of dried algal tissue was heated at 95 °C for 30 min and vortexed intermittently . Loci wesoftware .G. vermiculophylla (n = 28) and diploid tetrasporophytes (n = 30) collected at Elkhorn Slough, California, USA  statistics on allele size reveals a significant negative slope, then short allele dominance may be expected. We determined three to four allele size classes per locus and performed linear regressions using the STATS package in R  and expected heterozygosities (EH) were calculated using GenAlEx, ver. 6.501 .In summary, we have developed and characterized microsatellite markers for the haploid–diploid red seaweed 10.7717/peerj.1159/supp-1Figure S1Gracilaria vermiculophylla exhibits a complex haploid–diploid life cycle, including two free-living stages of different ploidy levels: the haploid dioecious gametophytes and the diploid tetrasporophytes. Fertilization takes place on the female gametophyte, and the female gametes, once fertilized, develop into cystocarps, an additional, though not a free-living, stage. The cystocarp is a macroscopic swelling on the surface of the female thallus, within which the zygote is mitotically copied producing thousands of diploid spores, called carpospores. Spores released from the cystocarps germinate into the diploid tetrasporophytes. (Photo credit: SA Krueger-Hadfield).Click here for additional data file.10.7717/peerj.1159/supp-2Table S1Ntall total number of alleles.Acc. No., GenBank accession number; locus; motif; primer sequences; Profile: one- or multi-locus genetic determinism, no amp. indicates non-amplification; Click here for additional data file.10.7717/peerj.1159/supp-3Table S2Frequencies were directly estimated in the haploid subpopulations, whereas frequencies in each of the diploid subpopulations at Akkeshi, Elkhorn Slough, Fort Johnson and Nordstrand were calculated using maximum likelihood and the software MLNullFreq .Click here for additional data file.10.7717/peerj.1159/supp-4Table S3No. of classes, and linear regression statistics. Loci Gverm_10367 and Gverm_2790 only exhibited two alleles in our sampled populations and consequently, short allele dominance analysis was not applicable (NA).This includes number of pooled size classes used in regression analysis .Darkened cells indicate pairs of loci that show significant linkage disequilibrium after Bonferroni correction ; mean allelic richness, EA, based on the smallest global sample size of 46 alleles  + SE; mean observed heterozygosity, OH, + SE; mean expected heterozygosity, EH, + SE.This includes: number of alleles at each locus, Click here for additional data file.10.7717/peerj.1159/supp-7Supplemental Information 1Click here for additional data file.