Hesperophycus and Pelvetiopsis), for which preliminary molecular tests revealed unexpected conflicts consistent with unrecognized cryptic diversity and hybridization.Molecular markers are revealing a much more diverse and evolutionarily complex picture of marine biodiversity than previously anticipated. Cryptic and/or endemic marine species are continually being found throughout the world oceans, predominantly in inconspicuous tropical groups but also in larger, canopy-forming taxa from well studied temperate regions. Interspecific hybridization has also been found to be prevalent in many marine groups, for instance within dense congeneric assemblages, with introgressive gene-flow being the most common outcome. Here, using a congeneric phylogeographic approach, we investigated two monotypic and geographically complementary sister genera of north-east Pacific intertidal seaweeds  and P. hybrida sp. nov. The congruence between the genotypic clusters and the mtDNA clades was absolute. Fixed heterozygosity was apparent in a high proportion of loci in P. limitata and P. hybrida, with genetic analyses showing that the latter was composed of both H. californicus and P. arborescens genomes. All four inferred species could be distinguished based on their general morphology.The three recovered mtDNA clades did not match a priori species delimitations. Hesperophycus/Pelvetiopsis, including the validity of the much endangered, modern climatic relict P. arborescens, and the identification of a new, stable allopolyploid species (P. hybrida) with clearly discernable ancestry , morphology, and geographical distribution. Allopolyploid speciation is otherwise completely unknown in brown seaweeds, and its unique occurrence within this genus (P. limitata possibly representing a second example) remains enigmatic. The taxonomic separation of Hesperophycus and Pelvetiopsis is not supported and the genera should be synonymized; we retain only the latter. The transitional coastline between Point Conception and Monterey Bay represented a diversity hotspot for the genus and the likely sites of extraordinary evolutionary events of allopolyploid speciation at sympatric range contact zones. This study pinpoints how much diversity (and evolutionary processes) potentially remains undiscovered even on a conspicuous seaweed genus from the well-studied Californian intertidal shores let alone in other, less studied marine groups and regions/depths.This study confirmed additional diversity and reticulation within NE Pacific The online version of this article (doi:10.1186/s12862-017-0878-2) contains supplementary material, which is available to authorized users. Phy. Phy26])Hesperophycus californicus and Pelvetiopsis limitata are the extant members of two monotypic genera of canopy-forming fucoid seaweeds  endemic to the temperate rocky-shores of the north-east Pacific. Both occur in mid/high intertidal assemblages, although with complementary biogeographical distributions. Warm-temperate H. californicus is present from Punta Eugenia (Mexico) to Monterey Bay , whereas cold-temperate P. limitata occurs from Point Conception  to Vancouver Island . A third putative species, P. arborescens, was described from just south of Monterey [Fucus [Fucus has been the focus of much physiological, ecological, and phylogeographical research [Hesperophycus-Pelvetiopsis have received considerably less attention, but preliminary tests for newly developed molecular markers immediately revealed multiple taxonomic and phylogenetic conflicts consistent with cryptic diversity and reticulate evolution.Monterey , but hasy [Fucus , a much research –43, but research , 44, 45.Pelvetiopsis) with four species, adding to this complex two narrow-endemic, pseudo-cryptic species from California (P. arborescens and P. hybrida sp.nov.), the latter of recent allopolyploid origin, a process so far unknown in brown seaweeds.This study aimed to identify all genetic entities present within this complex, and to describe their phylogenetic relationships, morphological differences and biogeographical ranges. We reveal a single genus  and Hesperophycus californicus was sampled along the entire ranges of these species in the NE Pacific. Preliminary results showed that P. arborescens and a fourth entity were genetically distinct, so additional populations  were sampled to include all the morphological and genetic diversity present in the complex. Whole individuals were also sampled for descriptions of the general morphology of each recovered genetic entity. These were collected at the sites where they had been genetically identified and morphometric measurements, photographs and herbarium pressings were made. In total, 36 population samples containing 16 individuals  from 30 sites spanning Punta Baja  to Cape Meares  were analysed , and diluted 1:100.A comprehensive panel of populations of Fucus, and some propensity to cross species barriers [Hesperophycus-Pelvetiopsis , as this marker was previously shown to provide good inter- and intra-specific resolution in the sister genus barriers , 31, 44.barriers  were usede novo for P. limitata. These were identified in silico by screening with MsatCommander [http://www.biocant.pt/) using shotgun 454 pyrosequencing. From these, 13 loci were selected to produce multi-locus genotypes for all Hesperophycus-Pelvetiopsis individuals . Microsatellite alleles were manually scored in STRand [Amplified fragments were analyzed in an ABI PRISM 3130xl automated capillary sequencer (Applied Biosystems) at CCMAR, Portugal. Amplicons were pre-cleaned with ExoSap  and sequences were aligned, proofread, and edited in GENEIOUS 4.8  using the Median-Joining (MJ) algorithm [6 generations, sampling trees and parameters every 102 generations. The number of substitution rates, among-site rate variation, and base frequency priors were set according to the substitution model selected, leaving the remaining options as default. Average standard deviation of split frequencies between runs and cold chains Log-likelihood stationarity were inspected to assess inter-run convergence and run length sufficiency, respectively. Based on the latter, 105 generations (1000 trees) were discarded as burn-in. The remaining 18 000 trees sampled were used to produce 50% majority-rule consensus trees and to calculate branch posterior probabilities. Maximum likelihood analyses were performed with PhyML 3 [http://www.atgc-montpellier.fr/phyml/). Nodal support was calculated using 1000 bootstraps. Trees were rooted with Fucus spp. Inter-specific divergence within Pelvetiopsis and within the two major lineages of Fucus was compared by estimating Kimura’s two parameter (K2P) distances between ancestral haplotypes defining each recovered species.Haplotype/sequence networks were constructed in Network 4 (lgorithm . Phylogelgorithm  and the lgorithm . Two par PhyML 3  using thA), Nei’s gene diversity (HE), observed heterozygosity (HO), and inbreeding coefficients (FIS) were calculated with Genetix 4 [Summary statistics of the microsatellite genetic diversity, including microsatellite allele frequencies, mean allelic richness , 2) P. arborescens, and 3) a new, undescribed cryptic species, P. hybrida sp. nov., genealogically closer to H. californicus. MtDNA clades were well resolved  but resolution was insufficient to establish the sequence of cladogenesis  and 0.122 , the same magnitude of that found between the two major lineages of Fucus  were recovered. The mtIGS recovered three main clades that partially conflicted with a priori species delimitations. H. californicus and P. arborescens typically exhibited homozygote excess  and P. hybrida (8/10)  in all their populations  was noticeable at loci Pl41 and Pl27 , or when amplification failed in one of them (Pl51). In P. limitata, alleles were shared with H. californicus or P. arborescens, or were species-specific. The congruence between the recovered genotypic clusters and the mtDNA clades was absolute  between H. californicus and P. arborescens. Contrastingly, allele states didn’t reveal any obvious genealogic relationship between P. limitata and its extant congeners.Patterns of microsatellite cross-amplification and polymorphism varied markedly between loci, with four  failing to produce clear products in at least one species, and four  revealing inter-specific differences only  distributions centered in the Monterey (Big Sur) and San Luis Obispo counties, respectively. The first was mostly found in the Carmel area, with the southernmost population  representing an isolated collection. P. hybrida was also found in remote San Miguel Island, and its distribution in other Channel Islands remains a possibility. No species were collected on the mainland between Mexico and Point Conception.ogy Fig. . A combiogy Fig. . The larogy Fig. . The souHesperophycus-Pelvetiopsis, notably the occurrence of 1) more genetic entities than the two initially recognized species, 2) species/gene conflicts, and 3) stable, genome-wide heterozygosity in two entities. Such conflicts often reflect incorrect taxonomy (e.g. cryptic species) or natural evolutionary processes such as incomplete lineage sorting and introgressive hybridization, among others [Our analyses revealed multiple taxonomic, phylogenetic and genomic inconsistencies within g others . Here, bH. californicus, genetic data clearly supported multiple entities within P. limitata sensu lato. One of these undoubtedly corresponds to the previously described P. arborescens, as confirmed by its characteristic morphology and occurrence at the type locality . All four entities corresponded to genetically discontinuous clusters that were found a posteriori to further exhibit distinctive morphological characteristics. All combinations of species pairs (with the exception of P. arborescens with P. hybrida) were found locally co-occurring in at least a few sites between Point Conception and Monterey Bay . Examples include P. limitata, P. arborescens and H. californicus all along the Monterey peninsula, P. hybrida and H. californicus throughout San Luis Obispo County, and P. limitata and P. hybrida in the same county around Hazards (Montaña de Oro State Park). Despite close proximity, local ongoing hybridization between species was never detected. Given their genetic and morphological integrity in sympatry, these four entities  clearly correspond to good biological species.In contrast with t Lobos, ). AnotheHesperophycus-Pelvetiopsis. Specifically, P. hybrida (and to a lesser extent P. limitata) exhibited fixed heterozygosity in a high proportion of microsatellite loci, with allele states clearly indicating the contribution of H. californicus and P. arborescens genomes. Genomic heterozygosity in sexually reproducing organisms is not expected beyond F1 hybrids, because independent (Mendelian) segregation of chromosomes and successive generations of mating necessarily generate a proportion of homozygotes while pulling genotypic frequencies of populations towards Hardy-Weinberg equilibrium. In selfing species and/or with very restricted gametic dispersal, inbreeding would actually promote an excess of homozygotes, as observed here in H. californicus and P. arborescens, and in other fucoids sharing similar life-history and reproductive traits [P. hybrida cannot result from the recurrent formation of F1 hybrids, since currently it doesn’t overlap its distribution with P. arborescens, one of its inferred ancestors. A clonal hybrid swarm is also unlikely because P. hybrida produces fully functional sexual gametes (pers. observ.) and also because vegetative propagation by Fucaceae has never been reported in intertidal wave-swept habitats. Clonal lineages are present only in sheltered environments such as salt-marshes, estuarine mudflats and land-locked bays, and exhibit distinctive morphologies characterized by dwarf and curly habit, vegetative proliferation and lack (or nearly so) of reproductive structures and anchoring holdfasts . Att. AttHespldfasts  in at least one locus indicates that most homeolog loci actually occur, as expected in low-dispersal selfing species, in the homozygous state.Under sexual reproduction (including selfing), only allopolyploidy can account for the generationally stable, genome-wide hybridity of  bridges , althougH. californicus and a male P. arborescens as the diploid ancestors of P. hybrida. The small mtDNA divergence and the (mainly) identical microsatellite allele states further argue for a relatively recent evolutionary origin. However, we show that mtDNA haplotypes are no longer shared between P. hybrida and H. californicus, and that the modern ranges of P. hybrida and P. arborescens do not overlap. Complete mtDNA lineage sorting and range shifts can occur on relatively short time-scales , buP. limitata had also an allopolyploid origin is striking because currently this cold-temperate species shows the widest latitudinal span of all recognized species (ca. 14°), and is the only species occurring north of Monterey Bay. By comparison, P. hybrida showed a much more restricted range in mainland California and in San Miguel Island (Northern Channel Islands). Low-dispersal organisms might take some time to migrate and colonize new habitats. It can be hypothesized that this rather recent species is still in the process of equilibrating with its climatic niche . Remarkably, its inferred paternal ancestor, P. arborescens, was only found in an even narrower, non-overlapping stretch of coastline spanning about 0.7° in latitude (<130 km). This degree of endemism is rare in temperate marine species, and in related fucoids is normally associated with newly emerged, extreme habitats . Notwithstanding its dynamic evolutionary history, currently this species may be better described as an extremely vulnerable climatic relict [H. californicus, also exhibited a fairly wide latitudinal range, although south of Point Conception this was very discontinuous and mostly restricted to offshore islands (e.g. Northern and Southern Channel Islands) and prominent capes (e.g Punta Baja) [The hypothesis that tic Sea, ) and/or tic Sea, ). Withinc relict . Finallyta Baja) , 69. ThiPelvetiopsis illustrates how much cryptic diversity may be hiding within more problematic groups and in more remote areas and depths. Remarkably, all Pelvetiopsis were present along the transitional coastline between Point Conception and Monterey Bay, either as relict (P. arborescens) or evolutionarily recent (P. hybrida) endemics, or, for more widespread species, overlapping the southernmost (cold-temperate P. limitata) and northernmost  parts of their ranges. This modern hotspot of biodiversity, refugial persistence and evolutionary opportunity probably results from its relatively stable climatic history and high spatial habitat heterogeneity. The narrow endemic species nevertheless raise conservation concerns and deserve finer detail monitoring, particularly P. arborescens. Its restricted range, rarity and ecologically marginal vertical distribution make it particularly vulnerable to demographic stochasticity and to extreme El Niño events and ongoing climatic changes.The rich coastal biota of California, including its luxuriant seaweed flora, has been extensively surveyed in the past decades; the intertidal fucoid assemblages in particular are conspicuous and easily accessible for amateurs and trained phycologists, and are routinely included in coastal monitoring programs (e.g MARINe consortium). The discovery of a new Hesperophycus and Pelvetiopsis, and a new combination for Hesperophycus californicus  is therefore proposed. In addition to the formal description of the allotetraploid P. hybrida, the practical recognition of rare P. arborescens is also warranted.The identification of three phylogenetic clades comprising four species (one of hybrid origin) calls for a taxonomic revision of the complex Table . SpecifiPelvetiopsis is certainly remarkable, but the allopolyploid nature of P. hybrida is truly unexpected and completely original both from a taxonomic (brown seaweeds) and ecological  perspective. Variance in genome size estimates in seaweeds have been attributed to palaeo-polyploidization events [P. hybrida, suspected Macrocystis x Pelagophycus allopolyploids could not be confirmed genetically based on this benchmark , some of which also display some propensity to hybridize. For instance, in the closely related and well-studied genus Fucus, hybridization between related species has been frequent and ongoing but the primary evolutionary consequence has only been introgressive gene-flow [The recognition of two narrow-endemic, pseudo-cryptic species within n events , 70, incn events , but dirn events , 72 and n events . Autopoln events , but notn events ). For invised in ). Surpriene-flow –31.Pelvetiopsis evolution while virtually unknown across its entire class? Globally, whole genome duplication appears to require relatively stringent conditions; hybridization for instance is taxonomically widespread and frequent [Fucus, chemotactic attraction of sperm by eggs is cross-specific [F. serratus x female (eggs) F. evanescens, presumably because sperm allocation is much higher in dioecious vs hermaphrodite species [P. hybrida on the other hand involved the interaction of two divergent self-compatible hermaphroditic species. This is unlikely to be a coincidence. Allopolyploids seem to originate more frequently when crosses involve genomically divergent species ([Pelvetiopsis vs intra-lineage (most likely homoploid) introgression in sister Fucus. It does not really offer, however, a general explanation for why allopolyploidy is otherwise so rare among brown seaweeds.Why is allopolyploidy featuring in recent frequent , whereasfrequent  and a fefrequent  but infrfrequent , 79, 80.frequent . In relaspecific , and thespecific ). Yet, hspecific , 30. For species . Speciatspecies  throughout much of its distribution. Niche differentiation is acknowledged as a pre-requisite, or at least facilitator, allowing initial establishment and long-term survival of viable  neopolyploids as stable species, by alleviating potential competition with longer-established diploid ancestors [Fucus, for instance, the integrity and vertical distribution of closely related species is maintained along strong vertical gradients by small differences in stress tolerance, even in the face of moderate gene-flow [Pelvetiopsis stands occurring between Point Conception and Monterey. The average vertical ranges of P. hybrida and P. arborescens were always above those of H. californicus and P. limitata where they co-occurred (unpublished data). Likewise, very local (e.g. boulder level) patterns of distribution suggest an important role for wave action in limiting their distributions. Whether these ecological differences are among the immediate effects of allopolyploidization is unclear, since they cannot be separated from other evolutionary processes  driving subsequent niche divergence [It should be noted that ncestors –90. Bothncestors . The impene-flow . Similarvergence .P. hybrida from its extant ancestors, this species is particularly suited to investigate how divergent parental genomes are transferred and functionally integrated in a new species and might contribute to adaptive diversification. The rocky intertidal zone provides one of the best natural laboratories for examining the relationships between abiotic constraints, biotic interactions, and ecological patterns in nature [Because of the very limited genomic differentiation of n nature . A few sPelvetiopsis than previously anticipated. In addition to the confirmation of the Big Sur endemic P. arborescens, molecular data allowed for the first time the identification of a stable allopolyploid marine species (P. hybrida) with clearly discernable phylogenetic relationships , morphology, and geographical distribution. Ongoing analyses of nuclear sequence data support our inferences regarding P. hybrida, and are consistent with the hypothesis of P. limitata corresponding to a second, cryptic paleopolyploid. Allopolyploid speciation remains otherwise unknown in brown seaweeds, and until some ecological, biological or reproductive originality is identified, its unique occurrence within Pelvetiopsis will remain enigmatic. The diversity hotspot between Point Conception and Monterey Bay likely reflects its relatively stable climatic history and its regionally high habitat heterogeneity. This conspicuous seaweed genus from the well-studied Californian intertidal illustrates how much diversity (and evolutionary processes) potentially remains undiscovered in other (less studied) marine groups and regions.This study revealed a much more diverse and evolutionarily complex picture of NE Pacific


Helwingia himalaica, the first in the distinctive family Helwingiaceae and only the second genus to be sequenced in the order Aquifoliales. We then combine this with 36 published sequences in the large  subclass Campanulidae in order to investigate relationships at the order and family levels. The Helwingia genome consists of 158,362 bp containing a pair of inverted repeat (IR) regions of 25,996 bp separated by a large single-copy (LSC) region and a small single-copy (SSC) region which are 87,810 and 18,560 bp, respectively. There are 142 known genes, including 94 protein-coding genes, eight ribosomal RNA genes, and 40 tRNA genes. The topology of the phylogenetic relationships between Apiales, Asterales, and Dipsacales differed between analyses based on complete genome sequences and on 36 shared protein-coding genes, showing that further studies of campanulid phylogeny are needed.Complete chloroplast genome sequences have been very useful for understanding phylogenetic relationships in angiosperms at the family level and above, but there are currently large gaps in coverage. We report the chloroplast genome for  Complete chloroplast genome sequences (plastomes) have been very useful for understanding phylogenetic relationships in angiosperms at the family level and above, and have been used to resolve previously recalcitrant nodes . HoweverHelwingia is the only genus in the campanulid family Helwingiaceae. It includes four species distributed in eastern Asia, from the Himalayas to Japan: Helwingia chinensis Batalin, H. himalaica Hook. f. & Thomson ex C.B. Clarke, Helwingia japonica (Thunb.) F. Dietr., and Helwingia omeiensis (W.P. Fang) H. Hara & S. Kurosawa . The specimen has been deposited in the herbarium of the Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences (HITBC). Total genomic DNA was extracted from fresh leaves using a modified CTAB method (B method . Each amB method .http://www.clcbio.com) by the de novo method using a k-mer of 63 and a minimum contig length of 1 kb. Outputted contigs were aligned with the chloroplast genome of the asterid Camellia yunnanensis (GenBank accession number: KF156838), which was the most similar genome identified via BLAST (http://blast.ncbi.nlm.nih.gov/), and ordered according to the reference genome. Genes in the assembled chloroplast genome were predicted using Dual Organellar GenoMe Annotator (DOGMA)  . The chlKX434807). REPuter was used to detect and assess repeats, including forward match, reverse match, complement match, and palindromic match repeats (http://www.rub.de/spezzoo/cm/cm_phobos.htm). Mauve v. 2.4.0 was used for determining the chloroplast genome rearrangements among the campanulid families   , was aliGenBank)  and manuhttp://people.sc.fsu.edu/~dswofford/paup_test/), using the heuristic searches with tree bisection-reconnection (TBR) branch swapping and the ‘Multrees’ option in effect. Bootstrap analysis was conducted with 1,000 replicates with TBR branch swapping.ML analyses were conducted in RAxML version 8.2.8 , using thttp://people.sc.fsu.edu/~dswofford/paup_test/), using the heuristic searches with TBR branch swapping and the ‘Multrees’ option in effect. Bootstrap analysis was conducted with 1,000 replicates with TBR branch swapping.In addition, 36 protein-coding genes  shared aycf). In addition, 40 genes (26 unique) encode for tRNAs and eight genes for rRNAs  have one intron and two genes have two introns .The total length of the chloroplast genome is 158,362 bp. Its quadripartite structure includes an LSC with 87,810 bp and SSC with 18,560 bp, separated by a pair of IR regions with lengths of 25,996 bp . The GC or rRNAs . All eigThirty repeated sequences were detected, with lengths ranging from 18 to 43 bp and sequence identity more than 90% . Among tAngelica acutiloba, Foeniculum vulgare, Ostericum grosseserratum and Petroselinum crispum) was contractive (Parthenium argentatum), only 36 coding genes were shared across all the campanulid families (P. argentatum) to 44 (Lactuca sativa and Trachelium caeruleum), while the number of rRNA was usually eight  is similar to that of Ilex  (see Helwingia (94 genes) has two fewer protein coding genes than Ilex (96) while both have the same number of tRNA (40) and rRNA (eight) genes.The only other published chloroplast genomes in the order Aquifoliales are for seven species of oliaceae . The len bp) see . HelwingH. himalaica from the others. Both H. himalaica and Helianthus annuus have ycf15 (Guizotia abyssinica does not (H. himalaica and L. sativa have trnE (H. annuus and G. abyssinica do not. Gene rps16 has one intron in G. abyssinica, H. annuus, and L. sativa, but none in H. himalaica, while gene ycf3 has two introns in H. himalaica, G. abyssinica, and H. annuus, but none in L. sativa. Gene ycf15 is between ycf2 and trnL-CAA in H. himalaica, but between rps7 and trnV-GAC in H. annuus. Gene ndhF is in the IRb-SSC junction in both L. sativa and H. himalaica, but in the SSC-IRa junction in G. abyssinica and H. annuus. The lengths of the published chloroplast genomes for the Campanulaceae range from 162,321 bp (T. caeruleum, Adenophora remotiflora, Minor differences among the published chloroplast genomes are common, including gene loss or gain events, but these do not separate ve ycf15 , but Guidoes not . H. himaave trnE  but H. aH. himalaica are potentially useful for studies of the evolutionary history of populations (Helwingia is similar to other published studies (Helwingia and related genera in the Aquifoliales.The many mononucleotide SSRs identified in ulations . The dom studies . It has  studies . Approxi studies , showingT. caeruleum, and inferred as the effects of recombination of repeats or tRNA genes (Arbutus unedo (Vaccinium macrocarpon (Massive rearrangements in the chloroplast genome have been identified in the Campanulaceae in comparison with other campanulid families . Except NA genes . Comparius unedo , Geraniaus unedo , Vaccinirocarpon  and cuprrocarpon . HoweverThe phylogenetic trees based on complete chloroplast genomes are incongruent with those from the protein-coding genes. Aquifoliales are basal in all phylogenetic analyses, but the phylogenetic relationships among the Asterales, Apiales and Dipsacales differ in different analyses  and 4. TH. himalaica as the first in the Helwingiaceae and the second genus in the Aquifoliales. It has the typical quadripartite circular structure, including an LSC with 87,810 bp and an SSC with 18,560 bp, separated by a pair of IR regions with 25,996 bp. In total, 142 genes were detected in this genome, consisting of 94 protein-coding genes, 40 tRNA, and eight rRNA. Repeated sequences are mainly distributed in intergenic regions. Comparisons among the available chloroplast genomes within the campanulids reveal massive chloroplast gene rearrangement in the Campanulaceae. The phylogenetic relationships among Apiales, Asterales and Dipsacales were incongruent between phylogenetic results produced from complete chloroplast genomes and the 36 shared protein-coding genes. The topology within Asteraceae also varied, which shows that further studies are still needed in these three orders. The results of this study will facilitate understanding of not only the family Helwingiaceae and its relationships with other taxa in the Aquifoliales, but also phylogenetic relationships within the angiosperms at higher levels.We report the chloroplast genome of 10.7717/peerj.2734/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.2734/supp-2Supplemental Information 2Click here for additional data file.


Pectobacterium carotovorum subsp. actinidiae is the causal agent of summer canker in kiwifruit plants in South Korea. We report here the draft genome sequences of two P. carotovorum subsp. actinidiae strains, ICMP 19971 and ICMP 19972, which were originally isolated from Actinidia chinensis with symptoms of summer canker. These genome sequences will aid in the identification of genetic traits associated with their unusual capacity to cause canker and help understanding of the threat these exotic enterobacteria pose to the New Zealand kiwifruit industry. Pectobacterium carotovorum subsp. actinidiae has been reported as the causal agent of canker-like symptoms of kiwifruit species Actinidia chinensis in South Korea  to cause disease and not producing the leaf spots characteristic of P. syringae pv. actinidiae (P. carotovorum subsp. actinidiae ICMP 19971 (OHJ3) and ICMP 19972 (SYJ3), isolated from yellow kiwifruit Actinidia chinensis cv. Hort 16A cane cankers extruding red rust ooze in South Korea (http://www.landcareresearch.co.nz/resources/collections/icmp).th Korea . Canker orldwide . Howevertinidiae . We annoP. carotovorum subsp. actinidiae genomes with the genomes of P. carotovorum subsp. actinidiae ICMP 19970 (KKH3) (accession no. NZ_JRMH00000000) and other pectobacteria, such as P. carotovorum subsp. brasiliensis, P. carotovorum subsp. carotovorum, Pectobacterium atrosepticum, and Pectobacterium wasabiae, will enable the taxonomic status of P. carotovorum subsp. actinidiae to be verified. Comparative analysis will also permit the identification of genetic traits associated with the capacity of P. carotovorum subsp. actinidiae to cause canker symptoms in kiwifruit plants, which could subsequently be used for risk-based diagnostics and risk assessments of similar exotic organisms at the New Zealand border.A comparison of the newly acquired P. carotovorum subsp. actinidiae strains ICMP 19971 and ICMP 19972 were incubated overnight in lysogeny broth medium at 28°C. Genomic DNA was isolated using a DNeasy blood and tissue kit (Qiagen). DNA samples of good quality were sequenced to generate 100-bp Illumina paired-end reads using an Illumina HiSeq 4000 system . The quality of the sequence reads was checked using FastQC , and low-quality reads (score of <Q30) were trimmed using Fastq-Mcf .Pure cultures of ion 2.21 , 6 to geP. carotovorum subsp. actinidiae strains and other bacteria that cause diseases of woody plants, suggesting they may be required for pathogenicity of P. carotovorum subsp. actinidiae on kiwifruit. A detailed assessment of the genetic traits shared by ICMP 19971, ICMP 19972, and the previously sequenced P. carotovorum subsp. actinidiae strain ICMP 19970 will follow.The estimated draft genome sizes for ICMP 19971 and ICMP 19972 were 4,891,911 and 4,886,946 bp, respectively. They comprised 50 and 49 scaffolds, respectively, with both genomes having a 51.53% G+C content. The PGAP annotations for each genome predicted 4,171 coding sequences for ICMP 19971 and 4,201 coding sequences for ICMP 19972. A number of loci were identified as common to the two MPUI00000000 and MPUJ00000000, respectively. The versions described in this paper are the first versions.The draft genome sequences of ICMP 19971 and ICMP 19972 are available in the GenBank database under accession numbers


Plasmodium knowlesi and Plasmodium cynomolgi are two malaria parasites naturally transmissible between humans and wild macaque through mosquito vectors, while Plasmodium inui can be experimentally transmitted from macaques to humans. One of their major natural hosts, the long-tailed macaque (Macaca fascicularis), is host to two other species of Plasmodium (Plasmodium fieldi and Plasmodium coatneyi) and is widely distributed in Southeast Asia. This study aims to determine the distribution of wild macaques infected with malarial parasites by examining samples derived from seven populations in five countries across Southeast Asia.Plasmodium knowlesi, P. cynomolgi, P. coatneyi, P. inui and P. fieldi, were detected using nested PCR assays in DNA samples from 276 wild-caught long-tailed macaques. These samples had been derived from macaques captured at seven locations, two each in the Philippines (n = 68) and Indonesia (n = 70), and one each in Cambodia (n = 54), Singapore (n = 40) and Laos (n = 44). The results were compared with previous studies of malaria parasites in long-tailed macaques from other locations in Southeast Asia. Fisher exact test and Chi square test were used to examine the geographic bias of the distribution of Plasmodium species in the macaque populations.Plasmodium-positive, with P. cynomolgi being the most common and widely distributed among all long-tailed macaque populations (53.3 %) and occurring in all populations examined, followed by P. coatneyi (20.4 %), P. inui (12.3 %), P. fieldi (3.4 %) and P. knowlesi (0.4 %). One P. knowlesi infection was detected in a macaque from Laos, representing the first documented case of P. knowlesi in wildlife in Laos. Chi square test showed three of the five parasites  with significant bias in prevalence towards macaques from Malaysian Borneo, Cambodia, and Southern Sumatra, respectively.Out of 276 samples tested, 177 were P. knowlesi infection in Laos, and the high prevalence of P. cynomolgi infections in wild macaques in general, indicate the strong need of public advocacy in related countries.The prevalence of malaria parasites, including those that are transmissible to humans, varied among all sampled regional populations of long-tailed macaques in Southeast Asia. The new discovery of  Plasmodium species [Plasmodium species: Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, and Plasmodium ovale [Plasmodium that typically infect wild macaques in Southeast Asia had been known to be transmissible to humans. This was first demonstrated by passage with blood infected with Plasmodium knowlesi [Plasmodium cynomolgi [Plasmodium inui [P. cynomolgi through mosquito bites were reported in 1960 [P. cynomolgi [P. inui [Most malaria parasites are considered to be host-specific, in that most of them infect only one host species, though a single host can be infected by multiple  species . Humans um ovale . Since 1knowlesi , and latynomolgi  and Plasium inui . Further in 1960 , 6 and 1 in 1960 , and alsynomolgi , 6, 8 anP. knowlesi using nested PCR assays [P. cynomolgi, was reported to have naturally infected a woman in Peninsular Malaysia [In 1965, the first case of a human naturally infected with knowlesi malaria was reported  and humaR assays . AdditioR assays , and in R assays . In 2014P. knowlesi and P. cynomolgi are long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (Macaca nemestrina) [Macaca mulatta) [The natural hosts of estrina) . Long-tamulatta) . Their nmulatta) .P. knowlesi and P. cynomolgi, long-tailed macaques are also natural hosts of three other Plasmodium species, namely P. coatneyi, P. fieldi and P. inui [P. knowlesi in Thailand, Peninsular Malaysia and Indonesia [Plasmodium mitochondrial genome in Malaysian Borneo [Plasmodium infections in long-tailed macaques is necessary to determine their potential as a public health threat. The present study utilizes sensitive molecular detection methods, and characterizes the prevalence of five Plasmodium species in seven long-tailed macaque populations across the geographic range of these non-human primates in Southeast Asia.Other than  P. inui , 17. The P. inui –20. Othendonesia –25, or bn Borneo . A thoroDNA samples of 276 wild-caught long-tailed macaques from seven populations in Southeast Asian countries were employed for this study. These samples had been collected for genetic studies that were reported elsewhere . These iPlasmodium species were used to amplify DNA from macaque blood samples [Plasmodium-specific primers and those that were positive were subsequently examined with species-specific primers. The PCR parameters and primers used to detect Plasmodium, P. knowlesi, P. cynomolgi, P. inui, P. fieldi and P. coatneyi have been described previously [P. knowlesi, P. cynomolgi, P. inui, P. fieldi and P. coatneyi were used as positive controls and were provided by the Malaria Research Centre at Universiti Malaysia Sarawak [Plasmodium infections of the samples were compared with published data in Plasmodium infections from wild long-tailed macaques in Sarawak, Malaysian Borneo, and multiple sites from Peninsular Malaysia, Thailand, Singapore and Indonesia [Malaria parasites have distinct small subunit ribosomal RNA (SSU rRNA) genes that are developmentally regulated . Nested  samples . Sampleseviously , 32. Pla Sarawak . The ampndonesia –26.P. knowlesi-specific primers, was sequenced using the Sanger dideoxy sequencing method [One of the PCR amplified fragments, generated from a macaque from Laos with g method  and analg method  with KimPlasmodium species is geographically biased, Fisher exact test was performed on software R (version 3.2.4) on the distribution table. The null hypothesis under the Fisher exact test is, “prevalence and region are independent, i.e., prevalence of malaria parasite does not change as geographic location changes”. For the test, 107 random tables with the same column and row totals as the observed data, assuming row and column independence, were generated. The P value cited is the sum of the probabilities of all generated tables that have individual probabilities smaller than those in the observed table. To compare each observed value with its expected value under null hypothesis, Chi square tests were performed in R on the observed table to generate each expected value under the null hypothesis.To determine whether the prevalence of each Plasmodium DNA using nested PCR assays, 177 were found to be positive  and most widespread distribution among macaques across Southeast Asia, followed by P. coatneyi (20.4 %), P. inui (12.3 %), P. fieldi (3.4 %), and P. knowlesi (0.4 %). The prevalence of each parasite species among the macaque populations varied by location , and the zoonotic malarias [Long-tailed macaques are widely distributed in both suburban areas and dense forests in Southeast Asia . Historimalarias –38. UndeP. cynomolgi, and P. inui, exist among all the 7 sampled regional populations of wild long-tailed macaques. Specifically, P. cynomolgi showed distinctively and uniformly high prevalence in the macaques, except for the two populations from the Philippines. The low prevalence of Plasmodium in macaques from Zamboanga in the southern Philippines and Batangas in the northern Philippines does not suggest that host genetic differences are responsible, because long-tailed macaques in the latter geographic region are assigned to a uniquely different subspecies (M. f. philippensis) than those (M. f. fascicularis) inhabiting all other regions studied [P. cynomolgi can also be transmitted to humans naturally and thus represents a potential new zoonotic malaria [P. cynomolgi in macaques from this study indicates the strong need of public advocacy in the affected areas.The present study demonstrates that two macaque malaria parasites naturally transmissible to humans,  studied . It is o malaria . The obsPlasmodium infections in natural hosts was noted in this study. Among the 177 malaria-positive macaques, 18.1 % were each found to have multiple (double and triple) Plasmodium species infections. More multiple infections, including co-infections with four or five species of Plasmodium in each long-tailed macaque were reported in other studies [Plasmodium parasites in macaques and other primates.A complex nature of  studies , 20. ThiPlasmodium infections in free-ranging macaques were low or zero, hypothetically due to the absence or limited presence of known competent vectors of malaria parasites [Plasmodium infections in macaques from other locations indicates that the prevalence of malaria and of the different species of Plasmodium vary according to the sampling site, even on a small island like Singapore [P. knowlesi infection. The exact location from where the macaques were trapped in Singapore is not known for the current study and the differences in the prevalence of P. knowlesi between the two studies could be due to different sampling locations in Singapore. In addition, in a study conducted in Sabah, Malaysian Borneo, Fornace et al. [P. knowlesi incidence depending on the land type. They found that the incidence of P. knowlesi varied between village, forests, and historical forest loss areas, which further indicates that environmental changes influence the transmission of P. knowlesi. More recently, Moyes and co-workers used a newly developed incidence–environment assessment model to investigate the distributions of reservoir hosts and Anopheline vectors of P. knowlesi in Southeast Asia and assessed their relationships with the environment [Plasmodium parasites.In previous studies, it was shown that in peri-domestic and urban areas of Singapore, Peninsular Malaysia, and Thailand, the parasitaemia and the presence of arasites , 25, 39.ingapore , 22–26. ingapore , there we et al.  have shoironment . Their aIt has been noted that the parasitaemia of malaria parasites in a human or non-human primate host could change on a daily basis . BecauseP. knowlesi infection from a long-tailed macaque from Laos is the first report of P. knowlesi infection in either humans or macaques in that country. This finding should encourage further investigations of P. knowlesi using molecular detection methods among macaques and humans in Laos.The detection of Plasmodium cynomolgi, which can be naturally acquired by humans, exists in all wild long-tailed macaque populations studied with varying prevalence. This is the first report of a natural infection of P. knowlesi in a macaque from Laos. The presence of malaria parasites underscores the risk of potential zoonotic infections in the local human populations.The prevalence of malaria parasites that naturally infect long-tailed macaques, including those that are transmissible to humans, varies among regional populations of wild long-tailed macaques in Southeast Asia.


Bacillus cereus sensu lato (s. l.) is an ecologically diverse bacterial group of medical and agricultural significance. In this study, I use publicly available genomes and novel bioinformatic workflows to characterize the B. cereus s. l. pan-genome and perform the largest phylogenetic and population genetic analyses of this group to date in terms of the number of genes and taxa included. With these fundamental data in hand, I identify genes associated with particular phenotypic traits , and quantify the degree to which taxa sharing common attributes are phylogenetically clustered.k-mer based approach (Mash) was used to create reduced representations of selected Bacillus genomes, and a fast distance-based phylogenetic analysis of this data (FastME) was performed to determine which species should be included in B. cereus s. l. The complete genomes of eight B. cereus s. l. species were annotated de novo with Prokka, and these annotations were used by Roary to produce the B. cereus s. l. pan-genome. Scoary was used to associate gene presence and absence patterns with various phenotypes. The orthologous protein sequence clusters produced by Roary were filtered and used to build HaMStR databases of gene models that were used in turn to construct phylogenetic data matrices. Phylogenetic analyses used RAxML, DendroPy, ClonalFrameML, PAUP*, and SplitsTree. Bayesian model-based population genetic analysis assigned taxa to clusters using hierBAPS. The genealogical sorting index was used to quantify the phylogenetic clustering of taxa sharing common attributes.A rapid B. cereus s. l. pan-genome currently consists of ≈60,000 genes, ≈600 of which are “core” (common to at least 99% of taxa sampled). Pan-GWAS analysis revealed genes associated with phenotypes such as isolation source, oxygen requirement, and ability to cause diseases such as anthrax or food poisoning. Extensive phylogenetic analyses using an unprecedented amount of data produced phylogenies that were largely concordant with each other and with previous studies. Phylogenetic support as measured by bootstrap probabilities increased markedly when all suitable pan-genome data was included in phylogenetic analyses, as opposed to when only core genes were used. Bayesian population genetic analysis recommended subdividing the three major clades of B. cereus s. l. into nine clusters. Taxa sharing common traits and species designations exhibited varying degrees of phylogenetic clustering.The B. cereus s. l. genomes. The best-performing methods were used to produce a phylogeny of all 498 publicly available B. cereus s. l. genomes, which was in turn used to compare three different classification systems and to test the monophyly status of various B. cereus s. l. species. The majority of the methodology used in this study is generic and could be leveraged to produce pan-genome estimates and similarly robust phylogenetic hypotheses for other bacterial groups.All phylogenetic analyses recapitulated two previously used classification systems, and taxa were consistently assigned to the same major clade and group. By including accessory genes from the pan-genome in the phylogenetic analyses, I produced an exceptionally well-supported phylogeny of 114 complete The online version of this article (doi:10.1186/s12862-017-1020-1) contains supplementary material, which is available to authorized users. Bacillus cereus sensu lato (s. l.) is an ecologically diverse bacterial group that comprises a growing number of species, many of which are medically or agriculturally important. Historically recognized and most well-sampled of the species are B. anthracis (the causative agent of anthrax), B. cereus sensu stricto (capable of causing food poisoning and other ailments), and B. thuringiensis (used to control insect pests). Other species are distinguished by rhizoidal growth patterns , thermootoxicus ), psychrhanensis  and B. wedmannii ), and utyonensis ). In addongensis , B. gaemmokensis , and B. ponensis ). In ordB. cereus s. l. genome contains ≈5500 protein-coding genes ; if taxa associated with the attribute form a monophyletic group, the gsi=1; otherwise, the greater the degree to which taxa associated with the attribute are dispersed throughout the tree (accounting for the number of taxa and the size of the tree), the smaller the gsi will be for that attribute.The genealogical sorting index  (gsi) wagsi was calculated for six B. cereus s. l. species that were sufficiently represented in the BCSL_498 phylogeny; all P-values were < <0.05 , and would have indeed been monophyletic except that one B. anthracis taxon (GCF_001029875) did not group with the others (but still placed in Group III). This might be a misannotation and should be investigated. B. weihenstephanensis was the species furthest from monophyly (gsi=0.15), primarily because it was represented by only six taxa, one of which (GCF_000518025) was found in Group IV — the remainder were found in Group VI. Again, the annotation of the Group IV taxon with regard to species affiliation should be scrutinized.The gsi was calculated for ten traits shared by various B. cereus s. l. taxa using the BCSL_114 phylogeny from the ML_6 analysis; all P-values with the exception of one were less than 0.05 , based on 114 complete genomes, is consistent with previous estimates [B. cereus s. l. can be accurately computed by relatively quick phylogenetic analyses based solely on the distribution of accessory genes among taxa .B. cereus s. l. with 100% bootstrap support for the first time  as well as analyses of pathogen evolution in an epidemic or outbreak scenario. However, the extremely high level of genomic conservation among closely related bacterial strains, especially in the core genome or in commonly typed conserved regions such as housekeeping genes, has limited the ability of previous analyses to make robust strain-level phylogenetic inferences. An important contribution of the current study is to show that bootstrap probabilities increase substantially when accessory genes are included in phylogenetic analyses along with core genes Table . Thus, IB. cereus sensu lato. Based on data from 114 complete genomes, I estimated that the B. cereus s. l. core and pan-genome contain ≈600 and ≈60,000 protein-coding genes, respectively. Pan-GWAS analysis revealed significant associations of particular genes with phenotypic traits shared by groups of taxa. All phylogenetic analyses recapitulated two previously used classification systems, and taxa were consistently assigned to the same major clade and group. By including accessory genes from the pan-genome in the phylogenetic analyses, I produced an exceptionally well-supported phylogeny of 114 complete B. cereus s. l. genomes. The best-performing methods were used to produce a phylogeny of all 498 publicly available B. cereus s. l. genomes, which was in turn used to compare three different classification systems and to test the monophyly status of various B. cereus s. l. species. The majority of the methodology used in this study is generic and could be leveraged to produce pan-genome estimates and similarly robust phylogenetic hypotheses for other bacterial groups. All scripts, software, and data products associated with this study are freely available.In this study, I used novel bioinformatic workflows to characterize the pan-genome and phylogeny of Additional file 1bcsl_498 taxa. (TXT 7 kb)RefSeq assembly accessions for the taxa used in this study. A list of RefSeq assembly accessions for the Additional file 2bcsl_114 taxa. The gene presence/absence spreadsheet lists all genes in the pan-genome and the taxa in which they are present, along with summary statistics and additional information. (CSV 38195 kb)Roary gene presence/absence matrix for Additional file 3bcsl_114 taxa, augmented with gene presence/absence information for bcsl_498 taxa. The gene presence/absence spreadsheet lists all genes in the pan-genome and the taxa in which they are present, along with summary statistics and additional information. (CSV 59187 kb)Roary gene presence/absence matrix for Additional file 4bcsl_498 taxa. Binary phenotypic trait matrix for bcsl_498 taxa, created using the isolate metadata obtained from PATRIC. (XLSX 61 kb)Binary matrix of phenotypic traits exhibited by Additional file 5B. cereus s. l. complete genomes. The resulting protein-coding gene annotations were provided as input to Roary, which constructed a pan-genome consisting of 59,989 orthologous protein sequence clusters. After filtering, which included mobile genetic element removal, the 8954 remaining clusters were aligned with MAFFT. Gene models were built from the multiple sequence alignments using the hmmbuild program from HMMER. The 8954 gene models, together with separately constructed reference taxon BLAST databases, constituted the hamstr_full_mges_removed HaMStR database. (PDF 15 kb)Construction of a HaMStR database. Prokka was used to annotate 114 Additional file 6B. cereus s. l. “query genomes”— i.e., draft genomes that were not included in bcsl_114. The resulting protein-coding gene annotations were provided as input to HaMStR, which used the hmmsearch program from HMMER followed by BLASTP to assign query sequences to HaMStR database gene models. Clusters of orthologous protein sequences from query and database taxa were aligned with MAFFT and converted to corresponding nucleotide alignments. The multiple sequence alignments were reduced to a single sequence per taxon with a consensus procedure that used nucleotide ambiguity codes to combine information from sequence variants where necessary. The individual alignments were then concatenated to produce the final data matrix. (PDF 15 kb)Construction of a concatenated data matrix. Prokka was used to annotate Additional file 7Bacillus. Phylogeny of 146 Bacillus genomes, computed with Mash and FastME. (PDF 34 kb)Mash-distance-based phylogeny of the genus Additional file 8B. cereus s. l. species, clades, and groups. Tables that provide number of taxa, average number of genes found among Roary clusters, and average number of genes present in mat_6 for B. cereus s. l. species, clades, and groups. (XLSX 44 kb)Attributes of Additional file 9Rarefaction curve: core vs. all genes. The rarefaction curve shows that after ≈35 genomes have been sampled , the number of core genes remains fairly constant at ≈600 genes, while the total number of genes in the pan-genome continues to increase almost linearly. (PDF 25 kb)Additional file 10Rarefaction curve: new vs. unique genes. The rarefaction curve shows that as genomes are sampled, genes never before observed continue to be found at a fairly steady rate, and the total number of unique genes discovered continues to increase, with no indication of soon approaching an asymptote. (PDF 15 kb)Additional file 11B. cereus s. l. clades include taxa with relatively few accessory genes included in the analysis, such as B. cytotoxicus, B. mycoides, and B. pseudomycoides. By contrast, the genomes with the most accessory genes present belong to the highly clonal clade of B. anthracis strains. (PDF 485 kb)Accessory binary tree and gene presence/absence visualization. The “accessory binary tree” and gene presence/absence information produced by Roary are plotted side-by-side. The outermost Additional file 12Scoary result summary, including enriched gene ontology biological processes. Positively or negatively trait-associated gene sets produced by Scoary were subsequently tested for possible enrichment of gene ontology biological processes. Complete Scoary results for eight traits, including gene annotations, are also given. (XLSX 423 kb)Additional file 13bcsl_114 phylogenetic results. Both the standard and normalized Robinson-Foulds distance is given. (XLSX 42 kb)Robinson-Foulds distance between all pairs of Additional file 14bcsl_114 taxa, computed with RAxML using 8954 genes . B. cytotoxicus was used to root the tree. Major B. cereus s. l. clades and groups are indicated, as are bootstrap probabilities. (PDF 40 kb)BCSL_114 maximum likelihood phylogenetic analysis results. Cladogram depicting the best estimate of the phylogenetic relationships among Additional file 15bcsl_498. Table providing clade, group and hierBAPS cluster affiliation for bcsl_498 taxa, along with the number of genes found among Roary clusters (complete genomes only) and the number of genes present in mat_6 . (XLSX 72 kb)Taxon metadata for Additional file 16bcsl_498 taxa, computed with RAxML using 8954 genes . B. manliponensis was used to root the tree. B. cereus s. l. species tested for monophyly with the gsi are color-coded. (PDF 71 kb)BCSL_498 maximum likelihood phylogenetic analysis results, color-coded by species. Phylogram depicting an estimate of the phylogenetic relationships among Additional file 17High resolution image of Figure Additional file 18High resolution image of Figure Additional file 19High resolution image of Figure Additional file 20High resolution image of Figure


Chronic kidney disease-mineral and bone disorder (CKD-MBD) is common in CKD patients and plays pivotal roles in the altered bone metabolism and vascular/valvular calcification. Several guidelines on the management of CKD-MBD have been published but large gaps between guidelines and clinical practice still exist. The field is swamped with information coming in the forms of basic and clinical research articles. Timely published review articles and focused special issues on this topic provide quick and valuable tools for the busy medical professionals looking after CKD patients.In this special issue, we offer a nexus for the most updated collection of reviews and clinical studies on CKD-MBD. This issue includes three review articles and six clinical studies. For better understanding the mechanisms of CKD-MBD, Dr. Y. Iwasaki et al. provide an intensive review on molecular abnormalities underlying bone fragility in chronic kidney disease. Dr. Y.-C. Hou et al. review roles of vitamin D in uremic vascular calcification. From practical viewpoints, Dr. N.-C. Chen et al. review strategies to prevent and regress the vascular calcification in dialysis patients.Clinical studies presented in this special issue collectively cover a broad spectrum of CKD-MBD. Dr. W.-H. Wang et al. investigate the association between parathyroid hormone, 25 (OH) vitamin D, and chronic kidney disease. Dr. Y.-P. Sun et al. analyze clinical epidemiology of mineral bone disorder markers in prevalent hemodialysis patients in Xinjiang and call for an urgent need to improve dialysis adequacy in the largest province in China. Dr. Y. Liu et al. examine the practical use of the KDIGO recommended target range of CKD-MBD markers in the real world and find that achievement of the suggested serum phosphorus level is the most important factor for lowering mortality in hemodialysis patients. In line with Dr. Y. Liu's findings, by looking at coronary artery calcification in peritoneal dialysis patients, Dr. D. Shang et al. address hyperphosphatemia and hs-CRP important initiators of vascular calcification in CKD-MBD. Parathyroidectomy (PTX) is indicated in CKD stages 3–5D with severe hyperparathyroidism that fail to respond to medical therapy. However, the impacts of PTX on serum fibroblast growth factor 23 (FGF23) and soluble Klotho levels and the optimal post-PTX intact parathyroid hormone (iPTH) levels are seldom investigated. By analyzing a larger patient cohort, Dr. S.-C. Liao et al. find no changes in serum concentrations of FGF23 and soluble Klotho in hemodialysis patients after total PTX. Dr. Q. P. Xi et al. look into the impact of post-PTX iPTH levels on all-cause mortality in dialysis patients.Articles presented in this special issue offer novel insights into the pathogenesis of CKD-MBD and inspire new ideas in clinical management on this complex problem.Wen-Chin LeeWen-Chin LeeXiong-Zhong RuanXiong-Zhong RuanYou-Ying ChauYou-Ying ChauJin-Bor ChenJin-Bor Chen


Plasmodium knowlesi is primarily responsible for zoonotic malaria in several Southeast Asian countries. Precise identification of the parasite in the blood of patients presently relies on an expensive and elaborate PCR procedure because microscopic examination of blood and other available field identification techniques lack adequate specificity. Therefore, the use of a simple and inexpensive dual-colour fluorescence in situ hybridization (FISH) assay, analogous to FISH assays recently described for Plasmodium falciparum and Plasmodium vivax, was investigated as a potential tool for identifying P. knowlesi.P. knowlesi 18S rDNA sequence-based DNA probe was used to test thin blood smears of P. knowlesi by FISH, and fluorescence viewed in a light microscope fitted with a light emitting diode light source and appropriate emission and barrier filters. The limit of detection in the P. knowlesi FISH assay was 84 parasites per μl in infected monkey blood and 61 parasites per μl for P. knowlesi cultured in human blood. The P. knowlesi-specific FISH probe detected only P. knowlesi and not P. falciparum, Plasmodium malariae, Plasmodium ovale, P. vivax or a panel of other human blood-borne pathogens. A previously described Plasmodium genus-specific probe used simultaneously in the P. knowlesi FISH assay reacted with all tested Plasmodium species.A P. knowlesi that is potentially useful for diagnosing infections in remote laboratories in endemic countries.To our knowledge, this is the first description of a FISH assay for The online version of this article (doi:10.1186/s13071-017-2273-7) contains supplementary material, which is available to authorized users. Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, transmitted between humans by anopheline mosquito vectors, have an ancient zoonotic origin [Plasmodium knowlesi and Plasmodium cynomolgi in Southeast Asia, the platyrrhine monkey parasites Plasmodium brasilianum and Plasmodium simium in South America, and Plasmodium vivax-like parasites from apes in West Africa [Plasmodium knowlesi malaria is increasingly recognized in Southeast Asia after the initial discovery of a focus of human infections in the Kapit division of the Sarawak state in Malaysian Borneo [Plasmodium knowlesi is normally a parasite of Macaca fascicularis (the long-tailed or crab-eating macaque), M. nemestrina (the pig-tailed macaque), Trachypithecus obscuras (the dusky leaf monkey or spectacled langur) and Presbytis melalophus (the banded leaf monkey or brown langur)  are being increasingly used worldwide to detect malaria but a reliable RDT for specifically detecting P. knowlesi is not yet available [Plasmodium species that infect humans, including P. knowlesi [P. knowlesi-endemic regions of Southeast Asia.The four common species of human malaria parasites, i.e. c origin . Zoonotic origin . Primatet Africa . The clin Borneo , subsequn Borneo –5 and thn Borneo . Plasmodlangur)  is a cytogenetic technique using short complementary probes labeled with fluorescent molecules to localize and detect specific target DNA or RNA sequences. Because there are many copies of ribosomal RNA (rRNA) in the cytoplasm able to react with specific probes, rRNA can be visualized without amplification of the target sequence. It is also possible to find short nucleotide stretches that are unique to a genus or species making rRNA suitable for differentiating closely related pathogens , 10. DNAP. knowlesi on pre-cleaned microscope slides for FISH assays were prepared from two different sources of the parasite: (i) frozen blood from a West Malaysian strain of P. knowlesi first isolated in 1962 from M. fascicularis and subsequently maintained by serial passage in monkeys ; and (ii) fresh in vitro cultures of the A1-H.1 clone, initially derived from the reference P. knowlesi H strain first isolated from a patient [P. knowlesi A1-H.1 strain parasites were grown in human blood obtained from the UK National Blood Transfusion Service using previously described culture conditions [Plasmodium knowlesi cultures were synchronized by centrifugation through a cushion of Nycodenz  as previously described [Thin smears of  patient  and then patient . The P. nditions . Plasmodescribed .Plasmodium falciparum, P. malariae and P. ovale blood smears from patients were provided by the Kenya Medical Research Institute/Walter Reed Project, Kisumu, Kenya, and P. vivax patient blood smears by the Kasturba Medical College Hospital, Mangalore, India, all being derived from anonymized left over patient samples as previously described [P. falciparum strain 3D7 in human red blood cells were obtained from the London School of Hygiene and Tropical Medicine, London, UK.escribed . In vitrUninfected EDTA-treated human blood used as a control in the PK-FISH assay was obtained from ID-FISH Technology Inc., Palo Alto, CA. Bacterial and non-plasmodial protozoan blood-borne pathogens from different sources were used as additional specificity controls in the PK-FISH assay and these are detailed in Table Plasmodium knowlesi FISH assay kit  used in the study was obtained from ID-FISH Technology Inc., Palo Alto, CA, USA. The kit contained Plasmodium genus- and P. knowlesi-specific DNA probes with hybridization buffer, smear preparation reagent (SPR), Plasmodium wash buffer, Plasmodium rinse buffer and Plasmodium mounting medium. The use of the Plasmodium genus-specific probe to detect the four common human Plasmodium species and also P. knowlesi by FISH has been previously described [P. knowlesi-specific probe in the PK-FISH assay kit  was based on a nucleotide sequence which has since been found to be conserved in all 18S rRNA sequences of different P. knowlesi isolates from humans and macaques deposited to date in GenBank. The PK-FISH assay was performed according to the manufacturer’s instructions provided with the kit.The escribed . The P. P. knowlesi-specific probe was labeled with Alexa 488 green fluorescent dye and the Plasmodium genus-specific probe with Atto 550 orange fluorescent dye. Therefore with LED illumination, only P. knowlesi parasites are expected to appear green under a green filter  in the assay. Under an orange filter , all Plasmodium species, including P. knowlesi, are expected to appear orange.In the PK-FISH assay kit, the P. knowlesi from monkey blood and in vitro culture in human blood, together with control smears from uninfected human blood, other human malaria parasite species derived from patients and the reference set of non-plasmodial blood-borne pathogens were tested in the PK-FISH assay to estimate its potential specificity for detecting P. knowlesi.Thin blood smears of P. knowlesi FISH assay procedure. EDTA-treated monkey blood containing parasites was mixed with SPR at 3 parts blood: 1 part SPR by volume. A set of four thin smears was prepared from each test sample. Each smear was prepared from 4 μl of the mixture, air-dried and fixed in methanol for subsequent testing in the PK-FISH assay. Briefly, after addition of the two probes in 12 μl of hybridization buffer to each methanol-fixed smear, the smear was covered with a plastic cover-slip and placed in a humid chamber at 37 °C for 15 min for hybridization. After 15 min, each smear was washed twice for 2 min each with 1× Plasmodium wash buffer at ambient temperature, followed by a rinse with 1× Plasmodium rinse buffer. After drying the smears in complete darkness, a drop of Plasmodium mounting medium was added to each smear. The smear was then covered with a glass coverslip and viewed at ×1000 magnification, in an Olympus light microscope with an attached LED unit containing the filter set. A laboratory microscope with a LED light source and filter attachment is illustrated in Additional file This was done first at the ID-FISH laboratories, Palo Alto, CA, USA, in order to establish the A1-H.1 parasites from in vitro cultures in human red blood cells and synchronised as described to enrich for ring, trophozoite and schizont stages in different cultures  were useBabesia duncani grown in hamster blood (ATCC PRA-302) were also tested with a Babesia genus-specific FISH probe conjugated with Alexa 488 provided by the IGeneX clinical testing laboratory in Palo Alto, CA, USA to confirm the presence of B. duncani parasites.Smears were prepared utilizing SPR treatment and then tested in the PK-FISH assay at the ID-FISH laboratories, Palo Alto, CA, USA, as described above for monkey blood. The presence of non-reacting cells in the test samples was confirmed by microscopy. Smears prepared from P. falciparum and P. vivax [P. knowlesi from monkey blood or human blood culture was determined as the LOD.In a manner previously described for P. vivax , the lowP. knowlesi-specific probe in all three replicate smears was considered to be the provisional LOD. Once a provisional LOD was determined, an additional 17 smears were tested at the same dilution for confirming the LOD.The parasitemia determined by Giemsa staining was 42,000 parasites per μl in the original monkey blood sample. This was serially diluted in EDTA-treated normal human venous blood  at 1:5, 1:10, 1:50, 1:100, 1:500 and 1:1000 dilutions. At each dilution multiple thin blood smears were made and three smears tested in the PK-FISH assay. The concentration of parasites at the greatest dilution at which parasites could be detected by the A1-H.1 culture at 3.9% parasitemia was diluted 1:10, 1:100 and subsequently at serial two-fold dilutions in EDTA-treated normal human blood at the London School of Hygiene and Tropical Medicine, London, UK, to prepare smears for determining the LOD. Initially two smears at 1:200 and greater dilutions were tested to determine a provisional LOD where both duplicate smears were positive in the PK-FISH assay. Based on the provisional LOD, 20 replicate smears at the same dilution as well as two-fold higher and two-fold lower dilutions were made and transported for testing by the PK-FISH assay in the ID-FISH laboratories, Palo Alto, CA, USA. The concentration of parasites at the greatest dilution at which all 20 replicate smears were positive in the assay was considered to be the LOD.P. falciparum affects the detection of P. knowlesi due to a possible cross-reaction of the P. knowlesi-specific FISH probe with P. falciparum, the PK-FISH assay was performed on serial dilutions of P. knowlesi containing a relatively high concentration of P. falciparum. In vitro cultures of P. falciparum 3D7 and P. knowlesi A1-H.1 at 4% parasitaemia were used as starting material for this purpose at the London School of Hygiene and Tropical Medicine, London, UK. Beginning at a 1:400 dilution in uninfected human red blood cells estimated to yield 500 P. knowlesi per μl, the P. knowlesi culture was serially diluted two-fold up to a dilution of 1:25,600 in a stock of 1:400 dilution of the original P. falciparum culture in fresh uninfected human red blood cells estimated to contain 500 P. falciparum per μl. A 1:1 v/v mixture of the 1:400 dilutions of the P. knowlesi and P. falciparum cultures at 4% parasitaemia was also prepared. Thin blood smears were then made from the mixed parasites at the London School of Hygiene and Tropical Medicine, London, UK, and triplicate blood smears at each dilution of P. knowlesi were tested by the PK-FISH assay at the ID-FISH laboratories, Palo Alto, CA, USA, using the procedures outlined above.To determine whether the concomitant presence of Plasmodium genus-specific and the P. knowlesi-specific probes in the PK-FISH assay showed that both probes reacted simultaneously with P. knowlesi present in the same microscopic field of monkey blood  procedure, but this has not yet been adapted for routine use in endemic areas [P. falciparum and P. vivax FISH assays [P. knowlesi malaria in peripheral and district-level diagnostic laboratories in Southeast Asian countries. It also has similar advantages in this respect to the dual colour fluorescence FISH assays recently described for identifying Mycobacterium tuberculosis and Mycobacterium avium complexes [Another technique that rapidly detects ic areas . The advH assays , and theH assays . Anotheromplexes .P. knowlesi in peripheral and district-level endemic area-laboratories is illustrated by two recent case reports of knowlesi malaria in (i) a person working in a mining development in Indonesian Borneo [The need for a simple and rapid assay for diagnosing n Borneo  and (ii)n Borneo . PCR-basn Borneo , 20.P. knowlesi-endemic areas. Investigations with pertinent patient blood samples in Southeast Asia are however needed to establish its value in the clinical diagnosis of knowlesi malaria.The PK-FISH assay described here has the potential for meeting an important diagnostic need in peripheral and district-level clinical laboratories in


Bdellovibrio spp. are predatory bacteria with great potential as antimicrobial agents. Studies have shown that members of the genus Bdellovibrio exhibit peculiar characteristics that influence their ecological adaptations. In this study, whole genomes of two different Bdellovibrio spp. designated SKB1291214 and SSB218315 isolated from soil were sequenced. The core genes shared by all the Bdellovibrio spp. considered for the pangenome analysis including the epibiotic B. exovorus were 795. The number of unique genes identified in Bdellovibrio spp. SKB1291214, SSB218315, W, and B. exovorus JJS was 1343, 113, 857, and 1572, respectively. These unique genes encode hydrolytic, chemotaxis, and transporter proteins which might be useful for predation in the Bdellovibrio strains. Furthermore, the two Bdellovibrio strains exhibited differences based on the % GC content, amino acid identity, and 16S rRNA gene sequence. The 16S rRNA gene sequence of Bdellovibrio sp. SKB1291214 shared 99% identity with that of an uncultured Bdellovibrio sp. clone 12L 106 (a pairwise distance of 0.008) and 95–97% identity (a pairwise distance of 0.043) with that of other culturable terrestrial Bdellovibrio spp., including strain SSB218315. In Bdellovibrio sp. SKB1291214, 174 bp sequence was inserted at the host interaction (hit) locus region usually attributed to prey attachment, invasion, and development of host independent Bdellovibrio phenotypes. Also, a gene equivalent to Bd0108 in B. bacteriovorus HD100 was not conserved in Bdellovibrio sp. SKB1291214. The results of this study provided information on the genetic characteristics and diversity of the genus Bdellovibrio that can contribute to their successful applications as a biocontrol agent. Bdellovibrio spp. are versatile predatory bacteria that specialize in preying upon a wide range of Gram-negative bacteria, utilizing the resulting molecules from their attack for growth and reproduction [Bdellovibrio, namely, B. bacteriovorus and B. exovorus. The former invade the periplasmic space of its prey while the latter attaches to the external surface (epibiotic) to derive its nutrients [Bdellovibrio are diverse with some of them exhibiting unique features that can influence their ecological adaptations. For instance, B. bacteriovorus strain W has the unique ability to develop a dormant structure called bdellocyst which can help them survive unfavorable conditions [B. bacteriovorus is an obligate predatory bacterium. However, a strain isolated from Tiber River (B. bacteriovorus strain Tiberius) has shown the unique ability to grow simultaneously in the presence and absence of prey [B. bacteriovorus strains that replicate and grow on nutrient-rich media without bacterial prey, usually called host-independent (HI) phenotypes often have mutations at a region of their genomes known as host interaction (hit) locus, tagged gene Bd0108 in B. bacteriovorus HD100. The hit locus has been proposed to regulate the formation of type IV pilus needed for prey attachment and invasion [Studies on predatory bacteria have received much attention recently because of the possibility to harness their potentials for the biocontrol of pathogenic bacteria. oduction . Based outrients , 3. Membnditions . B. bact of prey . B. bactinvasion .Bdellovibrio spp., and this may play a crucial role in their evolution probably leading to the development of unique features that can impact on their predatory lifestyle [Bdellovibrio spp. in turns helping in their successful application as biocontrol agents against bacterial pathogens. For instance, acquisition of pathogenic islands and alteration in their genomic structure via horizontal gene transfer may have an impact that can influence their application as biocontrol agents.Ancient and recent lateral gene transfers have been reported to occur in ifestyle , 7, 8. TBdellovibrio spp. found in soil are heterogeneous with different populations coexisting in the soil [Bdellovibrio spp. designated SKB1291214 and SSB218315 from soil samples in the same environment. The strains exhibited different phenotypes based on the time required to form plaque on Gram-negative bacteria prey lawns and prey range which was limited to some members of the family Enterobacteriaceae in Bdellovibrio sp. SKB1291214 [(hit) locus in Bdellovibrio sp. SKB1291214 using the PCR technique was unsuccessful. Therefore, we use whole-genome sequencing and comparative genomics as a tool to understand the genetic variations between these two strains and determine their relatedness with other reported genomes retrieved from the NCBI database.the soil . In our B1291214 . FurtherBdellovibrio spp. strains SKB1291214 and SSB218315 were isolated from soil samples obtained from different locations on a plot of land  within the Center for Genomic Biotechnology, National Polytechnic Institute (IPN as in Spanish) located in the city of Reynosa, Mexico. The Bdellovibrio spp. were cultured as described in our earlier report [https://www.ncbi.nlm.nih.gov/genome/annotation_prok/) and Rapid Annotation using Subsystem Technology (RAST) server [The pair-end reads generated from the Illumina sequencing were trimmed using the Sickle tool 1.33 , assembl) server . Prophag) server  and Isla) server  online aThe 16S rRNA gene sequences were aligned using the MUSCLE alignment tool with default parameters, and a phylogenetic tree was constructed using the maximum likelihood method based on the Kimura 2-parameter model. Bootstrap values were calculated to test the robustness of interior node support and were obtained by conducting 1000 pseudoreplicates using MEGA© 6.0 software . PairwisBdellovibrio spp. were retrieved from the NCBI database and compared with the genomes of the study Bdellovibrio strains (Bdellovibrio sp. SKB1291214 and B. bacteriovorus SSB218315). The retrieved genomes include that of the epibiotic B. exovorus JSS (NC_020813), B. bacteriovorus strains HD100 (NC_005363), W (NZ_CP002190), Tiberius (NC_019567), 109J (NZ_CP007656), R0 (LUKE00000000), EC13 (LUKD00000000), and BER2 (LUKF00000000).For the whole-genome comparative study, genomes of eight Bdellovibrio spp. These include B. exovorus JSS (NC_020813) and B. bacteriovorus strains HD100 (NC_005363), W (NZ_CP002190), Tiberius (NC_019567), and 109J (NZ_CP007656). BLASTP search and functional annotation analysis of the core and unique genes were done with the BLAST2GO pipeline [E value) of 1.0E − 3. The hit locus regions of Bdellovibrio spp. HD100, SKB1291214, and SSB218315 were compared by constructing a genome map using the KBase online software (https://kbase.us/), followed by BLASTP analysis of the hit regions in ExPASy Bioinformatics Resource Portal (https://www.expasy.org/). Alignment of the regions corresponding to the hit locus in the different Bdellovibrio strains was done using the multiple sequence alignment tool, Clustal Omega [The similarity among the genomes based on average amino acid identity (AAI) was inferred using the ANI/AAI-Matrix Genome-based distance matrix calculator . A pangepipeline  using thal Omega .Bdellovibrio sp. SKB1291214 (the version described in this paper is version NELQ01000000). The complete genome sequence of B. bacteriovorus SSB218315 was deposited in the same databases under accession number CP020946.The whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank databases under the accession NELQ00000000 for B. bacteriovorus strains SSB218315 and Bdellovibrio sp. strain SKB1291214 are summarized in B. bacteriovorus SSB218315 and Bdellovibrio sp. SKB1291214 is 3,769,537 bp and 3,730,590 bp, respectively. Bdellovibrio spp. are small but have large genomes (approximately 3.7 Mb) that encode predation factors presumed important to seek and lyse prey cells [Bdellovibrio sp. SKB1291214 (44.8%) is low compared to B. bacteriovorus SSB218315 (50.5%). Lambert et al. [B. bacteriovorus HD100. These include genes that are up- and downregulated at the early stage of B. bacteriovorus HD100 (30 minutes) infection as it switches from the motile prey-seeking attack stage to the intraperiplasmic phase, when it establishes itself in the prey cell. And because of the phenotypic differences observed between Bdellovibrio spp. SKB1291214 and SSB218315 [B. bacteriovorus SSB218315 have all the described gene equivalent , Bd0487, and Bd2298 equivalents in B. bacteriovorus HD100 were absent in the genome of Bdellovibrio sp. SKB1291214. The Bd1230 (lamb) gene encodes maltoporin, an outer membrane protein that is important for sugar transport in Gram-negative bacteria, and it is usually expressed when Bdellovibrio degrades its prey. The genes Bd0487 and Bd2298 are found only in the genome of Bdellovibrio, and they are significantly upregulated when Bdellovibrio enters the periplasmic phase of growth [Bdellovibrio were absent in the genome of SKB1291214. These genes include the equivalent of Bd3260, Bd2608, Bd2400, Bd0737, and Bd0992 (cwlJ) encoding putative membrane proteins and enzymes  that play a role in prey attachment and penetration. Two gene equivalents Bd0880 and Bd0931 encoding stress response proteins, a homologue of periplasmic adaptor protein CpxP and transcriptional regulator, and MerR family were also absent in the genome of Bdellovibrio sp. SKB1291214. B. bacteriovorus uses type IV pilus to attach and subsequently invade prey cells. The gene equivalent of Bd0108 which encode proteins that function in regulating type IV pilus secretion in Bdellovibrio was also not present in the genome of SKB1291214. The missing gene equivalents described above might be playing important roles during Bdellovibrio predation. And thus, the absence of these genes in the genomes of Bdellovibrio sp. SKB1291214 can affect its rate of predation.The genomic features of ey cells . The pert et al.  reportedSB218315  in the sf growth . FurtherBdellovibrio spp.  and SSB218315  as stress response proteins. Proteins that possess this GGDEF sequence secrete cyclic di-GMP, a signalling protein that controls Bdellovibrio to grow either as a predator that require prey for survival or a host-independent phenotype that can replicate on nutrient-rich medium. Four enzymatically competent GGDEF protein domains designated DgcA, DgcB, DgcC, and DgcD have been reported [dgcA mutants became a nonmotile host-independent strain that can grow axenically on nutrient-rich medium only. The ΔdgcA mutants can invade, replicate, and septate inside prey cells but cannot glide out of the prey cells to look for new prey. The ΔdgcB mutants became flagellated host-independent strains. The ΔdgcC mutants developed into predatory strains that are not capable of growing as host-independent (HI) strains. However, for ΔdgcC to grow axenically, they require additional or secondary mutation. Mutation of the dgcD gene did not result in any phenotypic alteration with mutants ΔdgcD growing both as host-dependent (HD) and HI. BLASTP analysis showed that GGDEF protein domains DgcA, DgcB, and DgcC are conserved in the two Bdellovibrio strains. However, the DgcD is not conserved in Bdellovibrio sp. SKB1291214, a similar result observed in B. bacteriovorus Tiberius [The rapid motility of location . From RAbiofilms , 28. TheBdellovibrio spp. have been described to be nonpathogenic to human [Bdellovibrio spp. SKB129124 and SSB218315 and other Bdellovibrio strains encode genes annotated as collagenase and hemolysin, virulence factors associated with some pathogens of human such as Staphylococcus aureus [Vibrio vulnificus [B. bacteriovorus SSB218315 also encodes genes annotated as RTX toxins, a factor that has different biological functions such as pore-forming leukotoxin, metalloprotease, and lipase activities [Bdellovibrio spp. will aid in their successful application as biocontrol agents against human pathogens. Analysis using the Pfam database [to human . Howeverlnificus . The gentivities . BLAST2Gtivities . Identifdatabase  revealedBdellovibrio strains SKB1291214 and SSB218315 include hypothetical proteins, peptidase, septation protein spoVG (in B. bacteriovorus SSB218315), and survival protein surA which can aid the survival of bacteria at the stationary growth phase  via horizontal gene transfer (HGT). These GEIs can confer adaptive features such as antibiotic resistance, survival features, and metabolic activities, metabolism of complex compounds on the bacteria . Some diBdellovibrio sp. SKB1291214 and B. bacteriovorus SSB218315 with sequences of other members of the genus Bdellovibrio and their relatives that belong to the genus Bacteriovorax, Peredibacter, and Halobacteriovorax (Bdellovibrio sp. clone12 L 106 (pairwise distance of 0.008) while strain SSB218315 shared 100% identity with other culturable terrestrial B. bacteriovorus which include B. bacteriovorus strain HD100 (pairwise distance 0.001) and Tiberius (pairwise distance 0.004). The phylogenetic tree showed that the two Bdellovibrio strains SKB1291214 and SSB218315 are phylogenetically different despite being isolated from soil samples in the same environment. Further species delineation was done to examine the AAI among the Bdellovibrio strains. For strains to belong to the same species, they must have ANI and AAI ≥ 95%, <10 Karlin genomic signature, and >70% in silico GGDH [B. bacteriovorus strains HD100, Tiberius, and 109J , and among these are three genes encoding type II CRISPR-associated endonuclease Cas1, CRISPR-associated Cas2, and type II CRISPR RNA-guided endonuclease Cas9 which usually act to defend prokaryotes against any invading foreign genetic material. These CRISPR genes are however absent in the genome of the periplasmic Bdellovibrio spp.A bacterial pangenome analysis (BPGA) tool was used to carry out pangenome analysis of eight  genomes . The BGPectively . The epiBdellovibrio spp. SKB1291214 and SSB218315. Homologue of genes encoding two protein families, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase  and indole-3-glycerol phosphate synthase , reported to be specific to nonpredatory bacteria was found among the unique genes of Bdellovibrio sp. SKB1291214 .A comparative genomic study by Pasternak et al.  identifiB. exovorus and B. bacteriovorus is the mechanism they use for prey attack [B. bacteriovorus HD100, three genes tagged Bd0816, Bd3459, and Bd3460 play an important role. [Bd0816 and Bd3459 encode D-alanyl-D-alanine carboxypeptidase usually expressed at the point of prey entry while Bd3460 encodes a protein called ankyrin which protects Bdellovibrio hydrolytic enzymes which it secretes during prey invasion.One of the differences between y attack , 3. The nt role. , 40, 41.Bdellovibrio sp. SKB1291214 (B9G69_09970 and B9G69_09965) and B. exovorus JJS (A11Q_2041)  which is involved in amylovoran biosynthesis. Amylovoran is an exopolysaccharide that plays a role in the pathogenesis of Erwinia carotovora [Genes encoding D-alanyl-D-alanine carboxypeptidase were present among the unique genes of rotovora .AmsF, and nonpredatory bacteria-associated 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (among the unique genes) suggests the occurrence of HGT in Bdellovibrio sp. SKB1291214. From the BLASTP analysis done with the BLAST2GO software using an E value threshold of 1E − 6, there is an indication that some of the unique genes are acquired horizontally from bacteria that belong to groups other than class Deltaproteobacteria. These groups include the Alphaproteobacteria , Betaproteobacteria , Gammaproteobacteria , Epsilonproteobacteria , Bacteroidetes , and Cyanobacteria . This result corroborates the earlier findings of Gophna et al. [Bdellovibrio spp. will provide useful information that can aid their successful application as biocontrol agents.The presence of genes encoding integrases (among predicted GEIs), transposases, phage-associated protein B. bacteriovorus has been described to have the ability to switch from being predatory usually referred to as host dependent (HD) to growing on nutrient-rich medium axenically, sometimes referred to as host independent (HI). Mutation at a region identified as host interaction (hit) locus has been reported to be responsible for the conversion from the HD to HI phenotypes. The hit locus has been described to be made up of an open reading frame (ORF) tagged Bd0108 and part of ORF tagged Bd0109 encoding a putative cell wall-associated protein in B. bacteriovorus HD100 [pil genes located upstream of the hit locus  and TadB (Bd0111) encode ATPase that provides energy for the type IV pilus secretion [hit locus are genes tagged BD_RS00505 (new locus tag for B. bacteriovorus HD100 genes) and Bd0103 in B. bacteriovorus HD100; both genes encode hypothetical proteins of unknown function. The above-described genes  are inserted in between two genes Bd0102 and Bd0121 encoding chemotaxis factors. In our previous study, hit locus was successfully amplified in B. bacteriovorus SSB218315 using the PCR technique. The negative result obtained from the PCR amplification of the hit locus in Bdellovibrio sp. SKB1291214 made us construct genomic maps to compare the hit locus region between Bdellovibrio spp. HD100, SSB218315, and SKB1291214  is not conserved in Bdellovibrio sp. SKB1291214. (Additional Bd0102 and BD_RS00505 in SKB1291214 (E value with BLASTP analysis. Comparative analysis revealed that the gene equivalent Bd0109 is conserved among the Bdellovibrio spp. including Bdellovibrio spp. SKB1291214 and B. exovorus JJS. Thus, this suggests that Bd0109 gene may have an important role in the predatory activities of Bdellovibrio spp. Also, variations in the sequence of Bd0108 may not be sufficient to hinder prey predation Bdellovibrio spp. Because Bdellovibrio spp. that have a mutation at the hit locus can be cultured axenically [Bdellovibrio spp. SKB1291214 and SSB218315 on nutrient-rich medium in the absence of prey using three different techniques described by Ferguson et al. [us HD100 . There ait locus . These pecretion . And dow Figures . From thB1291214 . This frenically , we atten et al. , Lambertn et al. , and Sein et al. . HoweverBdellovibrio have been reported to have potential applications as biocontrol agents against pathogens. This study focused on the whole-genome sequencing and comparative analysis of two Bdellovibrio spp. that showed phenotypic differences. The comparative analysis showed that B. bacteriovorus SSB218315 is genetically related to the soil-derived B. bacteriovorus HD100. We also observed that the Bdellovibrio sp. SKB1291214 is distinctively different from the epibiotic B. exovorus; although SKB1291214 showed traits associated with the intraperiplasmic predatory lifestyle, it is still different from SSB218315 and HD100 based on the 16S rRNA gene sequencing analysis, GC content, and AAI. The diversity was observed among the members of the genus Bdellovibrio thus suggesting the need to review the taxonomy of the genus Bdellovibrio in the nearest future. The pangenome analysis revealed that genomes of Bdellovibrio spp. have genes encoding different predation factors including signal transduction, hydrolytic, proteolytic, transport, and transport proteins that can help them survive as a bacterial predator. However, some factors such as hemolysin III and collagenase observed in the genomes need to be studied and characterized so that they will not have counterproductive effects when Bdellovibrio spp. are considered for applications as a biocontrol agent of pathogens in humans. Finally, Bdellovibrio sp. SKB1291214 have GEIs with atypical percent GC, AmsF protein, and a homologue of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase among its unique genes and an insertion of a 174 bp fragment in its hit locus region. These occurrences are presumptive indications of HGT in Bdellovibrio sp. SKB1291214.Members of the genus


A total of 15 populations of the genus were collected. Nine chloroplast haplotypes and two nuclear genotypes were detected. Both the nuclear and chloroplast data showed two lineages in Helianthemum songaricum, respectively, distributed in Yili Valley and western Ordos Plateau. A total of 66.81% (p < 0.001) of the genetic variation was supported by this lineage split. A Mantel test showed a significant correlation between genetic distance and geographical distance . Based on genetic analyses, cpSSRs data support strong genetic divergence between regions. We speculate that the climate change during the late Tertiary and early Quaternary isolated H. songaricum into their current distribution, resulting in interruption of gene flow, leading to isolation and genetic divergence between the two regions. Meanwhile, possible selfing would increase genetic drift in small fragmented populations, that might account for the observed genetic divergence in both regions. Given the loss of genetic diversity and genetic divergence in small populations of Helianthemum in northwestern China immediate conservation management steps should be taken on the species.Population genetic studies provide a foundation for conservation planning, especially for endangered species. Three chloroplast SSRs ( Helianthemum is a shrub or subshrub mostly distributed in the Mediterranean, extending to Central Asia  is essential to make effective conservation strategies for endangered species  are a highly polymorphic molecular tool in the population genetic analysis  and need to be further improved with nuclear genome data. In addition, population structure in the two regions are still unclear because of the limited polymorphism in the two chloroplast spacers  Whether populations of Helianthemum in western Ordos Plateau represent a distinct taxa, H. ordosicum? (2) if so, what is the genetic structure within the two species? (3) What are the conservation implications from the genetic structure analysis?The primary conclusion in our previous study, that was significant genetic divergence existed between Yili Valley and western Ordos Plateau, was only based on two chloroplast spacers , implemented in a Biorad T100 thermocycler (Biorad). The cpSSRs were amplified using three Helianthemum-specific polymorphic loci detected in regions trnL-trnF, trnL5-trnL3, and trnS-trnG: mtrnSf-trnGr, mtrnL2-trnF, and mtrnL5-trnL3 , total gene diversity (hT), genetic differentiation index  were calculated in the program HAPLONST, using U-test to determine whether NST is significantly larger than GST.The number of different alleles (Na), the effective number of alleles (Ne), and Nei's genetic distence Nei, , were caST) as the variance components  was 0.805 (SE 0.0632), and within-population gene diversity (hS) was 0.209 (SE 0.0679). Genetic differentiation index GST was 0.740 (SE 0.0784), and NST was 0.803 (SE 0.0617). As shown by the results of a U-test , NST was significantly higher than GST, suggesting a significant phylogeographical structure within Helianthemum. In Yili Valley, hT was 0.583 (SE 0.1501), hS was 0.04 (SE 0.0395), and genetic differentiation index was ; in western Ordos Plateau, hT was 0.634 (SE 0.0748), hS was 0.463 (SE 0.0836), and genetic differentiation index was . AMOVA analysis showed that 79.32% (p < 0.001) of the total variation occurred among populations. When populations were grouped by geographical region, 66.81% (p < 0.001) of the total variation occurred among the regions (Table p < 0.001) of the total variation occurred among populations; in western Ordos Plateau, 22.36% (p < 0.001) of the total variation occurred among populations. Mantel's test showed a significant correlation between genetic distance and geographical distance  was 1.267, and effective alleles number (Ne) was 1.150. Across the entire study area, total gene diversity , compared with other desert plants, such as Ammopiptanthus mongolicus (hT = 0.434), A. nunas (hT = 0.041)  , using two chloroplast intergenic spacers  For in situ conservation, all the natural habitat of Helianthemum populaton should be preserved by local governments. Nature reserves for H. ordosicum have been set up in western Ordos Plateau. However, in Yili Valley, conservation of H. songaricum has not been given enough attention, and we propose nature reserves for H. songaricum should be set up at once. In addition, for serious fragmentation in both species, extinct populations should be reestablished to connect remnant populations in each species, using progenies from populations with nearest geographic distance. Also, population sizes should be augmented by transplanting progenies propagated from original populations. (iii) For both species, ex situ conservation site should be established first. Seeds collection should capture all detected genetic variations to represent the genetic diversity of each species in maximum, avoiding artificially induced bottlenecks  and the rest of the populations should be tested ex situ to prevent potential outcrossing depression.Habitat fragmentation is a significant threat to the survival of plant species in many terrestrial ecosystems (Young et al., Conceived and designed the experiments: ZS, BR. Performed the experiments: ZS. Analyzed the data: ZS, BR, LZ, and XJ. Contributed reagents/materials/analysis tools: ZS. Wrote the paper: ZS.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.