Molecular Cloning,Expression and Characterization of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> Priming Glycosyltransferases

Oenococcus oeni is the main bacterial species that drives malolactic fermentation in wine. Most O. oeni strains produce capsular exopolysaccharides (EPS) that may contribute to protect them in the wine hostile environment. In O. oeni genome sequences, several genes are predicted to encode priming glycosyltransferases (pGTs). These enzymes are essential for EPS formation as they catalyze the first biosynthetic step through the formation of a phosphoanhydride bond between a hexose-1-phosphate and a lipid carrier undecaprenyl phosphate. In many microorganisms, mutations abolishing the pGT activity also abolish the EPS formation. We first made an in silico analysis of all the genes encoding putative pGT over 50 distinct O. oeni genome sequences. Two polyisoprenyl-phosphate-hexose-1-phosphate transferases, WoaA and WobA, and a glycosyltransferase (It3) were particularly examined for their topology and amino acid sequence. Several isoforms of these enzymes were then expressed in E. coli, and their substrate specificity was examined in vitro. The substrate specificity varied depending on the protein isoform examined, and several mutations were shown to abolish WobA activity but not EPS synthesis. Further analysis of woaA and wobA gene expression levels suggests that WoaA could replace the deficient WobA and maintain EPS formation.     

High genetic diversity and differentiation of an extremely narrowly distributed and critically endangered decaploid rose (<Emphasis Type="Italic">Rosa praelucens</Emphasis>): implications for its conservation

Rosa praelucens is a critically endangered decaploid alpine rose with an extremely narrow geographic distribution in Northwestern Yunnan, China. We sampled almost all the extant individuals (527 individuals in 31 natural locations and 56 individuals preserved in three local living collections) to assess the genetic variation and to probe the genetic connectivity among the individuals and populations based on three cpDNA intergenic spacers and six fluorescent amplified fragment length polymorphism (AFLP) markers. The morphological traits from seven populations were also measured. R. praelucens exhibited high levels of morphological variation, genetic diversity, and differentiation. The extant individuals were clustered into eight groups in neighbor-net networks, and subsequent Bayesian analysis assigned them into three larger gene pools, both in accordance with their morphological traits, especially flower color. The living collections embraced two private cpDNA haplotypes and included three out of the species’ total eight AFLP genotypes. Rhizome clonal growth, decaploid, and mixed breeding system may largely contribute to high genetic diversity and differentiation in R. praelucens. We concluded that the endangered status of R. praelucens may mainly be due to habitat fragmentation and loss and inherent reproductive difficulties, rather than low genetic diversity. The populations contributing higher cpDNA genetic diversity, representing more AFLP genotypes, and encompassing private cpDNA haplotypes should be given conservation priority by creating plant-micro reserves. The living collections should also be targeted for further ex situ conservation, population recovery, and reintroduction of R. praelucens plants.     

Comparison of molecular genetic utilities of TD,AFLP, and MSAP among the accessions of <Emphasis Type="Italic">japonica,indica</Emphasis>, and <Emphasis Type="Italic">Tongil</Emphasis> of <Emphasis Type="Italic">Oryza sativa</Emphasis> L.

Rice is one of the most important food crops in the world. Genetic diversity is essential for cultivar improvement programs. We compared genetic diversity derived from insertion–deletion (in–del) or base substitutions by amplified fragment length polymorphism (AFLP), from transposon transposition mutations by transposon display (TD), and from cytosine methylation by methylation-sensitive amplified polymorphism (MSAP) in japonica, indica, and Tongil type varieties of Oryza sativa L. Polymorphic profiles from the three marker systems allowed us to clearly distinguish the three types of varieties. The indica type varieties showed the highest genetic diversity followed by the Tongil and japonica type varieties. Of the three marker systems, TD produced the highest marker indices, and AFLP and MSAP produced similar marker indices. Pair-wise comparisons of the three marker systems showed that the correlation between the two genetic markers systems (AFLP and TD, r = 0.959) was higher than the correlations between the genetic and epigenetic marker systems (AFLP and MSAP, r = 0.52; TD and MSAP, r = 0.505). Both genetic marker systems had similar levels of gene differentiation (G _ST ) and gene flow (N _m ), which differed in the epigenetic marker system. Although the G _ST of the epigenetic marker system was lower than the genetic marker systems, the N _m of the epigenetic marker system was higher than in the genetic marker systems, indicating that epigenetic variations have a greater influence than genetic variations among the O. sativa L. types.     

Diversity and enzymatic potentialities of <Emphasis Type="Italic">Bacillus</Emphasis> sp. strains isolated from a polluted freshwater ecosystem in Cuba

Genotypic and phenotypic characterization of Bacillus spp. from polluted freshwater has been poorly addressed. The objective of this research was to determine the diversity and enzymatic potentialities of Bacillus spp. strains isolated from the Almendares River. Bacilli strains from a polluted river were characterized by considering the production of extracellular enzymes using API ZYM. 14 strains were selected and identified using 16S rRNA, gyrB and aroE genes. Genotypic diversity of the Bacillus spp. strains was evaluated using pulsed field gel electrophoresis. Furthermore, the presence of genetic determinants of potential virulence toxins of the Bacillus cereus group and proteinaceous crystal inclusions of Bacillus thuringiensis was determined. 10 strains were identified as B. thuringiensis, two as Bacillus megaterium, one as Bacillus pumilus and one as Bacillus subtilis. Most strains produced proteases, amylases, phosphatases, esterases, aminopeptidases and glucanases, which reflect the abundance of biopolymeric matter in Almendares River. Comparison of the typing results revealed a spatio-temporal distribution among B. thuringiensis strains along the river. The results of the present study highlight the genotypic and phenotypic diversity of Bacillus spp. strains from a polluted river, which contributes to the knowledge of genetic diversity of Bacilli from tropical polluted freshwater ecosystems.     

Genetic diversity and population structure of Chinese <Emphasis Type="Italic">Lentinula edodes</Emphasis> revealed by InDel and SSR markers

Genetic diversity and population structure of 88 Chinese Lentinula edodes strains belonging to four geographic populations were inferred from 68 Insertion-Deletion (InDel) and two simple sequence repeat (SSR) markers. The overall values of Shannon’s information index and gene diversity were 0.836 and 0.435, respectively, demonstrating a high genetic diversity in Chinese L. edodes strains. Among the four geographic populations, the Central China population displayed a lower genetic diversity. Multiple analyses resolved two unambiguous genetic groups that corresponded to two regions from which the samples were collected—one was a high-altitude region (region 1) and the other was a low-altitude region (region 2). Results from analysis of molecular variance suggested that the majority of genetic variation was contained within populations (74.8 %). Although there was a strong genetic differentiation between populations (F _ST ?=?0.252), the variability of ITS sequences from representative strains of the two regions (<3 %) could not support the existence of cryptic species. Pairwise F _ST values and Nei’s genetic distances showed that there were relatively lower genetic differentiations and genetic distances between populations from the same region. Geographic distribution could play a vital role in the formation of the observed population structure. Mycelium growth rate and precocity of L. edodes strains displayed significant differences between the two regions. Strains from region 2 grew faster and fructified earlier, which could be a result of adaptation to local environmental factors. To the best of our knowledge, this was the first study on the genetic structure and differentiation between populations, as well as the relationship between genetic structure and phenotypic traits in L. edodes.     

Multilocus sequence typing for phylogenetic view and <Emphasis Type="Italic">vip</Emphasis> gene diversity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains of the Assam soil of North East India

An agriculturally important insecticidal bacterium, Bacillus thuringiensis have been isolated from the soil samples of various part of Assam including the Kaziranga National Park. Previously, the isolates were characterized based on morphology, 16S rDNA sequencing, and the presence of the various classes’ crystal protein gene(s). In the present study, the phylogenetic analysis of a few selected isolates was performed by an unambiguous and quick method called the multiple locus sequence typing (MLST). A known B. thuringiensis strain kurstaki 4D4 have been used as a reference strain for MLST. A total of four the MLST locus of housekeeping genes, recF, sucC, gdpD and yhfL were selected. A total of 14 unique sequence types (STs) was identified. A total number of alleles identified for the locus gdpD and sucC was 12, followed by locus yhfL was 11, however, only 6 alleles were detected for the locus recF. The phylogenetic analysis using MEGA 7.0.26 showed three major lineages. Approximately, 87% of the isolates belonged to the STs corresponding to B. thuringiensis, whereas two isolates, BA07 and BA39, were clustered to B. cereus. The isolates were also screened for the diversity of vegetative insecticidal protein (vip) genes. In all, 8 isolates showed the presence of vip1, followed by 7 isolates having vip2 and 6 isolates for vip3 genes. The expression of Vip3A proteins was analyzed by western blot analyses and expression of the Vip3A protein was observed in the isolate BA20. Thus, the phylogenetic relationship and diversity of Bt isolates from Assam soil was established based on MLST, in addition, found isolates having vip genes, which could be used for crop improvement.     

Molecular characterization of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from various healthcare institutions in Nairobi,Kenya: a cross sectional study

Background

Staphylococcus aureus (S. aureus) has established itself over the years as a major cause of morbidity and mortality both within the community and in healthcare settings. Methicillin resistant S. aureus (MRSA) in particular has been a major cause of nosocomial infections resulting in significant increase in healthcare costs. In Africa, the MRSA prevalence has been shown to vary across different countries. In order to better understand the epidemiology of MRSA in a setting, it is important to define its population structure using molecular tools as different clones have been found to predominate in certain geographical locations.

Methods

We carried out PFGE, MLST, SCCmec and spa typing of selected S. aureus isolates from a private and public referral hospital in Nairobi, Kenya.

Results

A total of 93 S. aureus isolates were grouped into 19 PFGE clonal complexes (A–S) and 12 singletons. From these, 55 (32 MRSA and 23 MSSA) representative isolates from each PFGE clonal complex and all singletons were spa typed. There were 18 different MRSA spa types and 22 MSSA spa types. The predominant MRSA spa type was t037 comprising 40.6 % (13/32) of all MRSA. In contrast, the MSSA were quite heterogeneous, only 2 out of 23 MSSA shared the same spa type. Two new MRSA spa types (t13149 and t13150) and 3 new MSSA spa types (t13182, t13193 and t13194) were identified. The predominant clonal complex was CC 5 which included multi-locus sequence types 1, 8 and 241.

Conclusion

In contrast to previous studies published from Kenya, there’s marked genetic diversity amongst clinical MRSA isolates in Nairobi including the presence of well-known epidemic MRSA clones. Given that these clones are resident within our referral hospitals, adherence to strict infection control measures needs to be ensured to reduce morbidity and mortality associated with hospital acquired MRSA infections.

     

Diversity and symbiotic divergence of endophytic and non-endophytic rhizobia of <Emphasis Type="Italic">Medicago sativa</Emphasis>

Knowledge of rhizobium diversity is helping to enable the utilization of rhizobial resources. To analyze the phenotypic and genetic diversity and the symbiotic divergence of rhizobia of Medicago sativa, 30 endophytic and non-endophytic isolates were collected from different parts of five alfalfa varieties in three geographic locations in Gansu, China. Numerical analyses based on 72 phenotypic properties and restriction fragment length polymorphism (RFLP) fingerprinting indicated the abundant phenotypic and genetic diversity of the tested strains. According to the phylogenetic analysis of 16S RNA, atpD, glnII, and recA gene sequences, Rhizobium and Ensifer were further classified into four different genotypes: Rhizobium radiobacter, Rhizobium sp., Rhizobium rosettiformans, and Ensifer meliloti. The differences in architecture and functioning of the rhizobial genomes and, to a lesser extent, environment diversification helped explain the diversity of tested strains. The tested strains exhibited similar symbiotic feature when inoculated onto M. sativa cvs. Gannong Nos. 3 and 9 and Qingshui plants for the clustering feature of their parameter values. An obvious symbiotic divergence of rhizobial strains was observed in M. sativa cvs. Longzhong and WL168HQ plants because of the scattered parameter values. Their symbiotic divergence differed according to alfalfa varieties, which indicated that the sensitivity of different alfalfa varieties to rhizobial strains may differ. Most of the tested strains exhibited plant growth-promoting traits including phosphate solubilization and production of indole-3-acetic acid (IAA) when colonizing plant tissues and soil.     

Interaction between <Emphasis Type="Italic">Oenococcus oeni</Emphasis> and <Emphasis Type="Italic">Lactobacillus hilgardii</Emphasis> isolated from wine. Modification of available nitrogen and biogenic amine production

During the mixed culture of Lactobacillus hilgardii 5w, a common spoilage wine bacteria and Oenococcus oeni X₂L, an amensalistic growth response of the malolactic bacteria was produced due to a competition for nitrogenous nutrients, mainly peptides. Arginine was fully consumed and peptide concentration diminished 60% with respect to both pure cultures at the end of exponential growth. Histamine release increased 34% with respect to L. hilgardii single culture. Under the poor nutritional conditions present during winemaking, L. hilgardii could increase histamine production and adversely affect malolactic fermentation conducted by O. oeni and hence the quality of the final product.     

Quinolone resistance phenotype and genetic characterization of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Pullorum isolates in China,during 2011 to 2016

Background

Pullorum disease, caused by Salmonella enterica serovar Pullorum (S. Pullorum), is one of the most important bacterial infections in the poultry industry in developing countries, including China. To examine the prevalence and characteristics of S. Pullorum, the Multilocus Sequence Typing (MLST) genotypes, fluoroquinolones resistance, and biofilm-forming abilities of S. Pullorum isolates were investigated, collected from 2011 to 2016 in China.

Results

Thirty S. Pullorum isolates collected from 2011 to 2016 were analyzed. Quinolones susceptibility testing showed that 90% of the isolates were resistant to the first generation of quinolines nalidixic acid, but the resistance rates to different fluoroquinolones agents were lower than 13.3%; for some there was even no resistance. Multilocus sequence typing (MLST) showed that ST-92 was the dominating genotype, accounting for 90.0% of all S. pullorum strains. The remaining three isolates were of the new reported sequence type ST-2151. Interestingly, the Asp87Gly substitution in quinolone resistance-determining regions (QRDR) of GyrA was only observed in the three strains of ST-2151, suggesting a potential correlation between Asp87Gly substitution and sequence type (p?<?0.05). However, Asp87Gly substitution could not confer the resistant to ofloxacin and ciprofloxacin of these isolates. The plasmid-mediated quinolone resistance (PMQR) gene was not found in any of the tested isolates. Furthermore, an assay measuring biofilm-forming abilities showed that 46.7% of the isolates were non-biofilm producers, while 53.3% could form very weak biofilms, which might explain the relatively lower resistance to fluoroquinolones.

Conclusions

We reported a high resistance rate to the first generation of quinolines nalidixic acid and relatively low resistance rates to fluoroquinolones in S. Pullorum isolates. In addition, weak biofilm-forming abilities were found, which might be an important reason of the low fluoroquinolones resistance rates of S. Pullorum isolates. ST-92 was the dominating genotype demonstrated by MLST, and the new sequence type ST-2151 showed a potential correlation with Asp87Gly substitution in QRDR of GyrA. We believe the characterization of these S. Pullorum isolates will be helpful to develop prevention and control strategies.

     

AFLP,RAPD, and ISSR analysis of intraspecific polymorphism and interspecific differences of allotetraploid species <Emphasis Type="Italic">Aegilops kotschyi</Emphasis> Boiss. and <Emphasis Type="Italic">Aegilops variabilis</Emphasis> Eig

To evaluate genetic variation, 27 accessions of allotetraploid species Aegilops kotschyi and Ae. variabilis with the US genome were analyzed using the AFLP, RAPD, and ISSR methods. A total of 316 polymorphic RAPD fragments, 750 polymorphic AFLP fragments, and 234 polymorphic ISSR fragments were obtained. It was demonstrated that the analyzed species were characterized by a considerable level of nuclear genome variation. According to the data of ISSR and RAPD analysis, the average value of the Jaccard similarity coefficient for the accessions of Ae. variabilis from different geographical regions was slightly lower than that for the accessions of Ae. kotschyi. At the same time, AFLP analysis showed no considerable differences in the levels of intraspecific variation of the studied species. Analysis of the summarized RAPD, ISSR, and AFLP marking data in the Structure software program showed that most of the analyzed accessions with high degree of probability could be assigned to one of two groups, the first of which corresponded to Ae. kotschyi and the second corresponded to Ae. variabilis, thereby confirming the species independence of Ae. kotschyi and Ae. variabilis. Accessions k900, k907, k908, and v90 could not with a sufficiently high degree of probability be assigned to one of the species, which possibly was the result of interspecific hybridization. Analysis of the species diversity using different molecular markers made it possible to identify the accessions that were notably different from other accessions of its species.     

Correlations between the ages of<Emphasis Type="Italic">Alnus</Emphasis> host species and the genetic diversity of associated endosymbiotic<Emphasis Type="Italic">Frankia</Emphasis> strains from nodules

Nodule samples were collected from four alder species:Alnus nepalensis, A. sibirica, A. tinctoria andA. mandshurica growing in different environments on Gaoligong Mountains, Yunnan Province of Southwest China and on Changbai Mountains, Jilin Province of Northeast China. PCR-RFLP analysis of the IGS betweennifD andnifK genes was directly applied to unculturedFrankia strains in the nodules. A total of 21 restriction patterns were obtained. TheFrankia population in the nodules ofA. nepalensis had the highest genetic diversity among all fourFrankia populations; by contrast, the population in the nodules ofA. mandshurica had the lowest degree of divergence; the ones in the nodules ofA. sibirica andA. tinctoria were intermediate. A dendrogram, which was constructed based on the genetic distance between the restriction patterns, indicated thatFrankia strains fromA. sibirica andA. tinctoria had a close genetic relationship.Frankia strains fromA. nepalensis might be the ancestor ofFrankia strains infecting otherAlnus species. From these results and the inference of the ages ofAlnus host species, it is deduced that there was a co-evolution betweenAlnus and its microsymbiontFrankia in China.     

Modern State of Populations of Endemic <Emphasis Type="Italic">Oxytropis</Emphasis> Species from Baikal Siberia and Their Phylogenetic Relationships Based on Chloroplast DNA Markers

The genetic diversity and population structure of the endemic species of Baikal Siberia Oxytropis triphylla, O. bargusinensis, and O. interposita were studied for the first time on the basis of the nucleotide polymorphism of intergenic spacers psbA–trnH, trnL–trnF, and trnS–trnG of chloroplast DNA. All populations of these species were characterized by a high haplotype (0.762–0.924) and relatively low nucleotide (0.0011–0.0022) diversity. Analysis of the distribution of variability in O. triphylla and O. bargusinensis showed that there was no significant genetic differentiation between populations of each species; the gene flow was 4.43 and 8.91, respectively. The high level of genetic diversity in the studied populations indicates a relatively stable state of these populations. A study of the phylogenetic relationships of closely related species confirms the concept of the origin of O. bargusinensis and O. tompudae as a result of intersectional hybridization of the species of the sections Orobia and Verticillares.     

Evaluation of genetic and phenotypic consistency of <Emphasis Type="Italic">Bacillus coagulans</Emphasis> MTCC 5856: a commercial probiotic strain

Commercial probiotics preparation containing Bacillus coagulans have been sold in the market for several decades. Due to its high intra-species genomic diversity, it is very likely that B. coagulans strain may alter in different ways over multiple years of production. Therefore, the present study focuses to evaluate the genetic consistency and probiotic potential of B. coagulans MTCC 5856. Phenotypic and genotypic techniques including biochemical profiling, 16S rRNA sequencing, GTG 5″, BOX PCR fingerprinting, and Multi-Locus-Sequence typing (MLST) were carried out to evaluate the identity and consistency of the B. coagulans MTCC 5856. Further, in vitro probiotic potential, safety and stability at ambient temperature conditions of B. coagulans MTCC 5856 were evaluated. All the samples were identified as B. coagulans by biochemical profiling and 16S rRNA sequencing. GTG 5″, BOX PCR fingerprints and MLST studies revealed that the same strain was present over 3 years of commercial production. B. coagulans MTCC 5856 showed resistance to gastric acid, bile salt and exhibited antimicrobial activity in in-vitro studies. Additionally, B. coagulans MTCC 5856 was found to be non-mutagenic, non-cytotoxic, negative for enterotoxin genes and stable at ambient temperature (25 ± 2 °C) for 36 months. The data of the study verified that the same strain of B. coagulans MTCC 5856 was present in commercial preparation over multiple years of production.     

Genetic structure analysis of mantis shrimp <Emphasis Type="Italic">Oratosquilla oratoria</Emphasis> based on mitochondrial DNA control region sequence

The genetic relationships of mantis shrimp Oratosquilla oratoria between the coastal waters of China and Japan were not well studied. To reveal the genetic differentiation and genetic structure among populations, we collected populations of mantis shrimp O. oratoria from the coastal waters of China and Japan to analyze the mtDNA control region variation. A total of 309 individuals of O. oratoria were collected from 13 localities (11 from China and 2 from Japan) and a segment of mitochondrial DNA control region was sequenced. Three hundred nine haplotypes were defined, yielding a very high haplotype diversity and nucleotide diversity. Two lineages of O. oratoria were revealed and displayed strong differences in the geographical distribution. In the coastal waters of China, the geographic distribution of the two lineages was completely separated by the Yangtze River estuary; however, the lineages showed geographic sympatry in two populations from Japan. Based on the lineage distribution, three groups were defined. There was no significant genetic differentiation among the populations within the three groups, indicating high gene flow within each group. Significant and negative values for Tajima D and Fu’s Fs tests, and mismatch distributions for two lineages indicated population expansion. The present result confirmed that the freshwater outflow from the Yangtze River formed a physical barrier and affected gene exchange. The different distribution patterns of the two lineages in coastal waters of China and Japan indicated that the larvae of O. oratoria were transferred from China to the coastal waters of Japan with a one-way gene flow.     

The high diversity of <Emphasis Type="Italic">Lotus corniculatus</Emphasis> endosymbionts in soils of northwest Spain

The diversity of rhizobia that establish symbiosis with Lotus corniculatus has scarcely been studied. Several species of Mesorhizobium are endosymbionts of this legume, including Mesorhizobium loti, the type species of this genus. We analysed the genetic diversity of strains nodulating Lotus corniculatus in Northwest Spain and ten different RAPD patterns were identified among 22 isolates. The phylogenetic analysis of the 16S rRNA gene showed that the isolated strains belong to four divergent phylogenetic groups within the genus Mesorhizobium. These phylogenetic groups are widely distributed worldwide and the strains nodulate L. corniculatus in several countries of Europe, America and Asia. Three of the groups include the currently described Mesorhizobium species M. loti, M. erdmanii and M. jarvisii which are L. corniculatus endosymbionts. An analysis of the recA and atpD genes showed that our strains belong to several clusters, one of them very closely related to M. jarvisii and the remanining ones phylogenetically divergent from all currently described Mesorhizobium species. Some of these clusters include L. corniculatus nodulating strains isolated in Europe, America and Asia, although the recA and atpD genes have been sequenced in only a few L. corniculatus endosymbionts. The results of this study revealed great phylogenetic diversity of strains nodulating L. corniculatus, allowing us to predict that even more diversity will be discovered as further ecosystems are investigated.     

Population Identification of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Isolates from Russia

Using multilocus sequence typing (MLST), 22 Helicobacter pylori isolates from Russia have been characterized. All of the Russian strains were assigned to a single population, hpEurope.     

Assessing the genetic legacy of a rare,clonal Australian shrub <Emphasis Type="Italic">Grevillea infecunda</Emphasis> (Proteaceae)

Reliance on clonal reproduction is associated with an increased risk of extinction. Grevillea infecunda is a rare, putatively sterile shrub restricted to 11 populations in a localized coastal region of south-eastern Australia. We assessed the genetic diversity, clonal diversity and spatial distribution of clones in all populations of G. infecunda to guide conservation management. Eight chloroplast haplotypes were identified from the trnL – trnF and trnQ – rps16 intergenic spacer regions. All individuals belonged to a single maternal lineage dominated by one haplotype (95/111 samples). Minor haplotypes differed from the common haplotype only by single mutational steps. However, microsatellite markers revealed 89 multilocus genotypes (MLGs) in 38 multilocus lineages (MLLs) of variable size. MLLs were not shared among populations and ramets from different MLLs rarely intermingled physically. New shoots arising after fire were confirmed to belong to previously-existing MLLs, indicating that this species exhibits adaptation to fire. Genetically similar MLGs were more likely to be found in close proximity than less similar MLGs, resulting in significant spatial autocorrelation to ca 350 m. Genetic diversity was moderate but genotypic diversity was low once likely clonality was taken into account. Clonality appears to have arisen several times within the holly-leafed grevilleas and examining G. infecunda is a step towards understanding why and how often clonality occurs, and the long-term evolutionary outcomes of this life history.     

Conservation genetics and geographic patterns of genetic variation of endangered shrub <Emphasis Type="Italic">Ammopiptanthus</Emphasis> (Fabaceae) in northwestern China

Phylogeographic patterns of Ammopiptanthus in northwestern China were examined with internal transcribed spacer (ITS) and three chloroplast intergenic spacers (trnH–psbA, trnL–trnF, and trnS–trnG). Two ITS genotypes (a–b) and 8 chloroplast haplotypes (A–H) were detected. Both ITS genotypes and chloroplast lineages were split in two geographic regions: western Xinjiang and the Alxa Desert. This lineage split was also supported by AMOVA analysis and the Mantel test. AMOVA showed that 89.81 % of variance in Ammopiptanthus occurred between the two geographic regions, and correlation between genetic distances and geographical distances was significant (r = 0.757, p < 0.0001). All populations in western Xinjiang shared haplotype A with high frequency, and range expansion was strongly supported by negative Fu’s F_S value, and mismatch distribution analysis, whereas populations in the Alxa Desert had higher genetic diversity and structure. We speculate that the cold and dry climate during the early Quaternary fragmented habitats of the species, limiting gene flow between regions, and interglacial periods most likely led to the range expansion in western Xinjiang. The low genetic diversity of Ammopiptanthus indicate a significant extinction risk, and protective measures should be taken immediately.