Assessing bat droppings and predatory bird pellets for vector-borne bacteria: molecular evidence of bat-associated <Emphasis Type="Italic">Neorickettsia</Emphasis> sp. in Europe

In Europe, several species of bats, owls and kestrels exemplify highly urbanised, flying vertebrates, which may get close to humans or domestic animals. Bat droppings and bird pellets may have epidemiological, as well as diagnostic significance from the point of view of pathogens. In this work 221 bat faecal and 118 bird pellet samples were screened for a broad range of vector-borne bacteria using PCR-based methods. Rickettsia DNA was detected in 13 bat faecal DNA extracts, including the sequence of a rickettsial insect endosymbiont, a novel Rickettsia genotype and Rickettsia helvetica. Faecal samples of the pond bat (Myotis dasycneme) were positive for a Neorickettsia sp. and for haemoplasmas of the haemofelis group. In addition, two bird pellets (collected from a Long-eared Owl, Asio otus, and from a Common Kestrel, Falco tinnunculus) contained the DNA of a Rickettsia sp. and Anaplasma phagocytophilum, respectively. In both of these bird pellets the bones of Microtus arvalis were identified. All samples were negative for Borrelia burgdorferi s.l., Francisella tularensis, Coxiella burnetii and Chlamydiales. In conclusion, bats were shown to pass rickettsia and haemoplasma DNA in their faeces. Molecular evidence is provided for the presence of Neorickettsia sp. in bat faeces in Europe. In the evaluated regions bat faeces and owl/kestrel pellets do not appear to pose epidemiological risk from the point of view of F. tularensis, C. burnetii and Chlamydiales. Testing of bird pellets may provide an alternative approach to trapping for assessing the local occurrence of vector-borne bacteria in small mammals.     

From species to individuals: combining barcoding and microsatellite analyses from non-invasive samples in plant ecology studies

By means of DNA barcoding and microsatellite analyses, we studied the species and individuals of legitimate seed dispersers of the Mediterranean shrub Pistacia lentiscus, a keystone species that represents the main source of food in winter for frugivorous birds. We collected dropping of birds containing seeds, and after DNA extraction we amplified and sequenced a fragment of the mitochondrial COI gene. Through BLASTN queries of the sequenced fragments against registered sequences in the GenBank database we identified the bird species that are currently dispersing P. lentiscus seeds. Further, through the amplification of specific nuclear microsatellite loci we calculated standard genetic diversity parameters of the population of birds from the genus Sylvia (the blackcap and Sardinian warbler), the most important dispersers of P. lentiscus. Five bird species were identified as seed dispersers through their barcode match. Further, we found that S. melanocephala displayed slightly lower levels of genetic diversity than S. atricapilla. In this study we show how the genetic analyses of environmental faecal samples can be a useful and convenient tool for the study of plant-frugivore interactions through the ascertainment of the identity of the species involved and through the analyses of genetic variability of their populations.     

Development of an HRM-based,safe and high-throughput genotyping system for two <Emphasis Type="Italic">low phytic acid</Emphasis> mutations in soybean

Two low phytic acid (lpa) mutants, Gm-lpa-ZC-2 (ZC-lpa) and Gm-lpa-TW-1 (TW-lpa), resulting from a G → A mutation in GmIPK1 and a 2-bp deletion in GmMIPS1, respectively, were previously developed to increase the nutritional value and environmental friendliness of soybean meal. Two functional CAPS markers were subsequently developed for genotyping plants carrying the two mutant genes; however, both are costly and time consuming and hence unsuitable for large-scale breeding use. In the present work, by integrating a quick DNA extraction protocol with an optimized high-resolution melting curve (HRM) analysis, we developed a fast and high-throughput genotyping system for the two mutations. In this system, (1) DNAs are extracted within half an hour using a protocol that only requires freezing and heating of leaf disks in two non-toxic solutions and can be directly used for PCR; (2) for genotyping, asymmetric PCRs with competitive primers are performed, and the samples are then discriminated and grouped through HRM analysis; and (3) all steps are performed in a 96-well plate, and hence adaptable to high-throughput genotyping. Although the system was developed for two lpa mutations, the general principle should be applicable to any other genes in soybean.     

A genotyping platform assembled with high-throughput DNA extraction,codominant functional markers,and automated CE system to accelerate marker-assisted improvement of rice

The introgression of multiple genes into traditional cultivars using marker-assisted selection (MAS) in order to obtain favorable traits is an effective strategy to achieve improved rice lines. Genotyping of markers is a central component of the evaluation of germplasm and the selection of progeny lines. However, efficient DNA extraction and genotyping of large breeding populations still remain limiting factors in rice molecular breeding programs. This study has developed and validated a cost-effective, rapid (<1 h for 96 samples), and high-throughput (96-well format) total DNA-extraction method based on magnetic particle technology. To improve the grain-quality traits of two rice varieties, we have designed and employed an efficient codominant functional marker system (including Wx, ALK, Chalk5, and fgr genes), in combination with genotyping based on automated capillary electrophoresis. Rice lines with simultaneous improvement at multiple loci were obtained and found to have superior grain quality and to be fragrant. The genotyping pipeline established in this study represents an efficient, reliable, and precise platform for MAS.     

Community structure and diversity of endophytic bacteria in seeds of three consecutive generations of <Emphasis Type="Italic">Crotalaria pumila</Emphasis> growing on metal mine residues

Aims

We investigated the possible transgenerational transfer of bacterial seed endophytes across three consecutive seed generations of Crotalaria pumila growing on a metal mining site in Mexico.

Methods

Seeds were collected during three successive years in the semi-arid region of Zimapan, Mexico. Total communities of seed endophytes were investigated using DNA extraction from surface sterilized seeds and 454 pyrosequencing of the V5-V7 hypervariable regions of the 16S rRNA gene.

Results

The communities consisted of an average of 75 operational taxonomic units (OTUs); richness and diversity did not change across years. Methylobacterium, Staphylococcus, Corynebacterium, Propionibacterium and eight other OTUs constituted >60% of the community in each generation. The microbiome was dominated by Methylobacterium (present in >80% of samples). Functions associated with the microbiome were C and N fixation, oxidative phosphorylation and photosynthesis activity.

Conclusions

The bacterial endophytic communities were similar across three consecutive seed generations. Among the core microbiome Methylobacterium strains were the most abundant and they can contribute to nutrient acquisition, plant growth promotion and stress resilience to their host in metal contaminated mine residues. Identification of the seed microbiome of C. pumila may lead to novel and more efficient inoculants for microbe-assisted phytoremediation.

     

Alterations in gut bacterial and fungal microbiomes are associated with bacterial Keratitis,an inflammatory disease of the human eye

Dysbiosis, or imbalance in the gut microbiome, has been implicated in auto-immune, inflammatory, neurological diseases as well as in cancers. More recently it has also been shown to be associated with ocular diseases. In the present study, the association of gut microbiome dysbiosis with bacterial Keratitis, an inflammatory eye disease which significantly contributes to corneal blindness, was investigated. Bacterial and fungal gut microbiomes were analysed using fecal samples of healthy controls (HC, n?=?21) and bacterial Keratitis patients (BK, n?=?19). An increase in abundance of several anti-inflammatory organisms including Dialister, Megasphaera, Faecalibacterium, Lachnospira, Ruminococcus and Mitsuokella and members of Firmicutes, Veillonellaceae, Ruminococcaceae and Lachnospiraceae was observed in HC compared to BK patients in the bacterial microbiome. In the fungal microbiome, a decrease in the abundance of Mortierella, Rhizopus, Kluyveromyces, Embellisia and Haematonectria and an increase in the abundance of pathogenic fungi Aspergillus and Malassezia were observed in BK patients compared to HC. In addition, heatmaps, PCoA plots and inferred functional profiles also indicated significant variations between the HC and BK microbiomes, which strongly suggest dysbiosis in the gut microbiome of BK patients. This is the first study demonstrating the association of gut microbiome with the pathophysiology of BK and thus supports the gut–eye axis hypothesis. Considering that Keratitis affects about 1 million people annually across the globe, the data could be the basis for developing alternate strategies for treatment like use of probiotics or fecal transplantation to restore the healthy microbiome as a treatment protocol for Keratitis.     

Maternal obesity is associated with gut microbial metabolic potential in offspring during infancy

Children born to obese mothers are at increased risk for obesity, but the mechanisms behind this association are not fully understood. Our study aimed to investigate differences in the functions encoded by the microbiome of infants at 18 months of age when the transition from early infant-feeding to solid family foods is established. To investigate the impact of maternal prepregnancy body mass index on infants’ gut microbiome, faecal samples from infants born to normoweight (n = 21) and obese mothers (n = 18) were analysed by 16S rRNA gene sequencing and a functional-inference-based microbiome analysis. Our results indicated that Firmicutes was significantly enriched in infants born to normoweight mothers whereas Bacteroidetes was significantly enriched in infants born to obese women. In both microbiomes, the greatest number of genes (>50%) that were assigned a function encoded for proteins involved in “metabolism” among tier 1 KEGG Orthology (KO) categories. At lower KO functional categories, the microbiome of infants born to normoweight mothers was characterized by a significant enrichment in the abundances of “pentose phosphate pathway” (p = 0.037), “lysine biosynthesis” (p = 0.043), “glycerolipid metabolism” (p = 0.042), and “C5-branched dibasic acid metabolism” (p = 0.045). Notably, the microbiome of infants born to obese mothers was significantly enriched in “streptomycin biosynthesis” (p = 0.047), “sulphur metabolism” (p = 0.041), “taurine and hypotaurine metabolism” (p = 0.036), and “lipopolysaccharide biosynthesis” (p = 0.043). In summary, our study showed that maternal prepregnancy obesity may imprint a selective gut microbial composition during late infancy with distinct functional performances.     

Efficient isolation and observation of the most complex human commensal, <Emphasis Type="Italic">Demodex</Emphasis> spp.

Demodex spp. mites are an often neglected member of the human skin microbiome. Mostly they are commensals, although their pathophysiological role in rosacea, spinulosis folliculorum, and other skin diseases is recognized. Little is known about their life cycle, biology, and physiology. Demodex mites cannot be cultivated in vitro, thereby complicating research immensely. The manual extraction from human sebum is laborious and death can only be detected by surrogate markers like ceased movement or loss of fluorescence. Here we present a new approach for the extraction of larger mite numbers and the hitherto most precise way to detect death. The extraction of mites from sebum and debris by hand can be accelerated by a factor 10 using sucrose gradient centrifugation, which is well tolerated by the mites. Staining with propidium iodide allows for easy identification of dead mites, excluding frail mites that stopped moving, and has no negative effect on overall mite survival. We anticipate our methods to be a starting point for more sophisticated research and ultimately in vitro cultivation of Demodex spp. mites.     

Improving the standards for gut microbiome analysis of fecal samples: insights from the field biology of Japanese macaques on Yakushima Island

Fecal DNA-based 16S ribosomal RNA (rRNA) gene sequencing using next-generation sequencers allows us to understand the dynamic gut microbiome adaptation of animals to their specific habitats. Conventional techniques of fecal microbiome analysis have been developed within the broad contexts defined by human biology; hence, many of these techniques are not immediately applicable to wild nonhuman primates. In order to establish a standard experimental protocol for the analysis of the gut microbiomes of wild animals, we selected the Japanese macaques (Macaca fuscata yakui) on Yakushima Island. We tested different protocols for each stage of fecal sample processing: storage, DNA extraction, and choice of the sequencing region in the bacterial 16S rRNA gene. We also analyzed the gut microbiome of captive Japanese macaques as the control. The comparison of samples obtained from identical macaques but subjected to different protocols showed that the tested storage methods (RNAlater and lysis buffer) produced effectively the same composition of bacterial operational taxonomic units (OTUs) as the standard frozen storage method, although the relative abundance of each OTU was quantitatively affected. Taxonomic assignment of the detected bacterial groups was also significantly affected by the region being sequenced, indicating that sequencing regions and the corresponding polymerase chain reaction (PCR) primer pairs for the 16S rRNA gene should be carefully selected. This study improves the current standard methods for microbiome analysis in wild nonhuman primates. Japanese macaques were shown to be a suitable model for understanding microbiome adaptation to various environments.     

Herbarium tale: the utility of dry specimens for DNA barcoding Juncaceae

Phylogenetic and barcoding studies usually use fresh plant tissues as sources of DNA and have successfully amplified DNA for various loci. The use of dried samples, however, is often necessary due to the frequent inaccessibility of fresh rare plants or their parts for genetic analyses or barcoding. The difficulty in obtaining amplifiable DNA is a major restriction of the use of herbarium specimens for DNA analyses. Recent study has highlighted the crucial issues for comparing herbarium and fresh plants for barcoding. We analysed the performance of samples of the family Juncaceae from various herbarium specimens of different ages with fresh plant material in PCRs and the sequences of seven loci (rbcL, rpoC1, trnL-F intergenic spacer, trnL intron, and psbA-trnH from chloroplast DNA; atp1 from mitochondrial DNA; and ITS1-5.8S-ITS2 from nuclear DNA) using a combination of 28 primers. The herbarium specimens amplified well and may thus be successfully applied for both phylogenetic analyses and barcoding for the Juncaceae family. Amplifying DNA was more difficult from dried herbarium specimens than fresh samples but could be successful in most cases when appropriate internal primers were designed or methods were optimised. Using the set of universal primers recommended by the Consortium for the Barcode of Life and designing specific primers for a particular group of interest were both useful. Specimen age and amplicon length had limited detrimental effects on amplification success for most of the Juncaceae loci tested.     

<Emphasis Type="Italic">Spiroplasma</Emphasis> dominates the microbiome of khapra beetle: comparison with a congener,effects of life stage and temperature

Khapra beetle, Trogoderma granarium (Coleoptera: Dermestidae), is among the world’s most invasive and destructive pests of stored agricultural products. Its pest status is enhanced by the ability of the larvae to undergo diapause, which increases their tolerance to adverse conditions including insecticides and extreme temperatures. The ability of insects to tolerate extreme conditions can be influenced by their associated bacterial community (the microbiome). Understanding this relationship may lead to improved methods of pest control, but the microbiome of T. granarium is unknown. Here we use next-generation sequencing to address three main questions: 1) How similar are the microbiomes of the closely-related species T. granarium and T. variabile? 2) How does the microbiome change across life stage and physiological state? 3) How is the microbiome of adult T. granarium affected by extreme temperatures? Our results show that the core microbiomes of T. granarium and T. variabile are similar in composition. However, adults of former species have a microbiome dominated by Spiroplasma bacteria (99% of amplified sequences), whereas Spiroplasma in the latter species is almost absent (< 2%). The microbiome of T. granarium differs across life stage (feeding vs non-feeding life stages); its presence in eggs confirms the vertical transmission of Spiroplasma. High temperatures significantly reduced the relative abundance of Spiroplasma, but an effect of low temperatures on the microbiome of T. granarium was not detected. Given its dominance in a key pest species, further study of the interaction between Spiroplasma and its T. granarium host is warranted.     

Comparison of automated and conventional microbiological examination of donated human cardiac tissue in heart valve banking

Most tissue banks use the conventional method; however, the automated method has advantages over the conventional method. The aim of this study was to compare the conventional and automated methods of culture in human cardiac tissue using an artificial contamination model. Myocardial samples were contaminated with sequential concentration (10⁴ to 10^?1 CFU/mL) with Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Cultures were obtained from solution were the fragment was immersed and minced tissue, before and after the routine decellularization solution, with automated and conventional culture methods. Automated and conventional methods were compared and a p value ≤?0.05 was considered significant. Staphylococcus aureus presented a significantly higher growth in the automated method, as well as faster than the conventional (p?<?0.05). The positivity for growth in the automated method was higher in concentrated inoculum (>?10² CFU/mL) (p?<?0.05). The growth in the automated method was significantly faster than conventional when inoculum concentration was above 10³ CFU/mL. The automated culture method is faster than conventional method with a higher positivity in a contaminated model of myocardial and transport solution used in tissue banks.     

Taxonomic composition and species diversity of insect assemblages in grass–shrub cover of peat bogs in Belarus

The species composition and diversity of insect assemblages in the grass–shrub cover has been studied. A total of 374 species from 10 orders have been revealed. Coleoptera, Diptera, Heteroptera, and Auchenorryncha prevailed. Each order was dominated by two to eight species. These species are Cixius similis Kirschbaum, 1868; Neophilaenus lineatus (Linnaeus, 1758); Lepyronia coleoptrata (Linnaeus, 1758) (Auchenorryncha); Lygus pratensis (Linnaeus, 1758); Kleidocerys resedae (Panzer, 1797); Stictopleurus crassicornis (Linnaeus, 1758) (Heteroptera); Lochmaea suturalis (Thomson, 1866); Cyphon padi (Linnaeus, 1758); Plateumaris discolor (Herbst, 1795) (Coleoptera); etc. The assemblages were characterized by low species diversity and distribution of species abundance. The most similar entomocomplexes were those in biotopes dominated by herbs, on the one hand, and dwarf shrubs, on the other. The regression analysis has shown a significant relation between the species richness and diversity of insects and the species composition of plants and their projective cover. The multivariate analysis with various methods (CCA and PCA) demonstrated the influence of these factors on the spatial distribution of certain species and their preferences to particular habitats.     

DNA barcoding of selected UAE medicinal plant species: a comparative assessment of herbarium and fresh samples

It is commonly difficult to extract and amplify DNA from herbarium samples as they are old and preserved using different compounds. In addition, such samples are subjected to the accumulation of intrinsically produced plant substances over long periods (up to hundreds of years). DNA extraction from desert flora may pause added difficulties as many contain high levels of secondary metabolites. Herbarium samples from the Biology Department (UAE University) plant collection and fresh plant samples, collected from around Al-Ain (UAE), were used in this study. The three barcode loci for the coding genes matK, rbcL and rpoC1-were amplified. Our results showed that T. terresteris, H. robustum,T. pentandrus and Z. qatarense were amplified using all three primers for both fresh and herbaium samples. Both fresh and herbarium samples of C. comosum, however, were not amplified at all, using the three primers. Herbarium samples from A. javanica, C. imbricatum, T. aucherana and Z. simplex were not amplified with any of the three primers. For fresh samples 90, 90 and 80% of the samples were amplified using matK, rbcL and rpoC1, respectively. In short, fresh samples were significantly better amplified than those from herbarium sources, using the three primers. Both fresh and herbarium samples from one species (C. comosum), however, were not successfully amplified. It is also concluded that the rbcL regions showed real potentials to distinguish the UAE species under investigation into the appropriate family and genus.     

Common Cutaneous Bacteria Isolated from Snakes Inhibit Growth of <Emphasis Type="Italic">Ophidiomyces ophiodiicola</Emphasis>

There is increasing concern regarding potential impacts of snake fungal disease (SFD), caused by Ophidiomyces ophiodiicola (Oo), on free-ranging snake populations in the eastern USA. The snake cutaneous microbiome likely serves as the first line of defense against Oo and other pathogens; however, little is known about microbial associations in snakes. The objective of this study was to better define the composition and immune function of the snake cutaneous microbiome. Eight timber rattlesnakes (Crotalus horridus) and four black racers (Coluber constrictor) were captured in Arkansas and Tennessee, with some snakes exhibiting signs of SFD. Oo was detected through real-time qPCR in five snakes. Additional histopathological techniques confirmed a diagnosis of SFD in one racer, the species’ first confirmed case of SFD in Tennessee. Fifty-eight bacterial and five fungal strains were isolated from skin swabs and identified with Sanger sequencing. Non-metric multidimensional scaling and PERMANOVA analyses indicated that the culturable microbiome does not differ between snake species. Fifteen bacterial strains isolated from rattlesnakes and a single strain isolated from a racer inhibited growth of Oo in vitro. Results shed light on the culturable cutaneous microbiome of snakes and probiotic members that may play a role in fighting an emergent disease.     

Molecular analysis of the human faecal archaea in a southern Indian population

Archaea are an important constituent of the human gut microbiota, but there is no information on human gut archaea in an Indian population. In this study, faecal samples were obtained from different age groups (neonatal babies, pre-school children, school-going children, adolescents, adults and elderly) of a southern Indian population, and from a tribal population also resident in southern India). 16S rRNA gene sequences specific to Archaea were amplified from pooled faecal DNA in each group, sequenced, and aligned against the NCBI database. Of the 806 adequate sequences in the study, most aligned with 22 known sequences. There were 9 novel sequences in the present study. All sequences were deposited in the GenBank nucleotide sequence database with the following accession numbers: KF607113 - KF607918. Methanobrevibacter was the most prevalent genus among all the age groups accounting for 98% in neonates, 96% in post-weaning, and 100% each in preschool, school and adult population. In the elderly, Methanobrevibacter accounted for 96% and in tribal adults, 99% of the clones belonged to Methanobrevibacter genus. Other genera detected included Caldisphaera, Halobaculum, Methanosphaeraand Thermogymnomonas. Methanobrevibacter smithii predominated in all age groups, accounting for 749 (92.9%) of the 806 sequences. Archaea can be found in the faeces of southern Indian residents immediately after birth. Methanobrevibacter smithii was the dominant faecal archeon in all age groups, with other genera being found at the extremes of age.     

Usefulness of molecular markers in the diagnosis of occupational and recreational histoplasmosis outbreaks

Histoplasmosis is considered the most important systemic mycosis in Mexico, and its diagnosis requires fast and reliable methodologies. The present study evaluated the usefulness of PCR using Hcp100 and 1281–1283₍₂₂₀₎ molecular markers in detecting Histoplasma capsulatum in occupational and recreational outbreaks. Seven clinical serum samples of infected individuals from three different histoplasmosis outbreaks were processed by enzyme-linked immunosorbent assay (ELISA) to titre anti-H. capsulatum antibodies and to extract DNA. Fourteen environmental samples were also processed for H. capsulatum isolation and DNA extraction. Both clinical and environmental DNA samples were analysed by PCR with Hcp100 and 1281–1283₍₂₂₀₎ markers. Antibodies to H. capsulatum were detected by ELISA in all serum samples using specific antigens, and in six of these samples, the PCR products of both molecular markers were amplified. Four environmental samples amplified one of the two markers, but only one sample amplified both markers and an isolate of H. capsulatum was cultured from this sample. All PCR products were sequenced, and the sequences for each marker were analysed using the Basic Local Alignment Search Tool (BLASTn), which revealed 95–98 and 98–100 % similarities with the reference sequences deposited in the GenBank for Hcp100 and 1281–1283₍₂₂₀₎, respectively. Both molecular markers proved to be useful in studying histoplasmosis outbreaks because they are matched for pathogen detection in either clinical or environmental samples.     

Prevalence,quantitative load and genetic diversity of <Emphasis Type="Italic">Campylobacter</Emphasis> spp. in dairy cattle herds in Lithuania

Background

Campylobacteriosis is a zoonotic disease, and animals such as poultry, pigs and cattle may act as reservoirs for Campylobacter spp. Cattle shed Campylobacter spp. into the environment and they can act as a reservoir for human infection directly via contact with cattle or their faeces or indirectly by consumption of contaminated food. The aim of this study was to determine the prevalence, the quantitative load and the genetic strain diversity of Campylobacter spp. in dairy cattle of different age groups.

Results

Faecal samples of 200 dairy cattle from three farms in the central part of Lithuania were collected and examined for Campylobacter. Cattle herds of all three farms were Campylobacter spp. positive, with a prevalence ranging from 75% (farm I), 77.5% (farm II) to 83.3% (farm III). Overall, the highest prevalence was detected in calves (86.5%) and heifers (86.2%). In contrast, the lowest Campylobacter prevalence was detectable in dairy cows (60.6%). C. jejuni, C. coli, C. lari and C. fetus subsp. fetus were identified in faecal samples of dairy cattle. C. upsaliensis was not detectable in any sample. The high counts of Campylobacter spp. were observed in faecal material of dairy cattle (average 4.5 log₁₀ cfu/g). The highest numbers of Campylobacter spp. were found in faecal samples from calves (average 5.3 log₁₀ cfu/g), whereas, faecal samples from cows harboured the lowest number of Campylobacter spp. (average 3.7 log₁₀ cfu/g). Genotyping by fla A PCR-RFLP analysis of selected C. jejuni isolates showed that some genotypes were present in all farms and all age groups. However, farm or age specific genotypes were also identified.

Conclusions

Future studies are needed to investigate risk factors related to the degree of colonisation in cattle. Based on that, possible measures to reduce the colonisation and subsequent shedding of Campylobacter in cattle could be established. It is important to further investigate the epidemiology of Campylobacter in the cattle population in order to assess associated risks to public health.

     

<Emphasis Type="Italic">Bruguiera hainesii</Emphasis>, a critically endangered mangrove species,is a hybrid between <Emphasis Type="Italic">B. cylindrica</Emphasis> and <Emphasis Type="Italic">B. gymnorhiza</Emphasis> (Rhizophoraceae)

Bruguiera hainesii (Rhizophoraceae) is one of the two Critically Endangered mangrove species listed in the IUCN Red List of Threatened Species. Although the species is vulnerable to extinction, its genetic diversity and the evolutionary relationships with other Bruguiera species are not well understood. Also, intermediate morphological characters imply that the species might be of hybrid origin. To clarify the genetic relationship between B. hainesii and other Bruguiera species, we conducted molecular analyses including all six Bruguiera species using DNA sequences of two nuclear genes (CesA and UNK) and three chloroplast regions (intergenic spacer regions of trnL-trnF, trnS-trnG and atpB-rbcL). For nuclear DNA markers, all nine B. hainesii samples from five populations were heterozygous at both loci, with one allele was shared with B. cylindrica, and the other with B. gymnorhiza. For chloroplast DNA markers, the two haplotypes found in B. hainesii were shared only by B. cylindrica. These results suggested that B. hainesii is a hybrid between B. cylindrica as the maternal parent and B. gymnorhiza as the paternal one. Furthermore, chloroplast DNA haplotypes found in B. hainesii suggest that hybridization has occurred independently in regions where the distribution ranges of the parental species meet. As the IUCN Red List of Threatened Species currently excludes hybrids (except for apomictic plant hybrids), the conservation status of B. hainesii should be reconsidered.