Identification of Tenrec ecaudatus,a Wild Mammal Introduced to Mayotte Island,as a Reservoir of the Newly Identified Human Pathogenic Leptospira mayottensis

Leptospirosis is a bacterial zoonosis of major concern on tropical islands. Human populations on western Indian Ocean islands are strongly affected by the disease although each archipelago shows contrasting epidemiology. For instance, Mayotte, part of the Comoros Archipelago, differs from the other neighbouring islands by a high diversity of Leptospira species infecting humans that includes Leptospira mayottensis, a species thought to be unique to this island. Using bacterial culture, molecular detection and typing, the present study explored the wild and domestic local mammalian fauna for renal carriage of leptospires and addressed the genetic relationships of the infecting strains with local isolates obtained from acute human cases and with Leptospira strains hosted by mammal species endemic to nearby Madagascar. Tenrec (Tenrec ecaudatus, Family Tenrecidae), a terrestrial mammal introduced from Madagascar, is identified as a reservoir of L. mayottensis. All isolated L. mayottensis sequence types form a monophyletic clade that includes Leptospira strains infecting humans and tenrecs on Mayotte, as well as two other Malagasy endemic tenrecid species of the genus Microgale. The lower diversity of L. mayottensis in tenrecs from Mayotte, compared to that occurring in Madagascar, suggests that L. mayottensis has indeed a Malagasy origin. This study also showed that introduced rats (Rattus rattus) and dogs are probably the main reservoirs of Leptospira borgpetersenii and Leptospira kirschneri, both bacteria being prevalent in local clinical cases. Data emphasize the epidemiological link between the two neighbouring islands and the role of introduced small mammals in shaping the local epidemiology of leptospirosis.     

Prevalence and Genotype Allocation of Pathogenic Leptospira Species in Small Mammals from Various Habitat Types in Germany

Small mammals serve as most important reservoirs for Leptospira spp., the causative agents of Leptospirosis, which is one of the most neglected and widespread zoonotic diseases worldwide. The knowledge about Leptospira spp. occurring in small mammals from Germany is scarce. Thus, this study’s objectives were to investigate the occurrence of Leptospira spp. and the inherent sequence types in small mammals from three different study sites: a forest in southern Germany (site B1); a National Park in south-eastern Germany (site B2) and a renaturalised area, in eastern Germany (site S) where small mammals were captured. DNA was extracted from kidneys of small mammals and tested for Leptospira spp. by real-time PCR. Positive samples were further analysed by duplex and conventional PCRs. For 14 positive samples, multi locus sequence typing (MLST) was performed. Altogether, 1213 small mammals were captured: 216 at site B1, 456 at site B2 and 541 at site S belonging to following species: Sorex (S.) araneus, S. coronatus, Apodemus (A.) flavicollis, Myodes glareolus, Microtus (Mi.) arvalis, Crocidura russula, Arvicola terrestris, A. agrarius, Mustela nivalis, Talpa europaea, and Mi. agrestis. DNA of Leptospira spp. was detected in 6% of all small mammals. At site B1, 25 small mammals (11.6%), at site B2, 15 small mammals (3.3%) and at site S, 33 small mammals (6.1%) were positive for Leptospira spp. Overall, 54 of the positive samples were further determined as L. kirschneri, nine as L. interrogans and four as L. borgpetersenii while five real-time PCR-positive samples could not be further determined by conventional PCR. MLST results revealed focal occurrence of L. interrogans and L. kirschneri sequence type (ST) 117 while L. kirschneri ST 110 was present in small mammals at all three sites. Further, this study provides evidence for a particular host association of L. borgpetersenii to mice of the genus Apodemus.     

12 Novel clonal groups of Leptospira infecting humans in multiple contrasting epidemiological contexts in Sri Lanka

Leptospirosis is a ubiquitous zoonotic disease and a major clinical challenge owing to the multitude of clinical presentations and manifestations that are possibly attributable to the diversity of Leptospira, the understanding of which is key to study the epidemiology of this emerging global disease threat. Sri Lanka is a hotspot for leptospirosis with high levels of endemicity as well as annual epidemics. We carried out a prospective study of Leptospira diversity in Sri Lanka, covering the full range of climatic zones, geography, and clinical severity. Samples were collected for leptospiral culture from 1,192 patients from 15 of 25 districts in Sri Lanka over two and half years. Twenty-five isolates belonging to four pathogenic Leptospira species were identified: L. interrogans, L. borgpetersenii, L. weilii, and L. kirschneri. At least six serogroups were identified among the isolates: Autumnalis (6), Pyrogenes (4), Icterohaemorrhagiae (2), Celledoni (1), Grippotyphosa (2) and Bataviae (1). Seven isolates did not agglutinate using available antisera panels, suggesting new serogroups. Isolates were sequenced using an Illumina platform. These data add 25 new core genome sequence types and were clustered in 15 clonal groups, including 12 new clonal groups. L. borgpetersenii was found only in the dry zone and L. weilii only in the wet zone. Acute kidney injury and cardiovascular involvement were seen only with L. interrogans infections. Thrombocytopenia and liver impairment were seen in both L. interrogans and L. borgpetersenii infections. The inadequate sensitivity of culture isolation to identify infecting Leptospira species underscores the need for culture-independent typing methods for Leptospira.     

Characterization of Leptospira species isolated from soil collected in Japan

Leptospira were isolated from soil obtained from Hokkaido, the northernmost island, to Okinawa, the southernmost island, of Japan using sulfamethoxazole, trimethoprim, amphotericin B, fosfomycin, and 5‐ fluorouracil. Fifty of 132 soil samples (37.9%) were culture‐positive. On the basis of 16S‐rDNA sequences, 12 of the isolated Leptospira were classified into a pathogenic species clade that is closely associated with L. alstonii and L. kmetyi. Nine isolates were classified as intermediate species and were found to be similar to L. licerasiae. Twenty‐seven isolates were classified as non‐pathogenic species, of which 23 were found to be related to L. wolbachii. Non‐pathogenic Leptospira are commonly distributed in environmental soil.     

Mito‐nuclear discordance with evidence of shared ancestral polymorphism and selection in cactophilic species of Drosophila

The Drosophila serido haplogroup is a monophyletic group composed of the following four cryptic and cactophilic species that are endemic to eastern Brazil: D. borborema, D. gouveai, D. seriema and D. serido. Here, we investigate the mito‐nuclear discordance in these species found among the cytochrome c oxidase subunit I (COI) mitochondrial gene, the autosomal alpha‐Esterase‐5 (α‐Est5) and the X‐linked period gene (per). Our analysis indicates that shared polymorphisms in these three molecular markers may be explained by the maintenance of ancestral polymorphisms rather than introgressive hybridization. The primary structures of COI, per and α‐Est5 genes evolve primarily under purifying selection, but we detected some sites that evolved under positive selection in α‐Est5. Considering the high variability of cacti species in eastern Brazil and the role attributed to Drosophila esterases in digestion metabolism and/or the detoxification of several compounds found in cactus tissues, we conjecture about the role of natural selection triggered by host shifts as an important factor in the intraspecific diversification of the D. serido haplogroup.     

Distribution of Plasmids in Distinct Leptospira Pathogenic Species

Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups—pathogens, non-pathogens, and intermediates—based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological significance might contribute to our understanding of biology and infectivity of pathogenic spirochetes.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Concordance of bacterial communities of two tick species and blood of their shared rodent host

High‐throughput sequencing is revealing that most macro‐organisms house diverse microbial communities. Of particular interest are disease vectors whose microbiome could potentially affect pathogen transmission and vector competence. We investigated bacterial community composition and diversity of the ticks Dermacentor variabilis (n = 68) and Ixodes scapularis (n = 15) and blood of their shared rodent host, Peromyscus leucopus (n = 45) to quantify bacterial diversity and concordance. The 16S rRNA gene was amplified from genomic DNA from field‐collected tick and rodent blood samples, and 454 pyrosequencing was used to elucidate their bacterial communities. After quality control, over 300 000 sequences were obtained and classified into 118 operational taxonomic units (OTUs, clustered at 97% similarity). Analysis of rarefied communities revealed that the most abundant OTUs were tick species‐specific endosymbionts, Francisella and Rickettsia, and the commonly flea‐associated bacterium Bartonella in rodent blood. An Arsenophonus and additional Francisella endosymbiont were also present in D. variabilis samples. Rickettsia was found in both tick species but not in rodent blood, suggesting that it is not transmitted during feeding. Bartonella was present in larvae and nymphs of both tick species, even those scored as unengorged. Relatively, few OTUs (e.g. Bartonella, Lactobacillus) were found in all sample types. Overall, bacterial communities from each sample type were significantly different and highly structured, independent of their dominant OTUs. Our results point to complex microbial assemblages inhabiting ticks and host blood including infectious agents, tick‐specific endosymbionts and environmental bacteria that could potentially affect arthropod‐vectored disease dynamics.     

Host‐specific thermal profiles affect fitness of a widespread pathogen

Host behavior can interact with environmental context to influence outcomes of pathogen exposure and the impact of disease on species and populations. Determining whether the thermal behaviors of individual species influence susceptibility to disease can help enhance our ability to explain and predict how and when disease outbreaks are likely to occur. The widespread disease chytridiomycosis (caused by the fungal pathogen Batrachochytrium dendrobatidis, Bd) often has species‐specific impacts on amphibian communities; some host species are asymptomatic, whereas others experience mass mortalities and population extirpation. We determined whether the average natural thermal regimes experienced by sympatric frog species in nature, in and of themselves, can account for differences in vulnerability to disease. We did this by growing Bd under temperatures mimicking those experienced by frogs in the wild. At low and high elevations, the rainforest frogs Litoria nannotis, L. rheocola, and L. serrata maintained mean thermal regimes within the optimal range for pathogen growth (15–25°C). Thermal regimes for L. serrata, which has recovered from Bd‐related declines, resulted in slower pathogen growth than the cooler and less variable thermal regimes for the other two species, which have experienced more long‐lasting declines. For L. rheocola and L. serrata, pathogen growth was faster in thermal regimes corresponding to high elevations than in those corresponding to low elevations, where temperatures were warmer. For L. nannotis, which prefers moist and thermally stable microenvironments, pathogen growth was fastest for low‐elevation thermal regimes. All of the thermal regimes we tested resulted in pathogen growth rates equivalent to, or significantly faster than, rates expected from constant‐temperature experiments. The effects of host body temperature on Bd can explain many of the broad ecological patterns of population declines in our focal species, via direct effects on pathogen fitness. Understanding the functional response of pathogens to conditions experienced by the host is important for determining the ecological drivers of disease outbreaks.     

Penetration by Botryosphaeriaceae species in avocado,guava and persimmon fruit during postharvest

Botryosphaeriaceae species have a wide host range and a worldwide distribution. These fungal species can colonize several plant organs, such as the trunk, leaves and fruit. Some Botryosphaeriaceae species cause important diseases on persimmon, avocado and guava fruit. However, there is a lack of information regarding the mechanisms of penetration by Botryosphaeriaceae species on these tropical and subtropical fruits. This study aimed to better understand the mechanisms involved in fungal penetration, host specificity and aggressiveness of Botryosphaeria dothidea, Lasiodiplodia pseudotheobromae and Neofusicoccum parvum on avocado (Persea americana), guava (Psidium guajava) and persimmon (Diospyros kaki) fruit. Scanning electron microscopy (SEM) image analysis showed that in avocado fruit, the three studied Botryosphaeriaceae species penetrated through lenticels. In guava fruit, penetration through stomata was verified for Botryosphaeria dothidea and Neofusicoccum parvum. In persimmon fruit, an appressoria-like structure was observed for B. dothidea, which suggests direct penetration. Disease incidence in wounded fruit was 24% higher than in non-wounded fruit. L. pseudotheobromae and N. parvum showed differences in aggressiveness in guava fruit. The longest incubation period was observed for N. parvum inoculated on guava, with an average of 4.5 days, and the shortest incubation period was verified for B. dothidea inoculated on avocado, with an average of 2.8 days. The area under the disease progress curve (AUDPC) did not differ between Botryosphaeriaceae species on avocado, whereas on guava and persimmon fruit, the AUDPC was lower for B. dothidea. The information regarding penetration mechanisms and aggressiveness is important to improve postharvest disease control strategies.     

Causal or spurious relationship? Climate and the distribution of Phelsuma geckos on Grand Comoro Island

The volcanic island of Grand Comoro, Malagasy biogeographic region, is inhabited by three species of Phelsuma day geckos; two island‐endemic taxa (Phelsuma comorensis and Phelsuma v‐nigra comoraegrandensis) and the introduced Phelsuma dubia. Phelsuma comorensis is restricted to elevations of greater than 150 m above sea level on the northern of the island's two volcanoes and is the only Phelsuma above 300 m. The other species are widespread at low elevations but also reach levels above 900 m at the southern volcano. To investigate these divergent distribution patterns, we used environmental niche models based on climate and habitat data and tested whether predicted climate change may influence species distributions. Analyses of niche overlap did not show significant differences between present‐day and predicted future potential distributions of any Phelsuma species studied, which could be seen as an indicator of resilience towards climate change. Climate models reflected the restricted distribution of P. comorensis with precipitation of the wettest month detected as most important variable, whereas habitat models predicted an island‐wide distribution. While climate appears to determine the distribution of P. comorensis, we propose isolation by migration barriers as an alternative and discuss the detection of causal versus spurious relationships in ecological niche models.     

Pathogens of Ips amitinus: new species and comparison with Ips typographus

Ips amitinus and I. typographus are two serious pests of spruce in Europe, have similar bionomics and are likely to occur and meet on the same host trees. We therefore hypothesized that the two species support similar levels of similar pathogens. To test this hypothesis, we collected mature beetles from three trap trees at each of eight study sites and determined beetle numbers and pathogen infection levels. In total, 938 mature I. amitinus beetles and 3435 of I. typographus were dissected; five pathogens, as well as intestinal nematodes and endoparasitoids, were detected. The neogregarine Mattesia schwenkei is reported here for the first time as a new pathogen in 9.4% of I. amitinus individuals at one site. Average infection levels of most pathogens (Chytridiopsis typographi, Gregarina typographi, Mattesia schwenkei and parasitoids) were significantly higher in I. typographus than in I. amitinus. Metschnikowia typographi was confirmed only in Ips amitinus, while the microsporidium of Nosema typographi occurred only in I. typographus. Within‐season increases in G. typographi infection levels were documented in Ips amitinus.     

Mitochondrial introgressive hybridization following a demographic expansion in the tomato frogs of Madagascar,genus Dyscophus

Madagascar is a biodiversity hotspot with a unique fauna and flora largely endemic at the species level and highly threatened by habitat destruction. The processes underlying population‐level differentiation in Madagascar's biota are poorly understood and have been proposed to be related to Pleistocene climatic cycles, yet the levels of genetic divergence observed are often suggestive of ancient events. We combined molecular markers of different variability to assess the phylogeography of Madagascar's emblematic tomato frogs (Dyscophus guineti and D. antongilii) and interpret the observed pattern as resulting from ancient and recent processes. Our results suggest that the initial divergence between these taxa is probably old as reflected by protein‐coding nuclear genes and by a strong mitochondrial differentiation of the southernmost population. Dramatic changes in their demography appear to have been triggered by the end of the last glacial period and possibly by the short return of glacial conditions known as the 8K event. This dramatic change resulted in an approximately 50‐fold reduction of the effective population size in various populations of both species. We hypothesize these species' current mitochondrial DNA diversity distribution reflects a swamping of the mitochondrial genetic diversity of D. guineti by that of D. antongilii previous to the populations' bottlenecks during the Holocene, and probably as a consequence of D. antongilii demographic expansion approximately 1 million years ago. Our data support the continued recognition of D. antongilii and D. guineti as separate species and flag D. guineti as the more vulnerable species to past and probably also future environmental changes.     

Identification and Molecular Characterizations of Neoscytalidium dimidiatum Causing Stem Canker of Red‐fleshed Dragon Fruit (Hylocereus polyrhizus) in Malaysia

During the year 2008 to 2009, a new disease of stem canker was noticed in most red‐fleshed dragon fruit (Hylocereus polyrhizus) plantations in Malaysia. The symptoms observed were small circular sunken orange spot, black pycnidia and rotted stem. This study was conducted to determine the occurrence of the stem canker on H. polyrhizus in Malaysia, subsequently to isolate, identify and characterize the fungal pathogen based on morphology and molecular characteristics and pathogenicity test. From the surveyed 20 plantations in Malaysia, stem canker was detected in all the plantations. A total of 40 isolates of Scytalidium‐like fungus were isolated and identified as Neoscytalidium dimidiatum based on morphological characteristics and ITS region sequences, which showed 99% similarity to N. dimidiatum (FJ648577). From the phylogenetic analysis using maximum‐likelihood tree, isolates of N. dimidiatum from stem canker of H. polyrhizus were grouped together and did not show any sequence variation. From pathogenicity test, all 40 isolates of N. dimidiatum were pathogenic causing stem canker on H. polyrhizus. To our knowledge, this is the first report of stem canker of H. polyrhizus caused by N. dimidiatum in Malaysia.     

Molecular phylogeny,biogeography and chromosome evolution of Malagasy dwarf geckos of the genus Lygodactylus (Squamata,Gekkonidae)

We performed a phylogenetic analysis using nuclear (RAG‐1, RAG‐2) and mitochondrial (16S) markers, a statistical Bayesian reconstruction of ancestral distribution areas and a karyological analysis on most Malagasy species of the gekkonid genus Lygodactylus. The phylogenetic analysis largely confirms major basal branching pattern of previous molecular studies, but highlights significant differences concerning both the relationships between different species groups as well as those within groups. The biogeographic analysis supports a Malagasy origin of Lygodactylus, an oversea dispersal to continental Africa and a return to Madagascar. The L. madagascariensis group (also including a new candidate species identified herein) is the most basal clade in Lygodactylus, and the sister group of a clade with all the remaining species. The second most basal clade is the L. verticillatus group, placed as the sister group of a clade comprising African and Malagasy species. The sister lineage of the L. verticillatus group originated the African radiation through an oversea dispersal out of Madagascar. Eventually, the sister lineage of the L. capensis group originated secondary dispersals from Africa to Madagascar. In Madagascar, lineage diversification in different species groups mainly occurred from southern to northern and eastern regions. Dispersal, vicariance and paleoclimatic refugia probably played a relevant role in the evolutionary history of closely related taxa and in speciation mechanisms. The cytogenetic analysis evidenced a high karyotypic variability in Lygodactylus (from 2n = 34 to 2n = 40), which is at least partly consistent with the phylogenetic relationships and the composition of the various species group. Chromosome evolution occurred independently in different lineages, mainly through a reduction in the chromosome number and starting from a putative primitive karyotype of 2n = 40 with all telocentric elements.     

Cyst‐Theca Relationship and Phylogenetic Position of Impagidinium caspienense Incubated from Caspian Sea Surface Sediments: Relation to Gonyaulax baltica and Evidence for Heterospory within Gonyaulacoid Dinoflagellates

We investigate the cyst‐theca relationship of Impagidinium caspienense. Through an incubation experiment, we succeeded in examining the motile stage. Additional molecular analysis of single‐cyst PCR (LSU and SSU rDNA) reveal that the cyst is related to the species Gonyaulax baltica Ellegaard et al. ( 2002 ). The ability of this species to belong to two types of cyst‐based genera (spiniferate and impagidinioid) suggests that environmental (particularly salinity) and not genetic factors explain the formation of both morphotypes by G. baltica, which provides evidence for heterospory in this species. The affiliation to G. baltica demonstrates that I. caspienense is not endemic to the Caspian Sea. The phylogenetic position of several other gonyaulacoid species is also documented: Impagidinium pallidum, Ataxiodinium choane, Pyxidinopsis psilata, Spiniferites belerius, and Spiniferites ramosus. The LSU and SSU rDNA based phylogenies suggest that the genera Impagidinium and Spiniferites are not monophyletic, and that P. psilata and A. choane are close to Gonyaulax verior and Gonyaulax polygramma, respectively. In addition, this study accentuates the importance of cyst morphology in the classification of the Gonyaulacales.     

Population genetic structure,genetic diversity,and natural history of the South American species of Nothofagus subgenus Lophozonia (Nothofagaceae) inferred from nuclear microsatellite data

The effect of glaciation on the levels and patterns of genetic variation has been well studied in the Northern Hemisphere. However, although glaciation has undoubtedly shaped the genetic structure of plants in the Southern Hemisphere, fewer studies have characterized the effect, and almost none of them using microsatellites. Particularly, complex patterns of genetic structure might be expected in areas such as the Andes, where both latitudinal and altitudinal glacial advance and retreat have molded modern plant communities. We therefore studied the population genetics of three closely related, hybridizing species of Nothofagus (N. obliqua, N. alpina, and N. glauca, all of subgenus Lophozonia; Nothofagaceae) from Chile. To estimate population genetic parameters and infer the influence of the last ice age on the spatial and genetic distribution of these species, we examined and analyzed genetic variability at seven polymorphic microsatellite DNA loci in 640 individuals from 40 populations covering most of the ranges of these species in Chile. Populations showed no significant inbreeding and exhibited relatively high levels of genetic diversity (H_E = 0.502–0.662) and slight, but significant, genetic structure (R_ST = 8.7–16.0%). However, in N. obliqua, the small amount of genetic structure was spatially organized into three well‐defined latitudinal groups. Our data may also suggest some introgression of N. alpina genes into N. obliqua in the northern populations. These results allowed us to reconstruct the influence of the last ice age on the genetic structure of these species, suggesting several centers of genetic diversity for N. obliqua and N. alpina, in agreement with the multiple refugia hypothesis.     

Cytogeography and chromosomal variation of the endemic East Asian herb Lycoris radiata

Information on the spatial distribution of cytotypes and karyotype variation in plants is critical for studies of the origin and evolution of polyploid complexes. Here, the spatial distribution of cytological races and intraspecific variation in the karyotype of Lycoris radiata, an endemic species to East Asia, is investigated. Conventional karyotype analysis methods were used to determine ploidy level and karyotypical characteristics in 2,420 individuals from 114 populations of L. radiata nearly covering the whole distribution areas in China. Of 114 populations studied, 52 (45.61%), 58 (50.88%), and 4 (3.51%) are diploid, triploid, and mixoploid populations, respectively, with 1,224, 1,195, and 1 individuals being diploid, triploid, and tetraploid, respectively. The triploid possesses a much wider distribution range than the diploid, with the former almost occupying the entire range of this complex species in East Asia and the latter distributing in the middle and east regions of China. Triploids tend to occur at high altitudes, and the relationship between the ploidy and altitude is significantly positive but low (r² = 0.103, p < 0.01). About 98.6% of examined bulbs have a common karyotype consisting of 22 or 33 acrocentric (A) chromosomes. Some aberrant chromosomes which should be generated from A‐type chromosome have been found including metacentrics (m), small metacentrics (m′), and B‐type chromosome. The results can provide a fundamental cytogeographic data for further studies on the evolutionary origins and adaptive divergences of polyploids, especially the triploid, within L. radiata using molecular and/or ecological methods in the future.     

Evaluating the Qualitative Effectiveness of a Novel Pollinator: a Case Study of Two Endemic Hawaiian Plants

In situations where native mutualists have become extinct, non‐native species may partner with remnant native species. However, non‐native mutualists may differ behaviorally from extinct native mutualists. In the case of pollination, novel relationships between natives and non‐natives could differ both quantitatively and qualitatively from native–native relationships. In Hawai'i, the non‐native Japanese White‐eye (Zosterops japonicus) has largely replaced endemic birds as pollinator of the endemic Clermontia parviflora and C. montis‐loa. We surveyed Clermontia patches and found that they ranged from 106 to 1198 m in diameter. We performed manual pollination of flowers with pollen taken from plants at five distance categories, ranging from 0 (self‐fertilization) to 20 km, and examined the germination of resulting seeds. We used radiotelemetry to estimate daily Japanese White‐eye movement distances. Percent germination of seeds after short‐ to intermediate‐distance pollination crosses (i.e., 20–1200 m, or intra‐patch pollen transfer distances) significantly exceeded germination of seeds from selfed trials for C. parviflora. No significant differences in germination rates among treatments were detected for C. montis‐loa. The maximum daily movement distances of radio‐tracked birds were generally <1 km. Together, these results suggest that this novel pollinator may be an effective mutualist for both Clermontia species. This study serves as an example of research examining qualitative components of novel mutualism, which are generally neglected relative to quantitative components.