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    Ploidy variation in Rhododendron subsection Maddenia and its implications for conservation.
    (Oxford University Press on behalf of the Annals of Botany Company, 2023-06-01) Hu L; Tate JA; Gardiner SE; MacKay M; Loureiro J
    Polyploidy, which is common in plants, can confound taxon recognition and hence conservation assessments. In the taxonomically complex genus Rhododendron, 25 % of the over 1,300 taxa are considered under threat and 27 % Near Threatened or Data Deficient, with their taxonomy needing to be resolved urgently. Although ploidy levels of Rhododendron taxa range from diploid (2x) to dodecaploid (12x) according to previous reports, the extent of polyploidy across the genus has not been examined. We first summarized the taxonomic distribution of polyploids in the genus based on the literature. Then as a case study, we estimated ploidy levels of 47 taxa in subsection Maddenia (subgenus Rhododendron, section Rhododendron) using flow cytometry, together with verification of meiotic chromosome counts for representative taxa. The summary of reported ploidy in Rhododendron indicates that polyploidy is most common in subgenera Pentanthera and Rhododendron. In subsection Maddenia, all examined taxa are diploids except for the R. maddenii complex that shows a high ploidy variation (2-8x, 12x). We investigated ploidy level of 12 taxa in subsection Maddenia for the first time, and estimated genome sizes of two Rhododendron species. Knowledge of ploidy levels will inform phylogenetic analysis of unresolved species complexes. Overall, our study of subsection Maddenia provides a model for examining multiple issues including taxonomic complexity, ploidy variation and geographic distribution in relation to biodiversity conservation.
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    A novel, stain-free, natural auto-fluorescent signal, Sig M, identified from cytometric and transcriptomic analysis of infectivity of Cryptosporidium hominis and Cryptosporidium parvum.
    (Frontiers Media S.A., 2023-05-22) Ogbuigwe P; Roberts JM; Knox MA; Heiser A; Pita A; Haack NA; Garcia-Ramirez JC; Velathanthiri N; Biggs PJ; French NP; Hayman DTS; Xu R
    Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan Cryptosporidium. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the Cryptosporidium parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective in vitro system for Cryptosporidium culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the C. parvum-specific antibody Sporo-Glo™ to characterize infected cells 48 h following an infection with C. parvum or C. hominis. The Cryptosporidium parvum-infected cells showed higher levels of signal using Sporo-Glo™ than C. hominis-infected cells, which was likely because Sporo-Glo™ was generated against C. parvum. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both C. parvum and C. hominis cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing Cryptosporidium infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M's impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein-isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter® analysis to investigate the transcriptomic landscape for the two Cryptosporidium species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular Cryptosporidium gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to Cryptosporidium infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of Cryptosporidium infectivity.
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    Rhododendron taxonomy and diversity of ex situ collections for conservation : (subsection) Maddenia species as a case study : a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Agriculture and Horticulture at Massey University (Manawatū campus], Palmerston North, New Zealand
    (Massey University, 2024-02-01) Hu, Ling
    In the ‘big genus’ Rhododendron of over 1,000 species, more than half of the species are threatened, at risk, or lacking data for biodiversity conservation. Ex situ collections, especially those from the wild, are crucial for safeguarding the diversity of species. However, lack of knowledge on existing wild diversity in botanic garden collections, and complex taxonomy, are two main problems in species assessments and conservation decision making. This research studied subsection Maddenia, a group of ~65 taxa encountering the two problems but seldom studied, as an exemplar to investigate species taxonomy and ex situ diversity. An ex situ conservation gap analysis was undertaken, using ecogeographical representation as a proxy for genetic representation in current botanic garden collections worldwide. Fifty-five of the total 65 taxa were found in cultivation, with over 86% of the living collections conserved in 66% of global botanic gardens. Half of the 18 threatened taxa, and nine of the 12 Data Deficient taxa require further wild collection to achieve a minimum level of ecogeographical representation in ex situ collections. Occurrence of ex situ collections in countries of origin is limited, and the distribution of ex situ collections worldwide is northern hemisphere centric. The results highlight the necessity of having more ex situ collections in the 10 native countries, and the importance of inter-institutional data sharing and robust documentation of collections. Determination of ploidy level of species was the second study, as the presence of polyploid samples may affect phylogenetic analysis. Ploidy levels were estimated for 263 accessions of 47 taxa (including 135 wild accessions) using flow cytometry. Meiotic chromosomes were counted for representative species of both diploids and polyploids to verify the flow cytometry results. This study showed that all taxa except one were diploid. The exception was that polyploids (2–8x, 12x) occur in the R. maddenii complex, where only seven of the 62 accessions tested were diploid while the rest were polyploid. This high level of polyploidy, combined with (i) the wide geographical distribution of the R. maddenii complex, and (ii) the previous ‘lumping’ of 12 taxa into the two subspecies, suggests the possibility of either some cryptic species or the need to re-evaluate some of the synonymized species. If new species were revealed, some may require conservation action. However, a greater number of wild-collected accessions and of different geographic origins are needed to explore this possibility. Following the ploidy study, molecular phylogeny of 40 taxa, including diploids and polyploids, was analysed using target capture sequencing. Phylogenetic trees from maximum likelihood and Bayesian analyses largely supported the morphological groupings of the Maddenii Series, Ciliicalyx Subseries and Megacalyx Subseries, but not the Ciliatum Subseries as classified by Davidian (1982). Of particular interest was the clustering in one clade of all of the R. maddenii complex, including all polyploid samples. This occurred irrespective of the method of analysis; however, there was no clear pattern of relationships to ploidy levels within the clade. The molecular phylogeny delimited several species and suggested a revision of the boundary of ‘subsection Maddenia’, although further research, to include a wider range of species, is needed to determine whether the new boundaries should be wider or narrower than before. The feasibility of using controlled pollination for safeguarding germplasm of prioritised species in ex situ collections was studied. Fruit set and seed germination identified the self- and cross-incompatibility of R. excellens (Vulnerable), which requires methods other than controlled pollination to conserve the intraspecific diversity in botanic gardens. R. dalhousiae var. dalhousiae (Least Concern), R. dalhousiae var. rhabdotum (Vulnerable), R. lindleyi (Least Concern), and R. nuttallii (Near Threatened) were both self- and cross-compatible, but the compatibility between self and cross pollinations differed from taxon to taxon and from accession to accession. These results suggest the choice of intraspecific pollination should be tested for each species before a programme of propagation is initiated. These aspects studied for subsection Maddenia can be immediately applied to conservation of this group of plants by working with the Global Conservation Consortium for Rhododendron. Meanwhile, the methods used here provide an exemplar for investigating other Rhododendron species or plant groups that encounter similar problems, to guide conservation efforts.