Coffea canephora, C. eugenioides, C. liberica (var. dewevrei) and C. neoleroyi. Based on ground point data from various sources, survey of natural forests, and literature reviews we summarise taxonomy, geographical distribution, ecology, conservation, and basic climate characteristics, for each species. Using literature review and farm survey we also provide information on the prior and exiting uses of Uganda’s wild coffee resources for coffee production. Three of the indigenous species (excluding C. neoleroyi) represent useful genetic resources for coffee crop development , including: adaptation to a changing climate, pest and disease resistance, improved agronomic performance, and market differentiation. Indigenous C. canephora has already been pivotal in the establishment and sustainability of the robusta coffee sector in Uganda and worldwide, and has further potential for the development of this crop species. Coffea liberica var. dewevrei (excelsa coffee) is emerging as a commercially viable coffee crop plant in its own right, and may offer substantial potential for lowland coffee farmers, i.e. in robusta coffee growing areas. It may also provide useful stock material for the grafting of robusta and Arabica coffee, and possibly other species. Preliminary conservation assessments indicate that C. liberica var. dewevrei and C. neoleroyi are at risk of extinction at the country-level (Uganda). Adequate protection of Uganda’s humid forests, and thus its coffee natural capital, is identified as a conservation priority for Uganda and the coffee sector in general.Uganda is a major global coffee exporter and home to key indigenous (wild) coffee resources. A comprehensive survey of Uganda’s wild coffee species was undertaken more than 80 years ago (in 1938) and thus a contemporary evaluation is required, which is provided here. We enumerate four indigenous coffee species for Uganda:  Coffea canephora) and Arabica (C. arabica) coffee, at an estimated ratio of around 4:1, respectively  and thus the sector as whole  resistance for Ethiopian C. arabica (Gibberella xylarioides R. Heim & Sacca) resistance for Ugandan C. canephora (Hemileia vastatrix Berk. & Broome) resistance, globally, for C. arabica, through crossing with C. canephora (C. liberica (Perileucoptera coffeella Méneville) resistance  and particularly the hybrid ‘congusta’ (C. eugenioides (see main text), C. racemosa and C. zanguebariae (Ibo or Zanzibar coffee) (Coffea stenophylla was once exported from Upper West Africa (C. arabica (C. arabica (Coffea now known to science (Wild coffee (genetic) resources, both from within the two major crop species, as whole . Example arabica ; coffee anephora ; coffee anephora  and C. lliberica ; and cofsistance  and drousistance  in C. arracemosa . It is w onwards . Other congusta’ , C. euge coffee) . Coffea t Africa , and may arabica . Many sp arabica . There c arabica  amongst  science .C. eugenioides, C. canephora, C. excelsa (now known as C. liberica var. dewevrei), and C. spathicalyx, although the last of these species has since been transferred to the genus Calycosiphonia  (The most recent (and only) review of Ugandan wild coffee genetic resources was undertaken by A.S. Thomas in 1938, and published six years afterwards . Thomas hicalyx) . DespiteOur objectives were to review: (1) the eco-geography and climate requirements of Uganda’s indigenous coffee species; (2) the extinction risk of these species at the country level; and (3) the application of this natural capital for the development and sustainability of the coffee sector.Coffea, based on peer-reviewed taxonomic and systematic research, as summarised in global plant name checklists ; herbarium codes follow  were used to provide the data for the production of distribution maps and basic climate profiling analyses for s follow . LocatioC. canephora, C. liberica var. dewevrei, and C. eugenioides, and farm study visits for C. canephora and C. liberica var. dewevrei, were undertaken between 2015 and 2022. Location, habitat and ecology data were collected from forest sites, and basic agronomy observations were made during the farm visits. Herbarium specimens (see above) and literature were consulted for additional information . Information on the global distribution of coffee species was taken from published sources (Study of wild populations of  sources .https://gadm.org/). For mapping the protected areas, we used the World Database on Protected Areas data, obtained via the Protected Planet portal (https://www.protectedplanet.net/en/thematic-areas/wdpa?tab=WDPA) [accessed November 2022].A total of 583 data records were used to produce the distribution maps Figure 1To produce preliminary national conservation assessments, we followed the IUCN Red List of Threatened Species criteria . Area-baC. canephora, 80 for C. eugenioides, 10 for C. liberica var. dewevrei and 1 for C. neoleroyi. To understand the fundamental climatic requirements, the statistics package R  and miscellaneous notes.We enumerate four indigenous (wild) coffee species for Uganda: C. neoleroyiA. Leaves deciduous and/or restricted to short branches; flowers appearing terminal on short shoots; corolla tube much longer than wide …………………………………….. 4. A. Leaves evergreen, distributed evenly along the side branches; flowers distinctly axillary; corolla tube shorter or not much longer than wide …………………………………………… BB. Leaves 12–40 × 4.5–22 cm, with 8–17 pairs of secondary veins CC. eugenioidesB. Leaves 3–12 × 1–7 cm, with 5–7 pairs of secondary veins 2. C. liberica var. dewevreiC. Leaves thick (rather leathery), domatia present and usually obvious, on the secondary vein (at the base) or in the secondary vein-midrib axil ……… 3. C. canephoraC. Leaves thin (paper-like to slightly leathery), domatia absent or inconspicuous, if present in the secondary vein-midrib axil ………………………………………….. 1. Coffea canephora1.  Pierre ex A.Froehner, Notizbl. Bot. Gart. Berlin-Dahlem 1: 237 (1897) Global distribution: west Tropical Africa ; west-central Tropical Africa ; north-east Angola. Tropical Africa (southern South Sudan); east Tropical Africa ; south Tropical Africa (northern Angola). The exact limit of natural distribution is difficult to ascertain owing to introduction and naturalisation. Widely cultivated as robusta coffee across the tropical belt of the world and frequently as Conilon in Brazil; naturalised in Tropical Africa and other tropical areas (not listed here).Global IUCN conservation assessment: Least Concern (LC) (ern (LC) .Information for UgandaDistribution: Throughout Uganda but mainly in eastern and western parts of the country. Uganda is a centre of diversity for C. canephora , occurring with a wide range of dominant tree species. Often occurring in the same forests and in close proximity to C. eugenioides (C. liberica var. dewevrei (Elevation: 655–1570 m (observed and recorded); 675–1660 m (modelled).Preliminary country-level IUCN conservation assessment: Least Concern (LC). The Extent of Occurrence (EOO) is 128,922 km2 (LC); and Area of Occurrence (AOO) is 392 km2 (Endangered (EN)). Whilst the EOO for C. canephora in Uganda is substantial, the AOO calculation falls within the limits for EN, indicating that the preservation of this species should not be taken for granted and that careful monitoring is required. Over most the eastern part of its distribution in Uganda  and the EOO-based rating increased to Near Threatened (NT). Populations in the larger protected areas appear to be healthy, with a high density of individuals, but encroachment, deforestation and disturbance in some protected areas  are negatively affecting AOO, number of mature individuals, habitat quality, and population health.Main protected area occurrence: Budongo (CFR), Bugoma (CFR), Itwara (CFR), Kagombe (CFR), Kalinzu (CFR), Kasyoha - Kitomi (CFR), Kibale (NP), Kisangi (CFR), Mabira (CFR), Murchison Falls (NP), Queen Elizabeth National Park (NP/BR), Rwensama (CFR), Semuliki (NP), South Maramagambo (CFR), Tero (CFR), Zoka (CFR). Key: Central Forest Reserve (CFR), National Park (NP), UNESCO-MAB Biosphere Reserve (BR).Ugandan names: From (es: From : Mwanyi es: From : Mwanyi Other names: Wild robusta Coffee (a Coffee .Uses (other than beverage): As a masticatory-stimulant (due to the presence of caffeine), and snack, either fresh or dried. A traditional usage is to take a small number (c. 10) of prepared fruits  and package them in dried banana leaves, for sale in local shops and at roadsides  Global distribution: west-central Tropical Africa ; north-east Tropical Africa (southern South Sudan); east Tropical Africa .Global IUCN conservation assessment: Least Concern (LC) (ern (LC) .Information for UgandaDistribution: Throughout Uganda but mainly in eastern and western parts of the country. Uganda is a centre of diversity for C. eugenioides , occurring with a wide range of dominant tree species. Often occurring in the same forests and in close proximity to C. canephora (C. liberica var. dewevrei (Elevation: 910–1828 m (observed and recorded); 700–1693 m (modelled).Preliminary country-level IUCN conservation assessment: Least Concern (LC). The Extent of Occurrence (EOO) is 133,516 km2 (LC); and Area of Occurrence (AOO) is 372 km2 (Endangered (EN). Whilst the EOO for C. eugenioides in Uganda is substantial, the AOO calculation returns a rating of EN, indicating that the preservation of this species should not be taken for granted and that careful monitoring is required. Over most the eastern part of its distribution in Uganda  and the EOO based rating increased to Near Threatened (NT). Populations in the larger protected areas appear healthy, with a high density of individuals, but encroachment, deforestation, and disturbance in some protected areas  are negatively affecting AOO, number of mature individuals, habitat quality and population health.Main protected area occurrence: Budongo (CFR), Bugoma (CFR), Bwindi Impenetrable National Park (NP/WH), Itwara (CFR), Kagombe (CFR), Kalinzu (CFR), Kasyoha - Kitomi (CFR), Kibale (NP), Kitubulu (CFR), Luvunya (CFR), Mabira (CFR), Malabigambo (CFR), Mbale (CFR), Mpanga (CFR), Mukambwe (CFR), Namalala (CFR), North Maramagambo (CFR), Nsube (CFR), Queen Elizabeth National Park (BR/NP), Rwensama (CFR), Semuliki (NP), South Maramagambo (CFR), Zoka (CFR). Key: Central Forest Reserve (CFR), National Park (NP), UNESCO-MAB Biosphere Reserve (BR), World Heritage Site (WH).Ugandan names: From : From (e): From : the  97: 4 (1874).Global distribution: west Tropical Africa , Ghana, Guinea, Ivory Coast, Liberia, eastern Sierra Leone, Nigeria); west-central Tropical Africa ; north-east Tropical Africa (southern South Sudan); east Tropical Africa (Uganda); south Tropical Africa (Angola). Naturalised in Tropical Africa and other tropical areas (not listed here).Global IUCN conservation assessment: Least Concern (LC). (rn (LC). .Coffea liberica3a.  var. liberica (not indigenous in Uganda).Global distribution: west Tropical Africa ; west-central Tropical Africa ; south Tropical Africa (Angola). Naturalised in Tropical Africa and perhaps other tropical areas (not listed here); widely cultivated at small scale across the tropics.Global IUCN conservation assessment: Not Evaluated.Coffea liberica3b.  var. dewevrei (De Wild. & T.Durand) Lebrun, Mém. Inst. Roy. Colon. Belge, Sect. Sci. Nat. (8vo) 11(3): 168 (1941).Global distribution: west-central Tropical Africa ; north-east Tropical Africa (southern South Sudan); east Tropical Africa (western Uganda).Global IUCN conservation assessment: Not Evaluated.C. liberica var. dewevrei forma bwambensis (see note below).Represented in Uganda by the endemic taxon Coffea liberica3b(i).  var. dewevrei forma bwambensis Bridson, Kew Bull. 37: 314 (1982) Information for UgandaDistribution: east Tropical Africa (western Uganda). Endemic to Uganda. Restricted to western Uganda, adjacent to the border with the Democratic Republic of Congo (in Semuliki Forest) and in north-eastern Uganda, in Zoka and at Kilak (Killak) Central Forest Reserves). Comprehensive fieldwork in Itwara Forest (during the years 2020 and 2021) shows that this species does not occur at this location, contrary to previous reports  and lowland semi-deciduous humid forest (Semuliki Forest), with a diverse range of dominant tree species and various forest communities. In Semuliki Forest, C. liberica var. dewevrei occurs mainly (c. 90%) in swamp forest, and even in places that support truly riverine species such as Pandanus chiliocarpus (screw pine), although it is not exclusively confined to these habitats in Semuliki Forest, and it grows in drier (soil) areas of this forest reserve that are not associated with water. In Zoka Forest, this species is predominantly found in areas that are not associated with rivers and waterlogged areas.Elevation: 680–1200 m (observed and recorded); 686–1118 m (modelled).Preliminary country-level IUCN conservation assessment: Endangered (EN). The Extent of Occurrence (EOO) is 7,716 km2 (Vulnerable (VU); and Area of Occurrence (AOO) is 64 km2 (Endangered (EN)). Whilst the EOO for C. liberica var. dewevrei in Uganda is substantial, a large part of the EOO is without populations of this species, and a substantial area includes Lake Albert (in situ is urgently required for Uganda.Main protected area occurrence: Semuliki (NP), Zoka (CFR). Key: Central Forest Reserve (FR), National Park (NP).Ugandan names: From . Excelsa coffee ; Shari coffee [English; : Not known.Notes: The current consensus of taxonomic and systematic study  has been assigned to represent all indigenous Liberica coffee in Uganda, but the morphological circumscription may possibly also include populations in South Sudan , to the east of Zoka Forest, in riverine forest; fieldwork is required to ascertain whether this species still exists at this location.In 1941, Coffea neoleroyi4.  A.P.Davis, Phytotaxa 10: 43 (2010).Global distribution: north-east Tropical Africa ; east Tropical Africa (north-eastern Uganda).Global IUCN conservation assessment: Endangered (EN) (red (EN) .Information for UgandaDistribution: Restricted to Mt. Zulia in north-eastern Uganda Ecology: On riverbanks, and in seasonally dry Combretum-Terminalia savanna woodland, often amongst boulders.Elevation: c. 1200 m.Preliminary country-level IUCN conservation assessment: Critically Endangered (CR) or Data Deficient (DD). The Extent of Occurrence (EOO) cannot be calculated owing to a single data point; Area of Occurrence (AOO) is 4 km2 (CR), based on a single grid cell with a cell width of 2 km. Realistically, at the country-level, this would be better placed in the DD category, given that there is only a single collection, and the area where this species grows is isolated, and has not been the subject of detailed botanical survey; dedicated survey work for this species in north-eastern Uganda is urgently required.Protected area occurrence: Zulia (FR). Key: Forest Reserve (FR).Ugandan names: Not known.Other names: Not known.Uses (other than beverage): Not known.Notes: A rare and untypical Coffea species, formerly included in Psilanthus  other Coffea species have evergreen leaves, short-tubed flowers, and a long (excluded) style. Like all Coffea species, C. neoleroyi produces a fruit that contains two seeds, and each seed possesses the typical coffee-bean morphology. The seeds (coffee beans) of C. neoleroyi are much smaller than C. canephora and C. liberica, and smaller than C. eugenioides , and national cross-border trade (C. arabica) was believed to have been introduced into cultivation in Uganda in 1900 (C. canephora) had started to be promoted as a key agricultural export, gathering increased momentum from the 1910s onwards  was then considered a separate species to C. canephora, and that the variation across the two species included specific positive traits and qualities (C. canephora (which has a spreading habit) may have originated from a forest in Uganda, whereas the Erecta type (upright habit) was introduced from the Congo Basin. In their analyses C. canephora using directly sourced wild coffee , were unfavourable due to the small size of its seeds (coffee beans) , but no further development of the species seems to have been undertaken there. In south-eastern Kenya, small scale production of C. eugenioides has been underway for several decades, as Nandi coffee, although the current status of this crop is unclear. In Colombia, C. eugenioides is presently grown on a small scale  coffee), which sells at a substantial premium, and has been used in national and international coffee making competitions (i.e. the World Barista Championships) on account of its unique, complex flavour and intense natural sweetness. Coffea eugenioides is reported to be difficult coffee to grow, and low yielding . Based on the renewed interest, and high market price, preliminary trials of C. eugenioides are now underway in Uganda.Early trials of e beans) , suscepte beans) , poor que beans) . Early se beans) ; and …“te beans) . Outside clean.” . Trial pCoffea eugenioides has been used as a breeding partner, for imparting flavour qualities and other attributes via crosses with other species. Spontaneous diploid (2n=22) crosses between C. eugenioides and C. liberica have been identified (C. arabica (2n=44) and C. canephora (using the Timor Hybrid) to produce hybrids with the potential for commercial use  crossing with C. arabica and then backcrossing, have produced a line of high yielding hybrids with acceptable beverage quality and high productivity  variants of C. liberica var. liberica  but rather C. liberica var. liberica, as indicated by the large size of its seeds (C. liberica var. dewevrei (as C. excelsa) was more thoroughly assessed and received favourable reviews. Many identified considerable potential for excelsa as a coffee crop species (liberica (Liberica/Liberian coffee) and var. dewevrei (excelsa)  , as indiC. liberica var. dewevrei, either with C. canephora, or as the dominant coffee crop , and particularly the increasing occurence and severity of disease , pests (particularly stem/twig borers) and droughts. A similar upsacling of C. liberica var. dewevrei is also occurring in South Sudan  growing fee crop . The shid coffee . Accordierations , althougth Sudan . Farmersuit e.g. . As yet,uit e.g. , which il Africa .C. arabica and C. canephora, C. liberica var. dewevrei grows into a substantial, medium sized tree  is much more satisfactory (Unlike sfactory .Coffea liberica var. dewevrei (excelsa coffee) produced in Uganda and South Sudan yields a coffee that is smooth and easy-drinking, with low to medium acidity, low bitterness, possessing a range of positive flavour notes, and a caffeine content similar to C. arabica . This is partly due to confusion over the identity of excelsa  and because of convenience. A separate market for excelsa coffee (C. liberica var. dewevrei) does not exist in Uganda, although there have been limited exports of this coffee to Italy in recent years, as Kisansa coffee (https://www.fondazioneslowfood.com/en/ark-of-taste-slow-food/kisansa-coffee/) and in 2022 to the UK .In Uganda, Coffea liberica var. dewevrei may also have considerable utility in the Ugandan coffee sector as grafting stock. Coffea liberica sensu lato is used to improve resistance to root nematodes, and increase yield and survivability of grafted C. arabica, particularly in Hawaii  of C. canephora would likely benefit from the stout tap root and extensive, robust root system of C. liberica var. dewevrei, particularly under low soil moisture conditions. Further research is warranted, including the identification of the most suitable grafting stock, as undertaken for the grafting of C. arabica onto C. liberica var. liberica stock in Hawaii  hybrids, most notably the ‘Kalimas’ and ‘Kawisari’ hybrids, which also have a high degree of resistance to coffee leaf rust, and in some cases high yields and a fair market price (C. canephora and C. liberica (possibly var. dewevrei) have also been documented (C. eugenioides), C. liberica hybridizes with C. eugenioides, to form diploid and tetraploid hybrids. In India, these hybrids showed good yield potential and coffee leaf rust resistance, although the need for further development was identified . Thus, in Uganda, indigenous C. liberica var. dewevrei could offer potential as a breeding partner, for imparting required traits to new coffee crop plants, via interspecies crosses.d ‘S.26’ . In Indoet price . Diploidcumented . As mententified . In MadaC. neoleroyi from being a coffee crop species, including extremely low yields  size, diminutive stature and spindly growth form. It is possible that a coffee-like beverage could be made from the seeds of C. neoleroyi, but this remains untested. Given the differences in floral morphology between C. neoleroyi and coffee beverage crop species , this species remains poorly known.Several factors preclude  species  breeding species . Due to C. neoleroyi is included for illustrative purposes only (as there is only a single data point). Density plots for Bio1, Bio12, precipitation seasonality (Bio15), and elevation are given in C. neoleroyi). The modelled mean annual temperatures, annual precipitation and precipitation seasonality for the Ugandan populations are: C. canephora (21.8°C/1389 mm/44), C. eugenioides (21.3°C/1370 mm/42), and C. liberica var. dewevrei (24.4°C/1560 mm/51). These data  for C. liberica var. dewevrei (compared to C. canephora and C. eugenioides) is evident because this species is confined to lower elevation forests in western and northern Uganda , and precipitation seasonality (Bio15), as shown in C. canephora and C. eugenioides is due to the higher number of datapoints in drier locations, compared to wetter locations, biased by the higher number of datapoints for these species overall. For C. liberica var. dewevrei, the density distribution for Bio12 (i.e. number of wetter locations) is bimodal (C. liberica var. dewevrei (51), compared to C. canephora and C. eugenioides (44 and 42), but given the above-stated considerations (including the low number of samples) any firm interpretations are inadvisable. The global values (i.e. across the entire indigenous distribution) for C. canephora are higher [56;  Group 1;  as C. liberica var. dewevrei. It could be argued that the C. liberica var. dewevrei once occurred in this forest and has since been extirpated due to partial deforestation, but this seems unlikely. There are plentiful herbarium specimens for C. canephora and C. eugenioides collected from Itwara, and it seems likely that at least one specimen of C. liberica var. dewevrei would have been collected, especially given the relatively easy access to this forest. Coffea neoleroyi is only known from a single collection (There are four indigenous (wild) coffee species in Uganda: Group 1; . Coffea ed there ; dedicatC. canephora (Least Concern), C. eugenioides (Least Concern), C. liberica var. dewevrei (Endangered), and C. neoleroyi . Under a Least Concern rating, individual populations may still be at risk of extirpation, as is the case for C. canephora and C. eugenioides. In terms of genetic resources, and their value to the Ugandan coffee sector, this review shows that potentially useful attributes  are distributed across populations (as well as the species as a whole) and thus require conservation. The rating of Endangered for C. liberica var. dewevrei is of considerable concern, particularly given the level of forest clearance and land use change at Zoka Forest. Further data is required before a confident extinction assessment can be made for C. neoleroyi, but this is also undoubtedly also a species of concern.Our preliminary country-level IUCN Red List conservation assessments  for the C. canephora, C. eugenioides and C. liberica var. dewevrei occur (grow and reproduce) over the same range of basic climate variables  as summarised in Coffea liberica var. dewevrei occurs in much warmer locations  than C. canephora, C. eugenioides  on account of it being restricted to low elevation forests. The elevation restriction may not be due to inability, or lack of opportunity, for this species to exist at higher elevations, although there could be intrinsic factors in play. There is also the possibility that the distribution and elevation range of C. liberica var. dewevrei in Uganda may have been more extensive historically, prior to forest clearance by humankind. The bimodality in mean precipitation (Bio12) and higher precipitation seasonality (Bio 15) for C. liberica var. dewevrei  and can be close to rivers (Zoka Forest), and outside Uganda it has been often recorded in gallery forest in native habitats (C. liberica var. dewevrei is more drought tolerant than C. canephora. Drought tolerance assumptions and observations for C. liberica var. dewevrei (as C. excelsa) have been made by other workers  value = 44) C. liberica var. dewevrei (PS = 51) are not that far apart  infer that there are precipitation seasonality differences between these two species. This is supported by observation of wild and farmed populations of C. canephora, which generally occur in locations with lower precipitation seasonality than C. arabica , although these relationships are complex  and require careful evaluation. Coffea racemosa and C. zanguebariae, two species occurring in areas of extremely seasonal rainfall, can have Bio15 values of 90  are required to substantiate the analyses and observations presented here, and better understand the climatic tolerances of indigenous Ugandan coffee species, and cultivated Arabica coffee. Experimentation of this nature would be critical for understanding the value of Uganda’s wild species resources for crop development, across different agroecological conditions under a changing climate.In Uganda, indigenous habitats . Howeverhabitats  and farm workers . The modPS = 58; ; and indes of 90 , indicatC. canephora (robusta coffee) have provided [after selection  represents an interesting development, and may offer considerable potential, as a third coffee crop species for Uganda (after C. arabica and C. canephora). Excelsa coffee fulfils farmers requirements, as it is easy to grow, appears to possess resistance to many of the major pests and diseases of coffee, is high yielding, and has an acceptable conversion (outturn) ratio from fresh fruit to clean coffee, particularly compared to the large fruited, thickly pulped types of C. liberica var. liberica  of C. liberica var. dewevrei over C. canephora during drought conditions (in 2021 and 2022). Coffea liberica var. dewevrei is certainly more heat tolerant than wild and cultivated C. arabica, which has a mean annual temperature range of 18–22 °C  tolerance advantages over C. canephora. Under wet soil conditions in Uganda, the root-system of C. canephora has been reported to be superficial and shallow (C. liberica var. dewevrei is tolerant of a wide range of soils (Coffea liberica var. dewevrei (excelsa) is capable of producing economically viable, good quality coffee (see above) but further assessment is required to see how it will perform across the value chain. This species may also offer useful prospects as a rootstock for C. canephora and perhaps other coffee crop species, and for coffee crop development via breeding .The recent farming uptake of liberica . Coffea 18–22 °C . It is cort-term . Whilst  shallow , which iof soils . Coffea C. eugenioides may offer potential as a niche crop for the high-value sector of the coffee market, as it has in Central America. The development of C. eugenioides in Uganda would require investment and a proof-of-concept period, to test for commercial viability. The small seed (coffee bean) size and low yields represent key constraints, unless better performing variants can be found within wild populations, although this seems unlikely based on the field surveys we have carried out so far. Coffea eugenioides is likely to offer better potential as a breeding partner, for imparting flavour qualities and other attributes via interspecies crosses.Due to its highly desirable flavour qualities, C. eugenioides and C. canephora in Uganda could be of paramount interest, since they are the progenitors of C. arabica and could be used to produce Arabica analogues. Moreover, it has been shown that the C. canephora-derived sub-genome of C. arabica is closely related to the C. canephora accessions from northern Uganda, and in particular Zoka Forest  and C. neoleroyi) and provide new ecogeographical data summaries (and other information) for each of these species. Climate profiling, via simple modelling methods, shows overlap for basic climate requirements for three of these species (C. neoleroyi was excluded due to lack of data), although C. liberica var. dewevrei has a higher density of individual records in locations of higher temperature, and a higher precipitation seasonality. At the national level, a draft IUCN Red List assessment indicates that C. liberica var. dewevrei is Endangered, and that C. neoleroyi could be Critically Endangered. Many wild Coffea populations in Uganda are compromised due to land use change  and some populations may be threatened with extirpation. The considerable indigenous diversity reported for C. canephora, and assumed diversity for C. eugenioides and C. liberica var. dewevrei, based on number of populations and range, represents valuable natural capital for crop development (e.g. via breeding) and the sustainability of the Uganda coffee sector in general, particularly under changing climatic conditions. Wild populations of C. canephora have provided Uganda with the bulk of diversity for the establishment and sustainability of its thriving robusta coffee sector. Coffea liberica var. dewevrei (excelsa coffee) shows potential as stand-alone crop species, and as a source of grafting stock for C. canephora (robusta coffee) and other coffee species. The coffee natural capital of Uganda requires improved protection, in order to avoid the loss of genetic diversity and coffee crop development options.In this contribution we enumerate four indigenous coffee species for Uganda (The original contributions presented in the study are included in the article/supplementary materials. Further inquiries can be directed to the corresponding author.AD designed the research project, undertook the research and some of the analyses, and wrote the paper; CK designed the research project and undertook the research; AF designed the research, undertook the research and most of the analyses; JM provided ideas, guidance and logistic support; JK designed the research project and undertook the research. All authors contributed to the article and approved the submitted version.


Cryptococcus neoformans and Trichosporon asahii are known to produce melanin in the presence of phenolic precursors. Other species from the Trichosporonaceae family also produce this pigment, but the extent to this production among the clinically relevant species is unknown. For this reason, the aim of this study was to verify the production of melanin by different Trichosporonaceae species of clinical interest and to compare their pigments with the ones from C. neoformans and T. asahii, which are more prevalent in human infections. Melanin was produced in a minimal medium supplemented with 1 mM L-dihydroxyphenylalanine (L-DOPA). Pigment was evaluated using scanning electron microscopy, Zeta potential measurements, and energy-dispersive X-ray spectroscopy. It was found that, besides C. neoformans and T. asahii, Trichosporon japonicum, Apiotrichum montevideense, Trichosporon inkin, Trichosporon faecale, Cutaneotrichosporon debeurmannianum, and Cutaneotrichosporon arboriformis also produce melanin-like particles in the presence of L-DOPA. Melanin particles have negative charge and are smaller than original cells. Variations in color, fluorescence, and chemical composition was noticed between the studied strains. All melanins presented carbon, oxygen, sodium, and potassium in their composition. Melanins from the most pathogenic species also presented iron, zinc, and copper, which are important during parasitism. Biophysical properties of these melanins can confer to the Trichosporonaceae adaptive advantages to both parasitic and environmental conditions of fungal growth.Melanin is one of the most studied virulence factors in pathogenic fungi. This pigment protects them from a series of both environmental and host stressors. Among basidiomycetes,  Trichosporon was accounted for these infections. However, with the advent of the molecular biology, a taxonomic reclassification of this genus resulted in the distribution of some Trichosporon species to other fungal genera, such as Apiotrichum and Cutaneotrichosporon. The species in this family with a major clinical relevance include Trichosporon asahii, Trichosporon inkin, Trichosporon faecale, and Trichosporon asteroides  as a precursor -melanin is the major allomelanin type, and it is derived from 1,8-DHN generated by a series of reactions starting with the polymerization of endogenous acetyl-CoA or malonyl-CoA into 1,3,6,8-tetrahydroxynaphthalene by a polyketide synthase . MelaninTrichosporon species have been reported as agents of human infections, T. asahiistands as the major agent of trichosporonosis worldwide , Trichosporon japonicum (CFP00907), Trichosporon inkin , Trichosporon faecale (CFP00905), Apiotrichum montevideense (CFP00909 and CFP00950), Cutaneotrichosporon debeurmannianum (CFP00913), and Cutaneotrichosporon arboriformis (CFP00914). All strains were identified by molecular methods, as previously described (Cryptococcus neoformans var. grubii H99 strain (ATCC 208821) was included in the analyses as a positive melanization control.Ten different clinical isolates from the Trichosporonaceae family, all from the Coleção de Fungos Patogênicos, Fiocruz (WDCM 951) were analyzed in this study. They include escribed . The Cry4, 29.4 mM KH2PO4, 13 mM glycine, 3 μM thiamine, 1 mM L-DOPA, pH 5.5), in a final concentration of 5 × 104 cells/mL, as previously described  (pH 7.0), and washed in 1.0 M sorbitol, 0.1 M sodium citrate (pH 5.5). Cells were harvested, lytic enzymes from Trichoderma harzianum  were added at 10 mg/ml, and the suspension was incubated at 30°C for 1 h to generate fungal protoplasts. The protoplasts were collected by centrifugation, washed three times with PBS, and incubated with 4.0 M guanidine thiocyanate (Sigma-Aldrich), a denaturant agent, for 1 h, with frequent vortexing. Cell debris were harvested, washed three times with PBS, and then boiled in 6.0 M HCl (Sigma-Aldrich) for 1 h. Melanin particles were collected by centrifugation, washed extensively with PBS, and stored at 4°C. As per comparison purposes, 500 μl of each melanin suspension, including controls, were added to microcentrifuge tubes for a pigment intensity visual analysis, as described for other fungi (The cultures mentioned above were incubated in the dark for 14 days at 37°C under agitation (150 rpm). Fungal cells were collected by centrifugation at 10,842 × er fungi .The extracted melanins were quantified in 96-well microplates, in a final volume of 100 μL per well. For this, the optical density (OD) of the wells at 340 nm was determined using the Epoch microplate spectrophotometer , both for the melanin particles and for the negative control, medium with L-DOPA without cells. This experiment was performed in triplicate.The melanin particles were adhered to 12 mm diameter round glass coverslips  previously coated with 0.01% poly-L-lysine  for 60 min and dried in a nitrogen-rich atmosphere. Finally, they were mounted on stubs and coated with a thin layer gold-palladium (10–15 nm) using the sputtering method  and viewed on a scanning electron microscope (FEI Quanta FEG 450) at a voltage of 10 kV.A scanning electron microscopy with energy-dispersive X-ray spectroscopy (EDS) was performed to determine the fundamental composition of the melanin constituents of the samples. This consists of identifying the lines in the X-ray spectrum using energy tables or wavelengths and quantitative analysis of EDS (determination of the concentrations of the elements present) by measuring (run time was approximately 45 s) the line intensities for each element of the sample and for the same elements in the known composition calibration standards. As a control a carbon adhesive  mounting stub was analyzed without particles, and the resulting spectra showed no contributions from the stub, i.e., aluminum (Al) or sulfur (S).Microscopy was performed on the FEI Quanta FEG 450 with Apollo X – EDAX scanning microscope. Analyses were performed at a 10,000× magnification using the SEM’s backscattered electron (BSE) detector.These measurements were conducted in a Zeta potential analyzer NanoBrook Omni  as previously described . Ten meaMelanins were suspended in PBS after vigorous vortexing as the reference. These samples were distributed (100 μl) in a 96-well UV transparent microplate. The UV – visible absorption spectrum of the melanin was scanned in the wavelength range of 200–700 nm with the UV – visible spectrophotometer SpectraMax plus 384 . Absorbance values were used to construct the UV-light absorption spectrum of each melanin. Additionally, the logarithm of absorbance values were plotted against the 250–500 nm wavelengths and the slopes compared among the samples, as described . This exThe particles recovered from brown cells after treatment with enzymes, denaturant, and hot acid were visualized under a 1,000× magnification using a fluorescence microscopy  with DAPI, Alexa Fluor 488, Alexa Fluor 546, and Far-Red filters.p < 0.05 was considered significant.The software GraphPad Prism 7  was used for the analyses. Comparisons were made using the ANOVA test with Bonferroni’s multiple comparison test. Slopes of logarithmic spectral curves were compared after a linear regression analysis. A C. neoformans produced black melanin. Among the studied Trichosporonaceae strains, it was noticed that, like C. neoformans H99, one A. montevideense strain (CFP00909) produced black pigment. Four strains produced dark-brown pigment: A. montevideense (CFP00950), C. debeurmannianum (CFP00905), T. inkin (CFP00951), and T. japonicum (CFP00907). The other C. debeurmannianum strain (CFP00913) produced brown pigment. Three strains produced light-brown pigment: T. inkin (CFP00904 and CFP00946) and C. arboriformis (CFP00914). The T. asahii strain (CFP00944) produced black melanin particles . When compared to T. asahii, only the samples of Trichosporon inkin (CFP00904 and CFP00946) had no significant difference , A. montevideense (CFP00950), and T. faecale (CFP00905) presented six elements, while melanins from T. asahii (CFP00944) and T. inkin (CFP00951) presented 13 different elements. Overall, all samples presented high carbon, oxygen, and sodium concentrations. Potassium was also found, in lower proportion, in all samples. Nitrogen and sulfur were not found in any studied melanin. A few samples presented the heavy metals barium, vanadium, and titanium. Iron, copper, and zinc were observed in T. asahii and T. inkin melanins.C. neoformans was lower than most of members of the Trichosporonaceae family (p < 0.05), with the exception of the strains C. debeurmannianum (CFP00913) and A. montevideense (CFP00950). In addition, the zeta potential of melanin produced by T. asahii was lower than other Trichosporonaceae siblings, except C. debeurmannianum (CFP00913), T. inkin (CFP00946) and A. montevideense (CFP00950) (C. neoformans (T. asahii (766.1 ± 3.6 μS) and A. montevideense (108.4 ± 0.9 μS). All melanin samples exhibited UV spectra with absorption peaks around 200 nm followed by a progressive decrease for higher wavelengths  . The conoformans . Most Trelengths . Table 2elengths .Trichosporon genus are scarce, especially when considering the new taxonomic criteria of the Trichosporonaceae and its new genera. Our group described previously the production of eumelanin in T. asahii, T. asteroides, T. inkin, and T. mucoides  and A. montevideense (CFP00950) strains presented the most electronegative zeta potential among the Trichosporonaceae in this study. It has been described that Cryptococcus melanin is negative, which confers resistance against phagocytosis (T. asahii appears to be the most virulent species in the Trichosporonaceae family, as it is frequently found as the causative agent of trichosporonosis (C. debeurmannianum and A. montevideense are not often described as important agents of trichosporonosis (Other important biophysical aspect of all Trichosporonaceae melanin particles was their negative charge, as detected by zeta potential measurements. This is a typical characteristic for fungal melanins . The C. ividuals . One of ocytosis . T. asahoronosis . The speoronosis . Our resThe results of the present study show differences in melanin composition and/or structure among the strains studied. These differences could be observed through fluorescence and EDS scanning electron microscopy. Melanin is a moderately low quantum yield fluorophore when excited with UV or visible light, and this fluorescence can be weakened by its own broad spectral absorption . In addiSepia officinalis melanin composition revealed high concentrations of carbon, oxygen, sodium, and chlorine (T. asahii does not have a gene homologous to the polyketide synthase, the key gene for DHN-melanin production. In addition, the denaturant/hot acid treatment showed that the strains only produce melanin when L-DOPA is present. In fact, during the analysis of S. officinalis melanin using SEM-EDS, nitrogen was not detected in two of five replicates of the experiment, suggesting that the chemical composition is different depending on positions of the sample (Studies about chlorine . All butchlorine . This ele sample .T. japonicum strain. In addition, some basidiomycetes can accumulate high barium concentration (Fungal melanins have the ability to bind to various metals, participating in metal bio absorption from rocks and other environmental niches . Melaninntration . As basintration , our resT. asahii, T. inkin, and C. neoformans presented iron in their melanin composition. During parasitism, iron availability is limited, and fungal pathogens may develop strategies to acquire this element (Only the highly pathogenic species  element . The pre element .Characterization of the physicochemical properties of melanoid pigments has been carried out in different fungal species over the years. Magnetic and paramagnetic resonance studies, as well as the surface charge of this pigment, demonstrated the antioxidant capacity of melanin and its contribution with virulence . Our nexAt least seven human pathogenic species from the Trichosporonaceae family produces melanin when L-DOPA is available during growth. The composition and amount of melanin appear to vary within different strains. Biophysical properties of these melanins can confer to the Trichosporonaceae adaptive advantages to both parasitic and environmental conditions of fungal growth.The original contributions presented in the study are included in the article/IA: research design, data analysis, and writing-original draft preparation and review and editing. GA: research design, data analysis, and writing-review and editing. FB-S and SF: study design, data analysis, and writing-review and editing. MF-C: research design and writing-review and editing. RZ-O: study design, data analysis, writing-review and editing, and funding acquisition. RA-P: study design, data analysis, and writing-original draft preparation and review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Candida auris is considered to be an emerging fungal pathogen and is related to high mortality rates, persistent candidemia, inconsistencies in susceptibility testing results and misidentification by available commercial identification systems. Multidrug-resistant (MDR) and pandrug-resistant (PDR) strains are increasingly detected. In Europe, hospital outbreaks caused by C. auris have been reported in the United Kingdom (UK), Italy and Spain; however, several cases have been sporadically detected in all European countries. C. auris is difficult to control despite enhanced control measures due to its ability to survive for a long time in environments and colonize patients for prolonged periods. An adequate laboratory diagnostic capacity and national surveillance are fundamental to rapidly detect new C. auris cases and to apply the correct measures to circumscribe them and prevent their spread. Our narrative review aims to highlight the primary C. auris outbreaks and case reports that have occurred in Europe. Candida auris has been considered to be a rising healthcare emergency worldwide. C. auris infections are related to high mortality rates, persistent candidemia, inconsistencies in susceptibility testing results and misidentification by available commercial identification systems. All this must be considered alongside a high risk of treatment failure, which complicates its management [From 2009, nagement .C. auris was initially found in Japan [Candida strain found C. auris in South Korea in 1996 [C. auris emerged simultaneously and independently in four global regions . These four clades are genetically distinct [C. auris infections have increased worldwide [C. auris is now considered to be endemic [In 2009, in Japan . However in 1996 . Studiesdistinct . Most reorldwide . In many endemic . In addi endemic ,9,10.C. auris strains are increasingly detected worldwide. The most frequent resistance is to fluconazole (FLC), followed by amphotericin B (AMB) and voriconazole (VRC). Echinocandin remains the treatment of choice, but resistance can also affect this class of antifungal drugs [Multidrug-resistant (MDR) and pandrug-resistant (PDR) al drugs ,11.C. auris invasive infection are similar to other Candida infections. However, many studies have demonstrated the environmental persistence ability of C. auris, including in the air, on surfaces and bedding materials. Moreover, C. auris has been isolated from the skin of colonized patients  for several months [The clinical presentation and the risk factors of a l months .C. auris was first detected in the external ear canal of a 70-year-old Japanese woman. A 26S ribosomal DNA (rDNA) D1/D2 domain analysis, 18S internal transcribed spacer (ITS) rDNA region sequences and chemotaxonomic studies showed that the newly discovered Candida species (spp.) had a close phylogenetic relationship to the Metschnikowiaceae clade, particularly with C. ruelliae and C. haemulonii [C. auris strains were initially misidentified as C. haemulonii [C. auris belongs to the Metschnikowiaceae family within the Candida/Clavispora clade such as C. albicans, C. tropicalis, C. haemulonii and C. lusitaniae [emulonii . A retroemulonii . A genetsitaniae .C. auris as another yeast species using conventional phenotypic and biochemical methods can be common  and whole genome sequencing (WGS) analyses [The identification of yeasts by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analyses has the potential to quickly identify atabases ,13. In aanalyses .C. auris can express several virulence factors, including saps and lipases [C. auris is less virulent than C. albicans. That characteristic was shown in murine and invertebrate G. mellonella infection models. In murine models, it was demonstrated that C. auris was much more virulent than C. glabrata and C. haemulonii [C. albicans, depended on the inability of C. auris to develop virulence factors such as hyphae or pseudohyphae, which play a critical role in tissue invasion [C. auris is a haploid yeast whereas natural C. albicans isolates are diploid. This could have an essential role in the intrinsically low virulence of C. auris. In FLC-induced haploids, the C. albicans strain reduced their virulence compared with the diploid form [C. auris are poorly implicated in its virulence during systemic infections, but could play a role in skin and environmental surface colonization [ lipases . Howeveremulonii ,16. Thisinvasion . Furtheroid form ,17. The nization .C. auris strains have been reported in several cases, showing resistance phenotypes to FLC and AMB [FLC and echinocandins are the most used antifungal drugs to treat candidemia. Unfortunately, FLC (or other azole) resistance is common. A recent meta-analysis from Sekyere et al. showed that the most frequent resistance was to FLC (44.29%), followed by AMB (15.46%), VRC 12.67%), caspofungin (CAS) (3.48%), flucytosine (FC) (1.95%), itraconazole (ITZ) (1.81%), isavuconazole (ISA) (1.53%), posaconazole (POS) (1.39%), anidulafungin (AFG) (1.25%) and micafungin (MFG) (1.25%) ,12. MDR .67%, cas and AMB . Resista and AMB .C. auris is mainly related to alterations in the lanosterol demethylase enzyme, which is encoded by the ERG11 gene. C. auris can also encode ATP-binding cassette (ABC) and major facilitator superfamily (MFS) efflux pumps, which are essential mechanisms of antifungal resistance, especially during the initial stages of biofilm development. When resistance to echinocandins; occur, it is due to mutations in FKS genes that encode a subunit of the β-D-glucan synthase. Moreover, changes to the cell membrane sterol and/or a given point mutation are potential mechanisms of AMB resistance [The molecular mechanism for azole resistance in sistance ,20.C. auris are currently standardized for the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST). Therefore, the Centers for Disease Control and Prevention (CDC) defined a C. auris-specific antifungal susceptibility interpretation based on a close phylogenetic relationship to other Candida spp. The correlation between the microbiologic breakpoints and clinical outcomes is not known. The current breakpoints are summarized in Unfortunately, no antifungal susceptibility breakpoints for C. auris cases have escalated within the last few years. The reported isolates were mainly isolated in males (64.76%). No reason has been given for the C. auris distribution by gender. Local variables and the health diversity of countries could play a role in the increase in C. auris male case rates. Patients with C. auris infections frequently presented several other underlying health comorbidities such as diabetes, sepsis, pulmonary diseases, bacterial pneumonia, renal diseases, transplants, immunosuppression, solid tumors, cardiovascular diseases, chronic otitis media and liver diseases [Most diseases .C. auris infections are similar to other Candida spp. generic risk factors. Most frequently, infections occur in hospitalized patients, especially those admitted to the intensive care unit (ICU) or those who underwent surgery in the previous 30 days. Moreover, central venous catheters, hemodialysis catheters and permanent urinary catheters could be related to invasive C. auris infections [The risk factors for fections ,20,22.C. auris-case fatality rates. However, several authors have suggested that the mortality rate of invasive C. auris infections is comparatively higher than that of Candida spp. For C. auris, the crude mortality rate was estimated to be 30% to 72% [Even with an appropriate antifungal treatment, invasive candidiasis has a mortality rate of up to 30–40%. Currently, there is limited information on specific % to 72% ,18,23,24C. auris case was detected at San Martino Hospital (Genoa) in a patient with no history of recent travel abroad, hospital admission or close contact with other C. auris cases [C. auris was detected in 10 non-duplicate clinical isolates in the coronavirus disease-19 (COVID-19) ICU [In mid-July 2019, the first is cases . Since tC. auris reported cases occurred at San Martino Hospital; only 67 cases were distributed to 7 other healthcare facilities in Liguria [A subsequent increase in cases was observed throughout 2020 and 2021. In February 2022, 277 cases occurred at 8 healthcare facilities in Liguria and 11 patients were detected at facilities in the neighboring region of Emilia-Romagna. Most  Liguria .C. auris isolates from the first cases at San Martino Hospital between 2019 and 2020 were closely related to the South Asian clade and all isolates, except one, originated from the same cluster of the index case [C. auris cases were observed at AOU Città della Salute e della Scienza (Turin). All the patients were admitted for critically ill conditions to the ICU. The majority (75%) had severe respiratory diseases due to COVID-19 and presented with prolonged hospitalizations, multiple complications and the simultaneous or previous presence of other infections, especially from difficult-to-treat Gram-negative microorganisms [The rganisms .C. auris cases in continental Europe were identified at La Fe University Hospital and Polytechnic . All of them were admitted to the post-surgical ICU [C. auris colonizations were identified at the same hospital and 41 of them developed invasive fungemia. All isolates were FLC- and VRC-resistant, but echinocandin- and AMB-susceptible. A phylogenic analysis revealed that the Spanish isolates were clonal, with an overall similarity of >96%. In addition, all Spanish isolates seemed to be genotypically connected to the South African isolates and one was grouped in the Venezuelan cluster [Between April and June 2016, four ical ICU . From Ap cluster .C. auris was identified for the first time in the urine culture of a patient at the Consortium of the General University Hospital . As a result, the patient was admitted to another hospital. A month later, on 14 October 2017, the second case was diagnosed. From September 2017 to August 2019, 203 patients were colonized and 30 invasive infections were diagnosed: 29 candidemia and 1 meningitis. All strains were resistant to FLC [In September 2017, C. auris case to 2019, Spain reported 786 cases of C. auris colonizations/infections, with a reduction in cases from 2017 to 2019 [C. auris cases rose again (591 new cases from 2020 to 2021) [From the first Spanish  to 2019 ,31,32. UCurrently, Spain is the only European Union (EU) country where there is a reported regional endemicity .C. auris isolates were identified at the UK National Mycology Reference Laboratory (MRL) in 2013 from blood cultures from three unrelated patients from distant geographical localities. From 2013 to 2016, the MRL received 19 isolates from 6 hospitals [The first three ospitals ,31.C. auris cases were identified, with 61 infections (including 31 candidemia) across 22 hospitals. In addition, three significant outbreaks were reported in ICUs in London and Oxford [Between June 2013 and March 2017, 225 d Oxford ,34.C. auris was identified in a patient admitted to the medical-surgical adult ICU of Royal Brompton Hospital (London), a specialized cardiothoracic center. The yeast was initially cultured from a sternal wound. A total of 50 new C. auris cases were identified in the following 16 months from the index case. All isolates expressed a high-level FLC resistance. In most cases, C. auris exclusively colonized the skin or mucosa (56%). Only 9 patients had candidemia (16%). Nevertheless, 22 required an antifungal therapy. However, no deaths were directly attributed to infections by C. auris [The first hospital outbreak was in April 2015. C. auris .C. auris in the UK before 2013 suggested multiple independent introductions of the yeast [Phylogenetic analyses revealed that the UK isolates from the different outbreaks could be separated into three distinct clades, which contained isolates previously reported from India/Malaysia/Kuwait, South Africa and Japan/Korea. Furthermore, these data and the absence of he yeast .C. auris infections at the neuroscience ICU of Oxford University Hospitals (Oxford), Eyre et al. started an active screening period for C. auris colonization. Patients were tested on admission to the neuroscience ICU, weekly and on discharge. As a result, 70 patients were identified as being colonized and 7 had an invasive infection  [Between February 2015 and August 2017, after the identification of a case series of ingitis) .C. auris in ICUs. The study was conducted between May 2017 and April 2018. The study screened all ICU admissions who had no prior diagnosis of a C. auris colonization at eight adult hospitals. The results showed that C. auris screening for all 921 patients was negative. This finding demonstrated that in England, C. auris colonization among patients admitted to ICUs is rare [In 2017, England provided a national prevalence study on admission screening for  is rare .C. auris. From January 2018 to May 2019, the UK reported  48 cases only [Data from the UK showed that the early detection of outbreaks combined with isolation enhanced the infection control measures and screening could control the spread of ses only ,38.C. auris survey also counted cases from Germany (7), France (2), Belgium (1) and Norway (1). Most patients were considered to be colonized; fungemia or other invasive infections were less frequent [C. auris surveillance between January 2018 and May 2019 detected C. auris cases from eight other countries, including Austria (1), France (1), Germany (3), Greece (1), the Netherlands (2), Belgium (1), Norway (1) and Poland (1) [Between 2013 and 2017, the European Centre for Disease Prevention and Control (ECDC) frequent . Excludiland (1) .C. auris outbreaks in Europe were observed during the COVID-19 pandemic. The main reason for the spread of C. auris was an increase in the number of vulnerable patients requiring a high intensity of care combined with the concurrent overload of healthcare systems and the inability to maintain sufficient standards and contact precautions [Favorable conditions for cautions .C. auris outbreaks, defined as 2 or more cases with an epidemiological link [Sporadic cases were continuously identified scattered around Europe such as in Denmark, Switzerland, Finland, Ireland, Sweden and Russia ,41,42,43cal link .C. auris case was diagnosed in 2019 in a cystic fibrosis patient with no history of recent travel abroad or hospitalization [C. auris between 2020 and 2021 has been of particular concern. In 2021, 58 new cases were found, with a verified or plausible inter-facility spreading [In Greece, the first lization . Since tpreading .C. auris cases have been sporadically identified since 2015. To date, rare outbreaks with limited inter-facility spreading have been reported, with only 4 cases in 2020 and 4 cases in 2021 [In France, a number of  in 2021 .C. auris cases have been reported, most of which occurred in patients who had recently been in contact with hospitals/healthcare providers abroad. However, the number of cases documented by the National Reference Center for Invasive Fungal Infections (NRZMyk) has rapidly increased [C. auris can be contained with prompt control measures [C. auris case reports in Europe where C. auris was detected.In Germany, 27 ncreased ,44. Despmeasures . Figure C. auris cases (data not including the UK cases). A total of 44 (2.4%) cases were reported as imported and 10 (0.6%) as locally acquired. The origin was not always mentioned, but the most frequent countries of imported cases were Egypt, Ethiopia, Kenya, South Africa, Iraq, Kuwait, United Arab Emirates, India and Pakistan) [In the EU, between 2013 and 2021, there were 1812 identified akistan) .C. auris is isolated, robust actions are necessary to reduce the risk of developing hospital and regional outbreaks. A multidisciplinary approach is essential to implement infection prevention and control measures. A prompt identification and notification should trigger an investigation, including a detailed case review and a screening of close contacts. Infection control options include the application of contact precautions, single-room isolation or patient cohorting and, in a few cases, dedicated nursing staff. Screening of close contacts should be performed with axilla and groin swabs. Other samples  can be collected to implement the diagnostic tools. The regular cleaning of surfaces with chlorine-based disinfectants (at a concentration of 1000 ppm), hydrogen peroxide or other disinfectants with a documented fungicidal activity is needed to reduce environmental contamination. Single-use or dedicated equipment should be preferred. The cleaning and disinfection of reusable equipment according to the manufacturer’s instructions should be ensured. The environmental sampling or screening of healthcare workers is not routinely recommended [If ommended .C. auris has been isolated in multiple areas of the world and its capability to determine healthcare outbreaks raises significant global concerns [C. auris has been difficult to control, despite enhanced control measures. It is known that contaminated surfaces play an essential role as an environmental reservoir. Surveillance studies showed that C. auris could survive on surfaces for at least 14 days and on contaminated bedding for up to 7 days [C. auris can be isolated on the skin for several months [Since its initial discovery, concerns ,46. In to 7 days ,48. In tC. auris spreading in the hospital environment. For example, an analysis performed during the first UK outbreak at Royal Brompton Hospital showed that the minimum contact time required to acquire C. auris was four hours [C. auris case [Additionally, healthcare workers could be responsible for ur hours . In concris case .C. auris have occurred in the UK, Italy and Spain, but different case reports have been observed over almost all Europe. New C. auris cases were mainly diagnosed during the COVID-19 pandemic when the European healthcare system was subjected to continuous overload with an inability to maintain sufficient infection control measures and adequate antibiotic stewardship programs [C. auris cases and outbreaks [C. auris cases. It should be remembered that commercially available laboratory tests used by clinical laboratories may not be able to identify or could misidentify C. auris strains  died within 30 days after the beginning of late recurrent candidemia [C. auris [C. auris, the overall crude mortality ranged from 0 to 78%, with candidemia-associated mortality of 45% (vs. 21% in the non-candidemia group). A subanalysis of C. auris European mortality showed lower mortality rates (20%) [Although  unknown ,22,23. Aound 70% ,22,23,38e of 58% . High moe of 58% ,52,53. Indidemia . In contC. auris ,37. In aes (20%) .C. auris to generate hospital and inter-facility outbreaks, a local control protocol must be applied as soon as possible after the diagnosis of a new C. auris case. The possible laboratory misidentification of this yeast can delay the prompt application of all the measures that are necessary. Once C. auris has spread between facilities or regions, control is more difficult to achieve. For this reason, an adequate laboratory capacity for a correct fungal diagnosis is mandatory. This recommendation is valid, especially in the case of patients with a history of hospitalization in countries with a high C. auris prevalence. Currently, no antimicrobial stewardship program has evidenced the benefits of C. auris diffusion. A few European countries (Denmark and the UK) have shown that the spread of C. auris can be contained only with the planning of screening protocols, the isolation of suspected/confirmed cases and the application of additional hygiene measures.Considering the potential of C. auris cases represents an important wake-up call for all Europe. We should prepare to be increasingly confronted with this potentially fatal new pathogen.In conclusion, the increase in European


Sclerotinia sclerotiorum infection and longer ﬂowering-period for flower tourism.Oilseed rape plants with abscission-defective ﬂoral organs acquired through genome editing show less susceptibility to  Dear Editor,Brassica napus) is an important edible oil crop consumed worldwide. As an ornamental, its fascinating seasonal ﬂowers generate up to $1.57 million in flower tourism revenue per day in China , Inflorescence Deficient in Abscission (IDA) encodes a small secreted protein with an N-terminal signal peptide, and IDA knockouts retain floral organs indefinitely , and in other floral organs, including petals, sepals, and filaments. In contrast, the other three copies of BnIDA were barely expressed in floral organs , gRNA1 and gRNA2, were designed to specifically target two regions of the coding sequence (Tgt1 and Tgt2) of BnC06.IDA and BnA07.IDA  T0 plants exhibited editing events at the designed target sites   and bnc06.ida (line #3). To discriminate wild-type, heterozygous, and homozygous mutants, allele-speciﬁc PCR markers for BnA07.IDA and BnC06.IDA were developed (2 progeny with bnc06.ida bna07.ida double mutants within 6 months.Mutant plants were self-pollinated and the obtained T (J9712) , suggesteveloped . Marker-eveloped  allowed bna07.ida bnc06.ida plants (bna07.ida and bnc06.ida adhered to leaves or petioles (Wild-type and mutant plants were subsequently grown in a climate chamber. The sepals, petals, and stamens abscised at flower positions 10–11 in wild-type and a plants . Strikina plants . In contpetioles .bnc06.ida bna07.ida plants (To quantify the extent of floral organ abscission, the mechanical force required to remove petals from flowers was measured with a high-precision tension meter . The brea plants .bna07.ida bnc06.ida plants. At position 4, floral organs were forcibly removed, forming a fracture plane with broken cells at the floral AZ in both genotypes (bna07.ida bnc06.ida and wild-type plants were not substantially different (We further compared the morphological differences at the floral AZ between the wild-type and enotypes . After aenotypes , and theenotypes . Insteadenotypes , indicatifferent .Sclerotinia. In wild-type plants, inoculated petals were removed and adhered to leaves, while inoculated petals remained attached in the mutants. The majority of infected petals caused necrosis to the leaves of wild-type plants, whereas disease lesions were undetectable in the petals and leaves of the double mutants (To test sensitivity of the mutants to SSR, petals were inoculated with a mycelial suspension of  mutants .BnA07.IDA and BnC06.IDA are functionally redundant and are involved in the regulation of ﬂoral abscission in OSR. We successfully generated OSR plants with abscission-defective ﬂoral organs by simultaneously inactivating two BnIDA genes using CRISPR/Cas9 (Sclerotinia infection via abscised petals and stamens.In summary, our results demonstrate that SPR/Cas9 . The douThe following materials are available in the online version of this article. Petals adhered to leaves and petioles after abscission, spreading SSR. Fertility performances of bna07.ida bnc06.ida and wild-type plants. Sequences of the primers used in this study. Agronomic traits of wild-type and bna07.ida bnc06.ida plants grown in the climate chamber..kiac364_Supplementary_DataClick here for additional data file.


Cynopterus brachyotis (Müller 1838) is a generalist and widespread fruit bat species which inhabits different types of habitats in Southeast Asia. This species plays an essential role as a seed disperser and pollinator. Morphological study and phylogenetic analysis using mtDNA markers (cyt-b and D-loop) revealed that this species had two different forms in peninsular Malaysia and Borneo and six lineages in Southeast Asia that lead to new species formation. In addition, this species is also reported to have high genetic diversity in Malaysia and Thailand based on the D-loop sequence. However, a phylogenetic and genetic variation study of C. brachyotis in Indonesia has not been conducted yet. These two studies are important as additional information for taxonomic and population genetic studies of this species. Thus, we performed the phylogenetic and genetic diversity analysis of the C. brachyotis population collected from seven habitats on Java island, including open-fragmented habitats  and closed habitats  using the mtDNA D-loop marker.C. brachyotis from seven habitats on Java island overlapped between habitats and could not be distinguished according to their habitats and lineage. Intrapopulation and intraspecies analysis revealed high haplotype diversity of this species on Java island (Hd = 0.933–1.000). The haplotype network was split into two haplogroups, showing haplotype sharing between habitats. These phylogenetic and genetic variations analysis of C. brachyotis bats on Java island indicated that this species is widespread and adapt to different habitats.The phylogenetic tree using the Bayesian inference (BI) and genetic distance using the Kimura-2 parameter (K-2P) demonstrated that 33 individuals of C. brachyotis on Java island collected from seven different habitats has overlapped and genetically close and has high genetic variation. Our results provide the first reported study of C. brachyotis on Java island and provide data to understand the phylogenetic and genetic diversity of this species in Indonesia.This study of  Cynopterus brachyotis (Müller 1838) belongs to the family Pteropodidae and is widely distributed in Southeast Asia, including Indonesia. It has an important role in the ecosystem as seed dispersal and pollinator [C. brachyotis is generalis and eurytopic species where it can be found in many habitats (most frequently in the disturbed and fragmented area), such as the urban area, secondary forest, dipterocarp forest, gardens, mangroves, and strand vegetation [Lesser short-nosed fruit bat s Müller 838 belonllinator . C. bracgetation , 6.C. brachyotis  were revealed based on morphological study and inhabited two contrasting habitats in Peninsular Malaysia and Borneo. Moreover, the larger form (Sunda) was found to inhabit open areas (urban and plantation), whereas the smaller form (Forest) was confined to natural forests [This species in Southeast Asia has been reported to have morphological variations  and kary forests .D-loop demonstrated that C. brachyotis Sunda and Forest have different clades and separated population. Moreover, this analysis also revealed that C. brachyotis is a species complex with six distinct lineages: Sunda, Forest, India, Philippines, Myanmar, and Sulawesi [C. brachyotis species may occur [Phylogenetic analysis using mitochondrial DNA markers including cyt-b and the control region Sulawesi . Accordiay occur , 9. HoweC. brachyotis Sunda and Forest lineage can be found both in Peninsular Malaysia and Borneo and has contrasting habitats. A C. brachyotis sample was collected from Java island (Jakarta) and is included in the Sunda lineage [D-loop markers in Borneo, Thailand, and Peninsular Malaysia [Furthermore, according to that phylogenetic analysis,  lineage . UnfortuMalaysia , 10.D-loop is a hypervariable control region of mitochondrial DNA having high nucleotide variations and mutational hotspots [D-loop region was essential for understanding the genetic diversity and species population, including bats in particular habitats [D-loop can be used to estimate the impact of the anthropogenic process on an animal population. Genetic diversity can affect a species’ ability to adapt to environmental changes [C. brachyotis is fruit bat species closely related to habitat fragmentation [hotspots . Haplotyhabitats , 13. Gen changes . Besidesentation , 3.2 with a population projection is 56.1% of the Indonesian population makes this island the most densely populated island in Indonesia [D-loop in C. brachyotis have not been explored in Indonesia, especially on Java island Only karyotype and hematological studies of C. brachyotis were conducted on this island [C. brachyotis collected from different types of habitats on Java island inferred from the mitochondrial D-loop gene sequence. The results of this study are expected to be the basis for ecological management of C. brachyotis species and genetic research on other bat populations in Indonesia.Java island is an Indonesian island that has a very high and rapid population growth rate. This island belongs to the Greater Sunda islands alongside Peninsular Malaysia, Sumatra, and Borneo. A total area of about 128,297 kmndonesia . Thus, ts island , 17. TheThe ethical clearance for conducting this research was taken from the Research Ethics Committee of the Faculty of Veterinary Medicine, Gadjah Mada University, Indonesia . Animal handling and sampling (including tissue sampling) were carried out following their guidelines and supervision. This ethical clearance also covered other analyses, such as morphological measurements and hematological analysis of this species.C. brachyotis were collected from seven habitats including urban area, coffee plantation, rubber plantation, pine forest, secondary forest, natural forest, and mangrove forest in East Java, a special region of Yogyakarta, Central Java, and West Java  and H16498 (5′-CCTGAAGTAAGAACCAGATG-3′) [Genomic DNA extraction of membrane tissue was conducted using GS100 gSYNC™ DNA Extraction Kit  following manufacture instructions. Fragment of GATG-3′) . AmplifiGATG-3′) . PCR proD-loop. Alignment of D-loop sequence was performed using Opal in Mesquite v.3.51 [The ambiguous bases of chromatograms were corrected manually in GeneStudio software  to get a consensus sequence of e v.3.51  and Cluse v.3.51 .D-loop sequence nucleotides between habitats (intrapopulation) and all samples (intraspecies) were analyzed using the MEGA X software [Nucleotide composition analysis was used to determine the nucleotide differences between samples which indicated genetic variation. Differences in software .7 generations and a sampling frequency of 1000 generations.Reconstruction of the phylogenetic tree was performed using the Bayesian inference (BI) tree in BEAST 1.10 software . The besD-loop sequences of C. brachyotis from four distinct lineages, including Sunda , Forest , Myanmar (AY629073), and India (two accessions: AY629069 and AY629070) collected from GenBank were included in this analysis. Moreover, the D-loop sequence from two Rousettus bats, Rousettus amplexicaudatus (NC045044), and Rousettus leschenaultii (our data) were used as an outgroup. The analysis of genetic distance was conducted using the Kimura-2 parameter (K-2P) method in the MEGA-X software [A total of 48 software .Genetic variation analysis among habitat types including haplotype number (h), haplotype diversity (Hd), and nucleotide diversity (π) was performed using DnaSP 6.0 software . The levT = 27.29%, C = 29.17%, A = 30.16%, and G = 13.38%. The nucleotide composition analysis of intraspecies also revealed an AT-rich (57.45%) compared to GC (42.55%).The nucleotide composition analysis showed that the percentage of nucleotides T, C, A, and G of samples between habitats (intrapopulation) was varied. The average percentage range of T, C, A, and G nucleotides in seven habitats was 26.67–27.57%, 28.44–29.87%, 29.97–31.14%, and 12.57–13.87%, respectively. This analysis also showed AT-rich in all habitats with an average range of 57.39–58.35%. Meanwhile, the GC was 41.65–42.61%. The average nucleotide percentage of all sample (intraspecies) was D-loop sequence of 33 samples in this study and GenBank data yielded a fragment length of 412 bp. The phylogenetic tree demonstrated the D-loop sequence of C. brachyotis from seven habitats in this study, and GenBank data formed a single clade  had haplotypes ranging from 3 to 5 haplotypes with the number of samples of 3–6 individuals per habitat. The haplotype diversity and nucleotide diversity of seven habitats were 0.933–1.000 and 0.00980–0.01115, respectively . The first haplogroup consisted of H1-H22 (excluding H16 and H18) haplotypes originating from seven habitats . The second haplogroup consisted of H16 and H18 haplotypes from the secondary forest and natural forest habitats. Haplotype sharing in the first haplogroup, namely H1, H2, H4, and H11 haplotypes, was overlapping between habitats in different sampling locations. Meanwhile, other haplotypes were unique haplotypes . No previous studies explained the genetic distance threshold based on D-loop sequences in bats (Chiroptera). However, the genetic distance between samples in cattle  was less than 0.5 considered to have a close relationship [The phylogenetic tree reconstruction and genetic distance analysis using tionship .Miniopterus pallidus bat in Iran also showed a higher genetic distance within a population [The higher genetic distance within a habitat population than between populations in this study may indicate that genetic variation is more related to differences between samples in a habitat . In addipulation . Thus, hC. brachyotis bats on Java island could not be grouped into their habitat type and lineage based on the D-loop sequence marker. This finding is supported by the external morphology measurement data (data are not shown). Samples from seven habitats had no significant difference in external morphology measurement. The forearm length ranged from 58.69 to 62.55 mm, tibia length 20.79–24.18 mm, and body weight 30.17–36.08 g. In addition, the sample from open habitats (such as urban) had relatively the same average value of forearm length, tibia length, and body weight compared to secondary and natural forests. Further analysis using the cytochrome b (cyt-b) marker is needed to support species identification.The results showed that C. brachyotis D-loop sequence from seven habitats on Java island may describe the rapid evolutionary adaptation of this species. The higher nucleotide pair AT of the D-loop may cause the structure of C. brachyotis mitogenome to become less stable (due to the AT pair have two hydrogen bonds) and accelerate its evolutionary adaptation [C. brachyotis would be able to survive and adapt to different types of habitats on Java island.The average percentage differences in nucleotide composition between habitats in this study also indicated nucleotide variations of intrapopulation and may reflect the genetic variation. Nucleotide composition is a modest way to characterize the genome. Furthermore, the AT-rich of aptation . Evolutiaptation . Thus, iC. brachyotis on Java island has high haplotype diversity (Hd = 0.8–1.00). A previous study showed C. brachyotis population from Peninsular Malaysia and southern Thailand also has high haplotype diversity [C. brachyotis from the natural forest habitat in this study also have a higher haplotype diversity and nucleotide diversity than in other habitats. Those results are in line with this study. C. brachyotis Forest that inhabits closed habitat  has higher haplotype diversity (0.995 ± 0.0023) than C. brachyotis Sunda that inhabit the fragmented area (0.982 ± 0.0003). In addition, the nucleotide diversity of C. brachyotis Forest is about three times higher compared to C. brachyotis Sunda [According to Nei’s (1987) category, iversity . C. bracis Sunda .C. brachyotis population that inhabits different types of habitat, especially the natural forest on Java island, may represent a high genetic diversity and is considered capable of surviving environmental changes in any habitat types.High haplotype diversity could describe high genetic diversity. The genetic diversity of a population is essential for the adaptation process and long-term survival to environmental changes. Moreover, high genetic diversity in a population will degrade the risk of species extinction because they will be able to survive or adapt to environmental changes . TherefoC. brachyotis on Java island overlapped between habitats. This result was congruent with the phylogenetic tree. The samples of C. brachyotis in the haplotype sharing H1, H2, and H4 originated from different types of habitats in the East Java, Yogyakarta, Central Java, and West Java regions. The distance between these locations is more than 500 km, and geographical barriers such as mountains have existed. Therefore, sample connection or gene flow between the samples should not occur and lead to unique haplotype formation [A haplotype network analysis is used to analyze and describe the relationship between haplotypes or DNA sequences in populations (intrapopulation) or species (intraspecies) , 32. Anaormation , 34. HowPenthetor lucasi between the Miri and Kuching populations in Sarawak, Malaysia (the distance between populations of 348 km), based on the D-loop indicates that these two populations became the source of the P. lucasi population in Sarawak during the Pleistocene refugium [The haplotype sharing formation in this study is still unknown. However, according to previous studies, the haplogroups and haplotype sharing formations between fruit bat populations within long population area distances are related to demographic history during Pleistocene refugium. Furthermore, the formation of haplogroup and haplotype sharing in fruit bat species refugium , 36.Rattus rattus and Mus musculus. Several haplotypes of R. rattus and M. musculus in the Indochina region (including Java) were also shared based on the D-loop and cyt-b markers. It is due to the long-distance distribution that began in the mid-Pleistocene and is related to human activities in the modern era [C. brachyotis on Java island, but it is necessary to be studied further.The Pleistocene refugium theory explained that habitat fragmentation due to declining sea levels caused the isolation and diversification of taxons during the Pleistocene era, including bats , 37. It dern era , 39. TheC. brachyotis bats on Java island may indicate that this species is a generalist and widespread species and suspected can adapt to different types of habitats. Nevertheless, C. brachyotis species rely on their foraging activities and roosting habitat in the trees or forest stands [Java island is one of the islands in Indonesia that have the highest human population and massive habitat fragmentation . The fort stands , 45. TheC. brachyotis have been categorized as the least concern on the IUCN Red List, its existence is essential for the ecosystem’s sustainability and plant regeneration, especially for several trees in the forest and fragmented habitats. Previous studies showed that these species have a role in the pollination and seed dispersal of more than 16 plant species [C. brachyotis on Java island is needed.Although  species , 3. HencD-loop sequence, C. brachyotis collected from seven habitats on Java island formed a single clade, overlapped between habitats, and was genetically close. This species on Java island also had high haplotype diversity (Hd = 0.933–1.000). A total of 22 haplotypes from 33 samples were found and split into two haplogroups. Furthermore, the haplotypes shared among habitats were also found in this study. This study result provides D-loop sequence data of C. brachyotis from Indonesia inferred from the mitochondrial DNA D-loop.According to phylogenetic and genetic variation analysis using mtDNA