Extensive remodeling of sugar metabolism through gene loss and horizontal gene transfer in a eukaryotic lineage.

BACKGROUND: In yeasts belonging to the subphylum Saccharomycotina, genes encoding components of the main metabolic pathways, like alcoholic fermentation, are usually conserved. However, in fructophilic species belonging to the floral Wickerhamiella and Starmerella genera (W/S clade), alcoholic fermentation was uniquely shaped by events of gene loss and horizontal gene transfer (HGT). RESULTS: Because HGT and gene losses were first identified when only eight W/S-clade genomes were available, we collected publicly available genome data and sequenced the genomes of 36 additional species. A total of 63 genomes, representing most of the species described in the clade, were included in the analyses. Firstly, we inferred the phylogenomic tree of the clade and inspected the genomes for the presence of HGT-derived genes involved in fructophily and alcoholic fermentation. We predicted nine independent HGT events and several instances of secondary loss pertaining to both pathways. To investigate the possible links between gene loss and acquisition events and evolution of sugar metabolism, we conducted phenotypic characterization of 42 W/S-clade species including estimates of sugar consumption rates and fermentation byproduct formation. In some instances, the reconciliation of genotypes and phenotypes yielded unexpected results, such as the discovery of fructophily in the absence of the cornerstone gene (FFZ1) and robust alcoholic fermentation in the absence of the respective canonical pathway. CONCLUSIONS: These observations suggest that reinstatement of alcoholic fermentation in the W/S clade triggered a surge of innovation that goes beyond the utilization of xenologous enzymes, with fructose metabolism playing a key role.

Phylogenomic delineation of two new species of ascomycetous yeasts, Wickerhamiella koratensis sp. nov. and Wickerhamiella limtongiae sp. nov., and proposal of two synonyms, Wickerhamiella infanticola and Wickerhamiella tropicalis.

Two novel ascomycetous yeast species of the genus Wickerhamiella are proposed based on isolates obtained in Thailand from food waste and the fruiting body of a polypore fungus, and on a combination of conventional DNA-barcode sequence analyses and whole-genome phylogenies. We focus on a particular subclade of the genus Wickerhamiella that contains species found in anthropic environments and describe Wickerhamiella limtongiae sp. nov. (DMKU-FW31-5T=PYCC 9022T=TBRC 15055T), found on food waste samples. In an adjacent clade, we describe Wickerhamiella koratensis sp. nov. (DMKU-KO16T=PYCC 8908T=TBRC 14869T), which represents the closest relative of Wickerhamiella slavikovae and was isolated from the fruiting body of Bjerkandera sp. In the subclade of W. limtongiae sp. nov., we propose that Wickerhamiella infanticola should be regarded as a synonym of Wickerhamiella sorbophila and that Wickerhamiella tropicalis should be regarded as a synonym of Wickerhamiella verensis.

Taxogenomic analyses of Starmerella gilliamiae f.a, sp. nov. and Starmerella monicapupoae f.a., sp. nov., two novel species isolated from plant substrates and insects.

Four yeast isolates collected from flowers from different ecosystems in Brazil, one from fruit of Nothofagus alpina in Argentina, three from flowers of Neltuma chilensis in Chile and one obtained from the proventriculus of a female bumblebee in Canada were demonstred, by analysis of the sequences of the internal transcribed spacer (ITS) region and D1/D2 domains of the large subunit rRNA gene, to represent two novel species of the genus Starmerella. These species are described here as Starmerella gilliamiae f.a, sp. nov. (CBS 16166T; Mycobank MB 851206) and Starmerella monicapupoae f.a., sp. nov. (PYCC 8997T; Mycobank MB 851207). The results of a phylogenomic analysis using 1037 single-copy orthogroups indicated that S. gilliamiae is a member of a subclade that contains Starmerella opuntiae, Starmerella aceti and Starmerella apicola. The results also indicated that S. monicapupoae is phylogenetically related to Starmerella riodocensis. The two isolates of S. monicapupoae were obtained from flowers in Brazil and were probably vectored by insects that visit these substrates. Starmerella gilliamiae has a wide geographical distribution having been isolated in flowers from Brazil and Chile, fruit from Argentina and a bumblebee from Canada.

A glimpse at an early stage of microbe domestication revealed in the variable genome of Torulaspora delbrueckii, an emergent industrial yeast.

Microbe domestication has a major applied relevance but is still poorly understood from an evolutionary perspective. The yeast Torulaspora delbrueckii is gaining importance for biotechnology but little is known about its population structure, variation in gene content or possible domestication routes. Here, we show that T. delbrueckii is composed of five major clades. Among the three European clades, a lineage associated with the wild arboreal niche is sister to the two other lineages that are linked to anthropic environments, one to wine fermentations and the other to diverse sources including dairy products and bread dough (Mix-Anthropic clade). Using 64 genomes we assembled the pangenome and the variable genome of T. delbrueckii. A comparison with Saccharomyces cerevisiae indicated that the weight of the variable genome in the pangenome of T. delbrueckii is considerably smaller. An association of gene content and ecology supported the hypothesis that the Mix-Anthropic clade has the most specialized genome and indicated that some of the exclusive genes were implicated in galactose and maltose utilization. More detailed analyses traced the acquisition of a cluster of GAL genes in strains associated with dairy products and the expansion and functional diversification of MAL genes in strains isolated from bread dough. In contrast to S. cerevisiae, domestication in T. delbrueckii is not primarily driven by alcoholic fermentation but rather by adaptation to dairy and bread-production niches. This study expands our views on the processes of microbe domestication and on the trajectories leading to adaptation to anthropic niches.

Hannaella floricola sp. nov., a novel basidiomycetous yeast species isolated from a flower of Lantana camara in Portugal.

During a survey of floricolous yeasts in Portugal, a basidiomycetous yeast representing a novel species in the genus Hannaella was isolated in Portugal from the flower of Lantana camara, an ornamental exotic species native to Central and South America. A combination of phylogenetic analyses of DNA barcode sequences used in yeast molecular systematics, namely the D1/D2 domain and the complete internal transcribed spacer (ITS) region supported the recognition of a new species of Hannaella, that we designate Hannaella floricola sp. nov. (ex-type strain PYCC 9191T=CBS 18097T). Although the assignment of the new species to the genus Hannaella was evident, the detection of its closest relatives appeared more problematic. Nevertheless, our analyses suggested that H. floricola sp. nov. belongs a clade that also includes H. coprosmae, H. oryzae and H. surugaensis, together four candidate novel species. In addition we provide the molecular identification of several unidentified strains whose D1/D2 and ITS sequences are available from GenBank.

Do microbes evade domestication? - Evaluating potential ferality among diastatic Saccharomyces cerevisiae.

Certain lineages of the wine, beer and bread yeast Saccharomyces cerevisiae have diastatic activity. They contain the chimeric gene STA1 that codes for an extracellular glucoamylase which enables the strains to degrade starch and dextrins. Beer contaminations by diastatic yeasts can be dangerous because they can cause super-attenuation due to the consumption of otherwise non-fermentable oligosaccharides, gushing and off-flavours. Given that diastatic yeasts can be used for beer fermentation it is important to understand the relationship between production and contaminant strains, their natural reservoirs and entry routes into the brewery. Here, we analyze real cases of contamination in a Portuguese craft brewery over a period of 18 months. By analyzing with whole genome sequencing several contaminants, we show that recurrent contaminations by diastatic yeasts are caused by environmental strains. Moreover, some beer contaminants were closely related to diastatic environmental strains isolated in Botswana. We observed the widespread presence of domestication signatures in diastatic strains. Moreover, the combined phylogeny of STA1 and its ancestor, SGA1, suggested a single STA1 origin, as ancient as the entire lineage of diastatic yeasts. Together, our results suggest that diastatic yeasts isolated in natural settings could be escaping from domestication settings and becoming feral.

Global distribution of IRC7 alleles in Saccharomyces cerevisiae populations: a genomic and phenotypic survey within the wine clade.

The adaptation to the different biotic and abiotic factors of wine fermentation has led to the accumulation of numerous genomic hallmarks in Saccharomyces cerevisiae wine strains. IRC7, a gene encoding a cysteine-S-ß-lyase enzyme related volatile thiols production in wines, has two alleles: a full-length allele (IRC7F ) and a mutated one (IRC7S ), harbouring a 38 bp-deletion. Interestingly, IRC7S -encoding a less active enzyme - appears widespread amongst wine populations. Studying the global distribution of the IRC7S allele in different yeast lineages, we confirmed its high prevalence in the Wine clade and demonstrated a minority presence in other domesticated clades (Wine-PDM, Beer and Bread) while it is completely missing in wild clades. Here, we show that IRC7S -homozygous (HS) strains exhibited both fitness and competitive advantages compared with IRC7F -homozygous (HF) strains. There are some pieces of evidence of the direct contribution of the IRC7S allele to the outstanding behaviour of HS strains (i.e., improved response to oxidative stress conditions and higher tolerance to high copper levels); however, we also identified a set of sequence variants with significant co-occurrence patterns with the IRC7S allele, which can be co-contributing to the fitness and competitive advantages of HS strains in wine fermentations.

Phaffia brasiliana sp. nov., a yeast species isolated from soil in a Cerrado-Atlantic Rain Forest ecotone site in Brazil.

During studies of yeasts associated with soil in a Cerrado-Atlantic Rain Forest ecotone site in Brazil, three orange-pigmented yeast strains were isolated from samples collected in Minas Gerais state, Brazil. Molecular analyses combining the 26S rRNA gene (D1/D2 domains) and the internal transcribed spacer (ITS) sequences as well as whole-genome sequence data showed that these strains could not be ascribed to any known species in the basidiomycetous genus Phaffia, and thus they are considered to represent a novel species for which the name Phaffia brasiliana sp. nov. is proposed. The holotype is CBS 16121T and the MycoBank number is MB 839315. The occurrence of P. brasiliana in a tropical region is unique for the genus, since all other species occur in temperate regions. Two factors appear to contribute to the distribution of the novel taxon: first, the region where it was found has relatively moderate temperature ranges and, second, an adaptation to grow or withstand temperatures higher than those of the other species in the genus seems to be in place.

The Wickerhamiella/Starmerella clade-A treasure trove for the study of the evolution of yeast metabolism.

The Wickerhamiella and Starmerella genera form a clade (W/S clade) that branches close to Yarrowia lipolytica in the Saccharomycotina species tree. It comprises approximately 90 recognized species and 50 putative new species not formally described yet. The large majority of the members of the W/S clade are ecologically associated with flowers and floricolous insects. Many species exhibit unusual metabolic traits, like fructophily and the production of sophorolipids, which are glycolipids that can be used as environmentally friendly biosurfactants. Genomic data have not only firmly established the W/S clade but have also revealed a tumultuous evolution of metabolism marked by losses and gains of important metabolic pathways, among which alcoholic fermentation. Possibly the most surprising finding brought to light by comparative genomics concerned the large number of genes acquired by some species of the W/S clade from bacteria through horizontal gene transfer, many of which were shown to be functional in their new setting. This was facilitated by the genetic tractability of one species in the clade, Starmerella bombicola, which is used for the industrial production of sophorolipids. We suggest that high-density coverage of genome sequencing in this clade, combined with the possibility to conduct molecular genetics experiments in at least one species, has the potential to set the stage for yet more exciting discoveries concerning the evolution of yeast metabolism.

Cryolevonia gen. nov. and Cryolevonia schafbergensis sp. nov., a cryophilic yeast from ancient permafrost and melted sea ice.

A cryophilic basidiomycetous yeast unable to grow at 18 °C or higher temperatures was isolated from a subsurface permafrost layer collected in the Eastern Swiss Alps and from melted sea ice collected in the Artic at Frobisher Bay, Nunavut, Canada. Phylogenetic analyses employing combined sequences of the D1/D2 domain and ITS region indicated that the two new isolates belong to the family Camptobasidiaceae of the class Microbotryomycetes but are distantly related to any of the currently recognized species and genera. Consequently, the novel genus Cryolevonia, and the novel species Cryolevonia schafbergensis (type strain PYCC 8347T=CBS 16055T) are proposed to accommodate this cryophilic yeast. Although sparse hyphae and teliospore-like stuctures were observed upon prolonged incubation, a sexual cycle was not observed and therefore C. schafbergensis is documented solely from its asexual stage.

Adaptation of S. cerevisiae to Fermented Food Environments Reveals Remarkable Genome Plasticity and the Footprints of Domestication.

The budding yeast Saccharomyces cerevisiae can be found in the wild and is also frequently associated with human activities. Despite recent insights into the phylogeny of this species, much is still unknown about how evolutionary processes related to anthropogenic niches have shaped the genomes and phenotypes of S. cerevisiae. To address this question, we performed population-level sequencing of 82 S. cerevisiae strains from wine, flor, rum, dairy products, bakeries, and the natural environment (oak trees). These genomic data enabled us to delineate specific genetic groups corresponding to the different ecological niches and revealed high genome content variation across the groups. Most of these strains, compared with the reference genome, possessed additional genetic elements acquired by introgression or horizontal transfer, several of which were population-specific. In addition, several genomic regions in each population showed evidence of nonneutral evolution, as shown by high differentiation, or of selective sweeps including genes with key functions in these environments (e.g., amino acid transport for wine yeast). Linking genetics to lifestyle differences and metabolite traits has enabled us to elucidate the genetic basis of several niche-specific population traits, such as growth on galactose for cheese strains. These data indicate that yeast has been subjected to various divergent selective pressures depending on its niche, requiring the development of customized genomes for better survival in these environments. These striking genome dynamics associated with local adaptation and domestication reveal the remarkable plasticity of the S. cerevisiae genome, revealing this species to be an amazing complex of specialized populations.

Sporobolomyces agrorum sp. nov. and Sporobolomyces sucorum sp. nov., two novel basidiomycetous yeast species isolated from grape and apple must in Italy.

During a survey of yeast populations associated with grape and apple musts used for wine and cider fermentation, respectively, six pink-coloured ballistoconidia-forming yeasts belonging to the order Sporidiobolales (Basidiomycota) were isolated. Phylogenetic analysis inferred using sequences of the internal transcribed spacer (ITS), the D1/D2 domain of the large subunit rRNA gene, the small subunit (SSU) rRNA gene and DNA-directed RNA polymerase II subunit (RPB2) indicated that the six isolates were separated in two novel species. One of the new species, Sporobolomyces agrorum sp. nov., isolated from grape must, had Sporobolomyces roseus and Sporobolomyces metaroseus as its closest relatives, but showed four/two and 16 nucleotide substitutions in the D1/D2 and ITS regions, respectively, to these two species. The other novel species, Sporobolomyces sucorum sp. nov., was found in apple must and was closely related to Sporobolomyces pararoseus and Sporobolomyces patagonicus, but showed two/three and five substitutions in those two regions for its closest relatives. We detected additional representatives of this species, most of them isolated from grapes whose sequences were already available on public databases. A sexual stage could not be observed for the novel species.

Heterocephalacria sinensis sp. nov., Phaeotremella lacus sp. nov. and Solicoccozyma aquatica sp. nov., three novel basidiomycetous yeast species isolated from crater lakes.

The Arxan-Chaihe volcanic field of the Da Hinggan mountains in north-East PR China hosts various typical crater lakes. In this study we performed a yeast diversity survey using water sampled from five crater lakes and a total of 122 yeast strains belonging to 33 species of 25 genera were isolated. Three strains, TFL1-L, TFL2B and ATC4C, were identified as three novel species belonging to the Tremellomycetes based on a multiple gene phylogeny and on the comparison of physiological data. A phylogenetic study employing the sequences of seven genes indicated that the new species were more related to three separated phylogenetic lineages of the Tremellomycetes and their closest relatives were Heterocephalacria arrabidensis, Phaeotremella skinneri and Solicoccozyma keelungensis. The divergence values of the D1/D2 domain of LSU sequences of strains TFL1-L, TFL2B and ATC4C from H. arrabidensis CBS 8678T, P. skinneri CBS 5029T and S. keelungensisSN-82T were 4.8，3.4，2.1 %， respectively. The divergence values of the sequences of ITS regions between strains TFL1-L, TFL2B and ATC4C and their close relatives (H. arrabidensis, P. skinneri and S. keelungensis) were 16.1, 5.9 and 8.1  %, respectively. Moreover, the three strains differed from their phylogenetic neighbours by the ability to grow on distinct carbon and nitrogen sources. On the basis of these findings, it is suggested that these strains represent three novel species for which the names Heterocephalacria sinensis sp. nov. (holotype CGMCC 2.5595), Phaeotremella lacus sp. nov. (holotype CGMCC 2.5580) and Solicoccozyma aquatica sp. nov. (holotype CGMCC 2.5574) are proposed.

Genetic Dissection of Sexual Reproduction in a Primary Homothallic Basidiomycete.

In fungi belonging to the phylum Basidiomycota, sexual compatibility is usually determined by two genetically unlinked MAT loci, one of which encodes one or more pheromone receptors (P/R) and pheromone precursors, and the other comprehends at least one pair of divergently transcribed genes encoding homeodomain (HD) transcription factors. Most species are heterothallic, meaning that sexual reproduction requires mating between two sexually compatible individuals harboring different alleles at both MAT loci. However, some species are known to be homothallic, one individual being capable of completing the sexual cycle without mating with a genetically distinct partner. While the molecular underpinnings of the heterothallic life cycles of several basidiomycete model species have been dissected in great detail, much less is known concerning the molecular basis for homothallism. Following the discovery in available draft genomes of the homothallic basidiomycetous yeast Phaffia rhodozyma of P/R and HD genes, we employed available genetic tools to determine their role in sexual development. Two P/R clusters, each harboring one pheromone receptor and one pheromone precursor gene were found in close vicinity of each other and were shown to form two redundant P/R pairs, each receptor being activated by the pheromone encoded by the most distal pheromone precursor gene. The HD locus is apparently genetically unlinked to the P/R locus and encodes a single pair of divergently transcribed HD1 and HD2 transcription factors, both required for normal completion of the sexual cycle. Given the genetic makeup of P. rhodozyma MAT loci, we postulate that it is a primarily homothallic organism and we propose a model for the interplay of molecular interactions required for sexual development in this species. Phaffia rhodozyma is considered one of the most promising microbial source of the carotenoid astaxanthin. Further development of this yeast as an industrial organism will benefit from new insights regarding its sexual reproduction system.

Microbe Profile: Saccharomyces eubayanus, the missing link to lager beer yeasts.

Saccharomyces eubayanus was described less than 10 years ago and its discovery settled the long-lasting debate on the origins of the cold-tolerant yeast responsible for lager beer fermentation. The largest share of the genetic diversity of S. eubayanus is located in South America, and strains of this species have not yet been found in Europe. One or more hybridization events between S. eubayanus and S. cerevisiae ale beer strains gave rise to S. pastorianus, the allopolyploid yeasts responsible for lager beer production worldwide. The identification of the missing progenitor of lager yeast opened new avenues for brewing yeast research. It allowed not only the selective breeding of new lager strains, but revealed also a wild yeast with interesting brewing abilities so that a beer solely fermented by S. eubayanus is currently on the market.

Zygotorulaspora chibaensis sp. nov. and Zygotorulaspora danielsina sp. nov., novel ascomycetous yeast species from tree bark and soil.

Multiple isolates belonging to the ascomycetous genus Zygotorulaspora were obtained from forest soils and tree bark in Shiba Prefecture in Japan, and Lake Daniels, Lewis Pass, in New Zealand. Phylogenetic analyses employing combined sequences of the D1/D2 domain and ITS region support the recognition of two new species: Zygotorulaspora chibaensis sp. nov. (type strain PYCC 6970T=CBS 15364T) and Zygotorulaspora danielsina sp. nov. (type strain PYCC 6984T=CBS 15365T). Both species are able to grow on d-xylose and l-arabinose and at 35 °C, unlike Zygotorulaspora florentina and Zygotorulaspora mrakii, the other two species in the genus.

Moniliella sojae sp. nov., a species of black yeasts isolated from Vietnamese soy paste (tuong), and reassignment of Moniliella suaveolens strains to Moniliella pyrgileucina sp. nov., Moniliella casei sp. nov. and Moniliella macrospora emend. comb. nov.

The presence of yeasts at different steps of Vietnamese soy paste production was studied. Yeast growth occurred during primary soybean fermentation, with the cell density reaching 4.106 c.f.u. ml-1, and terminated during brine fermentation. The dominant species were Pichia kudriavzevii and Millerozyma farinosa. Over the span of 14 years, nine strains of Moniliella were isolated. The strains had identical PCR fingerprints generated with primer (GAC)5 and identical D1/D2 and internal transcribed spacer (ITS) sequences. A D1/D2-based phylogeny indicated that the strains were closest to a group of four previously assigned as Moniliella suaveolens strains. Together they form a new lineage that is well separated from all known species, including M. suaveolens (over 12.7 % divergence). ITS sequences indicated the presence of four species differing from each other by 9-57 nt. The name Moniliella sojae sp. nov. is proposed to accommodate the strains isolated from Vietnamese soy paste, Moniliella pyrgileucina sp. nov. is proposed for PYCC 6800 and Moniliella casei sp. nov. is proposed for CBS 157.58. An emended combination Moniliella macrospora is proposed for CBS 221.32 and CBS 223.32. The type strains and MycoBank numbers are: M. sojae sp. nov., SS 4.2T=CBS 126448T=NRRL Y-48680T and MB 822871; M. pyrgileucina sp. nov., PYCC 6800T=CBS 15203T and MB 823030; M. casei sp. nov., CBS 157.58T=IFM 60348T and MB 822872; M. macrospora emend. comb. nov., CBS 221.32T (=MUCL 11527T) and MB 822874.

Papiliotrema plantarum sp. nov., a novel tremellaceous sexual yeast species.

During a survey of the yeast community associated with the phylloplane of corn in Thailand, a basidiomycetous yeast strain belonging to the genus Papiliotrema was isolated. Analyses of the D1/D2 domains of the 26S (LSU) rRNA gene and complete ITS region supported the recognition of a novel species, for which the name Papiliotrema plantarum sp. nov. is proposed (type strain DMKU-CP801T=CBS 15220T=PYCC 7257T). Another strain of P. plantarum sp. nov., isolated in French Guiana, was found to be sexually compatible with the Thai isolate and mycelium with clamp connections, basidia and basidiospores were observed in culture. The basidial morphology of P. plantarum combined features previously observed for Papiliotrema bandonii and Papiliotrema fuscus, which represent the only sexual species hitherto known in the genus, i.e. transversely septate basidia, with sexual structures of the Tremella type.

Blastobotrys bombycis sp. nov., a d-xylose-fermenting yeast isolated from the gut of the silkworm larva Bombyx mori.

The gut of insects harbors a yeast community that is still poorly understood. Here, a novel species of the ascomycetous genus Blastobotrys is proposed based on a yeast strain isolated from the larval gut of the silkworm Bombyx mori (Order Lepidoptera). The novel species is closely related to Blastobotrys aristata and Blastobotrys elegans on the basis of the results of molecular phylogenetic analyses. A preliminary screening revealed that it produces 1.5 g l-1 ethanol by fermenting 5 % d-xylose. The novel species, that represents the first report, to our knowledge, of yeast isolation from silkworms, is described as Blastobotrys bombycis sp. nov. (type strain RAAB001T=CBS 15274T=PYCC 8105T=MCC 1427T; MycoBank accession number MB 825095).

Occultifur mephitis f.a., sp. nov. and other yeast species from hypoxic and elevated CO2 mofette environments.

An inventory of culturable yeasts from the soil and water of natural CO2 springs (mofettes) in northeast Slovenia is presented. In mofettes, CO2 of geological origin reaches the soil surface causing temporarily and spatially stable hypoxic environments in soil and water. In total, 142 yeast strains were isolated and identified from high CO2 and control meadow soil, meadow ground-water, forest pond and stream water. All water locations showed below-ground CO2 release. They were assigned to six basidiomycetous yeast genera (six species) and 11 ascomycetous genera (18 species). All ascomycetous yeasts, with the exception of Debaryomyces hansenii, were able to grow under elevated CO2 and fermented glucose. Candida sophiae-reginae, Pichia fermentans and Candida vartiovaarae were the dominating species in meadow and forest high CO2 exposed water. Meyerozyma guilliermondii and Wickerhamomyces anomalus predominated in high CO2 exposed soils. Using high dilution plating of a mofette soil sample, four strains of an unknown basidiomycetous species were isolated and are here newly described as Occultifur mephitis based on molecular phylogenetic and phenotypic criteria. The type strain of Occultifur mephitis is EXF-6436T[CBS 14611=PYCC 7049, LT594852 (D1/D2), KX929055 (ITS)]. An additional three isolated strains are EXF-6437 (LT594853, KX929056), EXF-6473 (LT594863, KX929057) and EXF-6482 (LT594867, KX929054), as well as a strain reported from previous studies isolated from a leaf of Cistus albidus in Portugal (CBS 10223=PYCC 6067), EU002842 (D1/D2), KY308183 (ITS).