Diversity and evolutionary patterns of bacterial gut associates of corbiculate bees

The animal gut is a habitat for diverse communities of microorganisms (microbiota). Honeybees and bumblebees have recently been shown to harbour a distinct and species poor microbiota, which may confer protection against parasites. Here, we investigate diversity, host specificity and transmission mode of two of the most common, yet poorly known, gut bacteria of honeybees and bumblebees: Snodgrassella alvi (Betaproteobacteria) and Gilliamella apicola (Gammaproteobacteria). We analysed 16S rRNA gene sequences of these bacteria from diverse bee host species across most of the honeybee and bumblebee phylogenetic diversity from North America, Europe and Asia. These focal bacteria were present in 92% of bumblebee species and all honeybee species but were found to be absent in the two related corbiculate bee tribes, the stingless bees (Meliponini) and orchid bees (Euglossini). Both Snodgrassella alvi and Gilliamella apicola phylogenies show significant topological congruence with the phylogeny of their bee hosts, albeit with a considerable degree of putative host switches. Furthermore, we found that phylogenetic distances between Gilliamella apicola samples correlated with the geographical distance between sampling locations. This tentatively suggests that the environmental transmission rate, as set by geographical distance, affects the distribution of G. apicola infections. We show experimentally that both bacterial taxa can be vertically transmitted from the mother colony to daughter queens, and social contact with nest mates after emergence from the pupa greatly facilitates this transmission. Therefore, sociality may play an important role in vertical transmission and opens up the potential for co‐evolution or at least a close association of gut bacteria with their hosts.     

Geographically widespread honeybee‐gut symbiont subgroups show locally distinct antibiotic‐resistant patterns

How long‐term antibiotic treatment affects host bacterial associations is still largely unknown. The honeybee‐gut microbiota has a simple composition, so we used this gut community to investigate how long‐term antibiotic treatment affects host‐associated microbiota. We investigated the phylogenetic relatedness, genomic content (GC percentage, genome size, number of genes and CRISPR) and antibiotic‐resistant genes (ARG) for strains from two abundant members of the honeybee core gut microbiota (Gilliamella apicola and Snodgrassella alvi). Domesticated honeybees are subjected to geographically different management policies, so we used two research apiaries, representing different antibiotic treatment regimens in their apiculture: low antibiotic usage (Norway) and high antibiotic usage (Arizona, USA). We applied whole‐genome shotgun sequencing on 48 G. apicola and 22 S. alvi. We identified three predominating subgroups of G. apicola in honeybees from both Norway and Arizona. For G. apicola, genetic content substantially varied between subgroups and distance similarity calculations showed similarity discrepancy between subgroups. Functional differences between subgroups, such as pectin‐degrading enzymes (G. apicola), were also identified. In addition, we identified horizontal gene transfer (HGT) of transposon (Tn10)‐associated tetracycline resistance (Tet B) across the G. apicola subgroups in the Arizonan honeybees, using interspace polymorphisms in the Tet B determinant. Our results support that honeybee‐gut symbiont subgroups can resist long‐term antibiotic treatment and maintain functionality through acquisition of geographically distinct antibiotic‐resistant genes by HGT.     

Diet‐related gut bacterial dysbiosis correlates with impaired development,increased mortality and Nosema disease in the honeybee (Apis mellifera)

Dysbiosis, defined as unhealthy shifts in bacterial community composition, can lower the colonization resistance of the gut to intrinsic pathogens. Here, we determined the effect of diet age and type on the health and bacterial community composition of the honeybee (Apis mellifera). We fed newly emerged bees fresh or aged diets, and then recorded host development and bacterial community composition from four distinct regions of the hosts’ digestive tract. Feeding fresh pollen or fresh substitute, we found no difference in host mortality, diet consumption, development or microbial community composition. In contrast, bees fed aged diets suffered impaired development, increased mortality and developed a significantly dysbiotic microbiome. The consumption of aged diets resulted in a significant reduction in the core ileum bacterium Snodgrassella alvi and a corresponding increase in intrinsic pathogen Frischella perrara. Moreover, the relative abundance of S. alvi in the ileum was positively correlated with host survival and development. The inverse was true for both F. perrara and Parasacharibacter apium. Collectively, our findings suggest that the early establishment of S. alvi is associated with healthy nurse development and potentially excludes F. perrara and P. apium from the ileum. Although at low abundance, establishment of the common midgut pathogen Nosema spp. was significantly associated with ileum dysbiosis and associated host deficiencies. Moreover, dysbiosis in the ileum was reflected in the rectum, mouthparts and hypopharyngeal glands, suggesting a systemic host effect. Our findings demonstrate that typically occurring alterations in diet quality play a significant role in colony health and the establishment of a dysbiotic gut microbiome.     

Hidden Diversity in Honey Bee Gut Symbionts Detected by Single-Cell Genomics

Microbial communities in animal guts are composed of diverse, specialized bacterial species, but little is known about how gut bacteria diversify to produce genetically and ecologically distinct entities. The gut microbiota of the honey bee, Apis mellifera, presents a useful model, because it consists of a small number of characteristic bacterial species, each showing signs of diversification. Here, we used single-cell genomics to study the variation within two species of the bee gut microbiota: Gilliamella apicola and Snodgrassella alvi. For both species, our analyses revealed extensive variation in intraspecific divergence of protein-coding genes but uniformly high levels of 16S rRNA similarity. In both species, the divergence of 16S rRNA loci appears to have been curtailed by frequent recombination within populations, while other genomic regions have continuously diverged. Furthermore, gene repertoires differ markedly among strains in both species, implying distinct metabolic capabilities. Our results show that, despite minimal divergence at 16S rRNA genes, in situ diversification occurs within gut communities and generates bacterial lineages with distinct ecological niches. Therefore, important dimensions of microbial diversity are not evident from analyses of 16S rRNA, and single cell genomics has potential to elucidate processes of bacterial diversification.     

Uncovering symbiont‐driven genetic diversity across North American pea aphids

Heritable genetic variation is required for evolution, and while typically encoded within nuclear and organellar genomes, several groups of invertebrates harbour heritable microbes serving as additional sources of genetic variation. Hailing from the symbiont‐rich insect order Hemiptera, pea aphids (Acyrthosiphon pisum) possess several heritable symbionts with roles in host plant utilization, thermotolerance and protection against natural enemies. As pea aphids vary in the numbers and types of harboured symbionts, these bacteria provide heritable and functionally important variation within field populations. In this study, we quantified the cytoplasmically inherited genetic variation contributed by symbionts within North American pea aphids. Through the use of Denaturing Gradient Gel Electrophoresis (DGGE) and 454 amplicon pyrosequencing of 16S rRNA genes, we explored the diversity of bacteria harboured by pea aphids from five populations, spanning three locations and three host plants. We also characterized strain variation by analysing 16S rRNA, housekeeping and symbiont‐associated bacteriophage genes. Our results identified eight species of facultative symbionts, which often varied in frequency between locations and host plants. We detected 28 cytoplasmic genotypes across 318 surveyed aphids, considering only the various combinations of secondary symbiont species infecting single hosts. Yet the detection of multiple Regiella insecticola, Hamiltonella defensa and Rickettsia strains, and diverse bacteriophage genotypes from H. defensa, suggest even greater diversity. Combined, these findings reveal that heritable bacteria contribute substantially to genetic variation in A. pisum. Given the costs and benefits of these symbionts, it is likely that fluctuating selective forces play a role in the maintenance of this diversity.     

The structured diversity of specialized gut symbionts of the New World army ants

Symbiotic bacteria play important roles in the biology of their arthropod hosts. Yet the microbiota of many diverse and influential groups remain understudied, resulting in a paucity of information on the fidelities and histories of these associations. Motivated by prior findings from a smaller scale, 16S rRNA‐based study, we conducted a broad phylogenetic and geographic survey of microbial communities in the ecologically dominant New World army ants (Formicidae: Dorylinae). Amplicon sequencing of the 16S rRNA gene across 28 species spanning the five New World genera showed that the microbial communities of army ants consist of very few common and abundant bacterial species. The two most abundant microbes, referred to as Unclassified Firmicutes and Unclassified Entomoplasmatales, appear to be specialized army ant associates that dominate microbial communities in the gut lumen of three host genera, Eciton, Labidus and Nomamyrmex. Both are present in other army ant genera, including those from the Old World, suggesting that army ant symbioses date back to the Cretaceous. Extensive sequencing of bacterial protein‐coding genes revealed multiple strains of these symbionts coexisting within colonies, but seldom within the same individual ant. Bacterial strains formed multiple host species‐specific lineages on phylogenies, which often grouped strains from distant geographic locations. These patterns deviate from those seen in other social insects and raise intriguing questions about the influence of army ant colony swarm‐founding and within‐colony genetic diversity on strain coexistence, and the effects of hosting a diverse suite of symbiont strains on colony ecology.     

Isolation and characterization of halotolerant Streptomyces radiopugnans from Antarctica soil

An actinomycete wild strain PM0626271 (= MTCC 5447), producing novel antibacterial compounds, was isolated from soil collected from Antarctica. The taxonomic status of the isolate was established by polyphasic approach. Scanning electron microscopy observations and the presence of LL‐Diaminopimelic acid in the cell wall hydrolysate confirmed the genus Streptomyces. Analysis of 16S rRNA gene sequence showed highest sequence similarity to Streptomyces radiopugnans (99%). The phylogenetic tree constructed using near complete 16S rRNA gene sequences of the isolate and closely related strains revealed that although the isolate fell within the S. radiopugnans gene subclade, it was allocated a different branch in the phylogenetic tree, separating it from the majority of the radiopugnans strains. Similar to type strain, S. radiopugnans R97^T, the Antarctica isolate displayed thermo tolerance as well as resistance to ⁶⁰Co gamma radiation, up to the dose of 15 kGy. However, media and salt tolerance studies revealed that, unlike the type strain, this isolate needed higher salinity for its growth. This is the first report of S. radiopugnans isolated from the Antarctica region. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Streptomyces radiopugnans MTCC 5447 is JQ723477 .

Significance and Impact of the Study

The study presents the first report of isolation of Streptomyces radiopugnans from Antarctica. To date, there is only one publication regarding S. radiopugnans R97^T isolated from radiation‐polluted soil. Like the type strain, Antarctica isolate was thermotolerant and radiotolerant, but in addition, it required salts for growth and did not degrade phenol. We envisaged that metabolic pattern of the same species varies based on acclimatization in its native ecological habitat. Additionally, Antarctica isolate had produced novel antibacterial compounds (patent‐US2012/0156295). The study highlighted that least explored extreme regions like Antarctica are rich resources of novel microbial strains producing novel bioactive compounds.     

Morphological and genetic diversity of Beaufort Sea diatoms with high contributions from the Chaetoceros neogracilis species complex

Seventy‐five diatom strains isolated from the Beaufort Sea (Canadian Arctic) in the summer of 2009 were characterized by light and electron microscopy (SEM and TEM), as well as 18S and 28S rRNA gene sequencing. These strains group into 20 genotypes and 17 morphotypes and are affiliated with the genera Arcocellulus, Attheya, Chaetoceros, Cylindrotheca, Eucampia, Nitzschia, Porosira, Pseudo‐nitzschia, Shionodiscus, Thalassiosira, and Synedropsis. Most of the species have a distribution confined to the northern/polar area. Chaetoceros neogracilis and Chaetoceros gelidus were the most represented taxa. Strains of C. neogracilis were morphologically similar and shared identical 18S rRNA gene sequences, but belonged to four distinct genetic clades based on 28S rRNA, ITS‐1 and ITS‐2 phylogenies. Secondary structure prediction revealed that these four clades differ in hemi‐compensatory base changes (HCBCs) in paired positions of the ITS‐2, suggesting their inability to interbreed. Reproductively isolated C. neogracilis genotypes can thus co‐occur in summer phytoplankton communities in the Beaufort Sea. C. neogracilis generally occurred as single cells but also formed short colonies. It is phylogenetically distinct from an Antarctic species, erroneously identified in some previous studies as C. neogracilis, but named here as Chaetoceros sp. This work provides taxonomically validated sequences for 20 Arctic diatom taxa, which will facilitate future metabarcoding studies on phytoplankton in this region.     

Morphological and molecular characterization within 26 strains of the genus Cylindrospermum (Nostocaceae,Cyanobacteria), with descriptions of three new species

Twenty‐six strains morphologically identified as Cylindrospermum as well as the closely related taxon Cronbergia siamensis were examined microscopically as well as phylogenetically using sequence data for the 16S rRNA gene and the 16S‐23S internal transcribed spacer (ITS) region. Phylogenetic analysis of the 16S rRNA revealed three distinct clades. The clade we designate as Cylindrospermum sensu stricto contained all five of the foundational species, C. maius, C. stagnale, C. licheniforme, C. muscicola, and C. catenatum. In addition to these taxa, three species new to science in this clade were described: C. badium, C. moravicum, and C. pellucidum. Our evidence indicated that Cronbergia is a later synonym of Cylindrospermum. The phylogenetic position of Cylindrospermum within the Nostocaceae was not clearly resolved in our analyses. Cylindrospermum is unusual among cyanobacterial genera in that the morphological diversity appears to be more evident than sequence divergence. Taxa were clearly separable using morphology, but had very high percent similarity among ribosomal sequences. Given the high diversity we noted in this study, we conclude that there is likely much more diversity remaining to be described in this genus.     

Molecular and morphological criteria for revision of the genus Microcoleus (Oscillatoriales,Cyanobacteria)

Ninety‐two strains of Microcoleus vaginatus (=nomenclatural‐type species of the genus Microcoleus Desmazières ex Gomont) and Phormidium autumnale Trevisan ex Gomont from a wide diversity of regions and biotopes were examined using a combination of morphological and molecular methods. Phylogenies based on the 16S rDNA and 16S‐23S ITS (partial) demonstrated that the 92 strains, together with a number of strains in GenBank, were members of a highly supported monophyletic clade of strains (Bayesian posterior probability = 1.0) distant from the species‐cluster containing the generitype of Phormidium. Similarity of the 16S rRNA gene exceeded 95.5% among all members of the Microcoleus clade, but was less than 95% between any Microcoleus strains and species outside of the clade (e.g., Phormidium sensu stricto). These findings, which are in agreement with earlier studies on these taxa, necessitate the revision of Microcoleus to include P. autumnale. Furthermore, the cluster of Phormidium species in the P. autumnale group (known as Group VII) must be moved into Microcoleus as well, and these nomenclatural transfers are included in this study. The main diacritical characters defining Microcoleus are related to the cytomorphology of trichomes, including: narrowed trichome ends, calyptra, cells shorter than wide up to more or less isodiametric, and facultative presence of sheaths. The majority of species are 4–10 μm in diameter. The possession of multiple trichomes in a common sheath is present facultatively in many but not all species.     

Deep Sequencing and Ecological Characterization of Gut Microbial Communities of Diverse Bumble Bee Species

Gut bacterial communities of bumble bees are correlated with defense against pathogens. Further understanding this host-microbe association is vitally important as bumble bees are currently experiencing global population declines, potentially due in part to emergent diseases. In this study, we used pyrosequencing and community fingerprinting (ARISA) to characterize the gut microbial communities of nine bumble species from across the Bombus phylogeny. Overall, we delimited 74 bacterial taxa (operational taxonomic units or OTUs) belonging to Betaproteobacteria, Gammaproteobacteria, Bacilli, Actinobacteria, Flavobacteria and Alphaproteobacteria. Each bacterial community was taxonomically simple, containing an average of 1.9 common (relative abundance per sample > 5%) bacterial OTUs. The most abundant and prevalent (occurring in 92% of the samples) bacterial OTU, based on 16S rRNA sequences, closely matched that of the previously described Betaproteobacteria species Snodgrassella alvi. Bacteria that were first described in bee-related external environments dominated a number of gut bacterial communities, suggesting that they are not strictly dependent on the internal gut environment. The ARISA data showed a correlation between bacterial community structures and the geographic locations where the bees were sampled, suggesting that at least a subset of the bacterial species may be transmitted environmentally. Using light and fluorescent microscopy, we demonstrated that the gut bacteria form a biofilm on the internal epithelial surface of the ileum, corroborating results obtained from Apis mellifera.     

Identification of Entamoeba polecki with Unique 18S rRNA Gene Sequences from Celebes Crested Macaques and Pigs in Tangkoko Nature Reserve,North Sulawesi,Indonesia

Unique species of macaques are distributed across Sulawesi Island, Indonesia, and the details of Entamoeba infections in these macaques are unknown. A total of 77 stool samples from Celebes crested macaques (Macaca nigra) and 14 stool samples from pigs were collected in Tangkoko Nature Reserve, North Sulawesi, and the prevalence of Entamoeba infection was examined by PCR. Entamoeba polecki was detected in 97% of the macaques and all of the pigs, but no other Entamoeba species were found. The nucleotide sequence of the 18S rRNA gene in E. polecki from M. nigra was unique and showed highest similarity with E. polecki subtype (ST) 4. This is the first case of identification of E. polecki ST4 from wild nonhuman primates. The sequence of the 18S rRNA gene in E. polecki from pigs was also unique and showed highest similarity with E. polecki ST1. These results suggest that the diversity of the 18S rRNA gene in E. polecki is associated with differences in host species and geographic localization, and that there has been no transmission of E. polecki between macaques and pigs in the study area.     

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

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

An interaction between host and microbe genotypes determines colonization success of a key bumble bee gut microbiota member

There has been a proliferation of studies demonstrating an organism's health is influenced by its microbiota. However, factors influencing beneficial microbe colonization and the evolution of these relationships remain understudied relative to host–pathogen interactions. Vertically transmitted beneficial microbes are predicted to show high levels of specificity in colonization, including genotype matching, which may transpire through coevolution. We investigate how host and bacterial genotypes influence colonization of a core coevolved microbiota member in bumble bees. The hindgut colonizing Snodgrassella alvi confers direct benefits, but, as an early colonizer, also facilitates the further development of a healthy microbiota. Due to predominantly vertical transmission promoting tight evolution between colonization factors of bacteria and host lineages, we predict that genotype‐by‐genotype interactions will determine successful colonization. Germ‐free adult bees from seven bumble bee colonies (host genotypic units) were inoculated with one of six genetically distinct strains of S. alvi. Subsequent colonization within host and microbe genotypes combinations ranged from 0 to 100%, and an interaction between host and microbe genotypes determined colonization success. This novel finding of a genotype‐by‐genotype interaction determining colonization in an animal host‐beneficial microbe system has implications for the ecological and evolutionary dynamics of host and microbe, including associated host‐fitness benefits.     

Specificity and stability of the Acromyrmex–Pseudonocardia symbiosis

The stability of mutualistic interactions is likely to be affected by the genetic diversity of symbionts that compete for the same functional niche. Fungus‐growing (attine) ants have multiple complex symbioses and thus provide ample opportunities to address questions of symbiont specificity and diversity. Among the partners are Actinobacteria of the genus Pseudonocardia that are maintained on the ant cuticle to produce antibiotics, primarily against a fungal parasite of the mutualistic gardens. The symbiosis has been assumed to be a hallmark of evolutionary stability, but this notion has been challenged by culturing and sequencing data indicating an unpredictably high diversity. We used 454 pyrosequencing of 16S rRNA to estimate the diversity of the cuticular bacterial community of the leaf‐cutting ant Acromyrmex echinatior and other fungus‐growing ants from Gamboa, Panama. Both field and laboratory samples of the same colonies were collected, the latter after colonies had been kept under laboratory conditions for up to 10 years. We show that bacterial communities are highly colony‐specific and stable over time. The majority of colonies (25/26) had a single dominant Pseudonocardia strain, and only two strains were found in the Gamboa population across 17 years, confirming an earlier study. The microbial community on newly hatched ants consisted almost exclusively of a single strain of Pseudonocardia while other Actinobacteria were identified on older, foraging ants in varying but usually much lower abundances. These findings are consistent with recent theory predicting that mixtures of antibiotic‐producing bacteria can remain mutualistic when dominated by a single vertically transmitted and resource‐demanding strain.     

A bridge too far in naming species: a total evidence approach does not support recognition of four species in Desertifilum (Cyanobacteria)

A population of Desertifilum (Cyanobacteria, Oscillatoriales) from an oligotrophic desertic biotope was isolated and characterized using a polyphasic approach including molecular, morphological, and ecological information. The population was initially assumed to be a new species based on ecological and biogeographic separation from other existing species, however, phylogenetic analyses based on sequences of the 16S rRNA gene and 16S–23S ITS region, placed this strain clearly within the type species, Desertifilum tharense. Comparative analysis of morphology, 16S rRNA gene similarity, 16S–23S ITS secondary structure, and percent dissimilarity of the ITS regions for all characterized strains supports placing the six Desertifilum strains (designated as PD2001/TDC17, UAM‐C/S02, CHAB7200, NapGTcm17, IPPAS B‐1220, and PMC 872.14) into D. tharense. The recognition of Desertifilum salkalinema and Desertifilum dzianense is not supported, although our analysis does support continued recognition of Desertifilum fontinale. Pragmatic criteria for recognition of closely related species are proposed based on this study and others, and more rigorous review of future taxonomic papers is recommended.     

Engineering fire blight resistance into the apple cultivar ‘Gala’ using the FB_MR5 CC‐NBS‐LRR resistance gene of Malus × robusta 5

The fire blight susceptible apple cultivar Malus × domestica Borkh. cv. ‘Gala’ was transformed with the candidate fire blight resistance gene FB_MR5 originating from the crab apple accession Malus × robusta 5 (Mr5). A total of five different transgenic lines were obtained. All transgenic lines were shown to be stably transformed and originate from different transgenic events. The transgenic lines express the FB_MR5 either driven by the constitutive CaMV 35S promoter and the ocs terminator or by its native promoter and terminator sequences. Phenotyping experiments were performed with Mr5‐virulent and Mr5‐avirulent strains of Erwinia amylovora, the causal agent of fire blight. Significantly less disease symptoms were detected on transgenic lines after inoculation with two different Mr5‐avirulent E. amylovora strains, while significantly more shoot necrosis was observed after inoculation with the Mr5‐virulent mutant strain ZYRKD3_1. The results of these experiments demonstrated the ability of a single gene isolated from the native gene pool of apple to protect a susceptible cultivar from fire blight. Furthermore, this gene is confirmed to be the resistance determinant of Mr5 as the transformed lines undergo the same gene‐for‐gene interaction in the host–pathogen relationship Mr5–E. amylovora.     

Taxonomic revision of oil‐producing green algae,Chlorococcum oleofaciens (Volvocales,Chlorophyceae), and its relatives

Historically, species in Volvocales were classified based primarily on morphology. Although the taxonomy of Chlamydomonas has been re‐examined using a polyphasic approach including molecular phylogeny, that of Chlorococcum (Cc.), the largest coccoid genus in Volvocales, has yet to be reexamined. Six species thought to be synonymous with the oil‐producing alga Cc. oleofaciens were previously not confirmed by molecular phylogeny. In this study, seven authentic strains of Cc. oleofaciens and its putative synonyms, along with 11 relatives, were examined based on the phylogeny of the 18S ribosomal RNA (rRNA) gene, comparisons of secondary structures of internal transcribed spacer 1 (ITS1) and ITS2 rDNA, and morphological observations by light microscopy. Seven 18S rRNA types were recognized among these strains and three were distantly related to Cc. oleofaciens. Comparisons of ITS rDNA structures suggested possible separation of the remaining four types into different species. Shapes of vegetative cells, thickness of the cell walls in old cultures, the size of cells in old cultures, and stigma morphology of zoospores also supported the 18S rRNA grouping. Based on these results, the 18 strains examined were reclassified into seven species. Among the putative synonyms, synonymy of Cc. oleofaciens, Cc. croceum, and Cc. granulosum was confirmed, and Cc. microstigmatum, Cc. rugosum, Cc. aquaticum, and Cc. nivale were distinguished from Cc. oleofaciens. Furthermore, another related strain is described as a new species, Macrochloris rubrioleum sp. nov.