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Candida carvajalis sp. nov., an ascomycetous yeast species from the Ecuadorian Amazon jungle

Stephen A. James, Enrique Javier Carvajal Barriga, Christopher J. Bond, Kathryn Cross, Norma C. Núñez, Patricia B. Portero, Ian N. Roberts
DOI: http://dx.doi.org/10.1111/j.1567-1364.2009.00518.x 784-788 First published online: 1 August 2009


In the course of a yeast biodiversity survey of different ecological habitats found in Ecuador, two yeast strains (CLQCA 20-011T and CLQCA20-014) were isolated from samples of rotten wood and fallen leaf debris collected at separate sites in the central region of the Ecuadorian Amazonia. These strains were found to represent a novel yeast species based on the sequences of their D1/D2 domain of the large-subunit (LSU) rRNA gene and their physiological characteristics. Phylogenetic analysis based on LSU D1/D2 sequences revealed this novel species to be most closely related to Candida asparagi, Candida fructus, Candida musae and two as yet undescribed Candida species, with the six yeast taxa collectively forming a distinct species group within the Clavispora clade. The species name of Candida carvajalis sp. nov. is proposed to accommodate these strains, with CLQCA 20-011T (NCYC 3509T, CBS 11361T) designated as the type strain.

  • Candida carvajalis
  • taxonomy
  • rRNA gene
  • LSU D1/D2 domain


Ecuador is located between 1°N and 5°S on the west coast of South America. Although relatively small in size, mainland Ecuador can be subdivided nevertheless into three different and quite distinctive climatic regions: the Pacific coastal plain, the Andean highlands and the Amazon basin. In addition, Ecuador possesses a fourth region, namely the Galapagos Islands. Climatically, the Pacific coastal plain is hot all year, with a rainy season between December and May. In the Andean highlands, the climate is markedly cooler, varying according to altitude. In contrast, the Amazon basin is hot, humid and wet all year round, while the Galapagos Islands are dry, with an annual average temperature of 25 °C (77°F).

To date, very little is known about the natural yeast diversity that exists in Ecuador. In an attempt to begin addressing this scientific shortfall, and to gain a better insight into the effects of contrasting habitats and climate variation on yeast species distribution, a survey was recently set up and initiated by the Colección de Levaduras Quito Católica (CLQCA) in Quito. The aim of the project is to catalogue, characterise and compare the indigenous yeast species found in the different ecological habitats of the four (climatic) regions of Ecuador.

In the early course of this survey, large-subunit (LSU) D1/D2 sequencing revealed that two of the yeast strains (CLQCA 20-011T and CLQCA 20-014), isolated from rotten wood and fallen leaf debris collected at separate sites in the central Amazonian region of Ecuador represented a novel species. Phylogenetically, this species belongs to the Clavispora clade and is closely related to Candida asparagi, Candida fructus, Candida musae and two as yet undescribed Candida species, with the six taxa collectively forming a distinct species group. The phylogenetic placement of this species, coupled with the fact that it could not be induced to sporulate in pure or mixed cultures on several media, led to the conclusion that CLQCA 20-011T and CLQCA 20-014 belong to a novel species of Candida. Here, we describe and propose Candida carvajalis sp. nov. to accommodate these two Ecuadorian strains.

Materials and methods

Sampling sites

The two C. carvajalis strains CLQCA 20-011T and CLQCA 20-014 were isolated from samples of rotten wood and fallen leaf debris collected at two separate sites located near the town of Dayuma, in Orellana province, in the central Amazonian region of Ecuador. CLQCA 20-011T was collected at oil well Auca 19 (GPS coordinates: 9924644N, 291407E), while CLQCA 20-014 was collected at oil well Auca 011 (GPS coordinates: 9931835N, 289886E), with the two oil wells separated from one another by a distance of 16.1 km (the name ‘Auca’ is the old name of the Huaorani people, an isolated tribe that once inhabited the Central Amazonia of Ecuador). The annual average temperature for this region of Ecuador is 26.5 °C, with very few fluctuations during the year. The oscillation in temperature is 2.2 °C between the hottest month (December, 27.4 °C) and the coldest month (July, 25.2 °C). This region has an annual average humidity of 79%, and is considered a high rainfall environment with an average annual rainfall, based on a multiannual measurement, of 3032.2 mm. Vegetation in this region is classified as tropical rainforest.

Yeast isolation

Yeast sampling was carried out using sterile cotton wool swabs. These were used to sample a variety of substrates, including rotten wood and fallen leaf debris, in a number of different geographic locations in the central Amazonian region of Ecuador. The sampled material was inoculated in 1.5-mL microtubes containing enrichment medium. The enrichment medium consisted of YPD (yeast extract, peptone, dextrose) broth supplemented with ampicillin (100 μg mL−1) to suppress bacterial growth. Inoculated broth cultures were incubated at 30 °C for 36 h, after which time 100-μL aliquots were removed and plated onto YPD agar, and incubated for a further 36 h (at 30 °C). Selected representative colonies were picked and replated onto YPD agar. If visible contamination was detected, a further round of colony purification was carried out. Samples of each purified yeast isolate were also examined by light microscopy to check for signs of possible bacterial and/or fungal contamination.

As well as being stored at the CLQCA, Quito, Ecuador, both C. carvajalis strains have been deposited with the National Collection of Yeast Cultures, Norwich, UK, and assigned the strain numbers NCYC 3508 (CLQCA 20-014) and NCYC 3509T (CLQCA 20-011T). In addition, the type strain (CLQCA 20-011T) has also been deposited with the Centraalbureau voor Schimmelcultures (CBS), Utrecht, the Netherlands, as CBS 11361T.

Yeast identification

The yeast strains were characterized morphologically, biochemically and physiologically according to the standard methods as described by Yarrow (1998). Growth temperature testing was determined by cultivation on YM (yeast extract–malt extract) agar. Sporulation tests were performed on corn meal agar, Gorodkowa agar, potassium acetate agar, potato dextrose agar (PDA) and YM agar, and plates were incubated at 25 °C for 1 month in pure and mixed cultures.

Molecular characterization

The D1/D2 domain of the LSU rRNA gene was PCR amplified directly from whole yeast cell suspensions following the method of James (1996), and using the primers NL1 and NL4 (O'Donnell, 1993). Initial amplification reactions were carried out on CLQCA 20-011T and CLQCA 20-014 cultures at the CLQCA, in Quito, while additional (confirmatory) amplification reactions were carried out on subcultures of these strains sent to the NCYC, in Norwich. Amplified fragments were checked by agarose gel electrophoresis, purified using a QIAquick PCR purification kit (Qiagen) as per the manufacturer's instructions, and cycle-sequenced directly using an ABI BigDye terminator cycle sequencing kit, version 3.1 (Applied Biosystems), with the external amplification primers NL1 and NL4. All purified sequence reaction mixes were sequenced using an ABI PRISM 3730 capillary sequencer (Applied Biosystems) at the John Innes Centre Genome Laboratory in Norwich. A sequence similarity search was conducted using EMBL fasta (Pearson & Lipman, 1988). Sequences of closely related taxa were retrieved and aligned using the clustal w program (Thompson, 1994), included in the dnaman software package, version 5.1.5 (Lynnon BioSoft). A phylogenetic tree, based on LSU D1/D2 sequences, was constructed using the neighbour-joining method (Saitou & Nei, 1987), with the Jukes–Cantor distance measure. Confidence limits were estimated by bootstrap analysis (1000 replicates) (Felsenstein, 1985), and only values of 50% or greater were recorded on the resulting tree.

Results and discussion

Phylogenetic analysis

The sequences of the D1/D2 domain of the LSU rRNA gene of CLQCA 20-011T and CLQCA 20-014 were found to be identical. A fasta sequence similarity search of the EMBL fungal sequence database revealed no other yeast taxon to have an LSU D1/D2 sequence identical to these strains. The closest species, in terms of sequence similarity, were Candida sp. BG02-7-21-004E-1-1 [95.9% (11 substitutions, 10 indels)], C. asparagi [95.3% (15 substitutions, 10 indels)], C. fructus [94.8% (19 substitutions, nine indels)], C. musae [94.8% (19 substitutions, nine indels)] and Candida sp. ST-451 [92.0% (30 substitutions, 14 indels)]. Although exceptions are known to exist [e.g. Clavispora lusitaniae (Lachance, 2003)], yeast strains belonging to the same species typically differ by <1% sequence divergence in the D1/D2 domain of the LSU rRNA gene (Kurtzman & Robnett, 1998). Thus, these results indicated that CLQCA 20-011T and CLQCA 20-014 belong to a novel and as yet undescribed species. A phylogenetic tree based on LSU D1/D2 sequences indicates that the novel species may be a sister species of Candida sp. BG02-7-21-004E-1-1. Collectively, these two species along with C. asparagi, C. fructus, C. musae and Candida sp. ST-451 form a distinct (bootstrap value, 94%) species group within the Clavispora clade, a clade that also includes Candida oregonensis, Candida sp. ST-451, Candida sp. VTT C-04530 as well as the two member species of the genus Clavispora, namely C. lusitaniae and Clavispora opuntiae (Fig. 1). Despite displaying a close phylogenetic relationship, examination of these yeast taxa from an ecological perspective failed to identify an obvious common substrate or source. Candida asparagi was isolated from Asparagus filicinus fruit collected in China (Lu, 2004), C. fructus and C. musae were both isolated from bananas collected in Japan (Nakase, 1971), Candida sp. BG02-7-21-004E-1-1 was isolated from the gut of a tenebrionid beetle (Suh, 2005), Candida sp. ST-451 was isolated from an unknown source in Thailand, while CLQCA 20-011T and CLQCA 20-014 were isolated from rotten wood and fallen debris collected in the central Amazonian region of Ecuador.

Figure 1

Neighbour-joining dendrogram based on sequences of the D1/D2 domains of the large-subunit rRNA gene, depicting the relationship of Candida carvajalis with closely related taxa. Bootstrap values ≥50%, determined from 1000 replicates, are shown. Scale bar=2 base substitutions per 100 nucleotides. Bold type represents novel species described in this paper.

Phenotypic characteristics

The novel species, C. carvajalis, can be differentiated from C. asparagi, C. fructus and C. musae, which represent some of its closest phylogenetic relatives (Fig. 1), by the phenotypic tests shown in Table 1. Unfortunately, Candida sp. BG02-7-21-004E-1-1 and Candida sp. ST-451 could not be included in the comparison, as no phenotypic data are currently publicly available for either of these yeasts.

View this table:
Table 1

Growth characteristics that distinguish Candida carvajalis (C.ca) from Candida asparagi (C.asp), Candida fructus (C.fr) and Candida musae (C.mu)

With 50% glucose+
  • * Data taken from Lu (2004), with additional data provided by an anonymous reviewer.

  • Data taken from Meyer (1998).

  • +, positive; −, negative; L, delayed positive (latent).

Based on the molecular and phenotypic data presented here, we concluded that the two Ecuadorian strains represent a novel species of Candida, and the name C. carvajalis sp. nov. is proposed to accommodate them.

Latin description of Candida carvajalis sp. nov. James et al.

In YMA ad 25 °C post dies duos cellulae globosae aut ovoidae (3–7 × 4–8 μm), cellulae singulae, binae et aggregatae, per gemmationem multipolarem reproducentes. Pseudohyphae formantur nec hyphae non formantur. Ascosporae non fiunt.

Glucosum, trehalosum (lente) et cellobiosum (variabile) fermentantur at non galactosum, sucrosum, maltosum, lactosum, raffinosum, inulinum, methyl α-d-glucosidum, melibiosum, melezitosum, d-xylosum nec amylum. Glucosum, sucrosum, galactosum, trehalosum, maltosum, melezitosum, methyl α-d-glucosidum (variabile), cellobiosum, salicinum, l-sorbosum, d-xylosum (exiguum), alcohol aethylicum (lente), glycerinum, ribitolum, d-mannitolum, d-glucitolum, acidum succinicum, acidum citricum et glucono-d-lactonum assimilantur at non inulinum, raffinosum, melibiosum, lactosum, amylum, l-rhamnosum, l-arabinosum, d-arabinosum, d-ribosum, methanolum, erythritolum, galactitolum, inositolum, acidum dl-lacticum nec d-glucosaminum. Ammonium sulfatum, ethylaminum, cadaverinum et l-lysinum assimilantur at non kalium nitricum.

Non crescit in 10% NaCl-5% glucosum. Non crescit in 0.01% cycloheximido. Crescit in 50% glucosum. Non crescit in 60% glucosum. Materia amyloidea non formantur. Non crescit in medio 1% acido acetico. Crescit in 30 °C, non crescit in 37 °C. Typus stirps CLQCA 20-011T (CBS 11361T, NCYC 3509T).

Description of Candida carvajalis sp. nov. James et al.

Candida carvajalis– this Latin-derived epithet refers to Enrique Carvajal, father of E.J.C.B. Although not a biologist himself, his passion for nature has nevertheless led him to become an active collaborator in the search for novel yeast species in Ecuador. He collected these as well as other yeasts while on a number of field trips to the central Amazonian region of Ecuador (Car.va.jal.is).

On YM agar, after 2 days at 25 °C, cells are spheroidal to ovoidal (3–7 × 4–8 μm), and occur singly, in pairs or in groups (Fig. 2). Budding is multilateral. No sexual state is observed from mixed or pure cultures plated on corn-meal agar, Gorodkowa agar, potassium acetate agar, PDA and YM agar. Pseudohyphae are formed (but only in CLQCA 20-011T), but true hyphae are not formed. A summary of the physiological and other growth characteristics of C. carvajalis is given in Table 2.

Figure 2

Scanning electron microscopic image of vegetative cells of Candida carvajalis strain CLQCA 20-011T grown in YM broth for 1 day at 25°C with agitation. Scale bar=1 μm.

View this table:
Table 2

Physiological characteristics of Candida carvajalis sp. nov.

Soluble starchSuccinate+
10%NaCl/5% glucose0.1% Cycloheximide
50% Glucose+Arbutin hydrolysis+
60% GlucoseGrowth at 30°C+
Starch formationGrowth at 37°C
GTYE/1% acetic acid
  • +, positive; w, weakly positive; L, delayed positive (latent); −, negative; v, variable.

The type strain is CLQCA 20-011T, isolated from rotten wood, collected near the town of Dayuma, in the central Amazonian region of Ecuador. Cultures of the type strain and CLQCA 20-014 have been deposited with the CLQCA, Quito, Ecuador, and the National Collection of Yeast Cultures (NCYC), Norwich, UK (CLQCA 20-011T as NCYC 3509T and CLQCA 20-014 as NCYC 3508). The type strain has also been deposited with the CBS, Utrecht, the Netherlands, as CBS 11361T.


E.J.C.B. would like to thank Donald McKenzie and Linda Fuller for their kind assistance and support during his visit to the NCYC and IFR in July 2007, and the IFR for helping to cofund the study as well as coordinate his UK visit. E.J.C.B. would also like to acknowledge the Pontificia Universidad Católica del Ecuador, for supporting his research in Ecuador and his visit to the United Kingdom. S.A.J. would like to thank Mary Parker of the Imaging and Microscopy Group (IMG) at the IFR for her assistance with scanning electron microscopic imaging. S.A.J., C.J.B. and I.N.R. were supported at the IFR by a BBSRC Competitive Strategic Grant.


  • Editor: Cletus Kurtzman


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