Nakazawaea odontotermitis f.a., sp. nov., a novel yeast isolated from the gut of Odontotermes horni in India

Three strains, SMT1.3, SMT1.10, and SMT2.2, representing a novel asexual ascomycetous yeast species, were isolated from the gut of a termite Odontotermes horni in Maharashtra, India. Phylogenetic analyses of the LSU, ITS, and SSU sequences revealed that they belonged to the genus Nakazawaea, with N. siamensis as the closest relative. The new species differed from the type strain of N. siamensis (DMKU-RK467T) by 11 substitutions in the D1/D2 region of the large subunit (LSU) rRNA gene and by 8 substitutions and one gap in the small subunit (SSU) rRNA gene. Notable biochemical and physiological differences were also observed between N. siamensis and the new species. Hence, the species Nakazawaea odontotermitis f.a., sp. nov. is proposed. The type strain is SMT1.3 T (MTCC 13,105 = NFCCI 5011 = PYCC 9153). GenBank accession numbers of the LSU, ITS and SSU sequences of Nakazawaea odontotermitis f.a., sp. nov. are MZ234240, MZ234239, and OK384663. The MycoBank number is MB 841926.


Introduction
The genus Nakazawaea of the order Saccharomycetales was first proposed in 1994 by Yamada et al. (1994), with Nakazawaea holstii as the type species. This ascomycetous genus was introduced to reassign Pichia holstii to a new genus, Nakazawaea, based on certain notable characteristics that distinguished it from the other hat-shaped, ascosporeforming and nitrate-assimilating species of the genus Pichia. However, phylogenetic analyses based solely on the partial sequences of the D1/D2 region of the LSU and SSU rRNA genes of all known species of Pichia did not support the creation of a new genus, since very few species were considered for rRNA analysis (Kurtzman and Robnett 1998). Later, a reclassification based on multigene phylogeny of the sequences of several protein-coding genes including actin (ACT1), largest subunit and second-largest subunit of the RNA polymerase II gene (RPB1 and RPB2), the second subunit of mitochondrial cytochrome oxidase (COX2), along with the D1/D2 region of the LSU rRNA gene provided sound support for the proposal of the genus Nakazawaea (Tsui et al. 2008). Combined analysis of the LSU (D1/D2) rRNA gene, translation elongation factor-1α (EF-1α) gene, SSU rRNA gene, RPB1 and RPB2 gene sequences separated the genus Pichia from Nakazawaea. In 2014, several asexual species of the genus Candida were transferred to the genus Nakazawaea, based on multigene phylogeny (Kurtzman and Robnett 2014). The species accommodated in the genus Nakazawaea were N. anatomiae, N. ernobii, N. ishiwadae, N. laoshanensis, N. molendini-olei, N. peltata, N. pomicola, N. populi, N. wickerhamii, and N. wyomingensis. Since this reorganization, only three new species, viz., N. siamensis, N. todaengensis, and N. ambrosiae, have been described (Kaewwichian and Limtong 2014;Polburee et al. 2017;Crous et al. 2019 The gut of insects, especially the xylophagous kind, is a niche for several ascomycetous and a few basidiomycetous yeasts, which may share a mutualistic relationship with insect hosts (Blackwell 2017). The exact role of yeast-insect associations is not entirely understood, but the most plausible explanation is that the yeast symbionts assist insects with nutrition, while the host insects protect yeasts from unfavorable environments and help in their dispersal to new habitats (Stefanini 2018). It is well known that the gut of beetles and wood roaches harbor many novel yeasts (Suh et al. 2005a, b). In recent years, the termite gut has also been established as a potential habitat for diverse yeast species, including various novel yeast taxa (Handel et al. 2016;Ali et al. 2017;Tiwari et al. 2021). Termites represent one of the most significant wood-degrading species, which can break down complex polymers into simpler monomers with the help of enzyme complexes secreted by gut symbionts in combination with the host's endogenous enzymes. In a recent survey of the termite gut-associated yeasts in India, yeasts belonging to the genera Vishniacozyma, Kodamea, Pseudozyma, Hannaella, and Cystobasidium were reported for the first time from the gut of termites Coptotermes heimi and Odontotermes javanicus (Tiwari et al. 2020). Termites of the genus Odontotermes are prevalent in tropical and subtropical regions of Africa and Asia, especially the Indian subcontinent (Shanbhag and Sundararaj 2013).
In the present study, while investigating yeast communities residing in the gut of wood-feeding termites, we isolated 30 ascomycetous yeast strains identified as Yamadazyma sp., Cyberlindnera bimundalis, Cy. fabianii, Candida silvanorum, and C. insectorum from the gut of Odontotermes horni. We also obtained three strains representing a novel species of the genus Nakazawaea, for which the name Nakazawaea odontotermitis sp. nov. is proposed.

Termite collection and yeast isolation
Worker termites (30 adults per sample) feeding on fallen pieces of wood were collected from Kapare, Maharashtra (17.551372° N, 73.434554° E), India, while investigating the yeast diversity of the termite gut in parts of the Western Ghats of India, in December 2020. Two separate termite-infested wooden twigs were sampled from the same area, but 50 m apart. The two samples were temporarily designated as SMT1 and SMT2. Molecular identification of the host termites was achieved by sequencing the mitochondrial 16S rRNA gene (Clark and Kambhampati 2003), and the sequences were deposited in NCBI GenBank. Previously described protocols were followed for termite dissection and preparation of homogenous gut suspensions (Tiwari et al. 2020). Thirty termite individuals (per sample) were surface sterilized with 95% ethanol, rinsed with 0.9% saline, and dissected with forceps or needles. Gut contents were transferred to a sterile tube containing 0.9% saline (500 µl) and crushed with a pestle. The gut mixture was briefly centrifuged to separate the debris, and 100 µl aliquots were spread on yeast-extract peptone dextrose (YPD) agar plates (1% yeast extract, 2% peptone, 2% dextrose, and 2% agar) containing antibiotics (100 µg ml −1 streptomycin and 100 µg ml −1 ampicillin). Three YPD plates per sample were inoculated and incubated at 25 °C until yeast colonies emerged. Single yeast colonies were picked and streaked for purification. YM agar (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, and 2% agar) was used for routine subculturing and maintenance at 25 °C. For long-term preservation, yeast cultures were stored at − 80 °C in broth culture supplemented with 20% (w/v) glycerol.

Phylogenetic analysis
Genomic DNA was isolated using a simple yet effective protocol (Aamir et al. 2015). The yeast isolates were screened by MSP-PCR (microsatellite-primed PCR) fingerprinting technique using the (GTG) 5 primer. The MSP-PCR was performed following standard reagents and cycling conditions (Ramírez-Castrillón et al. 2014). A few representative strains of each cluster (fingerprint-based grouping) were selected for molecular identification by sequencing the barcode DNA regions. The ITS region, SSU and the D1/D2 region of the LSU rRNA gene were amplified using the primers ITS4 and ITS5; SSU1f, SSU4r, SSU3f, and SSU2r; NL1 and NL4 (White et al. 1990;Kurtzman and Robnett 2003;Polburee et al. 2017). Amplicons were purified with the FavorPrep™ GEL/PCR Purification Kit (FAVORGEN Biotech Corporation, Taiwan) and sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) by Sanger sequencing. All sequences generated during the study were deposited in NCBI GenBank. The D1/D2 and SSU rRNA gene sequences of closely related species were retrieved from GenBank, and alignments were made using the MUSCLE program (Edgar 2004). A phylogenetic tree was constructed based on concatenated sequences of SSU rRNA and D1/D2 region of LSU rRNA genes, using the Maximum-Likelihood (ML) algorithm and GTR + F + R2 model in IQ-TREE software v. 1.6.7 (Nguyen et al. 2015).

Termite identification and yeast isolation
Termite heads were used for molecular identification. Mitochondrial 16S rRNA gene sequencing identified the host termites as Odontotermes horni (SMT1 OL629227, SMT2 OL629228). Several yeast colonies appeared on YPD agar plates after 2 days of incubation at 25 °C. Thirteen yeast strains were obtained from SMT1, while 17 were obtained from SMT2 (Table 1). Strains SMT1.3 and SMT1.10 were isolated from the SMT1 sample on two separate plates, while SMT2.2 was isolated from the SMT2 sample.

Sequence comparison and species delineation
All 30 yeast strains were subjected to MSP-PCR-based screening, and 11 representative yeast strains were processed for identification based on D1/D2 LSU rDNA sequencing (Table 1 and Fig. S1). The strains SMT1.3, SMT1.10, and SMT2.2 were isolated from the termite O. horni, among other ascomycetous yeast species. These three strains were identified up to species level by analyzing the D1/D2 LSU rDNA sequences (Table 1). When comparing sequences of the D1/D2 domains, the new species differed from the closely related species N. siamensis DMKU-RK467 T (NG_060235) by 1.9% sequence divergence (11 substitutions 0 gaps) and N. holstii CBS 6225 by 2.9% divergence (16 substitutions and 1 gap) in a 569 bp aligned region. The new species showed 0.6% divergence in SSU rDNA sequence compared to N. siamensis DMKU-RK467 T (8 substitutions and 1 gap) and 2.2% (29 substitutions and 1 gap) sequence divergence with N. holstii NRRL Y-2155 in a 1312 bp aligned region. The strains SMT1.3, SMT1.10, and SMT2.2 showed 100% sequence similarity with the same phenotypic characteristics, indicating that they are conspecific. The number of nucleotide substitutions in the D1/D2 region of LSU rDNA and SSU rDNA regions warrants considering these strains as a novel species in the genus Nakazawaea.
Moreover, the phylogenetic tree reconstructed from the combined sequences of the SSU rRNA and the D1/ D2 region of the LSU rRNA genes (Fig. 1) confirmed

Morphological and physiological characteristics
The colony of the new species appears circular, white with an opaque sector, shiny, and smooth textured (Fig. 2a). The cells of N. odontotermitis are globose to subglobose measuring 2.5-5 × 3-5 μm (Fig. 2b), while those of N. siamensis measured 3-5 × 3-5 μm. Both species show multilateral budding, but the buds are predominantly polar in N. odontotermitis sp. nov. (Fig. 2b, c). Ascospores, hyphae, or pseudohyphae formation were not observed in the new species. Furthermore, the new species can be discriminated from N. siamensis based on its ability to assimilate nitrate, nitrite, and d-glucosamine, while N. siamensis failed to assimilate these as sole nitrogen and carbon sources. Moreover, N. siamensis could assimilate inulin, methyl-α-d-glucoside, erythritol, and galactitol, while the new species could not utilize these compounds (Table 2). The new species could grow up to 37 °C, but N. siamensis could grow up to 40 °C. Members of the genus Nakazawaea have been isolated from various habitats, but are predominantly associated with plant material and insects. However, a few species like N. anatomiae, N. ishiwadae, and N. peltata have been isolated from some unusual habitats like corpses embalmed in formalin, deep core of stratigraphic drillings, and mastitis milk, respectively (Kurtzman 2011). Several strains of N. populi, N. wyomingensis and N. pomicola were recovered from sap fluxes of trembling aspen (Populus tremuloides), black cottonwood (Populus trichocarpa), birch (Betula sp.), red oak (Quercus rubra), and old fustic (Maclura tinctoria) (Lachance et al. 2011). Strains of N. wickerhamii were isolated from silage made of olive husks and olive oil wastewaters (alpechín), while N. molendini-olei was obtained from olive oil and its by-products (Lachance et al. 2011;Čadež et al. 2012). The species N. laoshanensis was recovered from decayed wood in China (Wang et al. 2010). Two species were discovered in Thailand; N. siamensis was isolated from the surface of sugar cane leaves, and N. todaengensis from peat in a swamp forest (Kaewwichian and Limtong 2014;Polburee et al. 2017). Three species of Nakazawaea, namely N. holstii, N. ernobii, and most recently N. ambrosiae, have been isolated from bark beetles infesting pine, spruce, and fir trees (Lachance et al. 2011;Crous et al. 2019). Similarly, in the present study, three strains of a novel species, N. odontotermitis, were isolated from the gut of termites (O. horni) feeding on wood. The occurrence of multiple isolates of this new species in the termite gut suggests that it may be a frequent inhabitant of the gut along with other symbiotic microbes and may contribute to host nutrition. A large proportion of the species in this genus have been isolated from wood materials or insects, indicating that these might be potential habitats for the isolation of Nakazawaea species.