Morphology and multigene phylogeny of Talaromycesamyrossmaniae, a new synnematous species belonging to the section Trachyspermi from India

Abstract A new Talaromyces species, T.amyrossmaniae, isolated from decaying fruit and litter of Terminalia bellerica, is described and illustrated. On the natural substrate, the new species produces determinate synnemata, with a well-defined, vivid orange red to orange red cylindrical stipe, and a greyish green capitulum. Conidiophores are typically biverticillate, or sometimes have subterminal branches, with acerose phialides that produce globose to subglobose, smooth to slightly roughened conidia. Multigene phylogenetic analyses based on the internal transcribed spacer region (ITS), and partial sequences of β-tubulin (BenA), calmodulin (CaM), and DNA directed RNA polymerase second large subunit (RPB2) genes, along with morphological characterization, revealed that these isolates are distinct and form a unique lineage of Talaromyces in section Trachyspermi, closely allied to T.aerius, T.albobiverticillius, T.heiheensis, T.erythromellis, and T.solicola. The new species T.amyrossmaniae is the first species in section Trachyspermi with determinate synnemata.


Morphology and multigene phylogeny of Talaromyces amyrossmaniae, a new synnematous species belonging
to the section Trachyspermi from India
During the 2009 monsoon season, routine surveys were conducted to explore microfungal diversity in natural forests of Lingmala waterfalls area (17.9218N; 73.6870E) of Mahabaleshwar, northern Western Ghats, India.A previously undescribed synnemaforming fungus with penicillate conidiophores and phialidic conidiogenous cells was collected from decaying fruits and litter of Terminalia bellerica (Combretaceae) fallen onto the ground near the Lingmala waterfalls.The fungus was isolated into pure culture on different culture media, microscopic characters were recorded and its classification studied using phylogenetic analysis of aligned DNA sequences from the nuclear ribosomal ITS region and BenA, CaM, and RPB2 partial gene sequences.This paper aims to resolve the taxonomy and phylogeny of this synnematous species, which is shown to represent a new species in Talaromyces section Trachyspermi, here named T. amyrossmaniae.

Isolation
Conidia were removed from synnemata directly from the surface of fallen fruits under a Nikon stereomicroscope (model SMZ1500 with Digital camera; Nikon, Tokyo, Japan) and placed on malt extract agar (MEA) media containing the antibiotic Streptomycin sulphate (100 mg/L) CMS220-5G (HIMEDIA Laboratories Pvt.Ltd, Mumbai, India).Methods and media used for examining colony characters, inoculating and incubating cultures, and microscopic examination followed those of Visagie et al. (2014), with the addition of Oatmeal Agar (OA), and Potato Dextrose Agar (PDA), with incubation occurring in a Bio Multi Incubator (Model LH-30-8CT, Japan).Herbarium specimens were deposited in the Ajrekar Mycological Herbarium (AMH); cultures were accessioned and preserved in the National Fungal Culture Collection of India (NFCCI; WDCM-932), Agharkar Research Institute, Pune, India.Reference and ex-type strains used in this study are listed in Table 1.

DNA extraction, amplification, and phylogenetic analyses
Colonies were grown on MEAbl plates, and genomic DNA was extracted following the rapid salt extraction method of Aljanabi and Martinez (1997).The ITS regions was amplified using primer pairs ITS5 and ITS4 (White et al. 1990).For the amplification of RPB2 gene region, primer pairs RPB2-5F and RPB2-7cR (Liu et al. 1999) were used with touch-up PCR conditions: 5 cycles with annealing temperature 48 °C followed by 5 cycles at 50 °C and final 25 cycles at 52 °C.The partial BenA gene was amplified with primer pair Bt2a and Bt2b (Glass and Donaldson 1995) with 50 °C as annealing temperature.The partial CaM gene was amplified using primer pair CF1M  1).

Phylogenetic analyses
The phylogenetic analysis showed that the new species described below as Talaromyces amyrossmaniae belongs to section Trachyspermi.The relationships of the new species with accepted species and its genetic coherence and phylogenetic consistency were analysed with single concatenated sequence datasets based on four loci (ITS, BenA, CaM and RPB2).The length of the data sets were 540 bp for ITS, 379 bp for BenA, 550 bp for CaM, and 851 bp for RPB2 loci.The best fitting models for the ML analysis were TPM2u+F+I+G4 for ITS, TIM2e+G4 for BenA, K2P+I+G4 for CaM, and K2P+I+G4 for RPB2.All trees were rooted with T. pinophilus (CBS 631.66).The single gene trees and the multigene phylogram are shown in Figures 1, 2.
Because of the limited resolution of the official fungal DNA barcode, the ITS (Schoch et al. 2012), in the Trichocomaceae, BenA was proposed as the secondary DNA barcode for Talaromyces (Yilmaz et al. 2014).The overall tree topologies of ITS and BenA phylogenies had relatively consistent association of species.However, the type species of section Trachyspermi, T. trachyspermus was well separated from T. assiutensis in the BenA analysis whereas strains of the two species were intermixed in the ITS analysis.Talaromyces ucraicinus was consistently a sister clade to T. trachyspermus and T. assiutensis.Our proposed new species, T. amyrossmaniae, was distinguished from other species both by ITS and other markers (Figs 1, 2).It is consistently included in a major clade along with T. aerius, T. albobiverticillius, T. erythromellis, T. heiheensis, and T. solicola in the ITS analysis.As with the ITS, in the concatenated phylogeny and RPB2 analyses, T. amyrossmaniae clustered with T. albobiverticillius, T. heiheensis, T. erythromellis, T. aerius, and T. solicola (Figs 1, 2).However, in the BenA phylogeny, T. amyrossmaniae was segregated from that major-clade.Talaromyces amyrossmaniae is clustered with T. austrocalifornicus in the CaM analyses (Fig. 1).Yilmaz et al. (2014) mentioned that amplification of CaM is difficult in section Trachyspermi.CaM data could not be analyzed critically because the new sequences generated through Sanger sequencing (ABI PRISM 3100 Genetic Analyzer) contained a homopolymer stretch of around 30 bp after base 320, resulting in poor quality and short sequences, even after many attempts using modified PCR conditions and primers.

Morphology
In this study, we introduce one new species, Talaromyces amyrossmaniae belonging to section Trachyspermi.Strains conform with the general morphological characters of this section.Talaromyces amyrossmaniae was compared with its close relatives, with the distinguishing characters mentioned in the note after the species description.Also, Table 2 compares the new species with the closely allied species in section Trachyspermi.The main character that differentiates T. amyrossmaniae from other synnemata-producing species in the genus Talaromyces is the length of the synnemata.Talaromyces amyrossmaniae has the shortest synnemata (up to 150 µm).A synopsis of comparative morphology and growth rate of synnema producing species of Talaromyces is given in Table 3.

Taxonomy
Talaromyces amyrossmaniae Rajeshkumar, Yilmaz & Seifert, sp.nov.MycoBank MB518601 Figure 3 Etymology.Latin, named after Dr Amy Y. Rossman, in honour of her career as a research leader in Systematic Mycology and Microbiology, USDA ARS, Beltsville, Maryland, USA.

Discussion
In our study, a novel Talaromyces species, T. amyrossmaniae is described based on two isolates from decaying fruits and litter of Terminalia bellerica (Combretaceae).We used ITS, BenA, CaM and RPB2 sequences to apply genealogical concordance phylogenetic species recognition (GCPSR; Taylor et al. 2000) to delineate the species, and a multigene phylogenetic analysis to place T. amyrossmaniae in Talaromyces section Trachyspermi.Talaromyces section Trachyspermi (as 'trachyspermus') was introduced by Yaguchi et al. (1996) based on ubiquinone systems, overriding the traditional morphology based classification of Talaromyces.Yilmaz et al. (2014) applied multigene phylogenies and morphology to redefine classification of Talaromyces and divided the genus into seven sections.They noted that Talaromyces section Trachyspermi includes species with generally biverticillate penicilli with acerose phialides and when ascomata are produced, they are creamish white or yellow.Colonies generally grow restrictedly on CYA, YES, CREA and DG18; some species have colonies with abundant red pigments.Morphologically, T. amyrossmaniae resembles the other species of section Trachyspermi and produces dark orange to red pigmentation on MEAbl, restricted growth on MEAbl, CYA, DG18, YES and CREA, symmetrical biverticillate penicilli with a minor proportion having sub-terminal branches, and acerose phialides that form globose to subglobose, smooth to slightly roughened conidia.Although, synnematous Talaromyces species also are found in section Purpurei, section Talaromyces, section Trachyspermi, and section Subinflati, T. amyrossmaniae is the first species in section Trachyspermi with determinate synnemata that are seen on fallen decaying fruits in nature and also on MEAbl after 7-14 d of incubation at 25 °C.Talaromyces systylus is another synnema producer in section Trachyspermi, but it produces indeterminate synnemata up to 4000 µm and grows at 37 °C (Romero et al. 2016).Talaromyces amyrossmaniae has the shortest synnemata in Talaromyces and it grows very restrictedly compared to the other synnema producing species on CYA and MEAbl.With these key characters, it is easy to distinguish the new species from the other synnemata producing species of Talaromyces.
Based on ITS, BenA, CaM, and RPB2 phylogenies, T. amyrossmaniae is part of the same clade as T. austrocalifornicus, T. convolutus, T. heiheensis, T. aerius, T. solicola, T. albobiverticillius, and T. erythromellis; however, it can be distinguished from all of these species by having determinate synnemata in nature and by differences in colony growth characteristics.Also, T. amyrossmaniae forms predominant concentric rings of synnemata on the different media used in our studies and even forms synnemata in vitro on MEAbl.
Many species of Talaromyces have been recorded as saprophytes, endophytes, and human pathogens from different geoclimatic regions and microhabitats across India.Most importantly, Talaromyces marneffei is a potentially pathogenic thermally dimorphic fungus causing systemic mycosis in HIV-infected patients; its dissemination was thoroughly studied from Manipur state of India (Singh et al. 1999;Ranjana et al. 2002).Recent studies on endophytic T. pinophilus isolated from the rhizomes of Curcuma amada from Karnataka revealed the production and partial characterization of L-asparaginase (Krishnapura and Belur 2016).Likewise, endophytic T. radicus, isolated from Catharanthus roseus, produces vincristine and vinblastine and was studied for induce apoptotic cell death (Palem et al. 2015).Talaromyces flavus is also recorded as an endophytic fungi isolated from ethno-medicinal plants in the sacred forests of Meghalaya having antimicrobial and antioxidant activity (Bhagobaty and Joshi 2012).Devi et al. (2014) reported a marine strain of T. verruculosus, from Andhra Pradesh, as a potent polyhydroxybutyrate degrader.Similarly, the stress-tolerant soil fungus T. funiculosus, isolated from the neem rhizosphere, was identified as a potential strain for phosphate solubilization (Kanseet al. 2015).Talaromyces flavus isolated from paddy rhizosphere of Darjeeling Hills exhibited phosphate solubilizing activity in vitro and positively influenced the growth of Oryza sativa, Cicer arietinum, and Vigna radiata under greenhouse conditions (Chakraborty et al. 2011).A keratin degrading strain of T. trachyspermus was isolated from the grounds of a gelatin factory in Jabalpur, Madhya Pradesh, and digested human hair in stationary culture (Rajak et al. 1991).Talaromyces trachyspermus was reported as a soil saprophyte in paddy fields of Orissa (Dutta and Ghosh 1965).Species identifications of these Talaromyces strains were mostly based on micro-and macro morphological characters in these studies.Because such approaches often underestimate species diversity, adoption of a polyphasic approach to authenticate such identifications will increase the number of Talaromyces species known from different eco-geographic zones of India.Further investigation is also needed to study the ecological importance of these species.

Table 1 .
(Nguyen et al. 2015)2013)gal strains and strains used for the phylogenetic analysis.Hubka et al. 2014)subjected to 32 cycles under the following temperature regime: first cycle at 95 °C for 3 min, 55 °C for 30 seconds, and 72 °C for 1 min; followed by 30 cycles at 95 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 1 min; and a final cycle at 95 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 10 min.PCR products were purified with StrataPrep PCR Purification Kit (Agilent Technologies, Reference sequences of Talaromyces section Trachyspermi were downloaded from Gen-Bank and aligned in MAFFT v. 7.305b(Katoh and Standley 2013)with the newly generated sequences.Alignments were manually adjusted in Geneious as needed.A Maximum Likelihood analysis was done in IQtree v. 1.6(Nguyen et al. 2015)after selecting the most suitable substitution model with the Modelfinder (Kalyaanamoorthy) algorithm built into the software.The trees were visualized in Figtree v. 1.4.3 ((http://tree.bio.ed.ac.uk/software/figtree) and edited for publication in Affinity Designer v. 1.6.1 (Serif Europe Ltd, UK).The new DNA sequences were deposited in GenBank (Table T: ex-type strain and CF4 (TX, USA) and sequenced using the BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, USA).Sequencing reactions were run on a ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems, USA).

Table 3 .
Synopsis of comparative morphology and growth rate of synnema producing species of Talaromyces.