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Identification of endophytic fungi from leaves of Pandanaceae based on their morphotypes and DNA sequence data from southern Thailand

Abstract The authors established the taxonomic status of endophytic fungi associated with leaves of Pandanaceae collected from southern Thailand. Morphotypes were initially identified based on their characteristics in culture and species level identification was done based on both morphological characteristics and phylogenetic analyses of DNA sequence data. Twenty-two isolates from healthy leaves were categorised into eight morphotypes. Appropriate universal primers were used to amplify specific gene regions and phylogenetic analyses were performed to identify these endophytes and established relationships with extant fungi. The authors identified both ascomycete and basidiomycete species, including one new genus, seven new species and nine known species. Morphological descriptions, colour plates and phylogenies are given for each taxon.


Introduction
Endophytic fungi are beneficial to their host plants and have the ability to produce bioactive compounds that have applied uses (Fisher et al. 1994;Strobel et al. 2004;Gunatilaka 2006;Arnold et al. 2007;Saikkonen et al. 2010;Aly et al. 2010;Lin et al. 2010;Rajulu et al. 2011;Chowdhary et al. 2015). Research on endophytic fungi began approximately 30 years ago and has intensified over the past 20 years (Thomson et al. 1997;Arnold et al. 2000;Stone et al. 2000;Hyde and Soytong 2008;Lumyong et al. 2009). This rising interest in endophytic fungi dates back to Bills' 1996 novel concept that mycelia sterilia isolates could be assigned to groups based on their degree of similarity in colony surface texture (Rodrigues 1994;Fisher et al. 1995;Lodge et al. 1996;Brown et al. 1998;Taylor et al. 1999;Umali et al. 1999;Fróhlich et al. 2000). Lacap et al. (2003) used molecular data to demonstrate the reliability of Bill's 1996 concept based on the cultural approach. Guo et al. (2000Guo et al. ( , 2003 found that morphological characteristics were insufficient to identify most endophyte isolates, especially when they do not sporulate and so DNA sequence data were used for identification of these taxa. Although this has been followed by numerous authors using ITS sequence data analysis, the use of ITS alone is not accurate (Promputtha et al. 2005). Subsequent studies have shown that multi-gene analyses are needed to identify endophytes (Ko et al. 2011).
Endophytic fungal strains have been isolated from many different plants including trees, vegetables, fruits, cereal grains and other crops (Rosenblueth and Martinez-Romero 2006). Dickinson (1976) published the first study of endophyte -leaf associations. However, there has been less research on the endophytic fungi associated with the leaves of tropical plants (Promputtha et al. 2007). The high species diversity of endophytic fungi makes their study a pressing research area. Globally, endophytic fungi were estimated to comprise 7 % of the 1.5 million species of fungi (Hawksworth 2001;Chowdhary et al. 2015). The actual numbers may be far higher. Recently, Hawksworth and Lucking (2017) estimated that there are 2.2 to 3.8 million fungal taxa. Endophytes are expected to be numerous because their host-specificity will drive diversification and they can occupy several niches, including that of pathogens and saprobes (Zhou and Hyde 2001). Several studies have investigated the relationships between endophytes and saprotrophs and also between endophytes and pathogens (Petrini 1991;Yanna and Hyde 2002;Ghimire and Hyde 2004;Photita et al. 2004;Hyde et al. 2006).
The objectives of the present study were to establish the endophytic fungal community on selected Pandanaceae collected in southern Thailand. The authors isolated 22 endophytic isolates and sorted them in morphotypes and identified the taxa based on DNA sequence analyses. Both ascomycete and basidiomycete genera were identified, including one new genus, seven new species and nine known species. The recommendations of Jeewon and Hyde (2016) were followed when introducing the new species based on molecular data.

Sample collection and fungal isolation
Healthy mature leaves of Pandanus and Freycinetia species (Pandanaceae, Figure 1) were collected from Chumphon (10°57'38.2"N 99°29'21.8"E) and Ranong (9°55'15.9"N, 98°38'30.7"E) provinces of southern Thailand during the rainy season (December) of 2016. Leaves with physical damage or showing signs of pathogenic infection were excluded from the study. In total, more than 100 healthy leaves were placed in Ziploc plastic bags, preserved with ice and transported to the laboratory. Leaves were randomly cut into 0.5 cm size pieces (10 pieces/leaf ) using a hole puncher under aseptic conditions. These sections were soaked in 95 % ethanol for 1 minute, then in 3 % sodium hypochlorite solution for 3 minutes and finally in 95 % ethanol for 30 seconds. All samples were rinsed with sterile distilled water and dried on sterile tissue paper. Leaf sections were placed in Malt Extract Agar (MEA), Potato Dextrose Agar (PDA) and Water Agar (WA). They were incubated at room temperature (25-30 °C) for 1-3 days. If hyphal tips of any fungal colony appeared during incubation, the colony was transferred to new PDA plates and incubated to obtain pure cultures.

Cultures and identification
The above methods resulted in 22 isolates which were separated into morphotypes based on visual assessment of the similarity of the cultures (Bills 1996;Umali et al. 1999;Fróhlich et al. 2000;Lacap et al. 2003). All of these cultures were grown on Potato Dextrose Agar (PDA). Growth rate measurements are shown in Table 1 with colony colour defined with the Methuen Handbook of Colour (Kornerup and Wanscher 1967). New taxa were examined in pure culture, allowing photographs, records of morphological characteristics and descriptions to be recorded. Herbarium specimens were prepared from cultures that were dried in silica gel. The holotypes were deposited in the Mae Fah Luang University Herbarium (Herb. MFLU), Chiang Rai, Thailand and in the Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China. The ex-types cultures were deposited in the Mae Fah Luang University Culture Collection (MFLUCC) with duplicates deposited in the BIOTEC Culture Collection Laboratory (BCC) and the Kunming Institute of Botany Culture (KMUCC). New taxa were registered in Facesoffungi (FoF) (Jayasiri et al. 2015) and MycoBank (Crous et al. 2004).

Phylogenetic analysis
The sequence data generated during this study were the subject of BLAST searches in the nucleotide database of GenBank (www http://blast.ncbi.nlm.nih.gov/) to determine their most probable closely related taxa. Sequence data were retrieved from Gen-Bank based on recent publications. Raw forward and reverse sequences were assembled using Geneious Pro.v4.8.5. Sequence alignments were carried out with MAFFT v.6.864b (Katoh and Standley 2016) and alignments were manually improved where necessary. The sequence datasets were combined using BioEdit v.7.2.5 (Hall 2004

Identification of morphotypes
Twenty-two fungal isolates from Pandanus and Freycinetia species were recovered and these mycelia sterilia were separated into eight morphotypes based on the similarity of their culture characteristics, as summarised in Table 2 (Bills 1996;Umali et al. 1999;Fróhlich et al. 2000;Lacap et al. 2003).

Phylogenetic analysis
Based on phylogenetic analysis, 22 fungal isolates were identified for 16 species. These include one new genus, seven new species and nine known species. All sequences obtained from this study are summarised in Table 3 (Kirk et al. 2008). According to Cannon and Kirk (2007), the species in this family are characterised by poroid, irregular or lamellate hymenophores and are saprobes. Recent phylogenetic analyses of Polyporaceae are by Binder Etymology. Named after its habitat as an endophyte of Pandanus. Type species. Endopandanicola thailandica Tibpromma & K.D. Hyde Culture characteristics. Colonies on PDA (PE60), superficial, initially white, later becoming yellow-white, smooth at the surface, irregular, with undulate margin, flossy to velvety; reverse white to yellow-white. Generative hyphae simple-septate, branched, sub-hyaline, thin-walled.
Notes. Endopandanicola formed a single, well-supported clade (100 % in ML, 100 % in MP), which is distinct as compared to other genera in Polyporaceae ( Figure 3). This genus comprises resupinate or crust polypores that live inside leaves or wood as endophytes and do not form fruiting bodies (sexual morph), but form flat mycelia. More collections of Pandanus are needed in the future to locate the sexual morph of Endopandanicola. Etymology. named after Thailand, the country where the fungus was first discovered.
Holotype. MFLU 18-0021 Culture characteristics. Colonies on PDA (Figure 2 PE10, FE42, FE43, FE46 and PE60), superficial, initially white, later becoming yellow-white, irregular, with un- Figure 3. Phylogram generated from maximum likelihood analysis based on ITS sequence data. Maximum parsimony (left) and maximum likelihood (right) bootstrap support values are given above/below the nodes. The newly generated sequences are in red text. The tree is rooted with Pirex concentricus. dulate margin, smooth with flossy to velvety; reverse white to yellow-white. Generative hyphae simple-septate, branched, with clamp connections, sub-hyaline, thin-walled, 1.5-3.5 μm wide.  Notes. Endopandanicola is introduced and typified by En. thailandica which is represented by six isolates and is described as a novel species based on its asexual morph. The phylogenetic analysis of ITS sequence data showed that this species clustered together with Panus, but there is a high level of statistical support for its separation (100% in ML, 100% in MP) ( Figure 3).
GenBank numbers. ITS=MG646957. Notes. Burdsall and Eslyn (1974) introduced Phanerochaete chrysosporium which was collected on dead wood of Platanus wrightii in the USA. Phylogenetic analysis of ITS sequence data shows this taxon groups with Phanerochaete chrysosporium (sequences obtained from GenBank) that had been collected from different hosts. The phylogenetic placement of this species is shown in Figure 3. Remarks. The order Botryosphaeriales was introduced by Schoch et al. (2006) with Botryosphaeriaceae as the type family. Botryosphaeriales is a diverse order with a worldwide distribution, comprising species that vary from endophytes to pathogens (Slippers and Wingfield 2007;Phillips et al. 2013;Chethana et al. 2016;Daranagama et al. 2016;Dissanayake et al. 2016;Konta et al. 2016a, b;Linaldeddu et al. 2016a, b, c;Manawasinghe et al. 2016;Zhang et al. 2017). Currently, nine families are recognised, namely, Aplosporellaceae, Botryosphaeriaceae, Endomelanconiopsisaceae, Melanopsaceae, Phyllostictaceae, Planistromellaceae, Pseudofusicoccumaceae, Saccharataceae and Septorioideaceae Figure 5. Phylogram generated from maximum likelihood analysis based on ITS, LSU and TEF1 sequenced data. Maximum likelihood bootstrap values are given above/below the nodes. The newly generated sequences are in red bold. The tree is rooted with Tiarosporella paludosa. Minnis et al. 2012;Wikee et al. 2013;Slippers et al. 2013;Wyka and Broders 2016;Dissanayake et al. 2016;Yang et al. 2017). In this study, Endomelanconiopsis freycinetiae is introduced as a new species and reports are provided on Phyllosticta capitalensis and Lasiodiplodia theobromae. Etymology. name referring to the host genus on which the fungus was found (Freycinetia).
Notes. Endomelanconiopsis freycinetiae is closely related to the endophytic fungus En. endophytica. Therefore, the culture characteristics of these two taxa were compared and it was found that, in En. endophytica, at first the hyphae are colourless, immersed, later becoming olivaceous in the centre with irregular concentric rings; aerial mycelia are dark olivaceous or grey when dense; shiny black when the aerial mycelia are loose (Rojas et al. 2008) whereas aerial mycelia of En. freycinetiae has dark olivaceous, circular rings and flossy surface (Figure 2, FE41). Nucleotide base pairs of ITS and TEF1 were also compared and it was found that there are differences (ITS 3 bp, TEF1 8 bp).
GenBank numbers. ITS=MG646954, LSU=MG646953, TEF1=MG646982. Notes. Phyllosticta capitalensis (Hennings 1908) is known as an endophytic taxon and a minor plant pathogen. It has a worldwide distribution and has been recorded on 70 plant families (Baayen et al. 2002;Okane et al. 2003;Motohashi et al. 2009;Wikee et al. 2013). The isolate recovered herein clusters with reasonable ML bootstrap support with other P. capitalensis isolates ( Figure 5). Morphological examination also depicts similar morphs and hence it is identified as P. capitalensis. Notes. Morphological and phylogenetic data supported placement of this isolate as Lasiodiplodia theobromae. The phylogenetic analysis showed the isolate groups with Lasiodiplodia theobromae. Nucleotide base pairs of published sequences of Lasiodiplodia theobromae (strain EucN188, CBS 111530, PHLO9, CDFA145) were also compared with the sequence and found that the nucleotide base pairs of the ITS gene are 100% similar. Remarks. The genus Cladosporium (Cladosporiaceae, Capnodiales) is a large genus of the Ascomycota (Wijayawardene et al. 2017). The genus comprises species that are saprobes, endophytes and pathogens. A few species have been documented as being etiologic agents in vertebrate hosts (David 1997;Bensch et al. 2012Bensch et al. , 2015Crous et al. 2014). In this study, a new species of Cladosporium is described, with high bootstrap support in the phylogenetic analysis (Figure 7).
Culture characteristics. Colonies on PDA (Figure 2, PE58), superficial, dark olivaceous with dark-grey centre, irregular, undulate with wrinkled and raised on surface media; reverse dark olivaceous. Generative hyphae simple-septate, branched, sub-hyaline, guttules, thick-walled (Figure 8) Notes. Cladosporium endophyticum was isolated as an endophyte from Pandanus sp. in Thailand. In the phylogenetic analysis of combined gene sequence data of ITS, LSU, SSU and TEF1, the new taxon Cladosporium endophyticum is sister to C. halotolerans (Figure 7), but well-separated with high bootstrap support (90% in ML). Moreover, the morphology of this new taxon was compared with Cladosporium halotolerans which has brown to dark  brown, subglobose to globose with verrucose, less often short-ovoid conidia, narrower at both ends (Zalar et al. 2007), while C. endophyticum has globose to ovoid, hyaline to pale-olivaceous conidia with rounded ends. Here, the authors introduce the new species C. endophyticum and provide an updated phylogenetic tree for the genus Cladosporium.
Pleosporales Luttr. ex M.E. Barr, 1987 Massarinaceae Munk. (1956) under Pleosporales together with Cucurbitariaceae and Didymosphaeriaceae. Later, Barr (1987) segregated Massarinaceae under Lophiostomataceae based on morphology, while based on multigene phylogenetic analysis Schoch et al. (2009) also showed Massarinaceae is a distinct family in order Pleosporales. Recently, Zhang et al. (2009Zhang et al. ( , 2012 recognised Massarinaceae as a distinct lineage based on both morphology and molecular phylogeny. In this study, a new species of endophytic Massarina, based on morphological and phylogenetic support, is introduced from Pandanus sp. in Thailand.

Massarina pandanicola Tibpromma & K.D. Hyde, sp. nov.
MycoBank number: MB823839 Facesoffungi number: FoF03904 Figure 10 Etymology. name referring to the host genus of the plant on which the fungus was first discovered (Pandanus).

Pleosporaceae Nitschke
Remarks. The family Pleosporaceae was introduced by Nitschke (1869) and is the largest family of the order Pleosporales Ariyawansa et al. 2015b;Liu et al. 2017). Members of this family can be endophytes, aquatic or terrestrial saprobes, plant pathogens or opportunistic animal pathogens (Sivanesan 1984;Carter and Boudreaux 2004). A backbone tree for Pleosporaceae was provided by Ariyawansa et al. (2015a). In this study, Alternaria burnsii is reported from a Pandanus sp. host in Thailand.  (Figure 2, PE26), superficial, white-orange to cream, circular, entire edge, smooth, flossy, velvety and raised on surface media; reverse yellow-white at the margin and yellow-brown in centre. Not sporulating in culture.

Alternaria burnsii
GenBank numbers. ITS=MG646973, LSU=MG646952, TEF1=MG646987. Notes. Alternaria burnsii was introduced by Uppal et al. (1938) from India on Cumnium cyminum. This species has a close phylogenetic relationship with Alternaria tomato and A. jacinthicola (Woudenberg et al. 2015). Results from phylogenetic analysis show that the authors' collection belongs to Alternaria burnsii with a relatively high bootstrap support (89% in ML) ( Figure 11). Nucleotides across the ITS regions of Alternaria burnsii CBS 108.27 and the isolates were compared and the authors noted that they are identical. Remarks. The family Diaporthaceae was introduced by von Höhnel (1917) and was placed in the order Diaporthales. This family comprised two Diaporthe genera (Phomopsis and Mazzantia) (Wehmeyer 1975;Castlebury et al. 2002). Later, Diaporthaceae was given the synonym Valsaceae (Barr 1978). Based on DNA sequence data, some other genera have been placed in Diaporthaceae (Dai et al. 2014;Voglmayr and Jaklitsch 2014). Recently, Maharachchikumbura et al. (2015) and  listed further genera that belong to Diaporthaceae. In this study, a new and a known species of Diaporthe from Pandanaceae hosts in Thailand is reported.
GenBank numbers. ITS=MG646974, β-tubulin=MG646930, ACT=MG646930. Notes. Diaporthe species are plant pathogens, endophytes or saprobes (Carroll 1986;Garcia-Reyne et al. 2011;Udayanga et al. 2011). Here, a new species Diaporthe pandanicola is introduced based on phylogeny support. Based on phylogenetic analysis, the new species was well-separated from closely related species of Diaporthe (61% in ML, 0.97 in PP). However, this isolate is an endophytic fungus and did not sporulate in culture during 5 months (Figure 13).
Notes. In the phylogenetic analysis, the authors' collection grouped with Diaporthe siamensis MFLUCC 10-0573 with high statistical values of 100% in ML and 1.00 in PP. Diaporthe siamensis is an endophytic fungus collected from a Pandanaceae host in Thailand.   Remarks. The family Glomerellaceae was introduced by Locquin (1984), but was invalidly published. To date, most Glomerellaceae have been recorded to be pathogens (Maharachchikumbura et al. 2016b). Earlier studies reported that the position of the family Glomerellaceae was not stable (Zhang et al. 2006;Kirk et al. 2001;Kirk et al. 2008). Réblová et al. (2011) resolved the placement of Glomerellaceae by using phylogenetic analysis of combined ITS, LSU, SSU and RPB2 sequence data. Recently, the family Glomerellaceae was established based on the genus Glomerella (Zhang et al. 2006), which had been given a synonym under its asexual morph Colletotrichum . Recently, Jayawardena et al. (2016) provided notes on currently accepted species of Colletotrichum. In this study, the authors introduce a new endophytic Colletotrichum species and report a known species of endophytic Colletotrichum from gloeosporioides species complex based on morphology and phylogenetic analysis.
Notes. The gloeosporioides species complex is mainly plant pathogens (Weir et al. 2012) and some species are endophytes . Colletotrichum fructicola has a wide host range (Weir et al. 2012) and was originally reported from coffee berries in Thailand (Prihastuti et al. 2009). In this study, the authors followed Jayawardena et al. (2016) and identify the collection as Colletotrichum fructicola which was isolated from a Pandanaceae host. Based on phylogenetic analysis, this taxon grouped with Colletotrichum fructicola with 90 % in ML and 1.00 in PP. The ITS, β-tubulin, GAPDH, CHS-1 and ACT DNA nucleotide comparison showed that the taxon and other strains of Etymology. name referring to the host genus (Freycinetia).
Culture characteristics. Colonies on PDA (Figure 2, PE09), superficial, white in the beginning and later becoming dark-grey, circular, entire edge, smooth, flossy, velvety and raised on surface media; reverse dark. Sporulating in culture after 1 month. Notes. Colletotrichum pandanicola is introduced here as a new species in the gloeosporioides species complex based on morphological and phylogenetic data. The phylogenetic analysis shows that this new taxon is well-separated from other known Colletotrichum species (Figure 14). The authors also compared nucleotides of β-tubulin, GAPDH, CHS-1 and ACT and found that there are differences between Colletotrichum tropicale and this new species (β-tubulin 7 bp, GAPDH 11 bp, CHS-1 7 bp and ACT 3 bp).

Magnaporthaceae P.F. Cannon
Remarks. The family Magnaporthaceae was introduced by Cannon (1994) and was placed as a family within the class Sordariomycetes (Kirk et al. 2001;Lumbsch and Huhndorf 2007). According to Thongkantha et al. (2009), the placement of the taxa Magnaporthaceae has long been problematic due to a lack of convincing morphological characteristics and inconclusive molecular data. Thongkantha et al. (2009) established a new order, Magnaporthales, to accommodate Magnaporthaceae, based on a combination of morphological characteristics and the phylogenetic analysis of combined sequence data. Maharachchikumbura et al. (2015) provided an updated outline of the family Magnaporthaceae with 20 genera, which included both sexual and asexual morphs. In this study, Mycoleptodiscus endophyticus is introduced as a new species. Etymology. Named after its original habitat as an endophytic fungus.
Material examined.   gorum. Most of these species were described without molecular data. In this study, a new species Mycoleptodiscus endophyticus is introduced, based on culture characteristics and phylogenetic analysis (100 % in ML). Mycoleptodiscus endophyticus was found as an endophytic fungus on leaves of Freycinetia sp; Mycoleptodiscus freycinetiae Whitton, K.D. Hyde & McKenzie was found as a saprobic fungus on the same host but there was no molecular data available to confirm this identification. The authors were unable to compare the morphological differences between the new taxon and Mycoleptodiscus freycinetiae, because only culture characteristics are presented here for this new taxon (Fig. 17).

Sporocadaceae Corda, 1842
Remarks. Sporocadaceae was introduced by Corda (1842) with Pestalotiopsis-like asexual morphs and confirmed by Senanayake et al. (2015). Members of Sporocadaceae are saprobes, endophytes or foliar pathogens in tropical and temperate regions (Jeewon et al. 2004;Tanaka et al. 2011). Pestalotiopsis can be found as saprobes or pathogens worldwide (Jeewon et al. 2002(Jeewon et al. , 2003Maharachchikumbura et al. 2011Maharachchikumbura et al. , 2012Maharachchikumbura et al. , 2013Maharachchikumbura et al. , 2014aMaharachchikumbura et al. , b, 2016a. Recently, Chen et al. (2017) provided updates for this genus based on morphology and phylogeny. In this study, two known species of Pestalotiopsis from Pandanaceae hosts were isolated. Figure 18. Phylogram generated from maximum likelihood analysis based on the combination of ITS, β-tubulin and TEF1 sequenced data. Maximum parsimony bootstrap is given above/below the nodes. The newly generated sequences are in red bold. The tree is rooted with Seiridium camelliae.

Pestalotiopsis jiangxiensis F. Liu & L. Cai, 2017
Culture characteristics. Colonies on PDA (Figure 2, PE05), superficial, white at the margin with yellow-white in the centre, with circular to undulate at the edge and raised and dense aerial mycelia on surface; reverse yellow-white. Sporulating in culture after 2 months.
Notes. The authors' collection from Pandanaceae host in Thailand was identified as Pestalotiopsis jiangxiensis. This taxon grouped with Pestalotiopsis jiangxiensis LC4399 which is collected from Eurya sp., with high bootstrap support of 100% in ML.

Debaryomycetaceae Kurtzman & M. Suzuki
Remarks. Debaryomycetaceae was introduced by Kurtzman and Suzuki in 2010 and was typified by Debaryomyces Klöcker. Meyerozyma belongs to family Debaryomycetaceae and was detailed in Kurtzman and Suzuki (2010). In this study, Meyerozyma caribbica was found on a Pandanaceae host as an endophytic fungus. Species identification was confirmed by DNA sequence data.  (Figure 2, PE75, 98), superficial, white to yellow-white, rings with irregular, undulate edge and curled, raised on the surface media; reverse yellow-white to yellow at the margin and dark-brown at the centre. Sporulating in culture after 2 months.
Notes. Meyerozyma caribbica collected in this study is represented by two endophytic isolates from Pandanaceae. Phylogenetic analysis also supported the identification of this sample as Meyerozyma caribbica. Figure 19. Phylogram generated from maximum likelihood analysis based on combined LSU and SSU sequence data. Maximum parsimony bootstrap is given above/below the nodes. The newly generated sequences are in red text. The tree is rooted with Schizosaccharomyces pombe.

Conclusion
In this study on fungal endophytes found on leaves of Pandanaceae, it was found that the taxa belonged to both Ascomycota and Basidiomycota. The majority of the taxa were Ascomycota, as found in most previous endophytic studies (Crozier et al. 2006;Selim et al. 2017). In classical mycology, most endophytic fungi were described based on their morphological features (Barseghyan and Wasser 2010). However, there are difficulties in identifying ascomycetes to the species level based only on morphological features (Lu et al. 2012), because they have only a small set of morphological characteristics and exhibit homoplasy (Barseghyan and Wasser 2010).
The 22 endophytic fungal strains found in this study were chiefly identified using their microscopic characteristics and DNA sequence data and holotype materials in the form of dried cultures. Future studies are however needed to recollect the taxa which are sporulating to describe sexual and asexual characteristics (sensu Lacap et al. 2003). In this study, 22 endophytes were isolated and sorted into eight morphotype based on colony characteristics. The authors, however, subjected all isolates to phylogenetic analysis and found they belong to 16 different taxa. The taxa were sorted roughly into morphotypes, but they did not reflect the actual species. Several isolates of this study did not sporulate, but are introduced as new species because DNA sequence comparison and multi-gene phylogenetic analyses provided sufficient evidence to show that they are distinct taxa (Jeewon and Hyde 2016). The new taxa are, however, code compliant, as they are provided with MycoBank numbers, full descriptions, colour photographss and illustrations.
The species composition of endophytic microorganisms is likely to depend on the plant age, genotype, sampled tissue, host type and season of isolation (Rosenblueth and Martinez-Romero 2006). Promputtha et al. (2007) showed that endophytic species can change their ecological strategies and adopt a saprotrophic lifestyle. However, it was found that for the cultures of some endophytic fungal species, mycelia are the only visible morphological structures. According to these conclusions, the authors agree with Petrini (1991), Yanna and Hyde (2002), Ghimire and Hyde (2004) and Hyde et al. (2006) regarding the relationships between fungal endophytes and saprobic fungi. However, the use of next-generation sequencing (NGS) (Shendure and Ji 2008) is another option for identification of fungal species that cannot be cultured in vitro and has now become popular. These methods have also been applied to large-scale culture-independent molecular biological methods (Zoll et al. 2016). Future developments in technology are likely to produce further novel methods that mycologists could apply to the field of taxonomy (e.g. Hawksworth and Lucking 2017).
ter E. Mortimer thanks the National Science Foundation of China (NSFC) project codes 41761144055 and 41771063. Fiona Worthy in the World Agroforestry Centre (ICRAF), Kunming Institute of Botany, China is thanked for English editing.