Three saprobic Dothideomycetes from the aerial parts of mangrove trees with polyphenism in Striatiguttula

Fungi inhabiting the aerial parts of two mangrove trees, Nypa fruticans, and Rhizophora apiculata, were studied from the central region of Thailand, utilizing morpho-molecular characteristics. Three different fungal taxa were isolated including Rhytidhysteron kirshnacephalus sp. nov., Lasiodiplodia citricola and Striatiguttula phoenicis. Sexual morphs are reported for these three taxa and the asexual morph of Striatiguttula phoenicis is identied based on molecular data. This is the rst asexual morph report for the genus Striatiguttula as well as the family Striatiguttulaceae. The new isolate of Striatiguttula phoenicis differs slightly from other extant species in the genus in terms of measurements of ascomata, asci, ascospores, and thickness of peridium. Also, a pigmented hamathecium was observed in this species. The morphological results are congruent to the phylogenetic results of previous studies and support Striatiguttula phoenicis as a new host record from Nypa fruticans. Rhytidhysteron kirshnacephalus was collected from dead twigs of a standing Rhizophora apiculata in Cha-am and it has signicant morphological and molecular differences to support its establishment as a novel taxon. Phylogenetically, Rhytidhysteron kirshnacephalus forms a sister clade to Rh. magnoliae, but has different ascomatal characters, including, smooth margins without striations and black pruina. Lasiodiplodia citricola is another species from Cha-am and a new record from Thai mangroves. Detailed descriptions of the isolates, along with their potential ecological roles, are provided. We have also provided the occurrence of fungi from the aerial parts of mangrove trees worldwide.


Introduction
Mangroves are salt-tolerant forest ecosystems consisting of woody trees, shrubs, and palms that grow in the intertidal zones of sheltered shores, estuaries, tidal creeks, backwaters, lagoons, marshes, and mud ats of tropical and subtropical coastal regions (Chaeprasert et al. 2010, Thatoi et al. 2013, Hamzah et al. 2018, Kumar et al. 2019a. Mangroves are hosts to many fungi, known as manglicolous fungi (Sarma and Hyde 2001, Sarma and Vittal 2001, Vittal and Sarma 2006, Sakayaroj et al. 2011. Mangroves are mainly evergreen forests, productive and rich in nutrients providing organic matter for fungal colonization Lee 1995, Besitulo et al. 2010) as indicated by the variety of species encountered in numerous studies (Hyde 1988a,b, 1990a,b, Sarma and Hyde 2001, 2018, Maria and Sridhar 2003, Jones and Abdel-Wahab 2005, Raveendran and Manimohan 2007, Alias and Jones 2009, Isaka et al. 2009, Nambiar and Raveendran 2009, Suetrong et al. 2010, Dayarathne et al. 2017, 2018, Devadatha et al. 2018a,b,c,d, Jones et al. 2019. Previous studies have concentrated on fungi isolated from the intertidal region of the mangrove forests and focused primarily on dead stems, leaves, or bark. However, fungi inhabiting the aerial parts of mangrove trees, such as leaves, branches, stems and aerial roots have rarely been considered in biodiversity studies or surveys (Hyde and Cannon 1992, Dayarathne et al. 2017, 2018, Devadatha et al. 2018a,b,c,d, Sarma 2018, Kumar et al. 2019b). These aerial parts form a separate niche for fungi in mangroves that are different from marine fungi occurring in the submerged parts. Studies have shown that aerial parts harbor diverse fungi and they are considered as terrestrial fungi. For instance, Chi et al. (2019) isolated 203 endophytic fungi from leaves of mangrove forests of Taiwan. In another study, Kumar et al. (2018Kumar et al. ( , 2019a isolated fungal taxa from the aerial parts of the mangrove trees Nypa fruticans and Rhizophora apiculata, which included the asexual morphs of Akanthomyces muscarius and Neopestalotiopsis alpapicalis and the sexual morph of Rhytidhysteron mangrovei. In Thailand, mangrove forests populate the southern and central coastal regions, where trees from Arecaceae (Nypa fruticans) and Rhizophoraceae, are the most abundant (Bamroongrugsa et al. 2013, Kumar et al. 2018, 2019b, Zhang et al. 2019). Nypa fruticans is an ancient palm that grows in the upper zone of mangroves stretching from the brackish water zone at river mouths to almost inland freshwater (Rozainah andAslezaeim 2010, Kumar et al. 2018). In a biodiversity study of fungi on N. fruticans, Loilong et al. (2012) reported 139 taxa from Southeast Asian countries, including Brunei, Malaysia, Philippines, Papua New Guinea, and Thailand. Most of the data included fungi reported from decomposing substrates in the intertidal zones. Recently, Sarma and Hyde (2018) listed 46 fungal species from decomposing frond and leaf samples of N. fruticans from Brunei, comprising 33 ascomycetes and 13 anamorphic taxa. In another study, fungi found from terrestrial habitats have also been recorded from the aerial and intertidal parts of N. fruticans, such as Fasciatispora petrakii, Astrosphaeriella nipicola, Oxydothis nypicola , Poonyth et al. 2000, Kumar et al. 2018). Rhizophora sp., another mangrove host genus growing in the same zone as N. fruticans, harbored a huge number of marine fungi including both saprobes and endophytes (Kohlmeyer 1979, Sarma and Vittal 2001, Schmit and Shearer 2003, Pang et al. 2010, Sakayaroj et al. 2011, Manimohan et al. 2011, Hamzah et al. 2018, Kumar et al. 2019a. Regardless of these studies, still, the species diversity and proper classi cation of fungi from N. fruticans and other mangrove trees are yet to be fully explored. This is more so from the aerial parts of these two mangroves.
Most fungi reported from mangrove hosts belong to Ascomycota (Dayarathne et al. 2020). Among them, studies on marine Dothideomycetes have increased exponentially in recent years (Suetrong et al. 2009, Pang et al. 2013, Loganathachetti et al. 2017, Devadatha et al., 2018a,b,c,2019, Kumar et al. 2019b, Zhang et al. 2019, Jones et al. 2019. These have shown that marine Dothideomycetes occur on a wide range of substrata, including mangrove wood, twigs, and leaves, sea and marsh grasses (Kohlmeyer et al. 1995, 1996, 1997, Suetrong et al. 2009, Kumar et al. 2019b, Zhang et al. 2019 show several new genera and species belonging to Dothideomycetes being recorded and it indicates that there is still a huge hidden diversity to be explored (Devadatha et al., 2017, 2018a,b,c,d, Jones et al., 2019.
During surveys of fungal species associated with the aerial parts of mangrove plants, Nypa fruticans, and Rhizophora apiculata, conducted in central and southern Thailand, three fungal species were recorded representing different orders of Dothideomycetes viz. Pleosporales, Hysteriales, and Botryosphaeriales. We introduce one new species Rhytidhysteron kirshnacephalus sp. nov., by comparing its morphology with existing Rhytidhysteron species and providing phylogenetic studies using LSU, ITS, and TEF markers. Two new host records for Lasiodiplodia citricola and Striatiguttula phoenicis are also introduced. An updated list of fungi occurring in the aerial parts of mangrove trees is lacking. Hence, we have provided a list of fungal diversity from the aerial parts of mangrove trees worldwide.

Collection and Isolation
Dead twigs of standing Rhizophoraapiculata tree were collected from Cha-am District, Phetchaburi Province in Southern Thailand (12°48'54.8"N 99°58'54.3"E). Dead rachides or lea ets of Nypa fruticans were collected from Samut Songkhram Province in Central Thailand (13°21'46.9"N 99°59'43.1"E). Fungi were isolated on potato dextrose agar (PDA) using single spore isolation method as described by Chomnunti et al. (2014). Germinating spores were transferred aseptically to fresh PDA plates and incubated at 27 °C ± 2 °C for 7-14 days to establish pure cultures. Morphological characteristics, such as mycelium color, shape, texture, and growth rate were recorded. Cultures were deposited in Mae Fah Luang University Culture Collection (MFLUCC).
Specimens (dry wood material with the fungal material) were deposited in the herbarium of Mae Fah Luang University (MFLU).
Specimens were observed and examined with a Motic SMZ 168 stereomicroscope. Micro-morphological characters of the taxon were examined with Canon EOS 750D and Leica. ImageJ software was used for measurements (Schneider et al. 2012). Faces of fungi numbers are provided as outlined in Jayasiri et al. (2015), and the species has been registered for Index Fungorum numbers (2020).

DNA isolation and ampli cation
Total genomic DNA was extracted, following the modi ed CTAB method, from freshly harvested mycelium (500 mg) (Thambugala et al. 2015, Zhang et al. 2019. and Zhang et al. (2019). The ITS region was ampli ed and sequenced with the primers ITS5 and ITS4 (White et al. 1990), the LSU was ampli ed using primers LROR andLR5 (Vilgalys andHester 1990, Rehner andSamuels 1994), NS1 and NS4 were used for SSU (White et al. 1990) and the TEF gene region was ampli ed using primers EF1-983F and EF1-2218R (Rehner and Buckley 2005). The PCR reactions were performed in a total volume of 25 μl. PCR mixtures contained 0.3 μl of TaKaRa Ex-Taq DNA polymerase, 12.5 μl of 2 × PCR buffer with 2.5 μl of dNTPs, 1 μl of each primer, 9.2 μl of double-distilled water, and 100-150 ng/ μl of DNA template. PCR reactions were run on a BIORAD 1000 Thermal Cycler (Applied Biosystems, Foster City, CA, U.S.A.) using the conditions described by Thambugala et al. (2015) and Zhang et al. (2019). The sequencing of the positive amplicons with primers used in the ampli cation reaction was carried out by Sun-biotech Company Sequencer (Beijing, China).
A combined LSU, ITS, and TEF dataset was used for the phylogenetic analysis of Rhytidhysteron strains. The dataset contained 28 taxa of Rhytidhysteron with Gloniopsis praelonga (CBS 112415) being the outgroup taxon. After trimming, the alignment had 2420 characters, whereby LSU contained 788 sites, ITS had 640 sites and TEF had 992 sites. The alignment has 522 distinct alignment patterns with 35.16% undetermined characters. The RAxML analysis for the combined dataset provided the best scoring tree ( Fig.   5) with a nal ML optimization likelihood value of -7391.602161. The new isolate, Rhytidhysteron kirshnacephalus resides in a distinct clade as a sister group to Rh. mangrovei (BS100%/1.0BI).
GenBank: ITS: MK106111 Notes: Lasiodiplodia, currently comprises 53 species. Both sexual and asexual morphs have been reported within the genus. The genus is a member of the family Botryosphaeriaceae, which is well-known and widespread as plant pathogens occurring mostly in tropical and subtropical regions (Punithalingam 1980). Cruywagen et al. (2017) suggested that hybridization between Lasiodiplodia species is widespread and further suggested that some of the currently recognized species may be hybrids, e.g., L. viticola, L. missouriana, L. laeliocattletae, and L. brasiliense. The fungal isolate understudy was obtained from a twig of a mangrove shrub and has been identi ed as a new host record for L. citricola with support from both morphology and phylogenic data (Fig. 7). This isolate clustered with type strain in the present multi-locus phylogeny (Fig. 7). Morphologically the species is slightly different from the ex-type species, L. citricola (IRAN 1522C) by having rough conidiogenous cells and smaller aseptate paraphyses. In L. citricola (IRAN 1522C), paraphyses are 125μm long, whereas, in L. citricola , paraphyses are 50-80 μm long, which might have occurred due to the change in the host and environment. While the conidial characters were overlapping for both the species. The untrimmed sequence of ITS had 6 bp differences when compared with that of L. citricola (extype IRAN 1522C and MFLUCC 19-0622). Based on its occurrence, the similarity in morphology, and inadequate differences in the molecular data, here we consider our isolate as a new host record for Lasiodiplodia citricola.
Diversity of fungi on the aerial parts of mangrove trees worldwide A list of fungi recorded from the aerial parts of the mangrove trees throughout the world is provided in Table 6. The occurrence of endophytic, pathogenic, and saprobic fungi from the aerial parts indicates their multi-level and variable relations with the host and their lifestyle. We have documented 268 fungi found on the aerial parts of 46 mangrove trees (  Table 6). A higher diversity on these mangrove hosts consists of fungi occurring in or on the leaves than branches and roots. In our data, fungi from the genus Aspergillus and Pestalotiod group were very common and have a wide distribution. Based on our data, we reckon that the fungal diversity from the aerial parts of the mangrove forests warrants a systematic survey for a correct estimation.

Discussion
In this study, three saprobic fungal species were isolated and identi ed from the aerial parts of mangrove trees, Nypa fruiticans, and Rhizophora apiculata, collected from two different provinces of Central Thailand, Cha-am (Phetchaburi) and Samut Songkhram. New isolates were identi ed by utilizing morpho-molecular techniques. They are as Striatiguttula phoenicis, Rhytidhysteron Kirshnacephalus sp. nov., and Lasiodiplodia citricola The three isolates belong to the class Dothediomycetes and fall within different orders viz. Pleosporales, Hysteriales, and Botryosphaeriales. The rst isolate, Striatiguttula phoenicis, falls in a newly circumscribed family Striatiguttulaceae. The second isolate, Rhytidhysteron kirshnacephalus sp. nov. is a member of Hysteriaceae and is characterized by its large, conspicuous ascomata. The third isolate belongs to the family Botryosphaeriaceae, and is a known pathogen, Lasiodiplodia citricola.  (Zhang et al. 2019). Striatiguttula is the type genus and is characterized by having immersed, erumpent to super cial stromata, with a papilla or a short to a long neck, trabeculate pseudoparaphyses, bitunicate asci, and hyaline to brown, fusiform to ellipsoidal, striate, guttulate, 1-3-septate ascospores, with paler end cells and surrounded by a mucilaginous sheath (Zhang et al. 2019).
The new strain of Striatiguttula phoenicis in this study was isolated from the midrib of the lea et of N. fruticans collected from Samut Songkhram. Morphologically our strain ts well within the species concept of Striatiguttula (Zhang et al. 2019). Previously, S. phoenicis was reported from Phoenix paludosa, which is known to be associated with mangroves and grows on the upper regions of mangrove forests (Teo et al. 2010, Zhang et al. 2019. In our study of fungi from aerial parts of mangrove trees, we isolated S. phoenicis from the midrib of a dead leaf from a standing N. fruticans in central Thailand. The present report extends the host range of this taxon. Since, in Zhang et al. (2019), the species was isolated from the submerged decaying rachis of P. paludosa in Southern Thailand. This suggests that S. phoenicis is not limited to one host species perhaps suggesting host jumping in the Arecaceae family though more extensive studies on coevolution are needed. In our isolate, there are slight morphological differences with S. phoenicis (MFLUCC 18-0266) viz. ascomatal size, the thickness of peridium, size of the asci, and size and septation of the ascospore (Fig. 1, Table 3). These morphological differences could be due to the occurrence on a different host plant i.e., N. fruticans. Through this study, we establish the anamorph to teleomorph connection for S. phoenicis through morphological and molecular studies. This is the rst report of an anamorph connection reported in the family Striatiguttulaceae (Fig. 2,3). During our study, we observed the asexual morph at rst then the sexual morph on the same substrate. This could indicate that S. phoenicis reproduces more frequently through the asexual mode of life than through sexual mode (Hyde et al. 2011, Jones et al. 2014).
Rhytidhysteron kirshnacephalus (MFLUCC 18-1111) was collected from the mangroves of Cha-am district, Thailand. It belongs to Hysteriales, which contains only one family, Hysteriaceae. There are three genera of marine or marine-derived fungi in the family: Gloniella, Hysterium, and Rhytidhysteron (Wijayaward. 2017, Jones et al. 2019, Kumar et al. 2019b). Rhytidhysteron was introduced by Spegazzini (1881) and is characterized by large, conspicuous ascomata, usually elongate and boat-shaped and features a prominent, perpendicularly striate margin, in combination with pigmented, sparsely septate to sub-muriform ascospores (Spegazzini 1881, Silva-Hanlin and Hanlin 1999, Thambugala et al. 2016, Kumar et al. 2019b). In the phylogenetic analysis, our isolate (MFLUCC 18-1111) was grouped with Rh. magnoliae as a sister taxon and the two can be separated based on the morphological differences, such as appearance and size of exciple, ascomata, pruina, hamathecium. In addition to the morphological differences, we have also observed DNA base-pair differences (5% in TEF from Rh. magnoliae) to establish Rhytidhysteron kirshnacephalus (MFLUCC 18-1111) as a new species in the genus (Table 4, Fig. 4,5). Although species of Rhytidhysteron are widely distributed in tropical and temperate countries such as Brazil, France, Ghana, Kenya, most of them have also been found in Thailand (Thambugala et al. 2016, Kumar et al. 2019b). This is not surprising given its tropical climate and mangrove forests, Thailand has one of the rich diversities of Rhytidhysteron. Rhytidhysteron mangrovei and Rh.bruguierae have also been reported from mangroves. Members belonging to Hysteriales are often reported from mangrove habitats, particularly the aerial parts of the mangrove plant substrate (Devadatha et al. 2018, Kumar et al. 2019b, Dayarathne et al. 2020. The super cial, well-protected wall layers of the ascomata of hysteriales seem to protect from desiccation and solar radiation for their occurrence in the upper parts of the mangrove plants. Lasiodiplodia citricola  was also found in Cha-am district as a new host record from Rhizophora apiculata. It belongs to Botryosphaeriales, which has nine families. Marine species have been found only in Botryosphaeriaceae and Phyllostictaceae, which belong to Botryosphaeriales (Wijayaward. 2017, Jones et al. 2019). Lasiodiplodia was introduced by Ellis and Everh (1896) and is characterized by the presence of pycnidial conidiomata and longitudinal striations on mature conidia (Sutton 1980, Zhou and Stanosz 2001, Slippers et al. 2004, Phillips et al. 2008, Prasher and Singh 2014. Lasiodiplodia citricola seems to be a cosmopolitan fungus, having a broad range of hosts and wide geographic distribution viz. Citrus latifolia, (Mexico), Citrus sp. (Iran), Juglans regia (California), Pistacia vera (California), Prunus persica (California), and Vitis vinifera (Australia, Italy) (USDA, https://nt.ars-grin.gov/fungaldatabases/). Mostly, L. citricola is known as a pathogenic fungus, but the new strain was observed as a saprobe on dead twigs of the mangrove tree, indicating the ability of L. citricola to adapt to the occurrence on new hosts and diverse lifestyles with saprophytism recorded in the present study. In our phylogenetic analyses, the TEF sequence of the new host record was not included as we were not successful in obtaining it.
The aerial parts of the mangrove trees in the marine environment are excellent habitat to study fungal diversity and ecology. However, when compared with the fungi from submerged marine/intertidal substrates, the number of fungi from the aerial parts is very small (Jones et al. 2019, Table 6). Poonyth et al. (2000) listed 163 fungi from mangrove and mangrove-associated trees. Whereas here we have listed 268 fungi (from 156 genera) from the aerial parts of 46 mangrove trees across 44 different locations (Table 6). Among them, Aspergillus (15), Pseudocercospora (13), and Pestalotiod fungi (15) are the top three genera to occur on the terrestrial part of the mangrove trees. Based on our data, woody substrata supported a greater number of fungi than leaves and most of them are saprophytes.
This study suggests that future studies should include the examination of fungi found from the aerial parts of mangrove forests and explore their signi cance. Also, investigations on the underlying mechanism of exhibiting both sexual and asexual morphs and lifestyle switching are required. Tables   Table 1 List of Pleosporalean taxa used in this study along with their GenBank Accession numbers. New sequences are given in bold typeface. T stands for the Type species of each genus.