Jurnal Biota Vol. 7 No. 1, 2021 ISSN: 2460-7746 (online); ISSN: 2528-262X (print) | 1

Study of Lentinus squarrosulus from West Java on The Basis of Molecular and Morphological Data

Rudy Hermawan

Department of Biology, Faculty of Mathematics and Natural Sciences, IPB University, Darmaga Campus, Bogor 16680, Indonesia. email: [email protected]

Article Info ABSTRACT Lentinus is a unique genus within Polyporales, because of Keyword: the lamellate basidiocarp. In Indonesia, Lentinus is First record commonly studied about their potential, rarely for their Lamellate basidiocarp taxonomy. BO 24427 specimen was found in West Java, Polyporales Indonesia, situated in the Landscape Arboretum of IPB Section Rigidi University. The specimen was identified using molecular study and supported by some morphological data of the fresh fruiting bodies. Molecular identification used ITS 4 Article history: and 5 regions. Phylogenetic tree was constructed using Received: 10/08/2020 MEGA Version X software. The morphology was based on Revised: 26/11/2020 macroscopic and microscopic observation. The result of Accepted: 26/11/2020 molecular analyses showed that BO 24427 specimen was identified as Lentinus squarrosulus with 99% bootstrap value and classified into section Rigidi. The morphological data of the fresh fruiting body supported the molecular identification. The important morphological data that were classified into Lentinus squarrosulus were scabrous cap and size of basidiospores. This study was the first record to publish the finding of Lentinus squarrosulus in Indonesia.

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Introduction (Steccherinum Gray) and smooth By naming Polyporales, it is an (Podoscypha Pat.) (Binder et al., 2013). order which generally has pores Some of those genera have the characteristic (Hawksworth et al., 1996). characteristics that are opposite by Nevertheless, this order is heterogeneous naming Polyporales, i.e. Lentinus with of basidiocarp types (Binder et al., 2005). lamellate basidiocarp. In the previous The basidiocarp types are bracket-shaped study, Lentinus has been placed in the (Fomitopsis P. Karst.; Trametes Fr.), agaric family as Tricholomataceae resupinate to effused-reflexed because of a lamellate hymenophore and (Ceriporiopsis Domański; Phlebia Fr., white spore print (Miller 1973). Newly, Antrodia (Fr.) Donk), stipitate (Polyporus Studies in Pegler (1975), Corner (1981), P. Micheli) and hymenophore and Singer (1986) put Lentinus within configuration such as poroid Polyporales. It was supported by (Perenniporia Murrill), lamellate molecular data by Hibbett & Vilgalys (Lentinus Fr.), hydnaceous (1993). Lamellate basidiocarp in

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Polyporales has two genera, i.e. Lentinus observation was conducted in the and Panus (Hibbett et al., 1993). mycology laboratory of Biology Hibbett et al., (2007) and Nilsson et Department, Mathematics and Natural al., (2014) suggested studying fungi Sciences, IPB University using Olympus using molecular analyses that are a very stereo and binocular microscope powerful tool to identify them until cs22LED. The sample was preserved in species. Schoch et al., (2012) FAA (Kottapalli et al., 2016) and recommended the Internal Transcribed deposited into Herbarium Bogoriense Spacer (ITS) area had been formally Indonesia. proposed as the main marker in the Identification of Mushrooms identification of fungi molecularly. It was The species was identified using agreed with Seelan et al., (2015) that molecular analyses. The genomic DNA study about Lentinus using molecular was extracted manually using CTAB study. The best single gene that could method (Hermawan et al., 2020a). Sterile clade the species within Lentinus was part of the fruiting body was mashed up ITS. until being a paste, 500 µL CTAB-buffer To date, Lentinus has 40 species (cetyl-trimethyl ammonium bromide) (Kirk et al., 2008), excluding the was added into fruiting body paste and synonyms, on the basis of 146 species incubated in 65 OC for 30 min. Then, 500 records in Index Fungorum (2020). Then, µL of chloroform isoamyl alcohol (24:1) many reports mentioned the wide was added and centrifuged at 12,000 rpm distribution in Asia, including Indonesia for 15 min. The supernatant was (Sulistiany & Sudirman, 2015), Thailand collected, 500 µL of phenol: chloroform: (Somchai, 2012), Malaysia (Bolhassan et isoamyl alcohol (25:24:1) was added and al., 2012), India (Manimohan et al., centrifuged at 12,000 rpm for 10 min. The 2004), and China (Nazura et al., 2010). In supernatant was collected, and 50 µL 2M India, Lentinus is becoming one of the NaOAc (Sodium Hypo-Acetate) and 500 potential mushrooms for edible µL absolute ethanol were added. This mushrooms containing many vitamins mixture was stored in -20 OC overnight, and good nutrition (Gulati et al., 2011), then centrifuged at 15,000 rpm for 30 antimicrobial (Sudirman, 2010), and min. The supernatant was removed. 300 antioxidant (Omar et al., 2011). One of µL of 70 % ethanol was added and the potential Lentinus is L. squarrosulus centrifuged at 10,000 rpm for 5 min. The (Mont.) Singer (Omar et al., 2011). supernatant was removed and the DNA Recently in this study, L. squarrosulus sample was in the pellet. The pellet was was found growing in West Java, air-dried using a Speed Vacuum on 30 OC Indonesia. Compared with many studies for 30 min. The pellet was resuspended in done on Lentinus in Indonesia, none has 50 µL of Tris-EDTA buffer and 10 µL 1 been done on its taxonomy. mg/mL of RNAse. Then, it was incubated at 37 OC for 10 min. RNAse was then Materials and Methods deactivated by incubating the mixture at Mushrooms Sampling Site 70 OC for 10 min. DNA quality and The sampling was conducted on 20 quantity were verified using a nanodrop January 2020 and located in Arboretum spectrophotometer. Landscape of IPB University. The After that, the DNA quality and mushroom was collected, documented, quantity were verified using a nanodrop and observed the morphological spectrophotometer. The amplification characters, such as size, color, shape, used Internal Transcribed Spacer (ITS) as ornamentation, and spores. The

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Jurnal Biota Vol. 7 No. 1, 2021 | 3 forward ITS 5 (5’-GGA AGT AAA AGT were sent to the 1st Base Malaysia for CGT AAC AAG G-3’) and reverse ITS 4 sequencing. (5’-TCC TCC GCT TAT TGA TAT GC- The Sequence was deposited in 3’) primers. PCR amplification was GenBank. This sequence, 28 reference performed in 30 µL total reaction sequences of updated Lentinus species containing 15 µL PCR mix from 2X (Seelan et al., 2015), and Polyporus Kappa Fast 2G, 1,5 µL of 10 pmol of each arcularius (outgroup) were used for primer, and 3 µL 100 ng template DNA, phylogenetic tree reconstruction (Table and 9 µL ddH2O. Amplification used a 1). Sequences were aligned and built the Thermoline PCR. The PCR condition was phylogenetic tree using MEGA Ver. X set as follows: initial denaturation at 94 software. The phylogenetic tree was built OC for 2 minutes, followed by 30 cycles by bootstrap analyses with 1000 of denaturation at 94 OC for 30 seconds, replicates. Maximum likelihood was annealing at 55 OC for 30 seconds, and chosen for phylogenetic tree construction extension at 72 OC for 1 minute. Then the with the best method based on MEGA final extension was set at 72 OC for 10 selection. Phylogenetic tree was built by minutes. The amplicons were estimated bootstrap analyses with 1000 replicates. on 1 % agarose gels and visualized by the Bootstrap (BS)≥60 was shown on the Gel DocTM XR system. PCR products branch.

Table 1. Collection code, species, GenBank accession numbers of Internal Transcribed Spacer used in this study. Collection code GenBank acc. no Species (Voucher/Cultures) ITS BO 24427 Lentinus squarrosulus MT815466 DED07668 Lentinus badius KP283480 PU00436 Lentinus badius KP283481 JSKT5858 Lentinus badius KP283479 JS0094 Lentinus badius KP283478 TFB11754 Lentinus bertieri GU207302 TFB11756 Lentinus bertieri GU207301 DSH9243C Lentinus crinitus KP283495 KM141387 Lentinus polychrous KP283487 AH00024 Lentinus polychrous KP283485 JS00054 Lentinus polychrous KP283486 SNP24989 Lentinus sajor-caju KP283493 JS0056 Lentinus sajor-caju KP283494 FRI62056 Lentinus sajor-caju KP283492 TFB11164 Lentinus scleropus GU207310 FRIM4180 Lentinus squarrosulus KP283483 CUI6513 Lentinus squarrosulus KP283482 BORHF0009 Lentinus squarrosulus KP283484 NG9 Lentinus striatulus KX580188 Tage Roland M0135 Lentinus striatulus GU207311 TENN19955 Lentinus suavissimus KP283504 ADD7 Lentinus suavissimus KP283501 LE0791625 Lentinus suavissimus KP283500 DSH2011 (AL57) Lentinus suavissimus KP283496 TFB6206 Lentinus swartzii GU207276 CCCT 17.106 Lentinus swartzii MN192922 DSH92D787 Lentinus tigrinus KP283488 ATCC 9406 Lentinus tigrinus GU207269 ATCC 28757 Lentinus tigrinus GU207271 DSH92-132 Polyporus arcularius KP283522

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Morphological Observation radiate growth. The hymenophores The species identification was within Lentinus can be divided into two confirmed by morphological data. All types (Seelan et al., 2015), i.e. lamellae characters of the fruiting body were and subporoid lamellae. observed and documented. The data were Phylogenetic tree important such as size, shape and Lentinus BO 24427 specimen was ornamentation of the fruiting body and identified as Lentinus squarrosulus by 99 basidiospores. The data will be compared % Bootstrap (BS) value (Fig 1). The with reference of species within Lentinus length of ITS 4 and 5 regions of this using a literature study. Describing fresh sequence was amplified for 634 base specimens used a description of the pairs of nitrogen bases. Lentinus fruiting body by Brundrett et al. (1996). squarrosulus was first reported to grow in Indonesia. This species had been found Results and Discussion and reported in Borneo, Malaysia, Specimen Thailand, Papua New Guinea, and South The fruiting body of Lentinus was Africa (Seelan et al., 2015). The Lentinus deposited into Herbarium Bogoriense squarrosulus was within the section with code BO 24427. It was collected by Rigidi. Rudy Hermawan on a rotten wood The phylogeny (Figure 1) showed substrate. All of the phases of the fruiting that the BO 24427 specimen was L. body were collected and preserved in the squarrosulus and classified within FAA. The fruiting body clearly had a section Rigidi. According to Seelan et al., stem and a cap. The cap was lamellate (2015) and Pegler (1983), there were type. The fruiting body was white milk three species inside section Rigidi, i.e. L. color. They were gregarious in their polychrous, L. squarrosulus, and L. environment. sajor-caju. Three of them are different Lentinus is classified into from each other, such as only L. sajor- Polyporales. The different hymenophore caju has annulus, L. squarrosulus has a type within Polyporales makes Lentinus slender stem, L. polychrous has a thick unique and as the exception in stem (Seelan et al., 2015; Corner, 1981; Polyporales which has lamellate type of Pegler, 1983). Some studies also reported its cap. Within Lentinus, there are many the comparison of L. squarrosulus and L. species that have been mentioned in other tigrinus that make them for similar publications. Despite Index Fungorum characters of the fruiting body (Dulay et (2020) mentioned 146 species, only 11 al., 2012; Bolhassan et al., 2012; Pegler, species were confirmed that were 1983). Between L. tigrinus and L. available for the sequences in GenBank squarrosulus are confused about making (Seelan et al., 2015). They are L. badius, a difference. A similar surface type of L. bertieri, L. crinitus, L. polychrous, L. them is the scabrous cap surface. But, the sajor-caju, L. scleropus, L. squarrosulus, phylogenetic of them is distinguished by L. striatulus, L. suavissimus, L. swartzii, separated clades. Based on phylogeny and L. tigrinus. Hibbet et al., (1993) (figure 1), between L. squarrosulus and mentioned and described in their study L. tigrinus is certainly placed as a sister that there are two ways Lentinus gills can clade. develop, i.e. descending growth and

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Figure 1. Lentinus phylogenetic tree based on the ITS4/ITS5 region using MEGA X with Maximum Likelihood using Kimura 2 Parameter + Gamma + Invariant Site. Bootstrap (BS)≥60 was shown on the branch

a b

c d e o

f g

Figure 2. Lentinus squarrosulus BO 24427. (a) young fruiting body; (b) mature fruiting body; (c) the stem; (d) the cap; (e) the lamellae; (f) basidiospores; (g) the hymenial hyphae. Scale bars: (a-b) 5 cm; (f) 5 µm; (g) 10 µm

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Morphology of L. squarrosulus BO al., (2015) which used the multigene for 24427 the phylogeny. Lentinus squarrosulus BO 24427 The morphology among young and was Basidiomycotina and within the mature fruiting bodies has a little Polyporales. The morphology of the different type of the cap. The young have young fruiting body (Fig 1a) has a exactly the circle shape of the cap and are circular cap incurved or enrolled inside. enrolled in cap margin, whereas the Then, the mature fruiting body (Fig 1b) mature has a widely circular shape than has a wider cap than the young. the young fruiting body and are not Mushroom cap shapes are central enrolled in cap margin. The mature has a depression (young fruiting body) or decurrent of cap and wavy of the margin. infundibuliform (mature fruiting body). Despite the difference in cap shape, the Mushroom caps are ornamented and the scabrous cap appears between the young entire cap surface and margin. The stem and mature fruiting bodies. Hermawan et is erected from bases (Fig 1c). The young al., (2020b) also found this pattern growth fruiting body has a pattern of universal among the young and mature fruiting veil remnant (scabrous) on the cap bodies. In Hermawan et al., (2020b), the surface (Fig 1d). But, in mature fruit Chlorophyllum mushroom had a different bodies, it may be absent or remain as type of cap among young and mature scales on the cap surface. Mushroom gills fruiting bodies. This normally happens are adnexed (young fruiting body) or when the development of the fruiting decurrent (mature fruiting body) of body is still growing at the time. attachment to the stem, crowded of gill Basidiospores of L. squarrosulus BO spacing (Fig 1e), and smooth (young 24427 are cylindrical and free of fruiting body) or wavy (mature fruiting ornamentation. It agrees with Lentinus' body) of gill margins. Mushroom stems description in Pegler & Young (1983). are equal in stem shape, bulbous of stem This shape was almost finely cylindrical, base, free of the membranous annulus, sometimes with the small appendage or and fragmented bands on the stem tube extension on the basis of surface. The cap is 1,5-3,7 cm (young basidiospore, namely sterigma fruiting body) and 5,3-6,9 cm (mature (Hawksworth et al., 1996). Basidia were fruiting body) in diam. The stem is 2,4- showing a well-developed sub hymenial 6,1 cm in height and 0,7-1,3 in diam. The layer and finely loose woven trama. basidiospores are cylindrical, smooth, Lentinus squarrosulus are popular and 6,6–8,9 × 2,5–3,3 µm (Fig 1f). The as an edible mushroom in central Africa hymenial hyphae is woven and well- (Watling, 1993), likewise L. sajor-caju development (Fig 1g). and L. strigosus (Chin, 1981). Lentinus Lentinus squarrosulus has scabrous has known as white root fungi (Sulistiany on the cap surface. This morphology is & Sudirman, 2015). Therefore, Lentinus the main character to identify the can be saprobic fungi at all time phases. Lentinus fruiting body to L. squarrosulus. Lentinus squarrosulus BO 24427 had Sometimes the morphology makes a been found on rotten wood. This was the difference to identify them specifically. first record for publishing L. squarrosulus Therefore, molecular identification is in Indonesia. Lentinus is not easy to more needed to make them sure of culture into a medium. But some species. The ITS is strong enough and researchers had been successful to culture valuable to identify within Lentinus. Our and domesticate them. De leon et al., phylogeny is a little similar to Seelan et (2013) had been successful in domesticating L. squarrosulus in

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Philippine. Current research, Omar et al., Brundrett., M. C., Bougher, N., Dell, B., (2011), studied the potential of L. Grove, T., & Malajczuk, N. (1996) squarrosulus. The water extract of the Working with Mycorrhizas in mycelium is successful as an antioxidant Forestry and Agriculture. ACIAR ingredient in some functional food and Monograph 32, Canberra. nutraceuticals. Chin, F. H. (1981) Edible and poisonous fungi from the forest of Sarawak. Conclusion Part 1. Sarawak Mus J, 50, 211– Lentinus BO 24427 specimen was 225. identified as L. squarrosulus using ITS Corner, E. J. H. (1981). The Agaric region with 99% bootstrap value on the genera Lentinus, Panus and phylogenetic tree. The Lentinus Pleurotus with particular reference squarrosulus was within the section to Malaysian species. Beihefte zur Rigidi. The morphology of L. Nova Hedwigia, 69, 1-189. squarrosulus BO 24427 central De Leon, A. M., Reyes, R. G., & Cruz, T. depression (young fruiting body) or E. E. D. (2013). Lentinus infundibuliform (mature fruiting body), squarrosulus and Polyporus ornamented as a scabrous cap, and free of grammocephalus: Newly the annulus on the stem. The Domesticated, Wild Edible basidiospores are cylindrical, smooth, Macrofungi from the Philippines. and 6,6–8,9 × 2,5–3,3 µm. This study was Philipp Agric Scientist, 96 (4), 411- the first record of finding L. squarrosulus 418. in Indonesia. Dulay, R. M. R., Cabrera, E. C., Kalaw, S. P., & Reyes, R. G. (2012). References Optimal growth conditions for Binder, M., Hibbett, D. S., Larsson, K. basidiospore germination and H., Larsson, E., Langer, E., & morphogenesis of Philippine wild Langer, G. (2005) The strain of Lentinus tigrinus (Bull.) phylogenetic distribution of Fr. Mycosphere, 3 (6), 926–933. resupinate forms across the major Gulati, A., Atri, N. S., Sharma, A. K., & clades of mushroom-forming fungi Sharma, B. M. (2011). Nutritional (Homobasidiomycetes). Syst studies on five wild Lentinus Biodivers, 3 (2), 113–157. species from North-West India. Binder, M., Justo, A., Riley, R., Salamov, World J Dairy & Food Sci, 6 (2), A., López-Giráldez, F., Sjökvist, 140-145. E., Copeland, A., Foster, B., Sun, Hawksworth, D. L., Kirk, P. M., Sutton, H., Larsson, E., Larsson, K. H., B. C., & Pegler, D. N. (1996). Townsend, J., Grigoriev, I. V., & Ainsworth & Bisby's dictionary of Hibbett, D. S. (2013) Phylogenetic the fungi. 8th ed. Oxford University and phylogenomic overview of the Press Inc, New York. Polyporales. Mycologia, 105 (6), Hermawan, R., Amelya, M. P., & Julia, Z. 1350–1373. R. (2020a). Trichaleurina javanica Bolhassan, M. H., Noorlidah, A., from West Java. J Mikologi Vikineswary, S., Hattori, T., Indonesia, 4 (2), 175-181. Sumaiyah, A., Noraswati, M. N. R., Hermawan, R., Imaningsih, W., & & Musa, M. Y. (2012). Diversity Badruzsaufari. (2020b). and distribution of Polyporales in Mushrooms assumed as peninsular Malaysia. Sains Malays, Ectomycorrhizal Fungi on South 41(2),155–161.

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