1. Rudolf V Kuhn.cdr BIOTROPIA Vol. 20 No. 1, 2013: 1 - 9 DIVERSITY OF PLASMODIAL MYXOMYCETES FROM ANDA ISLAND, PANGASINAN, PHILIPPINES RUDOLF V. KUHN , ANTON OLIVER M. JAVIER , COLEEN P. RODILLAS , CHRISTIAN M. PARRA , LIWAYWAY HIYAS M. CORPUZ , ANTHONY T. BUAYA , and THOMAS EDISON E. DELA CRUZ * Received 20 December 2011/Accepted 10 January 2013 The unique life cycle and fascinating fruiting bodies of myxomycetes make them ideal model organisms for the study of cellular differentiation and ecological patterns. Our research study then focuses on the diversity and abundance of myxomycetes found in Anda Island, Pangasinan in Northern Philippines. A total of 180 moist chambers were prepared from ground leaf litter and twigs collected from a 15 m quadrat within the study site. Twenty four species of myxomycetes belonging to 11 genera were collected and identified from the moist chambers: (2), (1) (1) (2), (1), (2) (1) (1), (1) (11), and (1) Of all moist chambers, 55% yielded myxomycetes. Ground leaf litter (29%) yielded more myxomycetes than twigs (26%). Assessment of species diversity (H =1.15), richness (H =5.33) and evenness (E=0.56) showed rich assemblage of myxomycetes. Among the collected species, one for each of the genera , , and were recorded to be abundant. Interestingly, three species of myxomycetes are new records for the Philippines: and . This is the first report of myxomycetes in Anda Island, Pangasinan, Philippines. myxomycetes, species abundance, diversity, biogeography, Philippines 1 1 1 1 1 2 1,2 2 Department of Biological Sciences, College of Science and Fungal Biodiversity and Systematics Group, Research Center for the Natural and Applied Sciences University of Santo Tomas España 1015 Manila, Philippines Arcyria Collaria , Comatricha , Craterium Diachea Diderma , Didymium , Elaeomyxa Perichaena , Physarum Stemonitis . Arcyria Craterium Diderma, Physarum, Craterium microcarpum, Physarum decipiens Elaeomyxa miyazakiensis 1 2 ABSTRACT INTRODUCTION S G Key words: The Philippines is a tropical archipelago of 7107 islands. The geographic isolation of many of these islands resulted in various unique flora and fauna. It has been estimated that more than 6 000 species of animals and plants are endemic to the country (Myers . 2000). Therefore, it is not surprising that new species of plants and animals are still being discovered recently in the country, e.g. forest mice ( et al Apomys * Corresponding author : tedelacruz@mnl.ust.edu.ph 1 aurorae A. banahao A. brownorum A. magnus A minganensis A. sierrae A. zambalensis) et al. (Varanus bitatawa) et al. (Nepenthes attenboroughii) et al. (Drosera ultramafica) et al. Craterium retisporum et al. et al et al. Hemitrichia serpula, Perichaena depressa, Physarella oblonga, Stemonitis fusca Arcyria Cribraria, Lycogala Stemonitis et al. et al. Arcyria globosa Comatricha robusta, Craterium atrolucens, Lamproderma cacographicum, Oligonema schweinitzii Perichaena microspora , , , , , , and (Heaney 2011), monitor lizard (Welton 2010), giant pitcher plant (Robinson 2009), sundew plant (Fleischmann 2011) including those belonging to the myxomycetes, e.g. (Moreno 2009). However, studies on island myxomycetes are relatively few in spite of their role in wood decay and nutrient cycling in temperate forests and in bio-accumulation of heavy metals (Zhulidov . 2002; Takahashi 2004). Rojas and Stephenson (2008) surveyed different habitat elevations and substrates types in Cocos Island, Costa Rica and reported 41 species belonging to 19 genera. Beltran (2010) also evaluated the species diversity of myxomycetes between arid and semi-arid zones in the Canary Islands in Spain and reported 63 species belonging to 21 genera. The study also highlighted the similarities between slime molds collected from the arid and semi-arid zones in the Canary Islands to the desserts of Central America. In the Philippines, and species of , and were reported from the islands of Palawan and Palaui, Cagayan (Reynolds 1981; Quimio 2002). Dela Cruz (2011) also reported 30 species of myxomycetes from Hundred Islands National Park, Pangasinan. Macabago (2012) identified 45 species and 13 genera from Lubang Island, Occidental Mindoro. Interestingly, this geographically isolated island yielded six new records for the Philippines: , and . In this research study, we reported 24 species including three new records of myxomycetes from specimens collected in Anda Island, Pangasinan, Philippines. This is the first report of myxomycetes in Anda Island, Pangasinan in Northern Philippines. Anda Island (16°17'00.16”N, 119°58'00.08”E; land area= 83.80 sq.km) is located near the town of Bolinao in the province of Pangasinan, Northern Philippines. Climate data indicate an average monthly rainfall range of 6.1 mm in February to 608.6 mm in August. The island has an average minimum and maximum temperature of 23.3 °C and 32.4 °C, respectively, and average monthly humidity between 71 - 85%. It has a type I climate season, i.e. wet from June to November and dry from December to May. The study site is characterized by a tropical secondary forest, with patches of rice fields and established residential houses. A small patch of this secondary forest along the local highway and near a rice field was used as the collection site. The island is also inhabited by man and may be useful in looking at the effects of human civilization on myxomycete assemblages. MATERIALS AND METHODS Study site 2 BIOTROPIA Vol. 20 No. 1, 2013 Collection of substrates and preparation of moist chamber set-ups and herbaria Characterization and identification of plasmodial myxomycetes Moist chamber productivity and ecological analysis Ground leaf litter (30) and twigs (30) were collected in brown paper bags within a 15 m quadrat. Prior to the preparation of moist chambers, the collected substrates were allowed to air-dry for few days. A total of 180 moist chambers were then set-up following the protocol of Stephenson and Stempen (1994). The moist chambers were placed inside wooden cabinets, not directly exposed to sunlight, and incubated at room temperature for up to 8 weeks. All moist chambers were observed regularly (at least twice a week) for the presence of plasmodia and/or fruiting bodies. The number of moist chambers with plasmodia and/or fruiting bodies was recorded and used in ecological analysis. Fruiting bodies with their respective substrate were removed from the moist chamber set-up, allowed to air dry and glued on previously prepared herbarium boxes. Herbarium boxes were then labeled with the collection number, substrate, collection sites and dates, the species name and the names of the collector. All herbaria were deposited at the Pure and Applied Microbiology Laboratory, Research Center for the Natural and Applied Sciences, University of Santo Tomas. To identify the collected myxomycetes, the specimens for each species were described based on their fruiting body descriptions and spore morphology under a dissecting and a compound light microscope. Identification was done following comparison of these morphological characters with published literature (Stephenson & Stempen 1994; Keller & Braun 1999), web-based identification keys (http://slimemold.uark.edu/) and an electronic, computer-based identification key, (Mitchell 2008). To check for the current valid names of the identified myxomycetes, an online nomenclatural information system (http://nomen. eumycetozoa.com) was used. The productivity of the moist chambers (MC) was computed by determining the percent yield for the collection site and each of the substrate types. Plasmodial/or fruiting body growth on a moist chamber was counted here as positive for myxomycetes and was recorded as one positive collection. Then, the total positive collection was divided by the total number of moist chamber prepared. Thus, the percentage yield (PY) was computed as follows: Percent Yield (PY) = (1) total number of MC prepared Then, the relative abundance (RA) was also determined for each of the collected species of myxomycetes. Initially, the number of moist chambers positive for a species of myxomycetes was counted and divided by the total number of collections. 2 SynKey number of MC positive for myxomycetes 3 Diversity of Plasmodial Myxomycetes from Anda Island Rudolf V. Kuhn– et al. The relative abundance was computed as follows: Relative Abundance (RA) = (2) total number of myxomycete collection Abundance Indices (AI) were then assigned to all of the species represented among the collections. Initially, the percentage of a particular species among the total number of collections, i.e. the Relative Abundance, was determined. Then, a “breaking point” was assigned based on these percentage values (S. L. Stephenson, pers. comm., 04 January 2009). Each species was then categorized as: (1) abundant (A) if their relative abundance (RA) is ≥10% of the total collections, (2) common (C) if RA is ≥5% but <10% of the total collections, (3) occasionally occurring (O) if RA is ≥3 but <5 of the total collections, and (4) rare (R) if the myxomycetes had an RA of <3% of the total collections. Finally, the myxomycete diversity of the study site was also calculated using different diversity indices as described by Dagamac . (2012). These indices were computed based on RA and as follows: Shannon Index Diversity (3) where p = is the proportional abundance of the th species Gleason Index of Species Richness (4) where N = the total number of species N = the total number of individuals in th species Pielou's Index of Species Evenness / (5) where H = Shannon Index of Diversity H = the maximum value of Hs Of 180 moist chambers set-up from ground leaf litter and decaying woody twigs collected from Anda Island, 55% yielded myxomycetes (Fig. 1A). Fruiting bodies were recorded more in moist chambers than plasmodia. Between the two collected substrates, ground leaf litter (29%) yielded more myxomycetes than twigs (26%) (Fig. 1B). Schnittler (2002) also found a higher occurrence of myxomycetes on the ground leaf litter, though Macabago (2010) recorded a higher yield in aerial than ground leaf litter. It was previously reported that myxomycetes associated with woody twigs are not necessarily same as those associated with ground litter in terms of their occurrence and abundance (Stephenson 2008). Stephenson (1989) implicated that the myxomycetes assemblage in ground leaf litter was mediated by factors that is favorable for plasmodial or amoeba development. These factors included acidic pH of the decaying leaf litter, soil nutrients, moist and the available food microorganisms, bacteria, yeast and algae. As shown in this study, the high myxomycetes yield recorded in ground leaf litter as compared to twigs indicated that ground leaf litter is a favorable substrate for myxomycetes. number of collections for a particular species of myxomycetes et al et al. et al. et al. e.g. (H ) = -∑ (p ln p ) (H ) = N -1/ln N E = H H Percent yield S i i i G p i S max i p i S max i i RESULTS AND DISCUSSION 4 BIOTROPIA Vol. 20 No. 1, 2013 5 B A Figure 1. Percent yield of myxomycetes in moist chambers (A) and between the two collected substrates (B) D B CA E F Figure 2. Representative species of myxomycetes recorded in Anda Island, Pangasinan: (A) (B) , (C) and (F) Craterium retisporum, Diachea leucopodia Perichaena pedata, (D) Elaeomyxa miyazakiensis, (E) Perichaena depressa Physarum bivalve Diversity of Plasmodial Myxomycetes from Anda Island Rudolf V. Kuhn– et al. 6 Species Occurrence, Abundance and Diversity Arcyria Collaria , Comatricha , Craterium Diachea Diderma , Didymium , Elaeomyxa Perichaena , Physarum Stemonitis . Physarum Phy. bivalve, Phy. bogoriense, Phy. cinereum, Phy. compressum, Phy. decipiens Phy. echinosporum Arcyria cinerea, Collaria arcyrionema Diachea leucopodia, Diderma hemisphaericum, Didymium nigripes, Elaeomyxa miyazakiensis, Per. depressa, Comatricha Stemonitis fusca Craterium Cra. retisporum Cra. microcarpum, Craterium microcarpum, Physarum decipiens Elaeomyxa miyazakiensis A. cinerea, Cr. microcarpum, Diderma hemisphaericum, Phy. echinosporum Arcyria Physarum Stemonitis et al. et al et al et al. Collaria arcyrionema Diachea leucopodia, Physarum decipiens Stemonitis fusca Comatricha Craterium retisporum, Diderma ., Didymium nigripes, Elaeomyxa miyazakiensis, Physarum bivalve, Phy. bogoriense, Phy. cinereum, Phys. compressum, Physarum Physarum Physarum Physarum Diderma Comatricha et al. Arcyria, Physarum, Diderma Stemonitis et al. Craterium Craterium leucocephalum Craterium et al , A total of twenty four species of myxomycetes were collected from Anda Island, Pangasinan (Table 1). The identified species belong to the following genera: (2), (1) (1) (2), (1), (2) (1) (1), (1) (11), and (1) Of the collected species, six were identified as and . , sp. and were also reported in Anda Island. Two species of , i.e. and were also noted among collected substrates. Interestingly, three species of myxomycetes are new records for the Philippines: and . These species have been previously reported in other countries but not in the Philippines until now. Of all collected myxomycetes, and were the most abundant (Table 1). Species of , and were also reported to be widely abundant in temperate and tropical forests, and in island habitats occurring in ground leaf litter or parts of living trees such as twigs and old bark (Schnittler 2002; Stephenson . 2004; Everhart & Keller 2008; Stephenson . 2008; Tran 2008; Adamonyte & Kastanje 2011). , and were recorded as common in this study, while most of the species were recorded as rarely occurring (Table 1). sp.1, sp sp.1, sp.2, sp.8, sp.9, and two unidentified myxomycetes were all recorded as rare. Similarly, species of and has been found to be abundant in temperate than in tropical forests (Stephenson 1989; Novozhilov 2000). The genera and are also abundantly diverse and distributed in island habitats (Beltran 2004; Rojas & Stephenson 2008). Rojas and Stephenson in 2008 described the genus as rare in Cocos Island, Costa Rica; however, Adamonyte and Kastanje in 2011 abundantly recorded in the island of Saaremaa, Estonia. Differences in climatic conditions in these two islands play a crucial part in the abundance and distribution of species. This may also be a factor for the differences in myxomycete distribution in Anda Island. Though Anda Island is inhabited by man, the influence of anthropogenic activities to myxomycete distribution was not correlated, and therefore, is recommended for future studies. In this study, the computed diversity indices of myxomycetes in Anda Island, Pangasinan showed high value for species diversity (H =1.15), species richness (H =5.33) and species evenness (E=0.56). Similarly, high species diversity was also recorded in Lubang Island, Occidental Mindoro (Macabago . 2012). However it was observed that geographic isolation does not greatly influence species composition, and endemism seems not to occur in for myxomycetes since species recorded in an island habitat also occur in mainland (Eliasson 1991; Rojas & S G BIOTROPIA Vol. 20 No. 1, 2013 7 Stephenson 2008). But the wide range of diverse habitats in island ecosystems provides diverse microhabitats for myxomycetes (Rojas & Stephenson 2008). Thus, the diverse assemblage of myxomycetes recorded in this study further supports other studies conducted in island ecosystems. A total of 24 species of myxomycetes belonging to 11 genera were collected and identified in Anda Island, Pangasinan Philippines: ( var. ) , , and CONCLUSIONS Arcyria cinerea A. cinerea digitata , Collaria arcyrionema, Craterium microcarpum, Cra. retisporum Diachea leucopodia, Diderma hemisphaericum Didymium nigripes, Elaeomyxa miyazakiensis, Perichaena depressa, Physarum bivalve, Phy. bogoriense, Phy. cinereum, Phy. compressum, Phy. decipiens, Phy. echinosporum, Table 1. Species abundance of myxomycetes collected from Anda Island, Pangasinan Taxa % Abundance Arcyria cinerea (A. cinerea var. digitata) A Arcyria sp. 1 O Collaria arcyrionema C Comatricha sp. 1 R Craterium microcarpum A Craterium retisporum R Diachea leucopodia C Diderma hemisphaericum A Diderma sp. R Didymium nigripes R Elaeomyxa miyazakiensis R Perichaena depressa O Physarum bivalve R Physarum bogoriense R Physarum cinereum R Physarum compressum R Physarum decipiens C Physarum echinosporum A Physarum sp. 1 R Physarum sp. 2 R Physarum sp. 3 O Physarum sp. 8 R Physarum sp. 9 R Stemonitis fusca C Unidentified Myxo 6 R Unidentified Myxo 7 R Note : Abundant Indices, where Abundant (A) = ≥10% of the total collections, Common (C) = ≥5% but <10% of the total collections, Occasional (O) = ≥3 but <5 of the total collections, and Rare (R) = <3% of the total collections a Diversity of Plasmodial Myxomycetes from Anda Island Rudolf V. Kuhn– et al. 8 BIOTROPIA Vol. 20 No. 1, 2013 Stemonitis fusca. Arcyria Comatricha Diderma Physarum Craterium microcarpum, Physarum decipiens, Elaeomyxa miyazakiensis, Physarum Arcyria Craterium Diderma One species each of , , and five species of were identified up to the genus level only. Three species, i.e. and are new records for the Philippines. Higher productivity was also recorded in the study as well as in ground leaf litter than twigs. Eleven species of and one species for each genus , , and were recorded abundantly. The authors would like to thank the Research Center for the Natural and Applied Sciences, University of Santo Tomas for the research grant, Prof. Dr. Steven L. Stephenson, University of Arkansas, USA for his assistance in the identification of our species, and Nikki Heherson A. Dagamac and Edward E. dela Cruz for their assistance in collecting our substrates. ACKNOWLEDGMENTS REFERENCES Adamonyte G, Kastanje V. 2011. Myxomycetes of the island of Saaremaa, Estonia. Folia Cryptog Estonica Fasc 48:1-4. 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