Philippine Journal of Science RESEARCH NOTE 150 (S1): 303-308, Special Issue on Biodiversity ISSN 0031 - 7683 Date Received: 30 Sep 2020

Rapid Survey of Anuran Species in - Area and Isolation of their Fungal Symbionts

Arthien Lovell Pelingen1,3*, Camille Andrea Flores2,3, Axel John Briz3, Roland Hipol3, and Celia Austria3

1Department of Biology, School of Science and Engineering Ateneo de Manila University City, National Capital Region 1108 2Institute of Biology University of the Philippines Diliman Quezon City, National Capital Region 1101 Philippines 3Department of Biology, College of Science University of the Philippines Baguio Baguio City, Cordillera Administrative Region 2600 Philippines

Baguio City is one of the fastest-growing centers of urbanization in the Philippines. As part of the Cordillera Mountain Range, it is a biodiversity hotspot that is largely unexplored. This study is a preliminary investigation using visual encounter surveys of anuran species in various localities of the Baguio-Benguet area (BBA). These include (LA), Camp (CJH), and Soroptimist Compound (SC) at the Military Cut Off, all in Baguio; Nangalisan, Tuba (NT), Benguet; and Master’s Garden (MG) at Puguis, La Trinidad, Benguet. We documented three Luzon-endemic species of frogs – namely, Kaloula rigida (Family Microhylidae), Sanguirana luzonensis (Family Ranidae), and Limnonectes macrocephalus (Family Dicroglossidae); one Philippine-endemic species, Kaloula picta (Family Microhylidae); and an invasive species, Rhinella marina (Family Bufonidae). The pathogenic fungi Batrachochytrium dendrobatidis was not detected in this study, but several cosmopolitan fungal species were isolated from the anuran integument – including Penicillium spp., Cladosporium sp., Talaromyces sp., Mucor sp., and Rhodotorula sp. The results of the study highlight the importance of regional surveys of urbanized areas in the country in aiding the conservation and preservation efforts for the remaining wildlife. Anthropogenic factors like logging and conversion of forested areas to commercial spaces, which lead to habitat degradation and catastrophic events, are still the leading threats to this understudied but presumed species-rich area. To date, this is the first update on anuran biodiversity in BBA after Inger’s study in 1954, and the first survey of fungi association with the amphibian integument in the Cordillera Administrative Region. Hence, further taxonomic and conservation research is recommended.

Keywords: biodiversity, conservation, Cordillera, frog, endemic, herpetofauna

*Corresponding Author: [email protected]

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Little is known about the herpetofauna in Baguio and Permission to collect was acquired from the authorities its vicinities. Recent efforts were mainly focused on in each site of interest below (Figure 1). Visual encounter central Cordillera (Diesmos et al. 2005) and in the surveys were conducted in the afternoons and evenings north (Brown et al. 2012). The last published account of during selected days and nights from November 2015 anuran sighting within Baguio was 60 years ago (Inger to February 2016. To avoid cross-contamination, sterile 1954). This study aids in updating species biodiversity procedures were observed as described by Phillott et assessments in the Cordillera that would contribute to the al. (2010), while swabbing of the anurans to determine baseline information for conservation efforts in the BBA. the fungal community followed that of Brem et al. Also, it promotes awareness and initiative to conduct (2007). Consequently, all anurans were safely released herpetofaunal studies in one of the more poorly understood immediately after swabbing and photo-documentation. areas in Northern Luzon concerning amphibian and reptilian fauna (Brown et al. 2012). Culture-dependent methods of fungal isolation were performed following the protocols described by Longcore In addition to a poor understanding of herpetofauna in (2000), Piotrowski et al. (2004), and Brem et al. (2007). the area, their role as host to microbes is also largely In brief, each anuran integument was swabbed using unknown. For instance, the pathogenic chytrid fungus B. sterile cotton tips then inoculated onto potato dextrose dendrobatidis greatly affects frog populations on a global agar (PDA) plates supplemented with chloramphenicol. scale (Wake and Vredenburg 2008). It was confirmed that After incubation, fungal colonies were subcultured to B. dendrobatidis is already present on Luzon (Smith et PDA slants and potato dextrose broth for morphological al. 2019). The possible occurrence of this pathogen, in characterization and DNA extraction (Liu et al. 2000). addition to anthropogenic practices leading to habitat DNA extracts were subjected to amplification targeting destruction especially in urban areas (e.g. logging and the ITS region using ITS1 and ITS4 primers. Amplicons conversion of forest patches into commercial spaces), were sent to First BASE Laboratories (http://www. make the local anuran species vulnerable to the threat of firstbaselab.com/) for sequencing. MEGA 6 was used in extinction (Wake and Vredenburg 2008). processing the sequences and initial fungal identification

Figure 1. Sampling sites in the BBA.

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(Tamura et al. 2013). The highest percentage identity FAMILY BUFONIDAE among BLAST (https://blast.ncbi.nlm.nih.gov/) hits were assumed putative genus identities later confirmed with Rhinella marina (Linnaeus, 1758) their morphology and colony characters. Common name: cane toad, marine toad We recorded three Luzon-endemic species, one Philippine- Rhinella marina (Figure 2A) was found at only two sites. endemic species, and one invasive species (Table 1), In NT, numerous individuals were seen in flooded rice which were also observed in other parts of Cordillera paddies. Several individuals were also seen in LA foraging (Brown et al. 2012). on the runway.

Table 1. Summary of anuran species encountered from all five collection sites. Anurans CJH SC MG LA NT Rhinella marinaa   Limnonectes macrocephalusb   Kaloula rigidab  Kaloula picta (first locality record)c  Sanguirana luzonensis (first locality record)b  aInvasive species; bLuzon-endemic; cPhilippine-endemic

Figure 2. Anuran species: (A) Rhinella marina, (B) Limnonectes macrocephalus, (C) Kaloula rigida, (D) Kaloula picta, and (E) Sanguirana luzonensis.

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Figure 3. Fungi from the anuran integument: (A–C) Penicillium spp., (D) Cladosporium sp., (E) Talaromyces sp., and (F) Mucor sp.

FAMILY DICROGLOSSIDAE Kaloula rigida (Figure 2C) was originally documented in Baguio City as early as 1922 (Brown et al. 2013). In this Limnonectes macrocephalus (Inger, 1954) study, this frog was recorded in SC, which is a partially Common name: giant Philippine frog disturbed habitat due to the presence of an establishment A brown morph of Limnonectes macrocephalus (Figure building with a man-made garden. 2B) was observed in CJH while a black morph was Kaloula picta (Duméril and Bibron, 1841) observed in MG. Common name: painted narrrowmouth frog Kaloula picta (Figure 2D) was seen in LA foraging on the runway like R. marina. FAMILY MICROHYLIDAE Kaloula rigida (Taylor, 1922) Common name: Luzon narrow-mouthed frog

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FAMILY RANIDAE Swabs. Retrieved on 02 Sep 2015 from http://www. amphibians.org Sanguirana luzonensis (Boulenger, 1896) Common name: Luzon frog BROWN RM, OLIVEROS CH, SILER CD, FER- NANDEZ JB, WELTON LJ, BUENAVENTE PAC, Sanguirana luzonensis (Figure 2E) was observed inside DIESMOS MLD, DIESMOS AC. 2012. Amphibians a water tank near a stream behind the administration and Reptiles, Northern Luzon Island (Philippines), building of LA. VII: Herpetofauna of Ilocos Norte Province, Northern From the swabbed frogs, several genera of fungal Cordillera Mountain Range. Check List 8: 469–490. symbionts were isolated, mostly ascomycetes. The isolates BROWN RM, SILER CD, OLIVEROS CH, WELTON LJ, were identified at the genus level – namely, Penicillium ROCK A, SWAB J, VAN WEERD M, VAN BEIJNEN spp. (96-100%), Cladosporium sp. (90%), Talaromyces J, JOSE E, RODRIGUEZ D, JOSE E, DIESMOS AC. sp. (100%), Mucor sp. (90%), and Rhodotorula sp. (94%) 2013. The amphibians and reptiles of Luzon Island, (Figure 3). Though the association of these fungi and Philippines, VIII: the herpetofauna of Cagayan and anuran integument are still not established, most were Isabela Provinces, northern Sierra Madre Mountain cosmopolitan species and are generally present in the Range. ZooKeys 266: 1–120. environment such as soil, air, and water, while some are endophytic in nature (Lanzafame 2001; Frisvad and DIESMOS AC, BROWN RM, GEE GVA. 2005. Pre- Samson 2004; Stewart and Munday 2005; Zalar et al. liminary report on the amphibians and reptiles of 2007; Yilmaz et al. 2012). Balbalasang-Balbalan National Park, Luzon Island, Philippines. Sylvatrop: The Technical Journal of Philip- Regional surveys are as important as major biogeographic pine Ecosystems and Natural Resources 13(1): 63–80. surveys, particularly in highly threatened urbanized areas FRISVAD JC, SAMSON RA. 2004. Polyphasic taxonomy where species richness is still understudied. In this study, of Penicillium subgenus Penicillium: a guide to identi- we have established that several Luzon- and Philippine- fication of food and air-borne terverticillate Penicillia endemic species thrive in vulnerable areas and the threat and their mycotoxins. Stud Mycol 49: 1–173. of B. dendrobatidis was not observed locally. However, anthropogenic factors that lead to habitat degradation INGER RF. 1954. Systematics and zoogeography of continue as threats to the survival of these endemic frog Philippine Amphibia. Fieldiana: Zoology 33: 182–531. species, and biodiversity conservation remain as urgent LANZAFAME M. 2001. Rhodotorula glutinis related imperatives. meningitis. Journal of Clinical Microbiology 39(1): 410–411. LIU D, COLOE S, BAIRD R, PEDERSON J. 2000. ACKNOWLEDGMENTS Rapid mini-preparation of fungal DNA for PCR. J Clin Microbiol 38: 471. We would like to extend our sincerest gratitude to Ms. Hilda Wayas and Ms. Jay-Ann Tallad for the laboratory LONGCORE J. 2000. Culture technics for amphib- assistance; to the MERIT Security Agency (from LA), ian chytrids: Recognizing, isolating, and culturing CJHMC Forest Rangers (from CJH), Mr. Pat Acosta and Batrachochytrium dendrobatidis from amphibians. Mr. Rodel Buan (from MG), as well as Mr. and Mrs. Ariel In: Proceedings of Getting the Jump! On Amphibian Emock (from NT) for their help during our fieldwork. Diseases Conference/Workshop; 2000 August 26–30; Great appreciation is also extended to the anonymous Cairns, Australia: James Cook University. p. 52–54. reviewers for their helpful comments in the manuscript. PHILLOTT AD, SPEARE R, HINES HB, SKERRATT The undergraduate studies of the first and second authors LF, MEYER E, MCDONALD KR, CASHINS SD, were supported by the Department of Science and MENDEZ D, BERGER L. 2010. Minimising exposure Technology–Science Education Institute (DOST-SEI) of amphibians to pathogens during field studies. Dis through the RA 7687 Scholarship Program. Aquat Organ 92(2–3): 175–185. PIOTROWSKI JS, ANNIS SL, LONGCORE J. 2004. Physiology of Batrachochytrium dendrobatidis, a chy- trid pathogen of amphibians. Mycologia 96(1): 9–15. REFERENCES SMITH SN, WATTERS JL, ELLSWORTH ED, DAVIS BREM F, MENDELSON IIII JR, LIPS KR. 2007. Field- DR, SILER CD. 2019. Assessment of Batrachochy- Sampling Protocol for Batrachochytrium dendrobatidis trium dendrobatidis and ranavirus among wild am- from Living Amphibians, Using Alcohol Preserved

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phibians from four Philippine islands. Herpetol Rev 50(4): 729–734. STEWART NJ, MUNDAY BL. 2005. Possible differ- ences in pathogenicity between cane toad-, frog- and platypus-derived isolates of Mucor amphibiorum, and a platypus-derived isolate of Mucor circinelloides. Med Mycol 43(2): 127–132. TAMURA K, STECHER G, PETERSON D, FILIPSKI A, KUMAR S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30: 2725–2729. WAKE DB, VREDENBURG V. 2008. Are We in the Midst of the Sixth Mass Extinction? A View from the World of Amphibians. Proceedings of the National Academy of Sciences of the of America 105: 11466–11473. YILMAZ N, HOUBRAKEN J, HOEKSTRA ES, FRISVAD JC, VISAGIE CM, SAMSON RA. 2012. Delimitation and characterisation of Talaromyces pur- purogenus and related species. Persoonia: Molecular Phylogeny and Evolution of Fungi 29: 39–54. ZALAR P, DE HOOG GS, SCHROERS HJ, CROUS PW, GROENEWALD JZ, GUNDE-CIMERMAN N. 2007. Phylogeny and ecology of the ubiquitous saprobe Cladosporium sphaerospermum, with descriptions of seven new species from hypersaline environments. Stud Mycol 58: 157–183.

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