TAXONOMY AND SPECIES COMPOSITION OF EPIPHYTIC ALGAE IN STA. CRUZ RIVER, LAGUNA (PHILIPPINES)

Algal epiphytes attached on three dominant aquatic macrophytes (Pistia striatotes, Eichhornia crassipes and Ipomoea aquatic) were investigated as part of a preliminary study on the ecological assessment of Sta. Cruz River, which has been used primarily for agriculture and fishery. In total, 22 algal taxa were identified on the studied macrophytes: 8 Chlorophyta, 4 Cyanobacteria, 7 Bacillariophyta, and 3 Euglenophyta. The taxa in this paper represent 8 classes, 16 orders, 20 families, 22 genera and 22 species based on the latest algal taxonomical nomenclature. Among these taxa, the existence of a rare filamentous cyanobacteria, Planktothrix compressa (Utermohl) Anagnostidis et Komarek is described for the first time in the Philippines. A species of diatom is also reported in this study for the first time based on recent algal taxonomic nomenclature and this is Ulnaria ulna (Nitzsch) Compere that is based on the former name of Synedra ulna (Nitzsch) Ehrenberg. These taxonomic records proved that aquatic macrophytes represents a unique habitat for novel and diverse types of algal flora, which demands further investigations in the country.


INTRODUCTION
Macrophytes are aquatic and amphibious plants found in the littoral zones of running waters (eg. rivers, streams, and the like) and shallow lakes contributing largely to the autochthonous carbon pool and oxygen budget of the aquatic ecosystem (Janauer & Dokulil, 2006). These plants have a significant function in water biocenoses, hydrology and sediment dynamics, biochemical cycles, structuring and altering the physico-chemical features (of an aquatic ecosystem) by photosynthesis, mineralization, and decomposition (Jeppesen et al., 2002;Joniak et al., 2007;Špoljar et al., 2012). Macrophytes have diverse species of microalgae attached to submerged plant parts which have been shown to be an important productive component of the aquatic ecosystem (Sheldon & Boylen, 1975). Algal epiphytes form a matrix system of microalgae and cyanobacteria attached to submerged aquatic macrophytes. These algae are regarded as primary species in different lotic water systems and are involved in maintaining ecological balance among the different groups of macrophytes and aquatic organisms (Hassan, et al., 2014;Fawzy, 2016). It is widely recognized that these organisms supply food to invertebrates, fish, and in other aquatic fauna in the littoral zone and contributes an estimated of 0.2% to 41% of the total primary production in an aquatic ecosystem (Laugaste & Ruemamen, 2005;Effiong & Inyang, 2015). Several observational and experimental studies on ecological status of bodies of water showed an increase in the population and diversity of algal epiphytes in response to nutrient loading and pollution (Gil et al., 2006;Peterson et al., 2007;Fawzy, 2016). This led to the consideration of these organisms as excellent bioindicators of environmental and water quality alterations because of its sensitivity to external sources of pollution (Lowe, 1996;Fawzy, 2016). Species of algal epiphytes associated with organically polluted enriched waters include Euglena, Phacus, Nitzschia, Chlorella, Scenedesmus, Chlamydomonas, Cladophora, and Closterium (Effiong & Inyang, 2015). The biological interconnection and relationship between algal epiphytes and host aquatic macrophyte in the aquatic environment are still limited and incompletely studied (Wetzel, 1996). Several reports suggested that biotic and abiotic factors such as physico-chemical characteristics of submerged parts (stem, leaves and roots) of macrophytes, availability of substrates, temperature, light intensity and grazing affects the diversity and prevalence of epiphytic algal community (Gil et al., 2006;Fawzy, 2016). Aquatic macrophytes are sometimes detrimental for algal epiphytes since they are capable of monopolizing light and assimilate nutrients within the vertical expanse of the water column, inhibiting algal epiphytes as well as other submerged macrophytes from acquiring enough resources for growth and survival (Schramm & Jirka, 1989). Moreover, morphology of several macrophyte species, specifically the length of stem and leaves, may limit the growth and proliferation of algal epiphytes by releasing inhibitory substances (allelochemicals) (Tunca et al., 2014). Thus, development of algal epiphyton community as well as its distribution and abundance depend largely on macrophyte host species. On the other hand, in situations where there is an evident increased in algal epiphyte population, it is observed that there is a reduction in the total macrophyte biomass density due to restriction in nutrient diffusion from the water to host plant (Fawzy, 2016). In the Philippines, taxonomic studies of algal epiphytes associated to different macrophytes were done only to those plant species inhabiting Laguna de Bay (Rañola, et al., 1990; Arguelles, 2019 a,b,c; Arguelles, 2020a). To date, a total of 82 taxa of algal epiphytes associated with four dominant aquatic macrophytes (Hydrilla verticilata, Nymphaea pubescens, Eichhornia crassipes and Ipomoea aquatic) found in Laguna de Bay were documented in in the Philippines. These taxonomic surveys reported the occurrence of five rare microalgaenamely, Cryptoglena skujae Marin and Melkonian, Pseudanabaena minima (G.S. An) Anagnostidis, Synechococcus nidulans (Pringsheim) Komárek, Chroococcus schizodermaticus West and Franceia amphitricha (Lagerheim) Hegewaldfor the first time in the Philippines. Also, some of the microalgal genera documented in these taxonomic studies (eg. Cryptoglena, Tetradesmus, Nitzschia, Euglena, Chroococcus, Oscillatoria, Phacus and Chlorella) are usually associated with organically polluted enriched waters (Arguelles, 2019 a,b,c; Arguelles, 2020a). These algal epiphytes are regarded as good indicators of environmental changes and water quality due to their sensitivity to external sources of fertilization which can be use in the assessment of ecological status of Laguna de Bay (Arguelles, 2020a).
The taxonomic records reported in these studies provided baseline information regarding distribution and diversity of Philippine epiphytic algae from aquatic macrophytes found in local freshwater habitat. Diversity and ecological studies of epiphytic microalgae of different aquatic macrophytes found in running waters (such as rivers and streams) in the Philippines remain poorly understood. To date, no documented taxonomic survey was conducted on these group of microalgae. Thus, additional taxonomic survey of algal epiphytes of different aquatic macrophytes found in these aquatic ecosystems are needed to deepen our understanding of the diversity and Algal epiphytes attached on three dominant aquatic macrophytes (Pistia striatotes, Eichhornia crassipes and Ipomoea aquatic) were investigated as part of a preliminary study on the ecological assessment of Sta. Cruz River, which has been used primarily for agriculture and fishery. In total, 22 algal taxa were identified on the studied macrophytes: 8 Chlorophyta, 4 Cyanobacteria, 7 Bacillariophyta, and 3 Euglenophyta. The taxa in this paper represent 8 classes, 16 orders, 20 families, 22 genera and 22 species based on the latest algal taxonomical nomenclature. Among these taxa, the existence of a rare filamentous cyanobacteria, Planktothrix compressa (Utermöhl) Anagnostidis et Komárek is described for the first time in the Philippines. A species of diatom is also reported in this study for the first time based on recent algal taxonomic nomenclature and this is Ulnaria ulna (Nitzsch) Compère that is based on the former name of Synedra ulna (Nitzsch) Ehrenberg. These taxonomic records proved that aquatic macrophytes represents a unique habitat for novel and diverse types of algal flora, which demands further investigations in the country. ecological roles of these microorganisms. The goal of this investigation is to account the species diversity of algal epiphytes associated to different aquatic macrophytes observed in Sta. Cruz River, one of the 21 major tributary rivers of Laguna de Bay. Also, morphotaxonomic description of each algal taxa were documented together with a brief description of the sampling sites and natural environment of its existence.

Study Site
Sta. Cruz River is considered as one of the main branch rivers of Laguna de Bay. It lies at 14.3001 o N and 121.4068 o E, specifically within the municipalities of Liliw, Magdalena, Nagcarlan, Pagsanjan, Pila, and Sta. Cruz ( Figure 1). The river is measured to be approximately 14.48 km in length, flowing towards Laguna de Bay. Currently, the principal use of this river is for fishery and agriculture (with coconut and rice production, livestock, poultry, and piggery raising as main activities). This body of water is known to be a receptacle for floodwaters coming from its small tributary rivers such as Maimpis river, Liliw river, San Diego river, Tipacan river, and Talahebeng river (Madamba et al., 1992).

Sampling and Specimen Preparation of Epiphytic Algae
A single preliminary collection of algal epiphytes from submerged aquatic macrophytes was done from the littoral zone of Sta. Cruz River. The plant parts were put into sterile autoclavable plastics filled with water for laboratory examination. A total of 20 aquatic macrophyte samples (each for Pistia striatotes, Eichhornia crassipes and Ipomoea aquatica) were collected and analyzed throughout the study period. Immediately after collection, these samples were washed several times with sterile distilled water. The algal epiphytes from submerged leaves, stems and roots on the collected aquatic macrophytes were set apart from the plant by gently scraping the attached algae on the plant material (Zimba & Hopson, 1997;Arguelles, 2021a,b,c). The collected scraped algal epiphyte was carefully mixed, and a portion of 50 mL was kept for taxonomic enumeration. The mixed algal epiphyte sample was transferred into a sterile beaker and left overnight to allow settling of the scraped algal samples. An aliquot of 45 mL of the liquid specimen was removed in the beaker after the settling period. The residual 5 mL of the collected scraped material was transferred into a sterile drum vial for taxonomic enumeration of algal epiphytes and were preserved using 2-3 drops of Lugol's iodine from the prepared specimens (Utermöhl, 1958

RESULTS AND DISCUSSION
Epiphytic algal flora of three dominant aquatic macrophyte (P. stratiotes, E. crassipes and I. aquatica) observed in Sta. Cruz River consisted of 22 taxa. Of these, 8 belong to the Chlorophyta, 4 to the Cyanobacteria, 7 to the Bacillariophyta, and 3 to the Euglenophyta divisions. Chlorophyta was dominant and comprised 36.36% of all recorded taxa. Bacillariophyta, Cyanobacteria and Euglenophyta represented 31.81%, 18.18% and 13.63% of all recorded taxa, respectively. The taxonomic list of the algal epiphytes determined in Sta. Cruz River is given in Table 1. All the taxa are systematically enumerated with morphotaxonomic descriptions along with a simple habitat information where the alga was observed. Illustrative photomicrographs of some of the algal species are given in Plates I-IV. Currently accepted algal taxonomic names were used based on the recent nomenclatural indices for algae of the International Association of Plant Taxonomy (IAPT) and Round et al. (1990). Cells spherical or irregularly spherical usually occurring as single or in cluster of 2-4 cells, bluish green to light green in color; colonies enclosed in an amorphous, colorless, homogenous mucilage diffluent at the margin; 5.0-7.0 μm in diameter with sheath and 3.0-5.0 μm in diameter without sheath; protoplast is smooth or slightly granulated.

Dichotomous Key
Found existing as a bluish green or light green layer associated on stems and roots of water lettuce and water hyacinth together with other green microalgae and filamentous cyanobacteria. Found existing as a greenish to blue green layer on stem and leaves of water spinach submerged in water together with other green algae and cyanobacteria. Found existing as a brownish layer on leaves and stem of water hyacinth and water spinach submerged in water together with cyanobacteria and green algae. Frustules with swollen middle and are bracket in shape (valve view), apices are sharply bent with convex margin; in girdle view, valves linearelliptical, inflated in median part with rounded poles; valves 45.5-53.5 μm long and 11.5-15.0 μm broad; ventral margin are usually straight and curve at the ends while the dorsal margin is convex; striae slightly radiate to parallel; striae 14-16 in 10 μm.

Bacillariophyta
Found existing as a brownish layer on stem and leaves of water hyacinth submerged in water together with several filamentous cyanobacteria and green microalgae. Found existing as a brownish layer on stem and leaves of water lettuce and water spinach slightly submerged in water together with diatoms, green microalgae, and filamentous cyanobacteria. Found existing as a brownish to greenish layer on leaves of water hyacinth submerged slightly in water together with several cyanobacteria. Cells are 2.0-5.0 μm in diameter, symmetrical; chloroplast bandshaped or lobed; pyrenoid is solitary associated with starch grains; cell reproduction is by formation of autospores released by rupture from the mother cell wall.
Found existing as a brownish to greenish layer on stem and leaves of water spinach submerged slightly in water together with other filamentous fungi and cyanobacteria. Found existing as a light greenish layer on stem and leaves of water hyacinth and water spinach submerged in water together with several filamentous blue green algae. Found existing as a light greenish layer on leaves and stem of water hyacinth submerged slightly in water together with other diatoms, green algae, and filamentous cyanobacteria. In the Philippines, little is known on the distribution, taxonomy and diversity of cyanobacteria and microalgae associated with aquatic macrophytes found in marine and freshwater ecosystems. A total of 22 epiphytic algal taxa from three dominant aquatic macrophyte (Pistia stratiotes, Eichhornia crassipes and Ipomoea aquatica) found in Sta. Cruz River were observed in the study. The taxonomic list present 8 taxa belonging to the Chlorophyta, 4 to the Cyanobacteria, 7 to the Bacillariophyta, and 3 taxa to the division Euglenophyta. This study reported and described for the first time in the Philippines the existence of Planktothrix compressa (Utermöhl) Anagnostidis et Komárek, a rare cyanobacteria first reported in submerged roots of Pistia stratiotes found in Sta. Cruz River. One species is also reported here for the first time in the Philippines based on recent algal taxonomic nomenclature and this is Ulnaria ulna (Nitzsch) Compère that is based on the former name of Synedra ulna (Nitzsch) Ehrenberg. In general, it was observed that the consortium of algal mats in submerged stem, leaves and roots of the dominant macrophytes are composed mainly of unicellular and filamentous type of the eukaryotic algae and cyanobacteria as well as other filamentous fungi. The algal groups observed on the macrophytes have been recorded to be similar in other taxonomic studies done for macrophytes in aquatic ecosystems found in the Philippines and other countries (Rañola et al., 1990; Sultana et al., 2004; Arguelles, 2019 a, b). Algal epiphytes associated with a macrophyte, Vallisneria americana in St. John Rivers in Florida (USA) documented a total of 122 taxa of algal epiphytes wherein thirteen genera (Cyclotella, Nitzschia, Synedra (Ulnaria), Rhopalodia, Ankistrodemus, Lacunastrum (Pediastrum), Chroococcus, Oscillatoria, Chlorococcum, Scenedesmus, Chlorella, Euglena and Trachelomonas) were observed to be similar to the taxa reported on the current investigation (Dunn et al., 2008). Also, taxonomic survey on the composition of algal epiphytes from three aquatic macrophytes (Typha latifolia, Nymphaea alba, and Ceratophyllum demersum,) at Acarlal floodplain forest in Northern Turkey reported the occurrence of similar algal taxa such as Phacus longicauda, Trachelomonas armata, Cyclotella meneghiniana, Nitzschia palea, Rhopalodia gibba, and Chroococcus minutus (Tunca et al., 2014). In running water environment, submerged aquatic macrophytes contribute to oxygen pool and are considered important habitats for several aquatic organisms (microbes as well small invertebrates and vertebrates). Macrophytes are abundant in the littoral zones of rivers worldwide, frequently considered as dominant species in the periphyton communities and sometimes associated in producing nuisance growths under eutrophic conditions. The environmental persistence of these organisms in an aquatic environment promotes the occurrence of high taxonomic diversity of cyanobacterial and microalgal epiphytes that can be use as good ecological indicators to determine the quality of water in a river system. In this study, several algal taxa were identified which can be use as biological indicator for aquatic ecosystem health assessment of Sta. Cruz River. Some of the algal taxa identified such as Aulacoseira granulata, Cyclotella meneghiniana, Nitzschia palea, Phacus longicauda, Lepocinclis acus, Trachelomonas armata, Oscillatoria tenuis, Planktothrix compressa, Chroococcus minutus, Scenedesmus quadricauda, and Chlorella vulgaris are indicative of nutrient pollution in a eutrophic system (Dunn et al., 2008;Effiong & Inyang, 2015;Arguelles, 2020a). The taxonomic data obtained on this survey will serve as baseline data in the evaluation of the current ecological status of Sta. Cruz River. Also, evaluation of the interaction of other environmental parameters (such as dissolved oxygen, pH, light intensity, and temperature) in relation to the spatial-temporal distribution of algal epiphytes in Sta. Cruz River is important to further understand the ecological state of the river.

CONCLUSION
The taxonomic survey done in this study shows diverse collection of microalgae and cyanobacteria associated with aquatic macrophytes found in Philippine freshwater ecosystem. Among these taxa, the existence of a rare filamentous cyanobacteria, Planktothrix compressa (Utermöhl) Anagnostidis et Komárek is described for the first time in the Philippines. The result of this survey suggests that further study on the diversity and ecological interactions of algal epiphytes in relation to other aquatic macrophytes found in both marine and freshwater habitat is needed since only few studies were reported in the Philippines. Also, the use of combined morphological and molecular techniques for future taxonomic studies of epiphytic algae in the Philippines is recommended. The current survey provided a quick assessment of some of the dominant algal epiphytes in a river ecosystem and provides basal information for further molecular studies.