81 Journal of Multidisciplinary Applied Natural Science Vol. 3 No. 1 (2023) Research Article The Identification of Euglenids (Euglenophyceae, Euglenophyta) from the Peat Waters of Palangka Raya, Indonesia Chaidir Adam* dan Agus Haryono Received : July 26, 2022 Revised : October 18, 2022 Accepted : October 21, 2022 Online : October 28, 2022 Abstract The aim of this study was to identify and describe the Euglenids (Euglenophyceae, Euglenophyta) from the peat waters of Palangka Raya, Central Kalimantan, Indonesia. This study revealed that 8 species of Euglenids were found in the study sites which belong to 3 genera, namely Euglena, Lepocinclis, and Phacus. The Euglenid species identified include Euglena gracilis G.A. Klebs 1883, Euglena mutabilis F. Schmitz 1884, Lepocinclis acus (O.F.Müller) B.Marin & Melkonian 2003, Lepocinclis ovum (Ehrenberg) Lemmermann 1901, Lepocinclis spirogyroides B.Marin & Melkonian 2003, Phacus cordatus (Pochmann) Zakryś & Lukomska 2015, Phacus helikoides Pochmann 1942, and Phacus orbicularis Hübner 1886. The eight Euglenid species found in this study have never been reported before, thus these findings provide additional new data regarding algae diversity in peat waters of Central Kalimantan, Indonesia. Keywords Euglena, Lepocinclis, microalgae, peat waters, Phacus, phytoplankton 1. INTRODUCTION Research on algae in Central Kalimantan, Indonesia, especially freshwater algae from peat waters has been intensively carried out since the last few years [1][2]. These studies have contributed the scientific data regarding the diversity of algae in the peat waters habitat. Adam [2] reported 10 species of desmids from the peat waters in Palangka Raya, consisting of 5 Cosmarium species and 5 Euastrum species respectively. The most recent study is related to the morphological study of Coelastrum cambricum, a microalgae species commonly found in peat waters. This study revealed the distinctive morphological characteristics of C. cambricum where 6 cells were observed in the center of the coenobium forming a pentagonal pattern that has the potential to be studied further as a key feature of Coelastrum species identification [1]. Similar studies still need to be done to develop scientific data related to the diversity of algae in Indonesia, especially Central Kalimantan which has extensive peat water habitats. Euglenophyceae is a group of flagellate green algae found in a variety of freshwater and marine environments [3][4]. Members of the Euglenophyceae are distinctive with red eyespots that are part of the eyespot apparatus [5]. The eyespot apparatus consists of the paraflagellar body, which connects the eyespot to the flagellum. The function of eyespot apparatus is as a photoreceptive organelle [6]–[8]. According to Algaebase [9], there are 1,007 algal species belonging to Euglenophyceae which lack references that provide data on their distribution in Indonesia, especially in Central Kalimantan. Recent studies regarding the diversity of microalgae in Indonesia have revealed several species of Euglenophyceae that are distributed in Bengkulu (Sumatra) [10], South Sumatra [11], East Java [12], Central Lombok [13], and West Kalimantan [14] which belong to the genera Euglena, Trachelomonas, Lepocinclis and Phacus. The aim of this study was to identify and describe the Euglenids (Euglenophyceae, Euglenophyta) from the peat waters of Palangka Raya, Central Kalimantan, Indonesia, to contribute to the development of scientific data on algae diversity. 2. MATERIALS AND METHODS 2.1. Study Site and Sample Collection Publisher’s Note: Pandawa Institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the author(s). Licensee Pandawa Institute, Metro, Indonesia. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution-ShareAlike (CC BY-SA) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.145 OPEN ACCESS https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.131 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.145&domain=pdf&date_stamp=2022-10-28 https://creativecommons.org/licenses/by-sa/4.0/ J. Multidiscip. Appl. Nat. Sci. 82 Table 1. The list of published works used to assist the identification phase. This research was conducted in shallow peat waters located in Palangka Raya, Central Kalimantan, Indonesia (Figure 1). Water samples were collected directly from peat waters along with sediment from the bottom of the trench, then stored in collection bottles. 2.2. Microscopic Observation and Microphotography The samples were observed microscopically using Olympus CX21 at 400× (10× ocular; 40× objective) magnification in the laboratory of Biology Education Program, University of Palangka Raya. Microphotography method explained as follows: specimen objects found that are suspected to be Euglenid species are immediately photographed using 64megapixel cellphone camera directly to the eyepiece of the microscope for further identification. 2.3. Image Processing The photographed specimen image was then processed for color and detail enhancement using Adobe Photoshop CC. Scale bar was added using ImageJ software version 1.53g [15] to estimate the object length. Scaling on ImageJ uses the method of estimating cell size by knowing the diameter of the field of view of the microscope [2][16], which in this study used a 400× magnification with a diameter of 0.4 mm or 400 μm. 2.4. Euglenid Identification The identification of Euglenid was carried out by analyzing the observed morphological characteristics (e.g., the presence of red eyespot & flagella; shape of the cells, and cell size) both qualitatively and quantitatively. Morphotaxonomic identification was done up to the species level using all available information. The following published works were used to assist the identification phase (Table 1). 3. RESULTS AND DISCUSSIONS This study revealed 8 euglenid microalgae species found in the peat waters of Palangka Raya, Indonesia, consisting of 3 genera, e.g., Euglena (2 species), Lepocinclis (3 species), and Phacus (3 species) as presented in the Table 2. All Euglenid species were found in shallow peat waters with the depth ranges from 30-50 cm and pH of 5. These characteristics of peat waters are in accordance with several references which describe that the color of peat water usually appears in blackish brown color, has acidity range between pH 3-5 and the organic matter content is relatively high [21][22]. 3.1. Euglena Ehrenberg, 1830 Euglena is spindle-shaped unicellular flagellate green algae with cylindrical or oval body; has a single flagellum embedded at the anterior end of the cell; red eyespots are clearly visible on the anterior area of the cells [5][9][20]. Currently, Euglena consists of 156 taxonomically accepted species names and 52 accepted varieties [9]. Taxonomic Enumeration Euglenozoa Class Euglenophyceae Order Euglenida Family Euglenidae Genus Euglena Ehrenberg 1830 No. Published Works Ref. 1 AlgaeBase. World-wide electronic publication. National University of Ireland, Galway. [9] 2 A Short Guide to Common Heterotrophic Flagellates of Freshwater Habitats Based on the Morphology of Living Organisms [17] 3 Algae identification lab guide: accompanying manual to the algae identification field guide [18] 4 A Beginner’s Guide to Freshwater Algae [5] 5 Easy Identification of the Most Common Freshwater Algae [19] 6 How to know the Freshwater Algae [20] J. Multidiscip. Appl. Nat. Sci. 83 3.1.1. Euglena gracilis G.A. Klebs 1883 Taxonomy: E. gracilis G.A. Klebs 1883 is currently taxonomically accepted species name according to Algaebase. First published in a paper entitled “Über die Organisation einiger Flagellatengruppen und ihre Beziehungen zu Algen und Infusorien” by Klebs in 1883 [9]. Description: E. gracilis were observed to have bright green color; a clearly visible red eyespot; a single flagellum; and known to be the largest species of Euglena found in this study. The shapes of the cell are dynamic due to the metabolic movement, can vary from oval or spherical to highly elongated shape. Cell length from anterior to posterior end ranged from 93-267 μm. Localities: Shallow peat waters in Palangka Raya, Central Kalimantan, Indonesia. Found in Site 1 (2°13'03.7"S 113°53'38.9"E) and Site 2 (2° 12'55.2"S 113°53'46.9"E). 3.1.2. Euglena mutabilis F. Schmitz 1884 Taxonomy: E. mutabilis F. Schmitz 1884 is currently taxonomically accepted species name according to Algaebase. First published in a paper entitled “Beitrage zur Kenntniss der Chromatophoren” by Schmitz in 1884 [9]. Description: E. mutabilis were observed as small Euglena cells with a single flagellum, narrow ends in the posterior and anterior areas of the cell, and clearly visible red eyespot. Cell length from anterior to posterior end ranged from 73-89 μm. Previously published work described E. mutabilis cells 57–65 µm long and 10–11 µm wide; cylindrical with narrowed ends; cells may be attached to substrate at posterior end; cells bend while swimming [23]. Localities: Shallow peat waters in Palangka Raya, Central Kalimantan, Indonesia. Found in Site 1 (2°13'03.7"S 113°53'38.9"E) and Site 3 (2° 12'58.0"S 113°53'48.8"E). Figure 1. Research Map: Palangka Raya, Central Kalimantan, Indonesia (Map Source: OpenStreetMap & Bing Aerial, QGis 3.16.3 Hannover). J. Multidiscip. Appl. Nat. Sci. 84 3.2. Lepocinclis Perty, 1849, nom. cons. Lepocinclis is photothropic flagellates; ovoid or spindle-shaped; with red eyespots that are clearly visible on the anterior region of the cells as in Euglena [4][9][20][24]. The cells are completely rigid and unflattened which distinguishes this genus from the genera Euglena and Phacus [9]. Currently, Lepocinclis consists of 88 taxonomically accepted species names and 66 accepted varieties [9]. Taxonomic Enumeration Euglenozoa Class Euglenophyceae Order Euglenida Family Phacaceae Genus Lepocinclis Perty, 1849, nom. cons. 3.2.1. Lepocinclis acus (O.F.Müller) B.Marin & Melkonian 2003 Taxonomy: Lepocinclis acus (O.F.Müller) B.Marin & Melkonian 2003 is currently taxonomically accepted species name according to Algaebase. Published in a paper entitled “Phylogeny and taxonomic revision of plastid- containing euglenophytes based on SSU rDNA sequence comparisons and synapomorphic signatures in the SSU rRNA secondary structure” by Marin et al. in 2003 [9]. This species has 4 homotypic synonyms and 10 heterotypic synonyms [9]. Description: L. acus were observed to have elongated spindle-shaped cells with narrow ends in the posterior and anterior areas of the cell; cell length around 200 μm. According to Kim et al. [24], L. acus cells 125-163 μm long and 9-77 μm wide; almost rigid; long-fusiform; the anterior and posterior ends are narrowed. Localities: Shallow peat waters in Palangka Raya, Central Kalimantan, Indonesia. Found only in Site 2 (2°12'55.2"S 113°53'46.9"E). 3.2.2. Lepocinclis ovum (Ehrenberg) Lemmermann 1901 Taxonomy: L. ovum (Ehrenberg) Lemmermann 1901 is currently taxonomically accepted species name according to Algaebase. First published in a paper entitled “Beiträge zur Kenntniss der Planktonalgen. XII. Notizen über einige Schwebealgen. XIII. Das Phytoplankton des Ryck und des Greifswalder Boddens” by Lemmermann in 1901 [9]. This species has 3 homotypic synonyms and 2 heterotypic synonyms [9]. Description: L. ovum were observed in the form of elliptical or spherical cells with clearly visible red eyespot; short caudal process on the posterior. L. ovum is the smallest Euglenid species found in this study with up to 25 μm long in diameter. Previously published works described L. ovum cells are citriform, fusiform, sub-globose, ovate to elliptical, and elliptical in shape [23]. Localities: Shallow peat waters in Palangka Raya, Central Kalimantan, Indonesia. Found in all three sampling sites: Site 1 (2°13'03.7"S 113° 53'38.9"E), Site 2 (2°12'55.2"S 113°53'46.9"E), and Site 3 (2°12'58.0"S 113°53'48.8"E). 3.2.3. Lepocinclis spirogyroides B.Marin & Melkonian 2003 Taxonomy: L. spirogyroides B.Marin & Melkonian 2003 is currently taxonomically accepted species name according to Algaebase. Table 2. Euglenid species found in this study. No Familia Genera Species Status of Species Name* 1 Euglenidae Dujardin 1841: 347 Euglena Ehrenberg, 1830 E. gracilis E. mutabilis accepted taxonomically accepted taxonomically 2 Phacaceae J.I. Kim, Triemer & W. Shin 2010: 1280 Lepocinclis Perty, 1849, nom. cons. L. acus L. ovum L. spirogyroides accepted taxonomically accepted taxonomically accepted taxonomically Phacus Dujardin, 1841, nom. et typ. cons. P. cordatus P. helikoides P. orbicularis accepted taxonomically accepted taxonomically accepted taxonomically *Based on Algaebase data 2022 [9] J. Multidiscip. Appl. Nat. Sci. 85 Published in a paper entitled “Phylogeny and taxonomic revision of plastid-containing euglenophytes based on SSU rDNA sequence comparisons and synapomorphic signatures in the SSU rRNA secondary structure” by Marin et al. in 2003 [9]. This species has 4 heterotypic synonyms [9]. Description: L. spirogyroides were observed to have that makes L. spirogyroides appear to have helical striated lines along its cells. According to Dawson et al. [25], the ornamentations on the cell wall named pellicular warts, iron-enriched and mineralized structures of the extracellular matrix. Localities: Shallow peat waters in Palangka Raya, Central Kalimantan, Indonesia. Found in Site 1 (2°13'03.7"S 113°53'38.9"E) and Site 2 (2° 12'55.2"S 113°53'46.9"E). 3.3. Phacus Dujardin, 1841, nom. et typ. Cons Phacus is a genus of green flagellates with rigid, compressed and flattened cells, and most appear in a leaf-shaped, folds or grooves running helically or longitudinally, giving the cells irregular or triradiate cross-sections; some species twisted into flat corkscrews [5][9]. Phacus consists of 167 taxonomically accepted species names and 73 accepted varieties [9]. Taxonomic Enumeration Euglenozoa Class Euglenophyceae Order Euglenida Family Phacaceae Genus Phacus Dujardin, 1841, nom. et typ. cons. Figure 2. Euglena: (A-H) E. gracilis; and (I-J) E. mutabilis. J. Multidiscip. Appl. Nat. Sci. 86 3.3.1. Phacus cordatus (Pochmann) Zakryś & Lukomska 2015 Taxonomy: P. cordatus (Pochmann) Zakryś & Lukomska 2015 is currently taxonomically accepted species name according to Algaebase. Published in a paper entitled “Delimiting species in the Phacus longicauda complex (Euglenida) through morphological and molecular analyses” by Lukomska-Kowalczyk et al. in 2015 [9]. This species has 2 homotypic synonyms that are Phacus longicauda subsp. cordatus Pochmann 1942 and Phacus longicauda f. cordatus (Pochmann) Popova 1955 [9]. Description: P. cordatus were observed to have a leaf-shaped; slightly shaped like a heart; red eyespots are clearly visible on the anterior region of the cells; long tail; cell length from the anterior to the caudal end is 133 μm and around 70 μm long from the anterior to posterior base. Localities: Shallow peat waters in Palangka Raya, Central Kalimantan, Indonesia. Found in Site 1 (2°13'03.7"S 113°53'38.9"E) and Site 2 (2° 12'55.2"S 113°53'46.9"E). 3.3.2. Phacus helikoides Pochmann 1942 Taxonomy: P. helikoides Pochmann 1942 2015 is currently taxonomically accepted species name according to Algaebase. First published in a paper entitled “Synopsis der Gattung Phacus” by Pochmann in 1942 [9]. This species has 1 homotypic synonym (Phacus tortus var. helikoides (Pochmann) Huber-Pestalozzi 1955) and 2 heterotypic synonyms (Phacus tortus var. tortuosus Skvortsov 1928 and Phacus sesquitortus Pochmann 1942) [9]. Description: P. helikoides were observed to have cells with helical folds; flagella and red eyespots; anterior to posterior (caudal end) length ranges from 62-89 μm. P. helikoides is actually helical in shape throughout the entire cell in Figure 3. Lepocinclis: (A-B) L. acus; (D) L. ovum; and (D) L. spirogyroides. J. Multidiscip. Appl. Nat. Sci. 87 contrast to most Phacus species which are flattened and leaf-shaped. Localities: Shallow peat waters in Palangka Raya, Central Kalimantan, Indonesia. Found in Site 1 (2°13'03.7"S 113°53'38.9"E) and Site 2 (2° 12'55.2"S 113°53'46.9"E). 3.3.3. Phacus orbicularis Hübner 1886 Taxonomy: Phacus orbicularis Hübner 1886 is currently taxonomically accepted species name according to Algaebase. First published in a paper entitled “Euglenaceen-Flora von Stralsund” by Hübner in 1886 [9]. This species has 14 heterotypic synonyms [9]. Description: P. orbicularis were observed to have ovoid cell shape; short curved caudal process; clearly visible red eyespot; anterior to posterior length ranges from 67-89 μm. Kosmala et al. [26], Kim et al. [24], and Łukomska-Kowalczyk [27] described that P. orbicularis are characterized by wide, ovoid cells ending with a sharp curved tail. Localities: Shallow peat waters in Palangka Raya, Central Kalimantan, Indonesia. Found in all three sampling sites: Site 1 (2°13'03.7"S 113° 53'38.9"E), Site 2 (2°12'55.2"S 113°53'46.9"E), and Site 3 (2°12'58.0"S 113°53'48.8"E). 4. CONCLUSIONS This study revealed that in the peat waters of Palangka Raya, Central Kalimantan, Indonesia, 8 species of Euglenids (Euglenophyceae, Euglenophyta) were found which belong to 3 genera. The Euglenid species identified include Euglena gracilis, Euglena mutabilis, Lepocinclis acus, Lepocinclis ovum, Lepocinclis spirogyroides, Phacus cordatus, Phacus helikoides, and Phacus orbicularis. In this study, there were several other specimens that were suspected to be Euglenid species but could not be identified due to lack of information needed to determine the species name, further research needs to be done using molecular identification methods for more accurate results. AUTHOR INFORMATION Corresponding Author Chaidir Adam — Biology Education Program, University of Palangka Raya, Palangka Raya – 74874 (Indonesia); Center for Development of Figure 4. Phacus: (A-B) P. cordatus; (C-E) P. helikoides; and (F-H) P. orbicularis. J. Multidiscip. Appl. Nat. Sci. 88 Science, Technology and Peatland Innovation (PPIIG), University of Palangka Raya, Palangka Raya – 74874 (Indonesia); orcid.org/0000-0002-8907-1117 Email: chaidir.adam03@gmail.com Author Agus Haryono — Biology Education Program, University of Palangka Raya, Palangka Raya – 74874 (Indonesia); Center for Development of Science, Technology and Peatland Innovation (PPIIG), University of Palangka Raya, Palangka Raya – 74874 (Indonesia); orcid.org/0000-0003-3725-201X Author Contributions C. A. study conception and design; sample collection, observation, and identification; and draft manuscript preparation. A. H. analysis and interpretation of results. All authors reviewed the results and approved the final version of the manuscript. Conflicts of Interest The author(s) declared no conflict of interest REFERENCES [1] C. Adam and A. Haryono. (2022). “Morphological Study of Coelastrum cambricum from the Peat Water of Palangka Raya, Indonesia”. 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