2025/05/06 by Muhammad Nasir, John M. Pfeiffer, Hanna Hartikainen +2 · 1 voice
Environmental Science · Agricultural and Biological Sciences · #Aquatic Invertebrate Ecology and Behavior #Fish Ecology and Management Studies #Mollusks and Parasites Studies
paper · pdf · doi:10.1093/mollus/eyaf008
openalex publication_date 2025/05/06 · openalex created_date 2025/06/26 · openalex updated_date 2026/07/31
There are six recognized species in the freshwater mussel genus Ensidens Frierson, 1911 (Unionidae: Gonideinae: Rectidentini), which is distributed across several major river basins in Indochina (i.e. Mae Klong, Chao Phraya, Bang Pakong, Chantaburi and Mekong) and the Malay Peninsula (Muanta et al., 2019; Pfeiffer et al., 2021). Ensidens dugasti (Morlet, 1892), E. jaculus (Rochebrune, 1882), E. sagittarius (Lea, 1856) and E. spiculus Pfeiffer et al., 2021 are restricted to the Mekong river basin. Ensidens telus Pfeiffer et al., 2021 is currently thought to occur in the Mekong river basin, as well as the Bang Pakong and Chantaburi basins (Pfeiffer et al., 2021). Lastly, E. ingallsianus (Lea, 1852) is considered the most widespread Ensidens species and is known from the Mekong, Mae Klong, Chao Phraya and Bang Pakong basins in Indochina and the Malay Peninsula. As such, E. ingallsianus is currently considered the only Ensidens species present south of the Isthmus of Kra (Pfeiffer et al., 2021: fig. 31) (Fig. 1). However, this assumption is based primarily on museum specimens that have been identified by morphology. This is problematic, because Ensidens species are notoriously difficult to identify using morphological characters alone and “are distinguished most easily by cohesive biogeographic and genetic patterns” (Pfeiffer et al., 2021: 449). The southernmost record of Ensidens confirmed with DNA data is from the Songkhla Lake basin, c. 390 km south of the Isthmus of Kra, and belongs to E. ingallsianus (Konopleva et al., 2022) (Fig. 1). As such, none of the Ensidens populations from south of the Kangar-Pattani Line—an important biogeographic barrier in the region (Parnell, 2013)—have been examined using DNA methods (Fig. 1). Distribution of Ensidens telus and E. ingallsianus. Updated map of E. telus and E. ingallsianus records, expanding on the dataset presented in Pfeiffer et al. (2021: fig. 31). The southernmost barcoded record of E. telus in the Pahang River, eastern coast of Peninsular Malaysia, is dealt with in this study. Solid black boundaries represent freshwater ecoregions (Abell et al., 2008; The Nature Conservancy & Esri Canada, 2014). Here, we provide the first DNA sequence data of Ensidens south of the Kangar-Pattani Line. Five specimens were collected from the Syed Mohammad River basin, very close to its confluence with the Pahang River basin (Figs 1–3). Due to its proximity to the South China Sea (<14 km distance to the coast), the river is tidally influenced. DNA was extracted from foot tissue snips using the DNeasy® Blood & Tissue Kit following the manufacturer’s protocol. A 658 bp fragment of the F-type cytochrome c oxidase subunit I (COI) region was amplified using standard primers LCO1490 and HCO2198 (Folmer et al., 1994) in 10 μl reactions containing 5.0 μl 10X Green Dye PCR Buffer, 0.5 μl 10 mmol l−1 of each primer, 1.0 μl DNA template and 3.0 μl nuclease-free water. Cycle parameters were initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C (50 s), annealing at 52 °C (50 s) and extension at 72 °C (50 s), and a final extension at 72 °C for 5 min. Amplified DNA fragments were sequenced by the commercial company Eurofins Genomics, Ebersberg, Germany, and deposited in GenBank under acc. nos PQ037208–PQ037212; voucher specimens are deposited at the South China Sea Repository and Reference Centre, University of Malaysia Terengganu (UMT), under acc. nos UMTMoll 3125–3129. To estimate the phylogenetic placement of the specimens, a COI alignment was constructed using the five newly sequenced individuals and all published E. ingallsianus and E. telus COI sequences (as available on 30 June 2024). Representatives of all other Ensidens species were also included: E. dugasti (MT905548), E. jaculus (MT905659), E. sagittarius (MT905530) and E. spiculus (MT905802). Rectidens sumatrensis (KX822664) was used as the outgroup. Sequences were aligned in AliView v. 1.28 (Larsson, 2014) using Muscle v. 3.8.31 (Edgar, 2004) and trimmed using trimAl V1.2 (Capella-Gutiérrez, Silla-Martínez & Gabaldón, 2009). A partitioned maximum-likelihood tree was generated using IQ-TREE v. 2.0.3 (Nguyen et al., 2015) with a model testing and partitioning scheme determined using the ModelFinder (MFP) algorithm and using the MERGE function to finds the best partitioning scheme (expressed as -m MFP + MERGE) (Chernomor, Von Haeseler & Minh, 2016; Kalyaanamoorthy et al., 2017), 1,000 ultrafast bootstrap replicates (UFboot; Hoang et al., 2018) and a minimum bootstrap support threshold of 0.5. The resultant 50% majority rule tree was edited using Adobe Illustrator. Syed Mohammad River basin near its confluence with the Pahang River, Malaysia, sampling site of the barcoded Ensidens telus. Specimens of Ensidens telus collected from the Syed Mohammad River basin near its confluence with Pahang River, Malaysia, and deposited at the South China Sea Repository and Reference Centre, University of Malaysia Terengganu (UMT). Molecular phylogenetic analysis recovered all the novel Ensidens sequences from the Malay Peninsula in the E. telus clade with strong support (Fig. 4). Ensidens telus was previously known only from the freshwater ecoregions (sensu Abell et al., 2008) Chao Phraya, Eastern Gulf of Thailand Drainages, Mekong Delta and Kratie-Stung Treng, but our new sequences expand its known distribution to include the Malay Peninsula Eastern Slope ecoregion (Figs 1, 4). Ensidens telus thus has a strongly disjunct distribution, occurring in the Chao Phraya, Mekong and adjacent river basins and in at least one basin of the Malay Peninsula (Fig. 1). Similar disjunct distributions are known from Hyriopsis bialata (Simpson, 1900) (Pfeiffer et al., 2021) and Pseudodon vagulus (Fischer, 1891) [= P. cambodjensis sensu Zieritz et al. (2016) (= Monodontina cambodjensis sensu Pfeiffer et al., 2021; Bolotov et al., 2023)]. Pfeiffer et al. (2021) offer two potential explanations for this disjunct distribution: (1) a temporary connection of the Mekong and Palaeo-Siam River basins followed by a major geographic expansion along the Paleo-Siam River with subsequent extinction across much of the species’ range or (2) non-native introduction to the Malay Peninsula. Phylogeny of Ensidens telus and E. ingallsianus. Maximum-likelihood tree of Ensidens based on cytochrome c oxidase subunit I. Branch support values (UFBoot) are shown near nodes. Terminals are coloured by freshwater ecoregions (inset). The scale bar indicates substitutions per site. Our finding raises questions on the distributions of Ensidens spp. across the Malay Peninsula and whether any or all of the historical E. ingallsianus records from the Malay Peninsula are misidentified E. telus. We are aware of six lots of Ensidens from Malaysia, all of which have been identified as E. ingallsianus: (1) NHMUK1902-5-24-1-5 (“Kielantan Malacca” = Kelantan State, Peninsular Malaysia), (2) NHMUK20240431 (= BMNHMPD280; “Market stall, Kota Bharu, East Coast Malaya”, Kelantan State), (3) NHMUK20240397 (= BMNHMPD275; “Kilantan Malay” = Kelantan State), (4) MNHNMP2748 (“Gunong Taekan Malakka Orient”; probably referring to Gunung Tahan in central Peninsular Malaysia, either in Pahang or Kelantan River basin), (5) UMMZ109728 (“Gunung Tukau, East Malacca”; again probably referring to Gunung Tahan) and (6) UMMZ109733 (“Kelantan”) (Graf & Cummings, 2023) [museum acronyms: NHMUK (also: BMNH), Natural History Museum, London UK; MNHN, Museum National d'Histoire Naturelle, Paris, France; UMMZ, University of Michigan Museum of Zoology). Re-examination of these historical location records in combination with our recent findings suggest that Ensidens on the Malay Peninsula is restricted to the river basins emptying into the South China Sea (i.e. on the east coast of the peninsula; Fig. 1), including the Kelantan and Pahang river basins. However, morphological identification of these specimens from available images is difficult due to the lack of conchological characteristics that allow unambiguous distinction between E. ingallsianus and E. telus (Pfeiffer et al., 2021). Further genetic sampling and DNA analyses are therefore needed to generate a better understanding of the morphological and geographic boundaries of these species and resolve questions of the number, identity and historical biogeography of Ensidens species south of the Kangar-Pattani Line. This may require considerable sampling effort when considering that historical records in this region are relatively scarce and dated (>47 years old), and that recent collecting expeditions have failed to detect any extant Ensidens populations (Zieritz et al., 2016; Zieritz & Lopes-Lima, 2018). Fieldwork was conducted under permit MBP.600(S)-1/1/1 jLD.5 (6) granted by the Pahang Biodiversity Agency (Majlis Biodiversiti Pahang). M.A.M.N. was funded by the Ministry of Higher Education Malaysia through a PhD research grant under the Fundamental Research Grant Scheme (FRGS) (FRGS/1/2019/WAB13/MUSM/03/1). The authors have no conflicts of interest to declare. All co-authors have seen and agree with the contents of the manuscript and there is no financial interest to report. The sequence data newly generated for this study are deposited in GenBank under acc. nos PQ037208–PQ037212; voucher specimens are deposited at the South China Sea Repository and Reference Centre, University of Malaysia Terengganu (UMT), under acc. nos UMTMoll 3125–3129.