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Che Engku Abdullah C E S M, Idris I, Mohd Fahmi A D, Flaxman B, Hutchings P (2024): new_marphysa_species_from_east_coast_peninsular_malaysia. v1.2. ZooKeys. Dataset/Occurrence. https://ipt.pensoft.net/resource?r=new_marphysa_species_from_east_coast_peninsular_malaysia&v=1.2

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Occurrence; Specimen

Four new species of Marphysa from Terengganu mangrove forest, east coast of Peninsular Malaysia, Malaysia, South China Sea

All organisms were identified to species

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Personas asociadas al proyecto:

Marphysa specimens were collected from the rivers, lagoon and estuary of the Terengganu mangrove forests during spring low tides from September 2021 until March 2022. A total of four mangrove areas were chosen, i.e. Setiu wetlands, Kuala Ibai, Merchang, and Kerteh. At each site, sediments were dug using a shovel to approximately 30 cm depth at several points along the river (upper course to lower course) and carefully broken into small pieces to search for the worms. Worms suspected to be Marphysa were fixed and preserved in 95% ethanol. Sediments where the Marphysa worms were found were also collected and kept in labelled plastic bags for sediment analysis.

Descripción de la metodología paso a paso:

1. Morphological analyses. Preserved specimens were examined under AmScope SM-2 Series stereo and 120 Series compound microscopes. Additionally, the specimens were also examined under Leica M165 C stereo and Nikon Labophot-2 compound microscopes, and photographed with a Nikon D610 camera at the Natural History Museum of Los Angeles County, USA (NHMLAC). Drawings of parapodia and pectinate chaetae were made using a Wacom Intuos Pro drawing tablet. Length at chaetiger 10 (L10) and width at chaetiger 10 (W10) without parapodia of all specimens were measured and recorded. Morphological terminology, including diagnostic features of Marphysa species, follows Molina-Acevedo and Carrera-Parra (2017). Terminology of pectinate chaetae is derived and modified from Carrera-Parra and Salazar-Vallejo (1998) for the relative length of outer and inner teeth, Zanol et al. (2014, 2016) for the thickness of the blade and Glasby et al. (2019) for the size of the inner teeth. Terminology of maxillary apparatus followed Molina-Acevedo and Carrera-Parra (2015). Several parapodia from the anterior, median, and posterior regions were removed from the type material of each species, dehydrated in ethanol and hexamethyldisilazane (HMDS), coated with 20 nm of silver-gold, examined under the scanning electron microscope JEOL JSM-6360LA and imaged with a secondary detector at SEM laboratories of Universiti Malaysia Terengganu and Macquarie University, Sydney, Australia.
2. Molecular Analyses. extractions of DNA were done using the xanthogenate method (Tillett and Neilan 2000). Approximately 600 bp of cytochrome oxidase subunit 1 (COI) gene were amplified using universal primer pair LCO1490 and HCO2198 (Folmer et al. 1994). Polymerase Chain Reaction (PCR) amplifications were carried out using 12.5 μL of OneTaq Quick-Load Master mix, 9.5 μL of biology grade water, 0.5 μL of primers (10 μM), 1 μL of 1% bovine serum albumin (BSA) and 1 μL DNA template. The temperature profile was as follows: 95 °C / 180 s – (94 °C / 20 s – 45 °C / 30 s – 72 °C / 60 s)*35 cycles and final extension time at 72 °C / 300 s. PCR success was verified by electrophoresis in a 1% p/v agarose gel stained with GelRed. Amplified products were sent to Apical Scientific Sdn. Bhd. for Sanger sequencing using forward primer (LCO1490). Extractions of DNA were also done with an ISOLATE II Genomic DNA kit (BIOLINE) following the protocol supplied by the manufacturers. Approximately 600 bp of COI gene were amplified using primers polyLCO and polyHCO (Carr et al. 2011). PCR was performed with Taq DNA Polymerase QIAGEN Kit in 20 μL mixtures containing: 2 μL of 10X CoralLoad PCR Buffer (final concentration of 1X), 1.5 μL of MgCl2 (25 Mm) solution, 1.5 μL of PCR nucleotide mix (final concentration of 0.2 mM each dNTP), 0.4 μl of each primer (final concentration of 0.2 μM), 0.1 μl of Taq DNA Polymerase (5U/μl), 1 μl template DNA and 13.1 μL of nuclease-free water. The temperature profile was as follows; 94 °C / 60 s – (94 °C / 40 s – 45 °C / 40 s – 72 °C / 60 s)*5 cycles – (94 °C / 40 s – 51 °C/ 40 s – 72 °C / 60 s)*35 cycles – 72 °C / 300 s. PCR success was verified by electrophoresis in a 1% p/v agarose gel stained with Gelred. Amplified products were sent to Macrogen Company for Sanger sequencing using the same set of primers used for PCR. Sixty-three COI sequences were downloaded from GenBank or obtained during this study; 60 COI sequences of Marphysa species and three outgroup species from closely related genera in the order Eunicida. All COI sequences were aligned in MEGA v. 11.0.10 using ClustalW plugin with default settings. The best DNA/ Protein Models (ML) test was conducted, and the GTR model of molecular evolution was chosen as the best evolutionary model for the COI gene alignment. The phylogenetic analysis was performed in MEGA v. 11.0.10 (Tamura et al. 2021). The analysis was run for 1000 replicates (sampled every 1000). Pair-wise Kimura 2-parameter (K2P) genetic distance was performed using MEGA v. 11.0.10.