Molecular Identification of Five Hormogaster Earthworm Isolates Newly Recorded in Iraq Using COX1 Gene Sequencing

Abstract

Background: Earthworms are highly valued as key organisms in ecosystems, playing an essential role in maintaining soil health and primary productivity. They are considered ecologically important species with complex and poorly understood genetic diversity. Since morphological methods of classification are often inaccurate, it is necessary to classify species based on molecular approaches using PCR techniques. Objectives: This study focused on molecular classification using the mitochondrial Cytochrome c oxidase I (COX1) gene as a DNA barcode, due to its genetic stability, low susceptibility to structural variation, and its importance in supporting traditional morphological taxonomy and clarifying differences among species. Materials and Methods: Earthworm specimens were collected from grazing areas and aquatic zones in Erbil Governorate, Iraq; genomic DNA was extracted, and the mitochondrial cytochrome oxidase I (COX1) gene was amplified using universal primers targeting a 710 bp fragment. PCR products were verified by agarose gel electrophoresis and sequenced. The obtained sequences were aligned with GenBank references using BLASTn. Phylogenetic trees were constructed in MEGA11 software using the Maximum Likelihood method (Tamura-Nei model) with 1000 bootstrap replicates to support taxonomic placement and to establish evolutionary relationships among earthworm species. Results: PCR amplification and sequencing of the mitochondrial COI gene identified two earthworm species in Iraq for the first time: Hormogaster castillana and Hormogaster samnitica. Five isolates were obtained, two belonging to H. castillana (PX257867, PX257868) and three to H. samnitica (PX257869, PX257870, PX257871). These sequences were deposited in the NCBI GenBank database under their respective accession numbers. Phylogenetic analysis using the Maximum Likelihood method (Tamura-Nei model, MEGA11, 1000 bootstrap replicates) revealed that the Iraqi isolates clustered within the same clades as their European and Asian reference strains, confirming their taxonomic placement and evolutionary relationships.