Molecular Identification

  1. Species identification of adult earthworms is possible by dissection of the anterior end. However, this method is labor intensive, time consuming and very difficult for non-specialists, particularly when dealing with field collections consisting of several different earthworm species.
  2. Furthermore, identification is limited to adult worms as most life stages are unidentifiable and, many morphological and anatomical characteristics of earthworms are variable, consequently the degree of variability can differ and features can overlap the taxa.
  3. The taxonomy of earthworms has always been hindered by shape of prostomium, arrangement of the setae, position and form of the clitellum, tubercula pubertatis, spermathecae, seta etc. that usually overlaps across taxonomically close and distant related taxa. This has led to morphologically similar species being lumped into single species with various morphotypes or as a species complex that includes various taxa of uncertain taxonomic category and to the creation of ‘catch-all genera’.
  4. The use of DNA barcodes, which are short sequences(COI gene) from standardized regions of the genome, a standard marker for DNA barcoding (e. molecular taxonomic identification), has been regarded as a promising approach to resolve this taxonomic dilemma in recent times and may be a useful supplement to traditional morphological taxonomists as well as non-experts in this area.
  5. Within earthworms COI is also the most highly sequenced marker and has proved to be effective for species identification, taxonomy and shallow (intrageneric) molecular systematics, ecology, phylogeography and for revealing cryptic speciation. The complete process involves following steps:
    1. Isolation and purification of DNA from earthworm tissue.
    2. Amplification of mitochondrial genome (COI).
    3. Cleaning and quantification of PCR products.
    4. Cycle sequencing
    5. Use of Basic Local Alignment Search Tool (BLAST) to identify sequence.
    6. Use multiple sequence alignment and tree-building tools to analyze phylogenetic relationship.
    7. Upload specimen details with sequence and trace file on BOLD to obtain DNA Barcode.