DNA Barcoding: Molecular Identification and Phylogenetic Analysis of Pheretimoid Earthworm (<i>Metaphire</i> sp. and <i>Amynthas</i> sp.) Based on Mitochondrial Partial COI Gene from Sialkot, Pakistan

Abstract

The extremely difficult and challenging process is identifying pheretimoid species, genus Metaphire and Amynthas involving increased homoplasy in various morphological characteristics. The molecular identification, phylogenetic relationships, and evolutionary divergence time of earthworms belonging to the pheretimoid complex were investigated in this study using partial mitochondrial COI (cytochrome C oxidase subunit I) gene sequences ranging from 550-680 bp. Results revealed that 86 pheretimoid earthworms were morphologically different from a total of 342 mature worms. Moreover, 11 pheretimoid species were molecularly identified, including Metaphire posthuma (02), M. anomala (01), M. houlleti (02), M. californica (01), M. birmanica (02), Amynthas minimus (01), A. morrisi (01), and M. bununa (01). A phylogenetic tree was constructed with bootstrap values of 95%, which supported a monophyletic lineage of two well-supported clades formed by 12 partial COI sequences and 48 GenBank sequences using Hirudo medicinalis as an outgroup. The monophyly of these obtained genera indicated overall similarity at species level. Today, species like Amynthas, Metaphire and Pheretima have worm diversity in the form of pheretimoid earthworms, which dates to the Late Miocene (11.2-5.3 Mya) and the Pliocene (5.3-2.4 Mya).Compared to all relevant pheretimoid species, genetic p-distance values ranged from 0.0% to 0.57% (less than 1%). These low range values demonstrated that both genera Metaphire and Amynthas, supported the theory, which states that there are shared similarities among the species, despite different morphology. The current study is the first attempt in Pakistan to identify earthworms through DNA barcoding thus providing a genomic stamp. The work explored the significance of COI gene sequences to construct molecular tools that will be useful to overcome the different obstacles in morphologically similar earthworm identification and their phylogenetic study.

Genome-Based Taxa Delimitation (GBTD): A New Approach

“…In particular, all individual specimens assigned to the genus Metaphire are clustered within the clade of the genus Amynthas in no discernible pattern. Other authors described similar clustering [37][38][39], and multiple synonymies were reported between these two genera [40], drawing to the conclusion that all these samples should belong to one genus. The same could be said about Duplodicodrilus schmardae KT429015, although in the case of a single specimen, misidentification may also be an explanation.…”

“…Simultaneous analysis of tree topology, genetic distances and divergence times allowed us to address some clear inconsistencies in taxonomic assignments within Clitellata. Some of them, like Amynthas-Metaphire synonymy are supported by literature sources [39,40]. Others, such as Whitmania assignment to the family Hirudinidae and dissolution of the genera Codonobdella and Baicaloclepsis clearly go against conventional systematics and require more careful investigation.…”

Investigating the impacts of agricultural land use on soil earthworm communities: A case study of northern Zagros Mountains of Iran

“…DNA barcoding is a useful method that has been widely used for studying biodiversity [ [32] , [33] , [34] , [35] , [36] ]. It is a powerful tool for identifying earthworms accurately using standard molecular protocols [ 32 , 37 , 38 ]. This method is a novel way to solve taxonomic problems with species discrimination earthworms identifying new taxa and juveniles, and detecting of cryptic diversity [ 35 , 39 ].…”