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

: 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.

vertebrates and vertebrate taxa. These sequences are approximately 530-680 bp in length 21,22 , responsible for the identification of cocoon, juvenile, and adult earthworms 17,23 .
Several genetic phylogenies of Megascolecidae have been constructed and published over the last few decades 24 to resolve systematic issues among closely related species with no apparent morphological differences 25,26 . Similarly, in earthworms belonging to the pheretimoid complex, the COI gene was used to distinguish morphologically different earthworms such as Metaphire houlleti, Amynthas arenulus, and A. longiculicaulatus 19 as well as to assess the phylogenetic relationships in Metaphire and Amynthas 27 . Phylogenetic analysis indicated that a single gene or multiple genes might be useful for identifying cryptic soil invertebrate species such as earthworms 15,28,29 . The divergence time of Lumbricidae was previously estimated, and its chronogram indicated a late Miocene 20.5 Mya origin 30,31 . However, the divergence time of pheretimoid species must be estimated to determine the time of diversification of Metaphire and Amynthas species within a clade.
The present study is the first attempt to determine DNA barcoding technique, phylogenetic analyses, and divergence time estimation to characterize earthworms at the molecular level. Unfortunately, despite abundant earthworm biodiversity in Pakistan, having little evidence regarding identifying earthworm species through morphological and physiological identification keys. Therefore, we aim to categorize the Asian pheretimoid earthworm complex Metaphire sp. and Amynthas sp. with wide genetic diversity within Zoo taxa in Pakistan on the basis of morphological and physiological aspects. Following categorization, phylogenetic trees based gene sequences were constructed to investigate inter and intraspecific relationships. In the end, molecular dating estimation for determining their origin with the time scale of the new and old clade was also performed using mitochondrial COI gene sequences.

Specimen sampling
Earthworms were collected for identification from eight localities Table 1 moist and shady along the bank sides of the Marala Ravi Link canal in the Sialkot region between May and October in 2020 Fig. 1 . Twenty soil quadruplets four at each sampling site were dug out with a spade with dimensions 30 35 40 cm, and unwounded, mature, clitellate earthworms were collected and sorted on the spot by hand 5, 32 . The 342 sorted earthworms were placed in labelled stainless-steel containers with porous lids containing moist soil from the same locations. Earthworm containers were kept moist with water showers and were immediately transported to Microbiology Lab, Department of Zoology, G.C. University Lahore, for further investigation.

Morphological taxonomic characterization
Collected species of earthworms were allowed to expel their gut material for 15 hours in a wide glass jar with a porous lid and then washed thoroughly with distilled water. These worms were preserved by immersing them in 98 alcohol for 5 minutes at room temperature 33 . Following alcohol treatment, these worms were stored in plastic containers at 4 morphological characterization. By observing the shape, colour, size, segmentation, clitellum, male and female genital pore, setae, prostomium, and dorsal pores at the family level Megascolecidae , and utilizing diagrams and identification keys, the specimens were characterized and separated from each other 34,35 .

DNA isolation, COI gene ampli cation and sequencing
Total DNA was isolated from 43 pheretimoid species for molecular identification using minor modifications 36 . To avoid soil contamination, one cm tissue closes to the anal region of each of the morphologically identified earthworms was collected in disposable Eppendorf tubes one for each specimen . At first, 180 μL of 1xTE buffer 1 M Tris HCl 0.5 M ethylene-diamine-tetra acetic acid EDTA double distilled water ddH 2 O to a volume of 1 liter was added in each Eppendorf containing earthworm tissue to solubilize and protect the DNA from degradation. Following that, 480 μL CTAB buffer 1 M Tris HCl 5 M Sodium chloride NaCl 0.5 M EDTA 20 CTAB cetyl-trimethyl-ammonium-bromide ddH 2 O to achieve a volume of 1 liter was added to the tubes. Proteinase K 25 mg/mL was immediately added and mixed using vertex to eliminate protein-based contaminants.
To eliminate RNA contaminants, 3 μL of RNase 15 mg/ mL was added. The mixture was placed at the aqueous phase and poured into a new disposable centrifuge tube. DNA templates from each specimen were amplified by using the procedure with minor modifications 37 . The mitochondrial

Phylogenetic and divergence time analysis
COI gene sequences were obtained from pheretimoid species and compared for similarity using BLAST at NCBI site before being deposited in GenBank and assigned accession numbers. The generated and alternate homologous sequences adapted from the GenBank database were uploaded in MEGA-X 10.1 version 39 for alignment using the BIC Bayesian information criterion with the lowest score. Multiple sequence alignment 40 was performed using the ClustalW program in MEGA-X, with a gap elongation penalty of 6.66 and a gap opening penalty of 1.5 for both multiple and pairwise alignments. Maximum likelihood 41 and neighbour-joining 37 methods were used to construct the phylogenetic tree, with 1000 bootstrap samples. Similarly, pairwise genetic distances of 12 pheretimoid species 2 Amynthas and 10 Metaphire were calculated using MEGA 7.0.20 42 .  Table 2 . Moreover, 43 COI mitochondrial gene sequences were successfully isolated from 50 of each morphologically screened earthworm species. Only 12 COI gene sequences were amplified using PCR and were thus used in subsequent studies. The failure in amplifications was attributed to DNA degradation or low-quality DNA templates due to the reaction mixture modification. All partial COI mitochondrial gene PCR amplicons were between 550-680 bp in length. PCR amplicons were sequenced and analyzed for homology using NCBI blast. These amplicons displayed a high degree of similarity to GenBank sequences for M. posthuma 02 , M. anomala

Phylogenetic analysis
In total, 11 earthworm species were phylogenetically identified by DNA barcoding. A phylogenetic tree of the COI partial gene sequences was constructed to investigate the evolutionary relationship between 12 newly identified earthworms and 47 closely resembled pheretimoid species published in GeneBank. Hirudo medicinalis, a non-earthworm Annelid HQ333518.1 Table 4 , was utilized as an out-group which was obtained from the NCBI database Fig. 2 . The Maximum likelihood phylogeny explored the monophyletic lineages for 2 well-supported clades, a simple A and complex B , along with 95 bootstrap support  Mya . This indicates that the Pheretimoid sp. were regarded as modern earthworm taxa. Additionally, it was concluded from this chronogram that all evolutionary events occurred between the mid and late Cenozoic eras Fig. 3 .

Pairwise P genetic distances
Calibrated genetic P-distances for pairwise species comparison were used to confirm the taxonomic positions of pheretimoid earthworms belonging to the Metaphire and Amynthas genera Table 3 . The pairwise distances between mitochondrial partial COI gene sequences from all concerned pheretimoid species ranged from 0.0 to 0.57 using the Tamura-Nei model on MEGA X 6.0 software and gamma distributions. The genetic p-distance values within and between species were found extremely low less than 1 . These lowered range values confirmed the similarities within and between Metaphire and Amynthas species. Likewise, slight differences were also observed in genetic p-distance values and were corroborated by morphological characters.

Discussion
Total 86 earthworm species were identified on the basis of morphological and physiological characteristics in the current study. The major morpho-differentiating characteristics of pheretimoid complex earthworms were observed in their body size 75-130 cm , colour which varies according to species , perichaetine setae, dorsal pore position, annular clitellum, epibolic prostomium, spermathecal and genital pores. Various authors have also analyzed similar characteristics to aid in identifying Amynthas and Metaphire species 35,43,44 . Members of the Amynthas  46 . Similarly, A. morrisi, was isolated and identified with M. posthuma, A. diffringens and A. minimus from different neighbouring districts of Sialkot i.e., Narowal , Punjab 6 . The distinguishing morphological characteristics were not sufficient shreds of evidence to categorize earthworms at the species level 25 . The literature revealed that during interrelationship study between Amynthas and Metaphire genus, it was difficult to characterize Amynthas genus due to a lack of major diagnostic traits 4, 7, 47 . This prompted us to use COI gene sequencing to characterize these worm species at the molecular level.
Through DNA barcoding, 11 earthworm species belonging to genera Metaphire and Amynthas were identified, while one was being unidentified. Previously researchers used COI mitochondrial gene-based DNA barcoding to molecularly characterize the Amynthas and Metaphire earthworm species in the United States 25 , Philippine 21 , Thailand 19 , and China 48 . Only eight species are classified as Metaphire, 3 to Amynthas, and one as unidentified Metaphire sp using 43 COI sequences. The remainder exhibited no homology to existing GenBank data. There could be several reasons for this; however, one critical is primer specificity, which is why the COI gene was not amplified successfully in the current study 49 . When the divergence in COI sequences of closely related pheretimoid species exceeds the intraspecific genetic divergence, species characterization is quite successful 50 19,24 . In history, some taxonomists successfully identified these two genera across the world 9, 34, 35 . Both of these genera are clustered according to the phylogenetic analysis 8,25 . The taxonomic characterization of these genera has been a source of intension and provoke insight interest. All morphological and anatomical characteristics are identical, but the only difference between the two genera is the presence of male pores on the surface of Amynthas. These are contained within the copulatory pouch in Metaphire 41 . Due to a lack of distinguishing morphological features, numerous reports of identical earthworm species are described using COI gene sequence data 25 .

Conclusion
In Pakistan, this is the first study to identify earthworms at molecular level and analyze their phylogeny with divergence time. The study provides genomic stamps to 11 species of pheretimoid earthworms: M. posthuma 02 , M. anomala 01 , M. houlleti 02 , M. californica 01 , M. birmanica 02 , A. minimus 01 , A. morrisi 01 and M. bununa 01 . The acquired data have supported the implementation of a hybrid taxonomy using classical morphology and modern DNA barcoding methods. Moreover, the phylogenetic tree has the monophyly relationship of all the species within both genera Metaphire and Amynthas by utilizing COI gene sequences. The evolutionary divergence time estimation results also revealed the diversification of pheretimoid species in modern taxa such as Amynthas, Metaphire, and Pheretima species during the late Miocene and Pliocene. However, a more detailed approach is required to examine additional samples to develop a robust taxonomy system for identifying pheretimoid earthworm species and construct a better resolved phylogenetic tree with molecular dating. It is also anticipated that mitochondrial COI sequences will help to resolve contrary taxonomy and investigate the diversity of earthworm pheretimoid in Pakistan.

Conflicts of Interest
The authors have reported that not a single person has a potential conflict of interest.