Using molecular methods in the identification of ectoparasites has enabled the identification and evolution of species with high morphological similarities . The present study provided morphometric, phylogenetic, and molecular divergence comparative data between fleas C. canis isolated from Western and Northwestern Iran. The morphological characteristics of family Pulicidae fleas in this study were in agreement with previous studies [29, 21, 25, 12, 4, 7].
The host specificity might affect the level of intraspecific genetic divergence because generalist parasite species will show a higher level of intraspecific genetic variation, enabling them to infest a broader host range . Hornok et al. (2018). studying mitochondrial sequences demonstrated divergence in some synanthropic flea species such as C.felis and Pulex irritans. In our study, morphological data indicated that there was no difference between C. canis specimens from Western and Northwestern of Iran. The result of the morphological study was in line with the results of the molecular study. Nucleotide sequences of C. canis from different regions showed 100% identity with. C. canis in Turkey: KY865411.1 (NCBI), they also had 95.59 % identity with C. orientis in India (KX 467332.1), 95.32 % with C. orientis in Iran (MF380397.1), 93.48 % with C. felis in Iran (MF380394.1). This study is in agreement with the results of Seyyedzadeh et al. (2018) which observed that there were no differences between the isolates of C. canis samples of different regions of West Azerbaijan, Iran. The results also in agreement with those of Zurita et al. (2019) showed no significant difference between the morphological data of P. irritans in Spain and Argentina. There was a considerable degree of intraspecific similarity between both populations based on mitochondrial genes. Our results agree the studies of Hornok et al. (2018), who observed no morphological differences between human and wild carnivorous P. irritans specimens in Hungary and Croatia. In contrast, the studies of Krasnov et al. (2015) showed that fleas species isolated from different hosts in different geographic regions have morphological differences that can indicate a high level of genetic diversity. We concluded that the cox1 region is a useful marker to approach intraspecific similarity in C. canis and confirming its diagnosis.
A phylogenetic tree based on the similarity between our sequences with registered sequences in
GenBank showed 2 subclades for C. canis: one subclade including C. canis of recent study and C. canis from Turkey and the other with C. canis of Iran, Urmia and, Iran, Makoo. C. felis, C. orientis and P. irritans were in separate clades (Fig. 8).
Studies have shown that ITS1 and ITS2 are one of the best molecular markers for analyzing phylogenetic relationships at the species level in fleas [27, 19]. In our study, a comparison of nucleotide sequences of the ITS1 gene in five different provinces showed 99.85% similarity. Comparison of nucleotide sequences also showed single-nucleotide transversion at position 294, which caused the substitution of adenine for thymine in the isolate from Kuhdasht, Urmia, and Kermanshah. This result is in agreement with the results of Ghawami et al. (2018), which compared the nucleotide sequence of the ITS1 gene in P. irritans in two different geographic regions and showed only one nucleotide difference with 99.85% similarity. Our results are similar to Vobis et al. (2004) study, which observed that the nucleotide sequence of the ITS1 gene was relatively constant in different populations of C. felis. Therefore, it can be concluded that ITS1 is a useful marker for the diagnosis of Ctenocephalides genus.
A phylogenetic tree based on ITS1 registered sequences in GenBank showed 2 subclades for C. canis: one subclade including C. canis isolated in Kamiyaran, Sanandaj, Mahabad, Ghilangharb, Hamedan, Bahar and Khorramabad and the other C.canis which was isolated in Kuhdasht, Urmia, and Kermanshah. P. irritans and C. felis were in separate clades (Fig. 9).
The ITS2 nucleotide sequence in different sampled regions showed 96.64% similarity. There are five isolates found.
The results of the current study agree with the survey by Zurita et al. (2015), which showed intraspecific variation in four clones of C. canis based on the ITS2 fragment is between 90.1% and 100%. Vobis et al. (2004) also used the ITS2 sequence to illustrate intra-species differences and identify different species of fleas.
A phylogenetic tree based on ITS2 in the present study showed 4 subclades for C. canis: one subclade including C. canis isolate in Kuhdasht, Mahabad, Urmia, Khorramabad and Kermanshah, next subclade including isolate in Kamiyaran, Sanandaj, and Bahar. The isolates of Ghilangharb and Hamedan were in separate subclades. P. irritans were in a separate clade, and C. felis were in separate subclades (Fig. 10).