Combination of nuclear and mitochondrial markers as a useful tool to identify Ctenophthalmus species and subspecies (Siphonaptera: Ctenophthalmidae)

Abstract

Ctenophthalmus is considered the largest genus within the Order Siphonaptera. From a morphological point of view, only males of this genus can be identified at species and subspecies levels using morphological keys, whereas there are no morphological criteria in order to classify females at these taxonomical levels. Furthermore, the amount of available molecular and phylogenetic data for this genus is quite scarce so far. The main objective of this work was to assess the utility of the combination of nuclear and mitochondrial markers with respect to their ability to differentiate among different subspecies within the Ctenophthalmus genus. With this purpose, we carried out a comparative morphological and molecular study of three different subspecies (Ctenophthalmus baeticus arvernus, Ctenophthalmus nobilis dobyi, and Ctenophthalmus andorrensis catalaniensis) in order to clarify and discuss its taxonomic status. In addition, our study complemented the molecular data previously provided for Ctenophthalmus baeticus boisseauorum and Ctenophthalmus apertus allani subspecies. We sequenced five different molecular markers: EF1-α, ITS1, ITS2, cox1, and cytb. Our results confirmed that morphological data by themselves are not able to discriminate among Ctenophthalmus female taxa; however, the combination of the nuclear marker EF1-α together with mtDNA markers cytb and cox1 constituted a useful taxonomical and phylogenetic tool to solve this issue. Based on these results, we consider that the use of this molecular approach should be gradually used within Ctenophthalmus genus in order to complement its classical taxonomy and clarifying the complex taxonomy of other congeneric species of fleas.

Morphological, molecular and phylogenetic characterization of Leptopsylla segnis and Leptopsylla taschenbergi (Siphonaptera)

“…Additionally, based on cyt b phylogenetic analysis, we could observe a geographical signal in L. segnis between specimens collected from mainland of Europe and those collected from Canary Islands, Spain. This fact again supports the molecular divergence observed in flea's populations isolated from island, contributing to the appearance of cryptic species (Zurita et al, 2015; Zurita & Cutillas, 2021). In addition, the phylogenetic position of L. nana remains incongruent in this work due to it seems to cluster within Leptopsylla species in cyt b and cox 2 analysis, but it was placed out this group based on EF1‐α phylogenetic results.…”

“…Therefore, the use of female genitalia as a diagnostic criterion for Leptopsylla species diagnosis, at least between L. segnis and L. taschenbergi , should be discarded, whereas the study of male genitalia must remain as a useful tool for this aim. This fact has just been observed in the differentiation of female and male of genus Ctenophthalmus (Zurita et al, 2020; Zurita & Cutillas, 2021).…”

“…For that reason, if we want to infer a robust and feasible phylogeny, we should include multiple independent loci combining nuclear and mitochondrial data (Edwards & Bensch, 2009). This fact has been largely proved in flea's field as for genus and species level (Lawrence et al, 2019; Zurita & Cutillas, 2021) as family and higher stratum (Whiting et al, 2008; Zhu et al, 2015).…”

Fleas as Useful Tools for Science

“…For flea samples that are considered to have been inadequately preserved in 70% ethanol for long periods, Multiple Displacement Amplification (MDA) of siphonapterid DNA holds promise as a valuable tool for studies concerning taxonomy, phylogeny, and epidemiology [36]. The combination of nuclear and mitochondrial markers has also been proposed as useful to identify the two sexes of species of the larger genus Ctenophthalmus, considering that only males can be identified using morphological data [37].…”