Molecular Phylogeny of Walterinnesia aegyptia (Reptilia, Elapidae) Isolated from Ha’il Province, Saudi Arabia

Ahmed Mohajja Alshammari¹, Ahmed Badry², Bander Hamad Aloufi¹, Eman El-Abd^3*

¹Biology Department, Faculty of Science, University of Ha’il, Ha’il, Saudi Arabia.
²Department of Zoology, Faculty of Science, Al-Azhar University, Cairo, Egypt.
³Head of the Department of Radiation Sciences, Medical Research Institute, Alexandria University, Alexandria, Egypt.

DOI: 10.4236/ojapps.2022.1210113                

Abstract

Walterinnesia aegyptia is one of the most venomous snakes belonging to the family Elapidae found in the Middle East and Africa. In addition to its ecological importance, it is accused of millions of deaths due to snakebites. Because molecular identification of snakes is crucial for the antivenom drug industry, mitochondrial genes are used to identify, characterize, and infer genetic diversity among different venomous snake species. Data of Walterinnesia collected from samples across Saudi Arabia were compared based on the mitochondrial 16S and 12S rRNA sequences with other Elapidae related taxa to assess the phylogenetic relationship. The phylogenetic analysis strongly supports the monophyly of the genus Walterinnesia based on two genes that represent different species of Elapidae. In addition, a close relationship between Walterinnesia aegyptia and W. morgani was found. Our molecular data showed that W. morgani from Riyadh, Saudi Arabia, is nearly genetically identical (D = 0) with W. aegyptia from Ha’il and Riyadh, Saudi Arabia, and Sinai, Egypt. Further study is required based on more material and detailed morphological and genetic analysis.

Keywords

Elapidae, MtDNA, Phylogeny, Saudi Arabia, Walterinnesia aegyptia

1. Introduction

The family Elapidae consists of about 389 species, including the cobras, mambas, and sea snakes. In Saudi Araba, two species Walterinnesia represent it, which include mainly two species W. aegyptia [1], W. morgani [2], and its relative species, which belong to the Naja genus including one species reported in Saudi Arabia, the N. haje arabica [3]. The geographical distribution of the Arabian cobra (N. haje arabica) is the southwestern region while W. aegyptia belongs to the central, northern, and western regions of Saudi Arabia. However, W. morgani is distributed along with the Eastern parts of Saudi Arabia [4] [5] [6] [7] [8].

Snakebites represent a neglected health problem worldwide as well as in Saudi Arabia (https://www.who.int/news-room/fact-sheets/detail/snakebite-envenoming), [9] [10] [11]. In a recent review, 1688 snake bite cases were reported in Arab countries from 1983 to 2010 [12]. W. aegyptia envenomation causes respiratory failure and muscle paralysis in mice and humans, followed by rapid death when lethal doses of the venom are injected [12] [13] [14]. To the best of our knowledge, only two previous studies on phylogenetic analysis (using venom and/or 16S and 12S rRNA mitochondrial genes) of Walterinnesia and Naja snake species samples collected from across Saudi Arabia [15] [16]. Therefore, the present study aimed to determine the phylogenetic relationships in Walterinnesia and Naja species in Saudi Arabia using the mitochondrial 16S and 12S rRNA gene sequences.

2. Materials and Methods

2.1. Materials

Two samples of W. aegyptia were collected from Hail, Saudi Arabia, and identified morphologically according to Egan [17] and Alshammari and Ibrahim [6] (Figure 1 and Table 1).

2.2. Methods

2.2.1. DNA Extraction, Amplification, and Sequencing

DNA was extracted using the Axy Prep Blood Genomic DNA Miniprep kit (Axygen Biosciences, USA), amplified, and sequenced for the 12S and 16S rRNA genes as described by Alshammari [18]. The obtained sequences were analyzed and submitted to GenBank (Table 1). Additional sequences of W. aegyptia from Saudi Arabia and Egypt, as well as available data sequences for other species of genus Walterinnesia from Saudi Arabia (Table 1) were downloaded from GenBank. Additional sequences of other genera were retrieved from GenBank to investigate the phylogenetic relationships of Walterinnesia within Elapidae. Thermophis baileyi [19] was used as an outgroup.

2.2.2. Phylogenetic Analyses

Finch TV 1.4.0 was used to screen and analyze the sequences, which were aligned using Clustal W [20] in Mega 6 using the default settings [21]. The aligned 12S and 16S sequences were concatenated and combined into a single alignment using the Mesquite v3.2 software [22], and the nucleotide composition was calculated. To estimate the sequence divergence for the whole data set, genetic distances were calculated using Mega v.6. Phylogenetic analyses were

Figure 1. Records and collection records of Walterinnesia sp. in Saudi Arabia and North Africa. Sources of records are given in Table 1.

Table 1. Records of Walterinnesia sp. reported over the period from 1930 to 2022 and their accession number submitted to GenBank (from Saudi Arabia) (HUM = Ha’il University Museum).

performed on the combined data set (n = 19), as well as separate analyses on the individual gene was performed to determine the signal in the individual gene. The maximum parsimony (MP) and neighbor-joining (NJ) analyses were performed with Paup v.4 [23] with heuristic searches using stepwise addition followed by tree bisection reconnection (TBR) branch swapping [24]. In all alignments, gaps were treated as missing characters. Confidence within the nodes was evaluated using 1000 bootstrap replicates [25] [26] with random addition of taxa. MrModeltest 2.3 [27] was used to select the best-fit models of nucleotide evolution supported by Akaike information criterion (AIC) [28]. The geographic structure was inferred using Bayesian inference (BI) implemented with MrBayes 3.1.2 [29]. Analyses were run for one million generations and the output parameters were visualized to determine stationarity and convergence using Tracer 1.4 [30].

3. Results

Across all combined sequences, there were 799 aligned nucleotides. Of these, 655 bases (80.9%) were constant; 144 (18.0%) were variable, and 89 (12.2%) were parsimony informative. Within the 799 bp, 141 polymorphic segregating sites were detected. Divergence among Walterinnesia and different Elapidae related taxa ranged from 0 to 0.06 (Table 2). For the 16S rRNA fragment, there were 468 aligned sites, of which 323 (69.0%) bases were constant, 142 (30.3%) bases were variable, and 111 (23.7%) were parsimony informative. The mean values of T, C, A, and G within the sequence data were 23.6%, 23.6%, 34.1%, and 18.7%, respectively. Within the 468 bp, 31 polymorphic segregating sites were detected. The sequence divergences among Walterinnesia and Elapidae related taxa lineages ranged from 0.05 to 0.05 as represented in Table 2. For the 12S rRNA fragment, there were 390 aligned sites, of which 258 (66.1%) bases were constant, 123 (31.5%) bases were variable, and 97 (24.8%) were parsimony informative. Within the 413 bp, 30 polymorphic segregating sites were detected. The mean values of T, C, A, and G within the sequence data were 19.8%, 25.0%, 36.9%, and 18.3%, respectively. Within the 390 bp, 26 polymorphic segregating sites were detected. The sequence divergences among Walterinnesia and Naja sp. lineages ranged from 0.00 to 0.07 as shown in Figure 2-4 and Table 2. The tree topologies were improved based on the 12 S rRNA and 16 S rRNA data sets. The NJ and MP analyses were comparable in defining two main clades. The first clade includes all taxa that belonged to the family Colubridae. Another clade encompassed all taxa belonging to the family Elapidae. In addition, the phylogenetic analysis revealed the monophyletic status of the genus Walterinnesia based on the 12s rRNA gene and the paraphyletic status between the genus Walterinnesia and Naja.

4. Discussion

The Saudi Arabia landscape has an extremely diverse topographic and bioclimatic profile resulting in the development of numerous habitats of diverse fauna and flora that is a blend of Indomalayan, Palearctic, and Ethiopian species [31] [32].

W. aegyptia, is known as the desert black snake or desert cobra. This species’ geographical distribution includes northern and southern Saudi Arabia, southern Israel, western Jordan, and possibly Lebanon [5] [33]. Our data showed a generally well-supported phylogenetic hypothesis for the Walterinnesia and different Elapidae snake species across Saudi Arabia. This provides the raw material for a discussion of the evolution of spitting in cobras and long-standing taxonomic issues. Phylogenetic analysis using 16 S and 12 S mtDNA gene sequences has produced distinctive clades for Walterinnesia and Naja snake species.

The phylogenetic analyses (NJ, MP and PI) weakly support the monophyly of the genus Walterinnesia, based on two combined genes representing different species of Elapidae taxa (Figures 2-4). When analyzed separately, the 12S rRNA and 16s rRNA genes support the monophyly of Walterinnesia, Naja, and Aspidelaps. However, the 16S rRNA gene showed a sister relationship between Walterinnesia and Aspidelaps (Supplemental material Figure 1, Figure 2). Wüster

Table 2. Uncorrected pairwise distances among Walterinnesia samples based on concatenated mitochondrial 12S rRNA and 16S rRNA sequences. Standard error estimates are shown above the diagonal. SA = Saudi Arabia.

Figure 2. Maximum parsimony phylogenetic tree of the Walterinnesia species and Elapidae related taxa based on the concatenated mitochondrial 12S rRNA and 16S rRNA sequences. The number above the branches indicates bootstrap values.

Figure 3. Neighbor-Joining phylogenetic tree of the Walterinnesia species and Elapidae related taxa based on the concatenated mitochondrial 12S rRNA and 16S rRNA sequences. The number above the branches indicates distance values.

et al. [34] confirmed the strongly supported monophyletic relationship between the core cobra clade, which includes Aspidelaps, Boulengerina, Hemachatus, Naja, Paranaja, and Walterinnesia. In addition, the two genera Walterinnesia and Aspidelaps formed two basal lineages to the other members of the cobra clade. However, the sister–group affinities between the above genuses remained uncertain [34] [35] [36] [37]. Our data strongly support the sister relationship between Walterinnesia and Aspidelaps based on the 16S and 12S rRNA genes.

Within the Walterinnesia, there is a strong support for the sister relationship between W. aegyptia and W. morgani based on 16S rRNA and 12S rRNA genes. The two species are closely similar and are related in their external morphology. W. morgani can be distinguished from W. aegyptia by its lower number of subcaudal and ventral scales in both sexes; a lower number of united subcaudals in females, by having a juvenile pattern of reddish crossbars on the back, and lower average ventral and subcaudal scale counts [4]. This information was originally described as W. aegyptia [1] from Egypt, or as N. morgani [2] or Atractaspis wilsoni [19] from Iran and they have appeared in the literature over time. Subsequently, the eastern populations represent a different species, W. morgani [4].

Figure 4. Bayesian inference tree of the Walterinnesia species and Elapidae related taxa based on the concatenated mitochondrial 12S rRNA and 16S rRNA sequences. The number above the branches indicates bootstrap values.

5. Conclusion

Our molecular data showed that W. morgani from Riyadh, Saudi Arabia, is nearly genetically identical (D = 0) to W. aegyptia from Ha’il and Ryiadh, Saudi Arabia, and Sinai, Egypt. As a result, it is certain that the samples identified as W. morgani from Riyadh, Saudi Arabia [16] are conspecific with W. aegyptia. Additional research based on more data and thorough morphological and genetic analysis is needed.

Acknowledgments

I would like to thank Dr. Ali Al-Odaib, Dr. Brian Meyer, Dr. Dorota Monies, and Mr. Mohamed Rajab (King Faisal Specialist Hospital & Research Centre, Riyadh, Kingdom of Saudi Arabia) for their assistance in providing the sequencing services.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

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