Genetic variations and phylogenetic relationship of genus Uromastyx from Punjab Pakistan

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract During the present study, specimens were collected from selected sites of Cholistan desert and Kalabagh Game Reserve, Punjab province, Pakistan. Each captured specimen was tagged with voucher number and morphometric measurements were taken. The average snout to vent length was 172.559±1.40 mm and average weight was 92.1±1.30 g. The DNA of Uromastyx hardwickii was amplified and sequenced using 16S rRNA primer set. The obtained DNA sequence has shown reliable and clear species identification. After trimming ambiguous bases, the obtained 16S rRNA fragment was 520 bp while 16S rRNA fragments aligned with closely matched sequence from NCBI comprised of 510 bp. Closely matched sequences of genus Uromastyx were retrieved from NCBI in blast searches. Neighbour-joining tree of genus Uromastyx was constructed based on p-distance using MEGA X. The mean intraspecific variation was 0.095±0.01 while intraspecific variation was ranging from 0-1%. Similarly, interspecific variation of Uromastyx hardwikii with Saara asmussi, Uromastyx alfredschmidti, Uromastyx geyri, Uromastyx thomasi, Uromastyx alfredschmidti was 0-12%, 0-19%, 0-19%, 0-20%, 12-19% respectively. The newly produced DNA was submitted to NCBI and accession number was obtained (MW052563.1). Results of current study provided information about the molecular and morphological identification of Genus Uromastyx. In our recommendation, comprehensive molecular based identification of Pakistan’s reptiles is required to report any new or subspecies from country.

the basis of external morphology and heir phylogenetic relationship is still under debate (Amer and Kumazawa, 2005;Wilms and Schmitz, 2007). Pakistan is represented by two species of genus Uromastyx namely Uromastyx hardwickii and Uromastyx asmussi. Present study was therefore planned to identify members of genus Uromastyx through mitochondrial genes and sort out taxonomy problems of these taxa.

Sample collection
A total of 10 specimens are collected from selected sites of Cholistan desert and Kalabagh Game Reserve, Punjab province. Each captured specimen was tagged with voucher number and morphometric measurements were taken following Ali et al.(2017). A few specimen of each sampling site (n=3) were euthanized and preserved in 75% ethanol for molecular characterization.

DNA extraction and amplification
DNA was extracted using phenol chloroform method . The quality of DNA was checked on 1.5% agarose gel in the post-graduate lab, Department of Wildlife and Ecology, University of Veterinary and Animal Sciences, Pakistan. The amplification was done by using cytochrome oxidase I (COI) and 16S rRNA primers sets. PCR reaction was performed in 0.2 ml PCR tube and 25µL reaction mixtures. To prepare 25 µL reaction mix 6 µL double distilled water, 1 µL (25 mM) Primer F,1µL (25 mM) primer R, 12 µL Master mix and 5 µL of DNA template was mixed. In each reaction a negative control was also run using sterilized water as the template. The following steps were performed forthe amplification of gene, 3 minutes denaturing at 94ºC followed by 40cycles of for 30 sec at 94ºC, primer annealing for 30sec at 42-55ºC depend on the primer's annealing temperature and elongation for 1 minutes at72ºC, with final 10 minutes at 72ºC and infinity at 4ºC Vences et al., 2005). PCR products were checked on 1.5% agarose gels and products were purified by using the Qiagen purification kit and all the samples were sequenced in both directions using dideoxy chain termination direct Sanger sequencing on ABI3730XL DNA Analyzer. The newly obtained DNA sequences were submitted to Gen bank for accession numbers.

Data analysis
The obtained DNA sequences were analyzed and edited in Bioedit software 7.0 and aligned using Clustal X

Introduction
The changes that reptiles faced over millions of years results in massive diversity in their morphology, behavior, ecology history of life and in strategies against their predators. Due to all these features reptiles in terms of evolutionary and ecological research now become the central model system (Rasmussen et al., 2011). We used the term paraphyletic group for reptiles which include all non-avian taxa such as Sphenodontia, Squamata, Testudines and Crocodylia in this group. The total numbers of living reptile species are approximately 9546, out of these the crocodiles are 25 (0.3%), turtles are 327 (3.4%) and tuatara is 1 (0.01%) and the remaining 9,193 (96.3%) are the squamates species (lizards, snakes and amphisbaenians) (Hay et al. 2010).
The identification and taxonomic classification of reptiles is very challenging task. Recently, a molecular technique which is used for the identification is known as DNA barcoding which is used for finding phylogenetic relationships. Before 2005 DNA barcoding of amphibians and reptiles was very difficult as compared to other taxa such as fishes, birds and mammals (Hebert et al., 2004). In DNA barcoding, a short sequence of DNA is compared with reference database for the identification of species (Hajibabaei et al., 2007). The genomic regions that are mostly used include 12S rRNA, 16S rRNA, Cytb and COI consisting on 600-1200 bp (Hebert et al., 2003;Nagy et al., 2012).
Habitat loss and illegal trading have effect the survival and existence of many reptile species worldwide especially in Pakistan. One of them is Indian spiny-tailed lizard (Uromastyx hardwickii). The U. hardwickii is medium to large size lizard reported from North Africa to North Western Indian desert. Spiny tailed lizard is ground dweller and herbivorous. In Pakistan, it is distributed in Southern Baluchistan, Indus valley, extended to Las bela and Chagai desert. The major threats to this species are its commercial exploitation for meat, skin and oil. About 367,000 specimens were legally traded between 1977 and 2005 (Knapp, 2004).
Genus Uromastyx is represented by 17 species. Most of species within genus Uromastyx were identify on Tamura et al., 2013).The consensus sequence of each sample were subjected to BLAST (basic local alignment tool search) analysis to compare the percentage identity of sequence by searching in the public DNA databases. The closely related sequences were obtained and incorporated in the neighbor-joining (NJ) tree analyses with bootstrap value of 100 replicates using MEGA 10. Genetic distances within and between species were calculated using Mega 6.0 based on p-distance.

Results
The specimens of Uromastyx hardwickii (Figure 1) were collected from selected sites of District Bahawalnagar and Kalabagh Game Reserve, Punjab province, Pakistan ( Figure 2) during field surveys extended from May to August, 2020.
The description of captured specimens from study area is as follows;

I. Genus Uromastyx (Merrem, 1820)
The subfamily Uromastycinae consist of two genera Saara Gray, 1845 and Uromastyx Merrem, 1820 Within the Uromastycinae. Uromastyx is the largest genus its habitat is desert and semi desert area with the passage of years it's taxonomy has been changed since 1980 it include four species and three subspecies. The members of Uromastycinae are commonly called spiny tail lizard as their tail is covered with spines. On the basis of presence and absence of inter-calaries between the tail and whorls two genera saara and Uromastyx is differentiated. Pakistan is represented by two species of genus Uromastyx namely Uromastyx hardwickii and Uromastyx asmussi. i. Uromastyx hardwickii Uromaastyx hardwickii commonly called spiny tailed lizard and found in dry regions of northwest India and Pakistan also found in Afghanistan its maximum SVL is 233 mm. Around the mid body 190-275 scales are found and 24-42 scales are present near the ear opening on the fourth left toe 15-21 scales are present. On the either side 12-19 pre-anofemoral pores are present. Between the gular-and inguinal fold 112-157 scales are present. The U. hardwickii is medium to large size lizard reported from North Africa to North Western Indian desert. Spiny tailed lizard is ground dweller and herbivorous. In Pakistan, it is distributed in Southern Baluchistan, Indus valley, extended to Las bela and Chagai desert. The major threats to this species are its commercial exploitation for meat, skin and oil. About 367,000 specimens were legally traded between 1977 and 2005.

Amplification and sequencing
During present study, DNA of Uromastyx hardwickii was amplified and sequenced using 16S rRNA primer set. The obtained DNA sequence has shown reliable and clear species identification. After trimming ambiguous bases, the obtained 16S rRNA fragment of Uromastyx hardwickii was 520 bp while 16SrNRA fragments aligned with NCBI sequences comprised 510 bp ( Table 2). The newly produced DNA was submitted to NCBI and accession number was obtained (MW052563.1).

Phylogenetic analysis
Recently few DNA barcoding studies of Asian amphibians and reptiles have been carried out and sequences for related species were available at NCBI. Closely matched sequences of genus Uromastyx were retrieved from NCBI in blast searches. Neighbour-joining tree of genus Uromastyx was constructed based on 16S rRNA sequence using MEGA X (Figure 3). The analysis involved 11 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were a total of 510 positions in the final dataset.

Discussion
The information related to DNA barcoding of reptiles is very less as compared to other animals. The genes that are used for this purpose is Cytochrome C Oxidase I (COI), cytochrome b (Cytb), 12S rRNA and 16S rRNA (Nicolas et al., 2012;Xia et al., 2012). In the past due to the problems that researcher faced in case of COI sequences amplification and evaluation of reptiles and amphibians prefer to use the 16 S ribosomal RNA gene for DNA barcoding. Vasconcelos et al. (2016) used COI gene to find the intra-specific of reptiles which was ranging between 13.8-54.4%. Sharma et al. (2018) isolate the DNA from the feces of Indian Spiny Tailed Lizard Saara hardwickii. They carried out research in Rajasthan in June 2016 and used the 16S rRNA gene in order to find out genetic variation in S. hardwickii. Phylogenetic relationship indicated that due to illegal trading, India and  global samples shared ancestry as at seven bp insertion (126-128) and deletion present at 2(49-252). Arida (2017) used different mitochondrial DNA regions for the identification of monitor lizard in Indonesia. Previous Studies on the Varanus genus shows that the mitochondrial markers that were used on the biogeography and on systematics were 12S rRNA and 16S rRNA. The combination of nuclear and mitochondrial genes also used by others (Ziegler et al., 2007;Fuller et al., 1998;Fitch et al., 2006;Douphty et al., 2014;Francis et al., 2010;Nagy et al., 2012). Adam et al. (2009) used mitochondrial DNA sequences of about 1,181 bp to described phylogeny of nineteen species of African lizards. The species of East and West Africa has monophyletic radiations which were supported along with a clade that contain 2 species of sahel region.

Conclusions and recommendations
During present study, different mitochondrial genes COI and 16S rRNA were used to identify genus Uromastyx from study area. The DNA of Uromastyx Hardwikii successfully amplified using 16S rRNA primer set. Results of current study provided information about the molecular and morphological identification of Genus Uromastyx. Comprehensive molecular based identification of Pakistan's reptiles is required to report any new or subspecies from country.