1 Introduction

Termites are members of the insect order Isoptera and are distinguished by their social behavioural traits. A termite colony typically includes a reproductive caste (a queen and a king) as well as workers and soldiers. Out of the 2650 identified termite species worldwide, around 300 species are serious pests that inflict significant damage to houses and timber structures (Edwards and Mill, 1986; Abe et al., 2000). Although various termite species assist in ecological processes, the cycling of nutrients, they are largely known for their economic importance and as a significant pest of agricultural crops (Ahmed et al., 2006). Most of termite research in the Arabian Peninsula, particularly in Saudi Arabia has reported by (Badawi et al., 1986).

DNA barcoding was proposed more than a decade ago as a quick, inexpensive, and simple method of identifying species based on the use of a short and unique gene region to generate the target specimens sequences (Hebert et al., 2003). Many taxonomists are impressed by DNA barcoding, which attempts to link a variety of biological samples with a specific portion of their DNA sequence, primarily the mitochondrial gene “ cytochrome c oxidase subunit I,“ also known as the COI gene (Ebach, 2011). Fragment size of COI has been shown to provide high resolution to identify cryptic species, thereby increasing taxonomy-based biodiversity estimates (Hebert et al., 2004) and its usefulness has been confirmed for several insect orders including Coleoptera (Löbl and Leschen, 2005), Diptera (Scheffer et al., 2006), Ephemeroptera (Ball et al., 2005), Hemiptera (Lee et al., 2011), Hymenoptera (Smith et al., 2008) and Lepidoptera (Hajibabaei et al., 2006).

Other molecular marker like, Random Amplified Polymorphic DNA (RAPD) has also been used to study genetic diversity like for order Neuroptera (Yari et al., 2014). DNA barcodes could aid in the routine identification of insects in applied settings by enabling the recognition of morphologically cryptic species, by associating immature forms with adults (pest management), and by identifying eggs (phytosanitary applications) and fragmentary remains (food quality, ecological analyses) (Park et al., 2011). A crucial evidence of DNA barcoding in animals is that genetic variation within species is lower than genetic variation among species (Hebert et al., 2003; Hebert et al., 2003; Yari et al., 2014). In other words, there is an existence of ‘barcoding gap’ which allow unknown specimens to be identified as an existing species or flagged as a putative undescribed taxon. The presence of global barcoding gap in birds, fish, butterflies (Hebert et al., 2003; Ward et al., 2005; Dincă et al., 2011) sometimes disregarding the importance of local barcoding gaps (Meier et al., 2008). DNA barcoding has proved to be particularly expedient in the study of taxonomically thought-provoking taxa, where morphology-based identifications are maddened due to cryptic diversity (Witt et al., 2006) or phenotypic plasticity (Adamowicz et al., 2004).

In fact, the Saudi termite fauna remains undiscovered, and there is no recent data in the literature. Identification of the pest is important for any successful integrated pest management program. The accuracy of species delimitation is critical to the identify and exploration of insect species. As a result, in present study cytochrome c oxidase subunit I (COI) based DNA barcode of A. ochraceus species of termites from different locations of Riyadh region have been analysed and significance of DNA barcode for accurate identification insect species has been discussed.

2 Materials and methods

3 Results

After the DNA extraction and PCR amplification all the amplified PCR products were analysed in 1% agarose gel electrophoresis and showed bright bands of approximately 700 bp size (Fig. 1). The agarose gel shows the samples purity and intensity. The sequenced samples accession numbers are presented in the Table 1.

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Fig. 1

PCR amplification of COI gene from sampled specimens of Anacanthotermes ochraceus resolved on 1% agarose gel electrophoresis.

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3.1

3.1 Phylogenetic analysis of Anacanthotermes ochraceus

COI gene sequences of 29 samples of A. ochraceus species were analyzed and barcode sequences were submitted into Genbank database with accession numbers ON529870-ON529872, ON529876-ON529877, ON529879-ON529887, ON529889-ON529892, ON682935-ON682937, ON682939-ON682946. Using basic local alignment search tool (BLAST), it was found that the studied samples belong to Hodotermitidae family of termites and there was no COI sequence of A. ochraceus species in the databases but those of two other member of the family Hodotermitidae i.e. Microhodotermes viator (EU253837) (Latreille, 1804) and Hodotermes mossambicus (EU253836) were retrieved from the NCBI database. COI gene sequence of a fruit fly Bactrocera zonata (Saunders, 1842) (MG770092) was used as an outgroup sequence. The sequences of closely species of termites were retrieved in fasta format. Those downloaded sequences along with present research samples sequences were subjected to multiple sequence alignment and phylogenetic tree was constructed using maximum likelihood algorithm. Phylogenetic analysis showed that all the specimens of present study grouped together into a single monophyletic clade, were confirmed to be highly closely related to one another, and proved to be members of the same species. Second closely related clade was the group of different members of genera Microhodotermes and Hodotermes which belong to the family Hodotermitidae. Species of genera Amitermes Silvestri, 1901 and Macrotermes Holmgren, 1910 form separate clades (Fig. 2). Pairwise sequence identity percentage analysis showed that studied samples barcode sequences show 88.5–100% nucleotide sequence identity with one another. This is showing highly conserved nucleotide sequence homology within the collected samples of the A. ochraceus species. Among the sequences retrieved from database M. viator (EU253837) showed highest nucleotide sequence identity of 88.9% with one of present research sample A. ochraceus because M. viator is the member of same family Hodotermitidae. Nucleotide sequence identity with members of other families of termites ranged 82.6–87.3% (Table 2).

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Fig. 2

Phylogenetic tree was created in MEGA 7.0 software using maximum-likelihood algorithm. The examination shows relationship of COI gene sequences of Anacanthotermes ochraceus with that of other closely related termite species from same family and other families of termites. All termite species used in the phylogenetic tree were labelled by their scientific name along with specific accession number. Numeric values at the base of each branch indicated the percentage of bootstrap value reiterated 1000.

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Table 2 Matrix of pairwise nucleotide sequence identity percentage of COI gene of 29 samples of Anacanthotermes ochraceus with other related sequences retrieved from NCBI database.

3.2

3.2 Intraspecific and interspecific sequence divergence

Degree of sequence divergence at intraspecific as well as interspecific level was studied using MegAlign software application of DNA Star (DNA Star Inc., Madison, WI, USA). A. ochraceus species, 29 samples were analyzed. Intraspecific sequence divergence among studied 29 specimens ranged from 0% to 7.8% indicating low divergence and highly conserved barcode sequence indicating all specimens belong to the same species. Interspecific sequence divergence between studied specimens and other species sequences retrieved from databases showed a high degree of divergence with a minimum interspecific value of 12% with M. viator (EU253837) (Table 3).

Table 3 Matrix of intraspecific and interspecific nucleotide sequence divergence of COI gene of 29 samples of Anacanthotermes ochraceus with other related sequences retrieved from NCBI database.

4 Discussion

Termites are abundantly present everywhere both in tropical and subtropical regions of the world and exhibit wide genetic diversity (Roy et al., 2006; Cheng et al., 2011). In the past, insect taxonomists were exclusively dependent on the morphological and histological features for the identification of the insect specimens which sometimes might be incorrect and which could lead to revisions of the taxonomic identification and classification of the same organisms. It is also important that specimen can be difficult to identify in different developmental stages (egg, larva, pupa and adult) of sample under study (Hussain et al., 2020). But this present era of the genomics and molecular biology has strengthened the field of taxonomy and systematic identification of species on the basis of DNA sequences (Seifert, 2009). The DNA barcoding has successfully delineated the species borders in insects proving itself a supportive technique for the accurate identification of specimen and discovering new species (Rasool et al., 2020; Sukirno et al., 2020; Wikantyoso et al., 2021; Zaman et al., 2022).

The present study includes 29 COI gene sequences which were amplified with LCO 1490 and HCO 2198 universal primers. These primers have already proved 100% success for insect DNA barcode amplification (Hebert et al., 2003). All the 29 specimens from the given study represented the A. ochraceus species. The sequence analysis of several termite specimens collected from different localities in the Riyadh region confirmed the morphological based identification of the species A. ochraceus, which were previously identified by Sharaf et al., (2021). DNA based identification of soldier caste of genus Coptotermes spp. has been done from Indonesia. The other mitochondrial genes 12S and 16S sequences were used to study phylogenetic relationships and genetic divergence among different species of genus Coptotermes (Wikantyoso et al., 2021). In line with these findings the 29 specimens of A. ochraceus analysis using maximum-likelihood algorithm for phylogenetic study showed highly conserved single clade indicating all specimens belong to same species confirming the morphological studies. Maximum likelihood algorithm has been previously used for phylogenetic relationships of termites by (Bourguignon et al., 2013). Similar results have been reported by Sharma et al., (2013) where several morphological and molecular studies of termites were performed and molecular data confirmed the morphological data. Moreover, the present pairwise nucleotide sequence identity as well as divergence percentage data also confirmed both phylogenetic and morphological information. All specimens identified as same species (intraspecific) showing higher nucleotide sequence identity percentage (88.5–100%) and lower divergence of (0–7.8%). On the other hand, interspecific nucleotide identity was as low as 82.6–87.3% and divergence was as high as 12%. Similar studies also confirmed the genetic divergence of termites at intraspecific as well as interspecific level (Cheng et al., 2011).

The habitat preference of A. ochraceus is remarkably diverse including the various niches in desert habitats (e.g. dead wood, dead date palm fronds, imported wood, dry grass, cartoon bags, dead Acacia, camphora, Cinnamomum and Tamarix, and sheep dung) (Cowie, 1989; Sharaf et al., 2021) which explains the wide geographical distribution records presented in the current study. It is likely that more species will be recorded from the Kingdom of Saudi Arabia by conducting surveys in poorly collected territories in different seasons and using various collection methods.

5 Conclusions

The specimens of termite species A. ochraceus already identified using morphological traits were revalidated deploying molecular analysis of a mitochondrial gene COI sequence. Molecular data analysis has confirmed morphological identification of all 29 studied samples of A. ochraceus. However, due to limited availability of data about COI barcode sequences about different species of termites in public databases may become hurdle in discovery of new species without the support of morphological data. However, this technology offers strong support for identification of cryptic species which are difficult to identify on the basis of morphological features. Further studies of complete mitogenome can be helpful for accurate identification of termites at species level.

Funding

This work was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdul-Aziz City for Science and Technology, Kingdom of Saudi Arabia (Award Number 13-BIO1410-02).

Ethical Statement

This article does not contain any studies with human participants performed by any of the authors.