COI-Based DNA Barcoding in Pakistan: Progress, Challenges, and Future Directions

COI-Based DNA Barcoding in Pakistan: Progress, Challenges, and Future Directions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Systematic Review COI-Based DNA Barcoding in Pakistan: Progress, Challenges, and Future Directions Sohail Anjum, Ikram Ilahi, Qaiser Zaman, Muhammad Salman Khan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7936079/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract DNA barcoding using the cytochrome oxidase I (COI) gene has revolutionized species identification and biodiversity research in Pakistan since 2006. This review evaluates the progress, trends, and challenges of COI-based DNA barcoding across various taxa, focusing on taxonomic coverage, genetic divergence, sampling methodologies, publication trends, and phylogenetic analyses. A total of 120 articles were downloaded, of which 91 were retained based on strict relevancy. Research efforts are unevenly distributed, with insects, particularly mosquitoes and fruit flies, receiving the most attention, followed by economically significant freshwater fish like Cyprinidae. However, mammals, amphibians, marine organisms, fungi, and microbes remain largely underrepresented, highlighting the need for broader taxonomic studies. Geographic coverage and sample sizes vary widely, affecting statistical reliability and species representation. Methodological inconsistencies, such as unreported collection sites and varying trapping techniques, limit reproducibility and comparative analysis. Genetic divergence data reveal inconsistencies, with conspecific distances typically between 0–2% but sometimes reaching extreme values (e.g., 67.10%), suggesting cryptic species or sequencing errors. Congeneric distances also vary significantly, emphasizing the need for taxon-specific barcoding thresholds instead of a universal cutoff. Phylogenetic analyses predominantly use MEGA software, with Neighbor-Joining and Maximum Likelihood methods being most common, while Bayesian inference remains underutilized. The publication trend was slow from 2006 to 2012 but showed steady growth from 2013 to 2021 and a sharp rise from 2022 onwards due to increased funding and technological advancements. Research is mainly published in international journals, with some contributions in national journals like the Pakistan Journal of Zoology. To enhance DNA barcoding in Pakistan, improvements such as expanded taxonomic and geographic coverage, standardized methodologies, increased data sharing, and integration with multigene approaches are necessary. Addressing these gaps will improve the accuracy and global relevance of COI-based DNA barcoding, supporting better conservation and sustainable management of Pakistan’s biodiversity. DNA barcoding COI gene Biodiversity Species identification Pakistan fauna Genetic divergence Review Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction DNA barcoding has revolutionized the field of taxonomy and systematics, enabling rapid and accurate identification of species. In the DNA barcoding approach, which is the use of a short and standardized fragment of DNA, such as the COI gene for species identification. It has facilitated the discovery of new species, invasive species, and monitoring of threatened populations(Antil, Abraham et al. 2023 ). Pakistan is blessed with significant faunal diversity, which further needs acceleration in DNA barcoding attempts (Baig and Al-Subaiee 2009 ). As Pakistan is located at the crossroads of the oriental and Palaearctic zoogeographic regions (Mirza 1975 ), it boasts a unique diversity. Tropical and temperate forests, deserts and grasslands, mountains and water bodies are rich with species both in diversity and richness. Despite the rich diversity, Pakistan faces challenges that hinder the application of DNA Barcoding (Joly, Davies et al. 2014 ). The limited availability of taxonomic expertise, limited resources, and lack of infrastructure limit the use of DNA barcoding. Furthermore, this biodiversity is facing threats like the destruction of habitat, climate change, overexploitation of resources, and pollution. These challenges can be covered with the valuable tool of DNA barcoding, lessening gaps and providing opportunities like cut off laboratory facilities at national level. International collaboration and knowledge sharing enables researchers to compare species from different regions. Globally, researchers have made considerable progress in applying COI-based DNA barcoding for species identification (Emerson 2025 ), however, research efforts in Pakistan remain limited and fragmented. Large portions of country’s rich fauna are still uncharacterized and limited to few taxonomic groups. Moreover, there is a lack of comprehensive assessment on the DNA barcoding status initiatives in Pakistan. Furthermore, little information is available on taxonomic coverage, institutional contributions and publication trends. The absence of coordinated research networks and centralized databases has further widened the gap. Therefore, these gaps highlight the need for a systematic review that compiles current knowledge, identifies achievements and limitations, and outlines future directions for strengthening DNA barcoding research in Pakistan. This study is needed because it brings together, for the first time, a systematic overview of COI-based DNA barcoding in Pakistan. Additionally, it documents history, limitations and achievements, and emerging trends in DNA barcoding. Earlier studies focused on isolated groups or limited to restricted locations; this review provides broader national-level perspective. Moreover, no published review study is available on COI-based DNA barcoding from Pakistan. The current study is review-based article which compiles, synthesizes and critically analyses published research between 2006 and 2025 on COI-based DNA barcoding in Pakistan. This review aims to provide a comprehensive overview of history, progress, challenges and limitations on COI-based DNA barcoding in Pakistan, with the focus of guiding future research priorities and acceleration of use of DNA barcoding as a tool for conservation of biodiversity and management in Pakistan. Materials and Methods The current review study has focused the COI-based DNA barcoding in Pakistan. The first published article is from the year 2006 which then stretched over the years to 2025. The data reveals that the COI-based DNA barcoding accelerated over the years. The study solely focused on articles published on DNA barcoding, specifying COI as gene of choice. The geographic focus is on Pakistan, including articles that barcoded animals from different regions including Provinces of Sindh, Balochistan, Punjab, Khyber Pakhtunkhwa and Gilgit Baltistan, further explored in the following sections. In the current review, a total of 120 articles were downloaded from research sites such as Google Scholar. Out of total, twenty-one articles were excluded because they were either on Cyt-b gene or 12S rRNA genes. Furthermore, some articles solely focused on review of a single animal and, finally, only 91 articles were retained for further study. The whole data was summarized in Microsoft Office Excel Sheet. Tables were also designed using the same Excel Sheet due to it’s easy to use. Figures were designed using R and GIS packages. Progress over the years DNA barcoding has evolved significantly over the past two decades in Pakistan. A closer examination reveals a development from an initial slow adoption to a recent surge in research activity ( Figure 1 ). It can be further observed through three main stages of development as following. Slow Progress (2006–2012) The first publication in the current dataset goes back to 2006 (Memon, Meier et al. 2006), marking the introduction of DNA barcoding in Pakistan. After that, no study appeared until 2010 (Ashfaq, Noor et al. 2010), suggesting isolated early efforts and large gap ( Figure 1 ). A lack of widespread consistency and very limited institutional support during this era can be observed, for example, evidenced from a study (Iftikhar, Ashfaq et al. 2016). Gradual Growth (2013–2021) The frequency of publications increased soon after 2013, with intermittent research activity leading to 2016 with a modest rise in output with five publications. The period shows an increase in interest in DNA barcoding, likely driven by global recognition in biodiversity conservation and species delimitation. During the years between 2017 and 2021, the output remained steady with multiple publications for each year. This duration shows the prominent research focus on DNA barcoding in Pakistan. Institutions and researchers recognized the importance of DNA barcoding during this phase. Rising Research Trends (2022-2025) The dataset shows a sharp increase in publication for 2022 and onward. Peak publication occurred during 2023 and 2024 ( Figure 1 ), witnessing to be the most productive years. This is possibly fuelled by increased funding, greater understanding of taxonomic and ecological applications and an advancement in sequencing technologies. The dataset marks the year 2024 an all-time high in research output with ten plus publications, while the momentum is being sustained for 2025 (Anjum, Ilahi et al. 2025) indicating a robust and expanding research community in the field. Factors Affecting Growth Several factors have aided to the recent acceleration in DNA barcoding in Pakistan. Bioinformatics tools and sequencing platforms have likely facilitated this domain. It further shows the increase in government and private funding to this sector as shown in funding section of many articles. Canadian Centre for DNA barcoding is credited with global influence and collaboration in Pakistan. Finally, as Pakistan is blessed with rich biodiversity (Baig and Al-Subaiee 2009), therefore, conservation efforts need rapid identification which is a growing factor for accelerated studies. This shift from foundational studies to more applied research indicates a promising future for molecular taxonomy and biodiversity assessment in the country (Akhtar and Ali 2016). Further investigation into specific research themes, institutional contributions, and funding trends would provide deeper insights into the progress and future trajectory of DNA barcoding research in Pakistan (Ruedi, Manzinalli et al. 2023). Research Across Journals The publication trend as viable from the dataset highlights the diverse range of journals as a closer look reveals key insights into the scope, impact and reach of DNA barcoding research in the country ( Table 1 ). International Dominance Internationally recognized high impact journals have published a significant portion of articles on DNA barcoding. A well regarded open-access platform PLOS One appears frequently, indicating it to have accepted well reputed research studies. Overview shows that PLOS One accepts articles with greater sample size (Ashfaq, Hebert et al. 2014, Iftikhar, Ashfaq et al. 2016). PeerJ, Scientific Reports, Frontiers in Physiology and Molecular Ecology Resources are the most appearing journals with high number of publication in DNA barcoding from Pakistan, helping in increasing visibility (Ashfaq, Khan et al. 2022). Other journals such as Mitochondrial DNA part A and B, and Molecular Biology Reports reflect strong focus in these studies. It is also evident that Mitochondrial DNA like journals should focus acceptance on DNA barcoding, a central theme of mitochondrial genome. National and local journals have contributed well to the research on DNA barcoding as clear from the inclusion of articles in Pakistan Journal of Zoology, Punjab University Journal of Zoology, Journal of Bioresource Management and others ( Table 1 ). Multidisciplinary and Applied Research Trends Some journals like Applied Ecology and Environmental Research, Journal of Freshwater Ecology and Journal of Materials and Environmental Science highlights the importance of DNA barcoding beyond pure taxonomy (Khan, Ali et al. 2023, Anjum, Ilahi et al. 2025). These journals have addressed ecological and environmental concerns such as species conservation, assessment of ecosystem health and biodiversity monitoring. More wide fields like veterinary sciences, livestock and agriculture are also on the focus as barcoding is used in these field while evidenced from publications in Journal of Applied Research in Plant Sciences and Frontiers in Veterinary Sciences (Ali, Khan et al. 2024) . The presence of greater data accessibility on open access platforms like Research Square suggests that data presentation is also necessary for broader studies within scientific community. (Asif, Naseem et al. 2023) Publication trends indicate that DNA barcoding should be a focus of interdisciplinary journals to be fruitful for wider scientific community. Both national and international journals are paying greater focus to DNA barcoding research in Pakistan ( Table 1 ). A shift toward biodiversity conservation, ecology and agriculture is an indicator of practical applications in the fields. Notably, the assessment of journal metrics like impact factor and citation metrics should be carried out by institutions to understand how Pakistan’s research is influencing global barcoding studies. This rapid growth should also be assessed to work out on the funding sources and affiliation of publications. Sources of DNA Extraction Two important aspects of DNA barcoding are the dependence of success on quality and reliability of DNA extracted from biological samples. In the current review, Table 1 summarizes diverse ranges of organs and tissues that have been observed for extracting DNA. The variation in samples is a reason for species type, objectives of research and ethical considerations. Arthropod Studies; Dominance of Leg Samples The dataset shows single or multiple legs of arthropods as the primary source of DNA ( Table 1 ). This organ yields sufficient genetic material for analysis while is nonlethal for insects. Many of the studies shows that only one or two legs of adults were used, an indication of minimum species damage and preservation of species for morphological assessment, a way to integrate genetic and morphological methods for identification. Vertebrate Studies: Muscle and Whole-body samples Muscles, dorsal muscles tissue, muscle pieces and pectoral fins were terms used by different articles for DNA barcoding. Fish, amphibians and mammals are shown to be barcoded from source taken from muscles. Insects, larvae and other certain aquatic species have been barcoded with tissues from the whole body. Some studies have used whole body except removal of 1cm of anal region, a possible focus to ensure uncontaminated DNA samples. Mammalian and Avian Barcoding: Blood and Internal Organs Several studies have utilized blood samples for DNA barcoding in birds and mammals(Rehman, Jafar et al. 2015, Ali, Rehman et al. 2016). It is a non-invasive or minimally invasive way of sampling. Many studies have used internal organs like spleen, liver and tail tip which possibly involve integration of disease studies, population genetics and molecular diagnosis. Chiropteran biodiversity has been taken into consideration extensively as evidenced from the DNA extraction from patagium and hind leg tissue samples, specific for this group. Keel tissues and feather follicles are extensively reported from birds, the later one shows the non-invasive use of birds for barcoding studies. Aquatic and Reptilian Species: Fins, Scales, and Skin Samples Studies involving fish and reptiles have been reported with DNA extraction from fins, scales and skin. DNA barcode studies have extensively used caudal and pectoral fins because it provides high quality DNA. Skin and scale samples are good sources of DNA if found fresh with less or no harm to species. Unspecified and Non-Traditional DNA Sources In the current review, a notable portion of studies have not provided DNA source, highlighting a potential gap in reporting and standardization of DNA barcoding ( Table 1 ) (Awan, Umar et al. 2013, Sajid, Zahid et al. 2021). Moreover, DNA extraction was carried out from animal derived products like tanned skins, coats and fur samples, ensuring the focus on illegal wildlife trade (Janjua, Fakhar-I-Abbas et al. 2017). Some studies have utilized faecal samples, hair and skin for DNA, which is a good application in non-invasive genetic monitoring of mammals (Naseem, Batool et al. 2020). In conclusion, a diverse range of sources have been used for DNA barcoding in Pakistan, including fins and scales from fish, muscles from vertebrates, legs from arthropods, coats and tanned skins from wildlife species ( Table 1 ). A more robust focus in DNA extraction should be the use of faecal matter, hair, blood drops etc for non-invasive sampling techniques. Primer Selection The effectiveness of COI gene as a standard for DNA barcoding largely depends on the selection of universal primers. Several primers utilized in Pakistan are being discussed in the text that follows. Universal Primers are designed to facilitate DNA barcoding across a broad range of taxa (Ullah, Ahmad et al. , Ali, Bukhari et al. 2024). These primers ( Table 5 ) were originally developed for arthropod’s barcoding while now applied extensively to fish, reptiles, insects and mammals because of high success. Some primers are used for samples that have degraded DNA such as museum specimen or forensic samples (Hussain, Bukhari et al. 2025). Lepidoptera is one of the most diverse orders of class insecta. Lepidopteran primers have yielded good results in moths and butterflies ensuring reliability in amplification and sequencing (Sajid, Zahid et al. 2021, Naz, Chatha et al. 2023) ( Table 5 ). In Pakistan, two sets of primers used successfully for DNA barcoding in Pakistan for fishes (Karim, Saif et al. , Sajjad, Jabeen et al. 2023). Birds, the drivers of ecosystem have been a focus of DNA barcoding in Pakistan. Several species have been identified and analysed using bird primers given in Table 5 (Awan, Umar et al. 2013). Mini barcodes are special primers which is the focus for museum specimen, environmental DNA (eDNA) and forensic studies (Khan, William et al. 2018). A known successful primer for degraded DNA barcoding is presented below. Notably, this primer is ideal for shorter DNA sequences. Specific primers designed for identification and characterization of sea turtles (Ali, Bukhari et al. 2024) are also the key focus of turtle-based studies. Some of the primer sets are specialized for only a known species and no universal study have declared them to be universal or applicable to other groups. Moreover, many of the studies have designed primer sets for their own. These were designed with Primer3 application (Akhtar and Ali 2016). The selection of primers plays a crucial role in the success of DNA barcoding. Universal primers like LCO1490/HCO2198 remain widely used across taxa, while group-specific primers (e.g., FishF1/FishR1 for fish, LepF1/LepR1 for Lepidoptera) offer higher amplification success for organisms. Mini-barcoding primers such as UniMinibarF1/UniMinibarR1 have expanded the application of DNA barcoding to degraded samples (Meusnier, Singer et al. 2008). The continuous development and optimization of primers will further enhance the effectiveness of DNA barcoding in taxonomy, biodiversity monitoring, and conservation efforts. Phylogenetic Models and Softwares One of the purposes of DNA barcoding is to find evolutionary linkages between species and to study their phylogenetic relationships. Several studies have used software packages and models for assessing evolutionary relationships and phylogenetic tree construction summarized in Figure 2 . Each of these has their own strengths and weaknesses. In the current review, the used packages include MEGA (various versions), Bayesian Inference, DNAStar, DnaSP, DNAMAN, and Fast Minimum Evolution Algorithm, along with phylogenetic models like Jukes-Cantor and Neighbor-Joining (NJ) methods illustrated in Figure 2 . MEGA Software In the current dataset MEGA is one of the most used software with multiple versions including MEGA 5, MEGA 6, MEGA 7, MEGA X, and MEGA 11(Kumar, Tamura et al. 1994). This software package is labelled with several user-friendly options. Firstly, it is user friendly, with a graphical user interface that is easy to navigate making it easy for beginners. Secondly, it supports comprehensive features like multiple sequence alignment, construction of phylogenetic tree, evolutionary model selection and provides ways for statistical validation. Thirdly, MEGA supports and offers multiple algorithms like Maximum Likelihood (ML), Minimum Evolution (ME), Neighbor-Joining (NJ) and Maximum Parsimony (MP). Fourthly, Muscle and ClustalW, both integrated alignment tools are supported by MEGA. Finally, it runs on Windows, Mac and Linuz systems, works offline and is updated regularly with new features and bug fixation (Keklik 2023). However, MEGA is not optimized for high performance computing, lacks flexibility, consumes significant RAM with large sequences, has no Bayesian inference and is less efficient for intensive analyses(Hall 2013). Bayesian Inference It is more sophisticated and alternative method for phylogenetic analysis. It is often implemented in MrBayes, BEAST, and PhyloBayes (Huelsenbeck and Ronquist 2001). It has several advantages such as robust statistics, suitable for handling evolutionary rate heterogeneity and can be utilized with complex evolutionary models. It is limited too, due to its intensive rate of computing and requires expertise in Markov Chain Monte Carlo (MCMC) methods Fast Minimum Evolution Algorithm Rapid phylogenetic tree can be constructed with this package. It is faster than ML and NJ methods and is sufficient for assessment of biodiversity on large scale (Desper and Gascuel 2002). Its use is limited due to the underestimation of evolutionary distances and its potential less accuracy than Bayesian inference and ML. Jukes-Cantor Model It is a simple substitution model that is fast and easy while making it easy only for basic phylogenetics. Its results are often unrealistic due to assumption of equal base frequencies and is less accurate than General Time Reversible models (Erickson 2010). DNAMAN and DNA Star These are commercial tools used for DNA sequence analysis. These are user friendly and supports multiple alignments, however they are not accessible publicly and are not feature-rich as MEGA or others. Analysis of Study Duration The aim of DNA barcoding is to analyse species richness and diversity, genetic variability and patterns of evolution over time. Therefore, duration of study or period of sampling is a crucial factor in reliability and robustness of DNA barcoding (Skalak, Sherwin et al. 2012). In the present dataset, a significant inconsistency has been observed such as the unviability or unevenness of duration ( Table 1 ). Importance of Study Duration DNA Studies Several aspects of the study on DNA barcoding are affected with duration of the study. Many species are available only during a specific time or season of the year. Species vary with respect to behaviour, population structure and genetic diversity. For example, insects and birds often have specific breeding seasons. Short-term research studies in DNA barcoding may lead to failure in capturing fluctuations and are biased. Moreover, long term study may help in detecting genetic changes over the years, adaptation and genetic drift. In the current review, studies that have spanned between 2010-2019 (Ashfaq, Khan et al. 2022) and 2017-19 ( Table 1 ) have provided more insights as compared to those that were limited to a season or so. Climate change and alteration of habitat are also factors of influence. They affect genetic diversity and species distribution. Studies that spanned from June 2014-October 2017 or August 2018-July 2022 allows researchers to actively study climate impacts and habitat factors while short term studies fail to do so. Short term studies provide limited snapshot of genetic diversity. While studies that samples for years provide a robust and reliable aspect of diversity. Gaps and Inconsistencies A significant number of research studies have not provided their study duration from the current review as indicated as (Not Provided) in Table 1 . It makes difficult for future researchers to replicate or validate current findings, and temporal trends cannot be compared to data from not mentioned time. Moreover, a standardized guidelines in DNA barcoding studies are necessary for ensuring methodological transparency. Based on the lack of duration, it becomes challenging to assess whether a study sufficiently covered seasons or years to avoid sampling bias such as the summer 2022 (Gul, Shah et al. 2024) or selected months of 2020. Patterns Observed With respect to duration of study from the current dataset, four major trends can be observed. Firstly, short-term studies lasted only a few months ( March-August 2023 , October-February , May and October in 2020 ) ( Table 1 ). These studies contribute valuable data but lack seasonal representation. Secondly, medium-term studies that lasted from one to three years offer more insights. Thirdly, long-term studies that extended from 2010 to 2019 and 2018 to 2022 provide stronger and more reliable datasets with multi-year genetic variations and environmental influences. Finally, some studies collected sampling at irregular intervals like Selected months from 2009, 2012, 2013, 2014, 2015 (Liu, Panhwar et al. 2017) which may affect comparability and consistency in DNA barcoding. Future studies may be equipped with mandatory reporting of study durations, long term monitoring, seasonal considerations and standardized methodologies. Analysis of Trapping Methods The choice of trapping method is always associated with sample diversity, data reliability and accuracy of species delimitation (Massey, Bronzoni et al. 2022). A wide range of trapping methods have been actively used by COI-based DNA barcoding studies in Pakistan ( Table 2 )(Hussain, Kakar et al. 2024). Despite wide range of trapping methods, the data reveals inconsistencies and gaps in research. Why are Trapping Methods Important? For collection of specimens trapping methods are necessary to minimize bias while ensuring a representative sample. To what extent a method is effective? This largely depends on the preservation of genetic material because some trapping methods may damage DNA, specialized techniques are required for habitat types like terrestrial, freshwater etc summarized in Table 2 with advantages and limitations. Moreover, some behaviours are specific for a species like insects are attracted to light traps while fish require nets. The present dataset reveals a combination of passive (trap-based) and active methods (manual collection), each of which is associated with advantages and limitations summarized and presented in Table 2 . Observations and Gaps Data Not Provided A notable number of studies did not report trapping methods which affects data comparability and reproducibility while questions reliability. It makes data difficult for future use by researchers to assess effectiveness of different trapping methods. Bias in Trapping Methods Light traps, malaise traps and hand collection are favourite methods for insect capturing ( Table 2 ) which possibly lead to underrepresentation of non-flying or nocturnal insects. Cast nets, gill nets and drag nets have been used for aquatic species, however, mesh size and sampling depth have not been mentioned which may influence species selectivity. In the present data set, researchers have relied greatly on hand collection and jugular vein blood sampling from mammals but there is lack of details on mammalian, amphibian and reptilian-specific traps like Sherman traps for rodents (Anthony, Ribic et al. 2005) and drift fences for amphibians. Moreover, there is lack of standardization in reporting like some studies have mentioned vague descriptions like nets and hand collection, which doesn’t reflect standardization in trapping mechanisms. Future Recommendations Future research should focus on standardizing trapping protocols, ensuring species-specific sampling strategies, and incorporating non-invasive genetic sampling methods to improve biodiversity assessments in Pakistan. Each publishing journal should set exact trapping technique as a requirement for publication along with mesh size, lure types and collection duration. Additionally, single method may be a way to mis or underrepresentation of species therefore, incorporation of techniques should be used. Ethical consideration should be a way out in studies where animals are involved, besides eDNA should be a focus for researchers on DNA barcoding. Analysis of Types of Articles The present review reveals that majority of the articles utilized are research articles while some are Rapid Communications, Short Communications, MITO Communications and Review Articles. Dominance of Research Articles The dataset is heavily skewed towards Research Articles, which are detailed studies presenting original findings based on field or laboratory investigations. These articles have presented data which lead to reproducibility, undergone peer review, are more reliable and accurate. However, these articles have focused on specific case studies and limited to few species in many cases and are limited to certain geographic area. Short and Rapid Communications These are types of articles which briefly discusses and focuses on new discoveries, methodological improvements, or preliminary results. The data set shows that these articles have provided timely insights, are focused and concise. However, these articles are with limited data and lack statistical analysis. Moreover, these are not peer-reviewed extensively which may lead to compromise quality control. MITO Communications In mitochondrial genome sequencing and its applications in phylogenetics, these two types of articles play a key role. These are highly relevant to DNA barcoding and provide genome-level insights. However, these have narrow focus as ecological and taxonomic discussions are missing and are in short format, lacking extensive methodological comparisons. Review Articles Critical analysis has been provided by review articles on specific perspective or section of DNA barcoding. These types of studies are limited in the present dataset and discusses only specific parts or aspects of DNA barcoding (Muhammad Tahir and Akhtar 2016, Hanif, Manan et al. 2023). A lack of multiple review articles reveals lack of multiple and broader meta-analysis. DNA Barcoding Sampling in Pakistan The currently available data on COI-based DNA barcoding reveals that extensive geographic coverage has been established across Pakistan. However, a critical analysis reveals notable gaps, biases and underrepresentation of specific taxa and ecosystems. Geographic Bias In DNA barcoding studies, unequal geographic distribution is observed in sampling ( Table 3 ). The dataset shows that Punjab dominated sampling efforts with significant locations from Lahore, Faisalabad, Sargodha and Multan ( Figure 3 ). However, several locations like Baluchistan, Gilgit-Baltistan, Khyber Pakhtunkhwa and most of the Sindh has gained less attention despite their ecological importance. Multiple cities and river systems like Indus, Ravi and Chenab has provided more data which could be due to accessibility in Punjab, so we can assume that it is Punjab-Centric research. Baluchistan despite being the largest province with unique ecosystems covering mountains, deserts and marine, only few locations in Quetta, Ziarat and Hazarganji-Chiltan National Park have been covered in DNA barcoding. There seems a lack of surveys due to logistical challenges, political instability and limited research funding. Broader Karakoram, Himalayas and Hindu Kush regions have been underreported despite of being biodiversity hotspots ( Table 3 ). Only Skardu has been given a little approach from Gilgit Baltistan. The omission of biodiversity hotspots such as distinct alpine and glacial regions is a significant research gap. Pakistan has relatively a smaller number of publications compared to neighboring countries like India, Iran, China and Bangladesh ( Figure 5 ). China is leading in this scenario with more than thousand publications. Underrepresentation of Certain Ecosystems Some of the critical ecological zones have been underrepresented in DNA barcoding studies. In Punjab, rivers like Indus, Chenab, Ravi, Jhelum and Satluj have been sampled widely, however coastal and marine biodiversity is underrepresented despite their importance with respect to mangroves, coral reefs, estuaries and deep-sea ecosystems. In Sindh, few sites like Sandspit (Karachi), Fish Harbour Road (Karachi), and Sindh Coast have been sampled while the nearby marine species are still underreported, showing a biasing towards certain ecosystem due to unviability of facilities and funds. Some of the most important spots like the Cholistan desert is sampled however Thar desert is still missing, despite its unique xeric biodiversity. Moreover, Kharan and Makran deserts are also missing from the dataset. When protected areas are compared to open areas, Hazarganji-Chiltan National Park, Juniper forests in Ziarat have been discussed and sampled while northern forests, alpine meadows and temperate ecosystems are overlooked. The Margalla Hills, Deosai National Park, Fairy Meadows, and other ecologically rich areas of Gilgit-Baltistan and Khyber Pakhtunkhwa remain absent from barcoding studies ( Figure 3, Table 3 ). Overrepresentation of some areas As compared to other ecosystems, agricultural and urban ecosystems have been widely studied and explored. Pests, pollinators and invasive species have been barcoded with Ayyub Agricultural Research Institute and PARS (UAF Faisalabad) being dominated. An overrepresentation has been shown for Lahore, Karachi and Faisalabad that thrive human-modified habitats. Taxonomic Bias and Underreporting In the current dataset, some species or ecosystem have been focused more as compared to others. For instance, there has been a more focus on economically significant species while holistic approach to biodiversity is missing. While talking taxonomically, some groups like insects, freshwater fish and agricultural pests have been reported widely while amphibians, reptiles, fungi and invertebrates have been underreported. Species of terrestrial vertebrates like birds, reptiles and mammals have been focused less as compared to occupied and protected places like national parks and zoos. Microbial DNA barcoding is nearly missing despite its importance and omnipresence in soil, water and gut that are considered crucial for ecological and health studies. Institutional and Logistical Constraints Punjab and Sindh have gained more focus that suggests the research facilities, funding and role of academic institutions. Security concerns, political instability and limited infrastructure may explain less exploration of Balochistan, Gilgit-Baltistan, and interior Sindh. Moreover, cross-border and multi-geographic sampling is rare which may limit regional comparative studies on biodiversity. Conservation and Policy Implications As DNA barcoding is successful when two major biases are removed, the geographic and taxonomic constrains which limit its applications in conservation biology. The dataset reveals that many endemic and threatened species remain unstudied in marine, desert and high-altitude biodiversity, affecting conservation policies. Biodiversity monitoring programs are negatively affected when less species is barcoded from national parks and protected areas. A more balanced DNA barcoding efforts are need of the day in Pakistan’s like rich cultural and ecologically diverse country. Future Recommendations To have a balanced focus on rich biodiversity barcoding of Pakistan, major gaps and biases should be addressed. Sampling should be expanded to underrepresented locations of Baluchistan, Gilgit-Baltistan, Khyber Pakhtunkhwa and parts of Sindh. Marine and high-altitude ecosystems should be focused with species focus on neglected groups like reptiles and mammals. Multinational collaborative research should be the key point for future research and international partnerships should be part of the study designs for cross-border studies. Sampling Effort and Species Identified Analysis of Number of Sampling Sites Substantial variation has been observed in the number of sampling sites across different studies utilized for this review ranging from multi-site investigations to studies with extremely limited geographical coverage ( Table 1 ). The inconsistency highlights several key points regarding the comprehensiveness, representativeness, and potential limitations of DNA barcoding efforts in the country. Some studies have demonstrated extensive sampling efforts with 1858, 491 and 329 sites ( Table 1 ). These studies and others like these provide robust datasets which helps in species identification and genetic diversity assessments. These types of studies are rare across 80 plus articles of the current review. In contrast to this, a high number of studies have reported few sampling sites like ten or below reaching to only one site in few studies. This can lead to underrepresentation of species and showcases biases in barcoding, reducing effectiveness of the said studies. A high number of studies have not mentioned the number of sites and labelled here as “Not Mentioned” ( Table 1 ). It raises concerns about scientific rigor and data reproducibility. Therefore, results can be questioned, and it is difficult to draw conclusions. Future research may be restricted due to data constrains and it will be difficult to compare, reproduce or validate data. Besides above, several studies have mentioned multiple sites which fall into the similar category of ambiguity. Missing of exact figures limits meaningful comparison and meta-analyses. Analysis of Number of Samples Number of samples were analysed across different studies on DNA barcoding in Pakistan. These numbers ranged from single-digit figures to over 60000 samples (Ashfaq, Khan et al. 2022). Some of the studies reported high number of samples such as 60,273, 53,092, 10,792, 10,653, and 8,641 ( Table 1 ) that provide robust dataset that enhances species identification and reporting, biodiversity assessments and increases reliability and validity of DNA barcoding databases. These studies have been supported by external funding that is why they supported high number of samplings. Besides these, many studies reported few numbers of sampling such as 1, 3, 4 or 5. These studies contribute well to DNA barcoding studies however they are often inadequate for biodiversity assessments and conclusions. They are often coupled with poor statistical analysis and potential misidentification and incomplete representation when at species-level biodiversity is considered. In several studies from Pakistan, number of samples is missing (Not Mentioned) which limits the impact of DNA barcoding. These studies conclude that there is a lack in transparency, data validation and reproducibility. Future studies should focus on standard number of samplings in DNA barcoding while more focus should be given to Large-scale sampling. Analysis of Number of Species identified The country’s DNA barcoding efforts are supported by studies that extended from one species to thousand number of species across different articles. These variations are associated with several factors like comprehensiveness, scope and methodological approaches. Large-Scale Studies: Comprehensive Biodiversity Assessment Extensive taxonomic coverages have been demonstrated in some of the studies like 1,364 species across 1,375 (Ashfaq, Khan et al. 2022) genera in a study while another with 379 (Ashfaq, Akhtar et al. 2017) species spanning 52 families. These studies contribute efficiently in terms of importance of DNA barcoding for species delimitation, evolutionary studies and conservation strategies. Another study with 254 families which fits in 17 orders also adds to broad taxonomic range. These studies demonstrate well designed sampling strategies which is crucial for DNA barcoding. These studies have improved large-scale studies and encourages others to follow in the same range and improved Pakistan’s representation at BOLD and GenBank level. Moderate Species Coverage studies: Limited Impact Many of the studies have reported handful of species, genera and families. Some of these have 1 to 5 species with certain studies even focusing on single species or a single genus ( Table 1 ). These are valuable for specific taxonomic groups; however, they provide limited contribution to overall efforts. Small sample sizes reduce the ability to assess intraspecific variation, that is crucial for differentiating cryptic species and understanding population dynamics. Moreover, some of the studies have focus on specific taxonomic groups like one limited to 86 pheretimoid species complex (Hussain, Liaqat et al. 2022) and the great gap between morphologically and molecular identified species as 50 species identified morphologically but only 8 species identified molecularly (Khan, Kakar et al. 2024). Some of the studies have not mentioned the exact number of species which produces ambiguity for taxonomic researchers. This lack of clarity reduces the usefulness of such studies for comparative analyses and biodiversity assessments. Additionally, the use of BINs (Barcode Index Numbers) instead of species names, as seen in the study reporting 15 BINs (Ashfaq, Hebert et al. 2014), may indicate that taxonomic validation was incomplete, limiting the practical application of the data. New Species: Promising but Underutilized Out of studied articles, few studies report the discovery of new species, such as one reported 11 species including 6 new ones and another study with 2 species with 1 new species ( Table 4 )(Memon, Meier et al. 2006, Sajid, Zahid et al. 2021). This highlights the potential of DNA barcoding in uncovering previously unrecognized biodiversity in Pakistan. However, the low number of articles that reports new species can be think of as underexplained or underexplored. There also seems a lack of expertise in species validation through DNA barcoding and molecular phylogenetics. In conclusion there is a need for expansion as well standardization of sampling protocols across the country. Smaller studies should focus on poorly studied taxa to fill the gap while extensive studies should be encouraged for biodiversity assessment. Efforts should be made towards expanding taxonomic coverage and species validation. Analysis of Most Abundant Species Many diversity indices like Shannon Diversity Index and Simpson’s Diversity index are extensively used to estimate species diversity and richness ( Table 4 ). However, in barcoding studies it is still scarce limited to few studies (Anjum, Ilahi et al. 2025). Therefore, it is needed to carry out biodiversity estimation parallel to identification. In the current scenario, reporting of most abundant species is inconsistent, with many studies failings to provide such information. Some of the studies have mentioned explicitly the dominant species such as such as Culex quinquefasciatus (61%) , Bactrocera zonata , Hypophthalmichthys molitrix , and Anopheles stephensi. Majority of list is labelled as Not Available ( Table 4 ). Species that are abundant in any ecosystem play a crucial role in ecosystem stability or functioning. For example, Culex quinquefasciatus and Aedes aegypti are not just dominant mosquito species but also significant vectors for diseases like dengue and malaria (Rodriguez 2005). Understanding their abundance and distribution can help in vector control and public health strategies. Similarly, dominant agricultural pests such as Bactrocera zonata can have serious economic implications. On the other end of the spectrum, less abundant species may indicate rare or threatened taxa requiring conservation attention. Species such as Gazella bennettii and Vulpes vulpes are notable mentions, as their population trends could indicate habitat disturbances or conservation success. If studies do not mention the most or least abundant species, critical information about species decline or dominance is lost. When dominant species are reported, it provides insights into potential biotic homogenization, where a few species outcompete and replace native biodiversity. The presence of invasive species such as Hypophthalmichthys molitrix (silver carp) and Oxya hyla hyla (a grasshopper species) suggests ecological shifts that could affect native species and ecosystems. Without data on species abundance, it is difficult to assess the impact of invasive species or take appropriate management actions. Accurate reporting improves DNA barcoding and its utility in DNA databases. Without mentioning the true status, the databases may not reflect true ecological importance of each species. Issues with "Not Available" Data The unavailability of data regarding species abundance may be coupled to several issues such as the focus of study on species identification rather than ecological significance. It may also be associated with incomplete sampling or bias in specimen collection. Conspecific Genetic Distance: Pakistan’s Perspective In the current review, the intraspecific divergence varies significantly across articles ( Table 4 ). Some studies have provided clear ranges or their mean values while others do not report it and labelled as “Not Available”. A broad range of genetic distances are available as low as 0.0% and as high as 67.10% ( Table 4 ). This genetic distance is a crucial metric in DNA barcoding and helps to provide and establish a threshold. Generally, this value remains less than 2% but depending upon evolutionary history, geographic separation and species, it can vary. Wide Variability in Reported Values Here, we report high inconsistency in intraspecific values from Pakistan based on COI gene. Many of the studies have reported values between 0-2.4% which aligns with standard barcoding expectations(Dinh, Ngatia et al. 2019). For instance, the studies reporting 0-1.6%, 0.0-2.26%, and mean values around 0.2%-0.7% suggest expected intraspecific variation ( Table 4 ). Some of the studies from the current scenario report high values as high as 32.18% which exceed normal values which may indicate presence of cryptic species (Dinh, Ngatia et al. 2019), taxonomic errors, variable genetic markers and geographic structuring. Some of the studies have reported no variation which reflect low genetic diversity and limited sampling. Several studies have not reported conspecific genetic distances. This lack of data reduces the chance of comparability and is a gap in establishing threshold for Pakistan’s biodiversity. Implications for Species Delimitation and Accuracy Absence of clear genetic distance may lead to misidentification and indicate overlooked taxonomic diversity. For species delimitation, the threshold is set to be 2%, however the variability in the dataset suggests that single universal threshold may not be sufficient therefore work on taxon-specific threshold is necessary. Future studies should adhere to standardized methods to improve the consistency and reliability of reported values. Congeneric Genetic Distance in DNA Barcoding In the whole dataset, interspecific genetic divergence is inconsistent ( Table 4 ). Few studies have provided well-defined ranges or their mean values from very low values of (0.0000–0.0067%) to exceptionally high distances (37.02%)(Islam, Qasim et al. 2018). Congeneric distances help to define the boundary between species. Wide Variation in Reported Values Many studies report expected values such as 2.3–17.8%, 2.8–23.2% (mean 8.8%), and 7.93% summarized and outlined in Table 4 . these values are in line with global thresholds and successfully defined species boundaries. Some of the studies have reported extreme values such as 37.02% which may be attributed to deep genetic divergence, misidentification and presence of cryptic species. Some of the values are less than 1% which may indicate recent speciation, hybridization or taxonomic issues. Many of the studies have not presented values for congeneric distances, which limits the comparison. In the current review, this gap may be due to focus on identification instead of evolutionary relationships, incomplete sampling and use of various markers instead of one universal primer. The Barcode Gap Challenge The barcode gap-the distance which separates species is crucial for distinguishing species. Congeneric distance should be more than conspecific distance. Some studies have reported overlapping values which results in blurring identification. Therefore, there is a need for taxon-specific threshold adjustment, co-validation of morphological and molecular identification and the integration of mitochondrial markers alongside nuclear markers. Focused vs. Ignored Taxa, Limitations, and Recommendations In Pakistan, a wide range of taxa including insects, fishes, birds, mammals, arachnids, reptiles, and amphibians have been covered through DNA barcoding. However, this tremendous focus during the past two decades is uneven across different groups and geographies. Groups Received More Attention A significant portion of studies have focused on insect species including species from Diptera, Hemiptera, Lepidoptera, Hymenoptera and Orthoptera of which pests and disease vectors are dominant. Species of mosquitoes like Aedes aegypti , Culex quinquefasciatus , Anopheles mosquitoes, pests like Thrips ( Thysanoptera ), whiteflies ( Hemiptera: Aleyrodidae ), stink bugs ( Hemiptera: Pentatomidae ), fruit flies ( Bactrocera spp. ), cotton bugs ( Dysdercus spp. , Oxycarenus hyalinipennis ), and mealybugs ( Phenacoccus solenopsis ) and beneficial insects like Apis mellifera (honeybee) and Coccinellidae (ladybird beetles, important for biological control) have gained special attention. Spiders and arachnids belonging to order Araneae and have been documented well with respect to DNA barcoding. While talking about fishes, more focus has been given to economically important species like members from Cyprinidae family, marine fishes ( Sillago indica ) and endangered species like Tor putitora. Few studies on Doves, pigeons, pheasants and starlings, few mammals like ungulated including Gazella bennettii , Moschus cupreus , Muntiacus muntjac , Capra hircus , Bos taurus and bats like Vespertilionidae, Emballonuridae, Pipistrellus coromandra have been barcoded. Some studies have examined turtles ( Lissemys punctata ) and toads ( Duttaphrynus spp. ), however, herpetofauna remain understudies largely. Ignored or Understudied Taxa When studied and understudied groups are compared to each other, Pakistan’s coastal and deep-sea biodiversity remains largely unexplored. Corals and molluscs and crustaceans have been ignored. Studies on carnivores such as foxes, hyenas, jackals and leopards are missing. Rodents, shrews and hedgehogs have not been studies still. Pakistan is blessed with snake and lizard diversity (Khalid, Attaullah et al. 2019), however only few studies have focused them. Molecular work on fungi and microorganisms is scarce across Pakistan. Limitations in DNA Barcoding Studies One of the major limitations on DNA barcoding in Pakistan is the focus on economically important species while ignoring the ecologically significant species. Many of the studies are restricted to specific regions like Punjab. However, diverse regions like Balochistan and Khyber Pakhtunkhwa have been ignored. Moreover, many of the studies have focused on few species while other taxa are largely ignored. Finally, availability of the universal or species-specific markers is also a significant problem. Comparison of Pakistan’s Biodiversity and Molecular Work Currently over 198 fish species, 198 mammal species, 666 bird species, 177 reptile species, and 22 amphibian species have been identified in Pakistan ( Figure 4 ) which is far more than the barcoded species and there is still a large gap in between the two. Moreover, approximately 6,000 species plant species but very few DNA barcoding studies exist. While microorganisms are Extremely underexplored at the molecular level. Conclusion The current study concludes that COI-based DNA barcoding in Pakistan has faced with multiple challenges. These challenges include biased geographic studies, limited resources in terms of financial as well as laboratory, fragmented studies, ignored taxa (e.g., fungi, microbes, reptiles, and marine organisms), missing barcode library, and limited availability of biodiversity data, therefore these should be the focus of next studies. Multinational collaboration should be strengthened to enhance DNA barcoding progress. Barcoding outcomes should be linked to policy and conservation strategies. Furthermore, ignored taxa can be studied if global and local collaboration between institutions and governments are established. The study further concludes the unavailability of expertise in COI-based DNA barcoding at institutional level. Recommendations To address these taxonomic gaps, taxonomic coverage should be expanded to neglected taxa including carnivores, rodents, small mammals, snakes, geckos, skinks and frogs and to aquatic life across waters of Pakistan.Geographic sampling should be prioritized in underepresented region, specifically Balochistan, Khyber Pakhtunkhwa and Gilgit-Baltistan. COI gene should be coupled with other genes as well as molecular markers should be assessed alongside morphological characters. Moreover, barcoding outcomes should be linked to policy management and conservation strategies. Conservation strategies should be focused and eDNA should be the top priority of DNA barcoding researchers with the use of Artificial intelligence for species delimitation and sequencing as well. We develop and propose a standardized checklist (Table 6 ) to enhance reproducibility and comparability of future DNA barcoding studies in Pakistan. 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J Mammal 34(1):54–61 Sajid M, Zahid M, Shah M, Rasool M, Ullah I, Ahmad R, Habibullah P, Majeed N (2021) IDENTIFICATION OF THE ORB WEAVING SPIDER (ARANEAE: ARANEIDAE) FAUNA OF DIR LOWER (PAKISTAN) THROUGH DNA BARCODING. JAPS. J Anim Plant Sci 31(4) Sajjad A, Jabeen F, Ali M, Zafar S (2023) DNA barcoding and phylogenetics of Wallago attu using mitochondrial COI gene from the River Indus. J King Saud University-Science 35(6):102725 Skalak SL, Sherwin RE, Brigham RM (2012) Sampling period, size and duration influence measures of bat species richness from acoustic surveys. Methods Ecol Evol 3(3):490–502 Ullah S, Ahmad H and M. A. Rafi CO1 based DNA barcoding of some pentatomomorpha bugs (Hemiptera: Heteroptera) from Swat, Pakistan Tables Table 1 to 6 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Table2.docx Table3.docx Table4.docx Table5.docx Table6.docx GraphicalAbstractCOI.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7936079","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Systematic Review","associatedPublications":[],"authors":[{"id":536941286,"identity":"a2d81a54-5ecc-4d7a-b469-895db621773d","order_by":0,"name":"Sohail 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18:02:45","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":32598,"visible":true,"origin":"","legend":"","description":"","filename":"Table4.docx","url":"https://assets-eu.researchsquare.com/files/rs-7936079/v1/c49b466b28c06a339c38464f.docx"},{"id":94790700,"identity":"d44b6c57-a0ec-42b3-bee3-1525e0bc0a72","added_by":"auto","created_at":"2025-10-30 18:02:45","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":19651,"visible":true,"origin":"","legend":"","description":"","filename":"Table5.docx","url":"https://assets-eu.researchsquare.com/files/rs-7936079/v1/8d2c1537a29b97a028dd5b53.docx"},{"id":94824851,"identity":"a187daf8-b45a-433c-87ee-a3f41b6667a9","added_by":"auto","created_at":"2025-10-31 06:49:27","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":17245,"visible":true,"origin":"","legend":"","description":"","filename":"Table6.docx","url":"https://assets-eu.researchsquare.com/files/rs-7936079/v1/3c9cfedbbe70a8544d6d141d.docx"},{"id":94825976,"identity":"f75724a9-c793-4a03-9f7a-d48e157c3b41","added_by":"auto","created_at":"2025-10-31 06:50:51","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":201784,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstractCOI.png","url":"https://assets-eu.researchsquare.com/files/rs-7936079/v1/a6fadf0982edcd94fb5ae9f8.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"COI-Based DNA Barcoding in Pakistan: Progress, Challenges, and Future Directions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDNA barcoding has revolutionized the field of taxonomy and systematics, enabling rapid and accurate identification of species. In the DNA barcoding approach, which is the use of a short and standardized fragment of DNA, such as the COI gene for species identification. It has facilitated the discovery of new species, invasive species, and monitoring of threatened populations(Antil, Abraham et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Pakistan is blessed with significant faunal diversity, which further needs acceleration in DNA barcoding attempts (Baig and Al-Subaiee \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). As Pakistan is located at the crossroads of the oriental and Palaearctic zoogeographic regions (Mirza \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1975\u003c/span\u003e), it boasts a unique diversity. Tropical and temperate forests, deserts and grasslands, mountains and water bodies are rich with species both in diversity and richness.\u003c/p\u003e\u003cp\u003eDespite the rich diversity, Pakistan faces challenges that hinder the application of DNA Barcoding (Joly, Davies et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The limited availability of taxonomic expertise, limited resources, and lack of infrastructure limit the use of DNA barcoding. Furthermore, this biodiversity is facing threats like the destruction of habitat, climate change, overexploitation of resources, and pollution. These challenges can be covered with the valuable tool of DNA barcoding, lessening gaps and providing opportunities like cut off laboratory facilities at national level. International collaboration and knowledge sharing enables researchers to compare species from different regions.\u003c/p\u003e\u003cp\u003eGlobally, researchers have made considerable progress in applying COI-based DNA barcoding for species identification (Emerson \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), however, research efforts in Pakistan remain limited and fragmented. Large portions of country\u0026rsquo;s rich fauna are still uncharacterized and limited to few taxonomic groups. Moreover, there is a lack of comprehensive assessment on the DNA barcoding status initiatives in Pakistan. Furthermore, little information is available on taxonomic coverage, institutional contributions and publication trends. The absence of coordinated research networks and centralized databases has further widened the gap. Therefore, these gaps highlight the need for a systematic review that compiles current knowledge, identifies achievements and limitations, and outlines future directions for strengthening DNA barcoding research in Pakistan.\u003c/p\u003e\u003cp\u003eThis study is needed because it brings together, for the first time, a systematic overview of COI-based DNA barcoding in Pakistan. Additionally, it documents history, limitations and achievements, and emerging trends in DNA barcoding. Earlier studies focused on isolated groups or limited to restricted locations; this review provides broader national-level perspective. Moreover, no published review study is available on COI-based DNA barcoding from Pakistan. The current study is review-based article which compiles, synthesizes and critically analyses published research between 2006 and 2025 on COI-based DNA barcoding in Pakistan. This review aims to provide a comprehensive overview of history, progress, challenges and limitations on COI-based DNA barcoding in Pakistan, with the focus of guiding future research priorities and acceleration of use of DNA barcoding as a tool for conservation of biodiversity and management in Pakistan.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe current review study has focused the COI-based DNA barcoding in Pakistan. The first published article is from the year 2006 which then stretched over the years to 2025. The data reveals that the COI-based DNA barcoding accelerated over the years. The study solely focused on articles published on DNA barcoding, specifying COI as gene of choice. The geographic focus is on Pakistan, including articles that barcoded animals from different regions including Provinces of Sindh, Balochistan, Punjab, Khyber Pakhtunkhwa and Gilgit Baltistan, further explored in the following sections. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the current review, a total of 120 articles were downloaded from research sites such as Google Scholar. Out of total, twenty-one articles were excluded because they were either on Cyt-b gene or 12S rRNA genes. Furthermore, some articles solely focused on review of a single animal and, finally, only 91 articles were retained for further study. The whole data was summarized in Microsoft Office Excel Sheet. Tables were also designed using the same Excel Sheet due to it\u0026rsquo;s easy to use. Figures were designed using R and GIS packages.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690147\"\u003eProgress over the years\u003c/p\u003e\n\u003cp\u003eDNA barcoding has evolved significantly over the past two decades in Pakistan. A closer examination reveals a development from an initial slow adoption to a recent surge in research activity (\u003cstrong\u003eFigure 1\u003c/strong\u003e). It can be further observed through three main stages of development as following. \u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690148\"\u003eSlow Progress (2006\u0026ndash;2012)\u003c/p\u003e\n\u003cp\u003eThe first publication in the current dataset goes back to 2006 (Memon, Meier et al. 2006), marking the introduction of DNA barcoding in Pakistan. After that, no study appeared until 2010 (Ashfaq, Noor et al. 2010), suggesting isolated early efforts and large gap (\u003cstrong\u003eFigure 1\u003c/strong\u003e). A lack of widespread consistency and very limited institutional support during this era can be observed, for example, evidenced from a study (Iftikhar, Ashfaq et al. 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690149\"\u003eGradual Growth (2013\u0026ndash;2021)\u003c/p\u003e\n\u003cp\u003eThe frequency of publications increased soon after 2013, with intermittent research activity leading to 2016 with a modest rise in output with five publications. The period shows an increase in interest in DNA barcoding, likely driven by global recognition in biodiversity conservation and species delimitation. During the years between 2017 and 2021, the output remained steady with multiple publications for each year. This duration shows the prominent research focus on DNA barcoding in Pakistan. Institutions and researchers recognized the importance of DNA barcoding during this phase.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690150\"\u003eRising Research Trends (2022-2025)\u003c/p\u003e\n\u003cp\u003eThe dataset shows a sharp increase in publication for 2022 and onward. Peak publication occurred during 2023 and 2024 (\u003cstrong\u003eFigure 1\u003c/strong\u003e), witnessing to be the most productive years. This is possibly fuelled by increased funding, greater understanding of taxonomic and ecological applications and an advancement in sequencing technologies. The dataset marks the year 2024 an all-time high in research output with ten plus publications, while the momentum is being sustained for 2025 (Anjum, Ilahi et al. 2025) indicating a robust and expanding research community in the field.\u003c/p\u003e\n\u003cp id=\"_Toc208690151\"\u003eFactors Affecting Growth\u003c/p\u003e\n\u003cp\u003eSeveral factors have aided to the recent acceleration in DNA barcoding in Pakistan. Bioinformatics tools and sequencing platforms have likely facilitated this domain. It further shows the increase in government and private funding to this sector as shown in funding section of many articles. Canadian Centre for DNA barcoding is credited with global influence and collaboration in Pakistan. Finally, as Pakistan is blessed with rich biodiversity (Baig and Al-Subaiee 2009), therefore, conservation efforts need rapid identification which is a growing factor for accelerated studies. This shift from foundational studies to more applied research indicates a promising future for molecular taxonomy and biodiversity assessment in the country (Akhtar and Ali 2016). Further investigation into specific research themes, institutional contributions, and funding trends would provide deeper insights into the progress and future trajectory of DNA barcoding research in Pakistan (Ruedi, Manzinalli et al. 2023).\u003c/p\u003e\n\u003cp id=\"_Toc208690152\"\u003eResearch Across Journals\u003c/p\u003e\n\u003cp\u003eThe publication trend as viable from the dataset highlights the diverse range of journals as a closer look reveals key insights into the scope, impact and reach of DNA barcoding research in the country (\u003cstrong\u003eTable 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp id=\"_Toc208690153\"\u003eInternational Dominance\u003c/p\u003e\n\u003cp\u003eInternationally recognized high impact journals have published a significant portion of articles on DNA barcoding. A well regarded open-access platform PLOS One appears frequently, indicating it to have accepted well reputed research studies. Overview shows that PLOS One accepts articles with greater sample size (Ashfaq, Hebert et al. 2014, Iftikhar, Ashfaq et al. 2016). \u003cem\u003ePeerJ, Scientific Reports, Frontiers in Physiology\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Molecular Ecology Resources\u003c/em\u003e are the most appearing journals with high number of publication in DNA barcoding from Pakistan, helping in increasing visibility (Ashfaq, Khan et al. 2022). Other journals such as \u003cem\u003eMitochondrial DNA part A and B,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Molecular Biology Reports\u0026nbsp;\u003c/em\u003ereflect strong focus in these studies. It is also evident that Mitochondrial DNA like journals should focus acceptance on DNA barcoding, a central theme of mitochondrial genome. National and local journals have contributed well to the research on DNA barcoding as clear from the inclusion of articles in \u003cem\u003ePakistan Journal of Zoology, Punjab University Journal of Zoology, Journal of Bioresource Management\u003c/em\u003e and others (\u003cstrong\u003eTable 1\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690154\"\u003eMultidisciplinary and Applied Research Trends\u003c/p\u003e\n\u003cp\u003eSome journals like \u003cem\u003eApplied Ecology and Environmental Research, Journal of Freshwater Ecology\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Journal of Materials and Environmental Science\u003c/em\u003e highlights the importance of DNA barcoding beyond pure taxonomy (Khan, Ali et al. 2023, Anjum, Ilahi et al. 2025). These journals have addressed ecological and environmental concerns such as species conservation, assessment of ecosystem health and biodiversity monitoring. More wide fields like veterinary sciences, livestock and agriculture are also on the focus as barcoding is used in these field while evidenced from publications in \u003cem\u003eJournal of Applied Research in Plant Sciences\u003c/em\u003e and \u003cem\u003eFrontiers in Veterinary Sciences\u0026nbsp;\u003c/em\u003e\u003cem\u003e(Ali, Khan et al. 2024)\u003c/em\u003e. The presence of greater data accessibility on open access platforms like \u003cem\u003eResearch Square\u003c/em\u003e suggests that data presentation is also necessary for broader studies within scientific community.\u0026nbsp;(Asif, Naseem et al. 2023)\u003c/p\u003e\n\u003cp\u003ePublication trends indicate that DNA barcoding should be a focus of interdisciplinary journals to be fruitful for wider scientific community. Both national and international journals are paying greater focus to DNA barcoding research in Pakistan (\u003cstrong\u003eTable 1\u003c/strong\u003e). A shift toward biodiversity conservation, ecology and agriculture is an indicator of practical applications in the fields. Notably, the assessment of journal metrics like impact factor and citation metrics should be carried out by institutions to understand how Pakistan\u0026rsquo;s research is influencing global barcoding studies. This rapid growth should also be assessed to work out on the funding sources and affiliation of publications.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690155\"\u003eSources of DNA Extraction\u003c/p\u003e\n\u003cp\u003eTwo important aspects of DNA barcoding are the dependence of success on quality and reliability of DNA extracted from biological samples. In the current review, \u003cstrong\u003eTable 1\u003c/strong\u003e summarizes diverse ranges of organs and tissues that have been observed for extracting DNA. The variation in samples is a reason for species type, objectives of research and ethical considerations.\u003c/p\u003e\n\u003cp id=\"_Toc208690156\"\u003eArthropod Studies; Dominance of Leg Samples\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe dataset shows single or multiple legs of arthropods as the primary source of DNA (\u003cstrong\u003eTable 1\u003c/strong\u003e). This organ yields sufficient genetic material for analysis while is nonlethal for insects. Many of the studies shows that only one or two legs of adults were used, an indication of minimum species damage and preservation of species for morphological assessment, a way to integrate genetic and morphological methods for identification.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690157\"\u003eVertebrate Studies: Muscle and Whole-body samples\u003c/p\u003e\n\u003cp\u003eMuscles, dorsal muscles tissue, muscle pieces and pectoral fins were terms used by different articles for DNA barcoding. Fish, amphibians and mammals are shown to be barcoded from source taken from muscles. Insects, larvae and other certain aquatic species have been barcoded with tissues from the whole body. Some studies have used whole body except removal of 1cm of anal region, a possible focus to ensure uncontaminated DNA samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMammalian and Avian Barcoding: Blood and Internal Organs\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSeveral studies have utilized blood samples for DNA barcoding in birds and mammals(Rehman, Jafar et al. 2015, Ali, Rehman et al. 2016). It is a non-invasive or minimally invasive way of sampling. Many studies have used internal organs like spleen, liver and tail tip which possibly involve integration of disease studies, population genetics and molecular diagnosis. Chiropteran biodiversity has been taken into consideration extensively as evidenced from the DNA extraction from patagium and hind leg tissue samples, specific for this group. Keel tissues and feather follicles are extensively reported from birds, the later one shows the non-invasive use of birds for barcoding studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAquatic and Reptilian Species: Fins, Scales, and Skin Samples\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eStudies involving fish and reptiles have been reported with DNA extraction from fins, scales and skin. DNA barcode studies have extensively used caudal and pectoral fins because it provides high quality DNA. Skin and scale samples are good sources of DNA if found fresh with less or no harm to species.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690158\"\u003eUnspecified and Non-Traditional DNA Sources\u003c/p\u003e\n\u003cp\u003eIn the current review, a notable portion of studies have not provided DNA source, highlighting a potential gap in reporting and standardization of DNA barcoding (\u003cstrong\u003eTable 1\u003c/strong\u003e) (Awan, Umar et al. 2013, Sajid, Zahid et al. 2021). Moreover, DNA extraction was carried out from animal derived products like tanned skins, coats and fur samples, ensuring the focus on illegal wildlife trade (Janjua, Fakhar-I-Abbas et al. 2017). Some studies have utilized faecal samples, hair and skin for DNA, which is a good application in non-invasive genetic monitoring of mammals (Naseem, Batool et al. 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, a diverse range of sources have been used for DNA barcoding in Pakistan, including fins and scales from fish, muscles from vertebrates, legs from arthropods, coats and tanned skins from wildlife species (\u003cstrong\u003eTable 1\u003c/strong\u003e). A more robust focus in DNA extraction should be the use of faecal matter, hair, blood drops etc for non-invasive sampling techniques.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690159\"\u003ePrimer Selection\u003c/p\u003e\n\u003cp\u003eThe effectiveness of COI gene as a standard for DNA barcoding largely depends on the selection of universal primers. Several primers utilized in Pakistan are being discussed in the text that follows.\u003c/p\u003e\n\u003cp\u003eUniversal Primers are designed to facilitate DNA barcoding across a broad range of taxa (Ullah, Ahmad et al. , Ali, Bukhari et al. 2024). These primers (\u003cstrong\u003eTable 5\u003c/strong\u003e) were originally developed for arthropod\u0026rsquo;s barcoding while now applied extensively to fish, reptiles, insects and mammals because of high success. Some primers are used for samples that have degraded DNA such as museum specimen or forensic samples (Hussain, Bukhari et al. 2025).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLepidoptera is one of the most diverse orders of class insecta. Lepidopteran primers have yielded good results in moths and butterflies ensuring reliability in amplification and sequencing (Sajid, Zahid et al. 2021, Naz, Chatha et al. 2023) (\u003cstrong\u003eTable 5\u003c/strong\u003e). In Pakistan, two sets of primers used successfully for DNA barcoding in Pakistan for fishes (Karim, Saif et al. , Sajjad, Jabeen et al. 2023). Birds, the drivers of ecosystem have been a focus of DNA barcoding in Pakistan. Several species have been identified and analysed using bird primers given in \u003cstrong\u003eTable 5\u003c/strong\u003e (Awan, Umar et al. 2013).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMini barcodes are special primers which is the focus for museum specimen, environmental DNA (eDNA) and forensic studies (Khan, William et al. 2018). A known successful primer for degraded DNA barcoding is presented below. Notably, this primer is ideal for shorter DNA sequences. Specific primers designed for identification and characterization of sea turtles (Ali, Bukhari et al. 2024) are also the key focus of turtle-based studies. Some of the primer sets are specialized for only a known species and no universal study have declared them to be universal or applicable to other groups. Moreover, many of the studies have designed primer sets for their own. These were designed with Primer3 application (Akhtar and Ali 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe selection of primers plays a crucial role in the success of DNA barcoding. Universal primers like LCO1490/HCO2198 remain widely used across taxa, while group-specific primers (e.g., FishF1/FishR1 for fish, LepF1/LepR1 for Lepidoptera) offer higher amplification success for organisms. Mini-barcoding primers such as UniMinibarF1/UniMinibarR1 have expanded the application of DNA barcoding to degraded samples (Meusnier, Singer et al. 2008). The continuous development and optimization of primers will further enhance the effectiveness of DNA barcoding in taxonomy, biodiversity monitoring, and conservation efforts.\u003c/p\u003e\n\u003cp id=\"_Toc208690160\"\u003ePhylogenetic Models and Softwares\u003c/p\u003e\n\u003cp\u003eOne of the purposes of DNA barcoding is to find evolutionary linkages between species and to study their phylogenetic relationships. Several studies have used software packages and models for assessing evolutionary relationships and phylogenetic tree construction summarized in \u003cstrong\u003eFigure 2\u003c/strong\u003e. Each of these has their own strengths and weaknesses. In the current review, the used packages include MEGA (various versions), Bayesian Inference, DNAStar, DnaSP, DNAMAN, and Fast Minimum Evolution Algorithm, along with phylogenetic models like Jukes-Cantor and Neighbor-Joining (NJ) methods illustrated in \u003cstrong\u003eFigure 2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp id=\"_Toc208690161\"\u003eMEGA Software\u003c/p\u003e\n\u003cp\u003eIn the current dataset MEGA is one of the most used software with multiple versions including MEGA 5, MEGA 6, MEGA 7, MEGA X, and MEGA 11(Kumar, Tamura et al. 1994). This software package is labelled with several user-friendly options. Firstly, it is user friendly, with a graphical user interface that is easy to navigate making it easy for beginners. Secondly, it supports comprehensive features like multiple sequence alignment, construction of phylogenetic tree, evolutionary model selection and provides ways for statistical validation. Thirdly, MEGA supports and offers multiple algorithms like Maximum Likelihood (ML), Minimum Evolution (ME), Neighbor-Joining (NJ) and Maximum Parsimony (MP). Fourthly, Muscle and ClustalW, both integrated alignment tools are supported by MEGA. Finally, it runs on Windows, Mac and Linuz systems, works offline and is updated regularly with new features and bug fixation (Keklik 2023). However, MEGA is not optimized for high performance computing, lacks flexibility, consumes significant RAM with large sequences, has no Bayesian inference and is less efficient for intensive analyses(Hall 2013).\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690162\"\u003eBayesian Inference\u003c/p\u003e\n\u003cp\u003eIt is more sophisticated and alternative method for phylogenetic analysis. It is often implemented in MrBayes, BEAST, and PhyloBayes (Huelsenbeck and Ronquist 2001). It has several advantages such as robust statistics, suitable for handling evolutionary rate heterogeneity and can be utilized with complex evolutionary models. It is limited too, due to its intensive rate of computing and requires expertise in Markov Chain Monte Carlo (MCMC) methods\u003c/p\u003e\n\u003cp id=\"_Toc208690163\"\u003eFast Minimum Evolution Algorithm\u003c/p\u003e\n\u003cp\u003eRapid phylogenetic tree can be constructed with this package. It is faster than ML and NJ methods and is sufficient for assessment of biodiversity on large scale (Desper and Gascuel 2002). Its use is limited due to the underestimation of evolutionary distances and its potential less accuracy than Bayesian inference and ML.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690164\"\u003eJukes-Cantor Model\u003c/p\u003e\n\u003cp\u003eIt is a simple substitution model that is fast and easy while making it easy only for basic phylogenetics. Its results are often unrealistic due to assumption of equal base frequencies and is less accurate than General Time Reversible models (Erickson 2010).\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690165\"\u003eDNAMAN and DNA Star\u003c/p\u003e\n\u003cp\u003eThese are commercial tools used for DNA sequence analysis. These are user friendly and supports multiple alignments, however they are not accessible publicly and are not feature-rich as MEGA or others.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690166\"\u003eAnalysis of Study Duration\u003c/p\u003e\n\u003cp\u003eThe aim of DNA barcoding is to analyse species richness and diversity, genetic variability and patterns of evolution over time. Therefore, duration of study or period of sampling is a crucial factor in reliability and robustness of DNA barcoding (Skalak, Sherwin et al. 2012). In the present dataset, a significant inconsistency has been observed such as the unviability or unevenness of duration (\u003cstrong\u003eTable 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp id=\"_Toc208690167\"\u003eImportance of Study Duration DNA Studies\u003c/p\u003e\n\u003cp\u003eSeveral aspects of the study on DNA barcoding are affected with duration of the study. Many species are available only during a specific time or season of the year. Species vary with respect to behaviour, population structure and genetic diversity. For example, insects and birds often have specific breeding seasons. Short-term research studies in DNA barcoding may lead to failure in capturing fluctuations and are biased. Moreover, long term study may help in detecting genetic changes over the years, adaptation and genetic drift. In the current review, studies that have spanned between 2010-2019 (Ashfaq, Khan et al. 2022) and 2017-19 (\u003cstrong\u003eTable 1\u003c/strong\u003e) have provided more insights as compared to those that were limited to a season or so. \u0026nbsp;Climate change and alteration of habitat are also factors of influence. They affect genetic diversity and species distribution. Studies that spanned from \u003cem\u003eJune 2014-October 2017\u003c/em\u003e or \u003cem\u003eAugust 2018-July 2022\u003c/em\u003e allows researchers to actively study climate impacts and habitat factors while short term studies fail to do so. Short term studies provide limited snapshot of genetic diversity. While studies that samples for years provide a robust and reliable aspect of diversity.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690168\"\u003eGaps and Inconsistencies\u003c/p\u003e\n\u003cp\u003eA significant number of research studies have not provided their study duration from the current review as indicated as (Not Provided) in \u003cstrong\u003eTable 1\u003c/strong\u003e. It makes difficult for future researchers to replicate or validate current findings, and temporal trends cannot be compared to data from not mentioned time. Moreover, a standardized guidelines in DNA barcoding studies are necessary for ensuring methodological transparency. Based on the lack of duration, it becomes challenging to assess whether a study sufficiently covered seasons or years to avoid sampling bias such as the summer 2022 (Gul, Shah et al. 2024) or selected months of 2020.\u003c/p\u003e\n\u003cp id=\"_Toc208690169\"\u003ePatterns Observed\u003c/p\u003e\n\u003cp\u003eWith respect to duration of study from the current dataset, four major trends can be observed. Firstly, short-term studies lasted only a few months (\u003cem\u003eMarch-August 2023\u003c/em\u003e, \u003cem\u003eOctober-February\u003c/em\u003e, \u003cem\u003eMay and October in 2020\u003c/em\u003e) (\u003cstrong\u003eTable 1\u003c/strong\u003e). These studies contribute valuable data but lack seasonal representation. \u0026nbsp;Secondly, medium-term studies that lasted from one to three years offer more insights. Thirdly, long-term studies that extended from 2010 to 2019 and 2018 to 2022 provide stronger and more reliable datasets with multi-year genetic variations and environmental influences. Finally, some studies collected sampling at irregular intervals like \u003cem\u003eSelected months from 2009, 2012, 2013, 2014, 2015\u003c/em\u003e (Liu, Panhwar et al. 2017) which may affect comparability and consistency in DNA barcoding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFuture studies may be equipped with mandatory reporting of study durations, long term monitoring, seasonal considerations and standardized methodologies.\u003c/p\u003e\n\u003cp id=\"_Toc208690170\"\u003eAnalysis of Trapping Methods\u003c/p\u003e\n\u003cp\u003eThe choice of trapping method is always associated with sample diversity, data reliability and accuracy of species delimitation (Massey, Bronzoni et al. 2022). A wide range of trapping methods have been actively used by COI-based DNA barcoding studies in Pakistan (\u003cstrong\u003eTable 2\u003c/strong\u003e)(Hussain, Kakar et al. 2024). Despite wide range of trapping methods, the data reveals inconsistencies and gaps in research.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690171\"\u003eWhy are Trapping Methods Important?\u003c/p\u003e\n\u003cp\u003eFor collection of specimens trapping methods are necessary to minimize bias while ensuring a representative sample. To what extent a method is effective? This largely depends on the preservation of genetic material because some trapping methods may damage DNA, specialized techniques are required for habitat types like terrestrial, freshwater etc summarized in \u003cstrong\u003eTable 2\u003c/strong\u003e with advantages and limitations. Moreover, some behaviours are specific for a species like insects are attracted to light traps while fish require nets. The present dataset reveals a combination of passive (trap-based) and active methods (manual collection), each of which is associated with advantages and limitations summarized and presented in \u003cstrong\u003eTable 2\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690172\"\u003eObservations and Gaps\u003c/p\u003e\n\u003cp id=\"_Toc208690173\"\u003eData Not Provided\u003c/p\u003e\n\u003cp\u003eA notable number of studies did not report trapping methods which affects data comparability and reproducibility while questions reliability. It makes data difficult for future use by researchers to assess effectiveness of different trapping methods.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690174\"\u003eBias in Trapping Methods\u003c/p\u003e\n\u003cp\u003eLight traps, malaise traps and hand collection are favourite methods for insect capturing (\u003cstrong\u003eTable 2\u003c/strong\u003e) which possibly lead to underrepresentation of non-flying or nocturnal insects. Cast nets, gill nets and drag nets have been used for aquatic species, however, mesh size and sampling depth have not been mentioned which may influence species selectivity. \u0026nbsp;In the present data set, researchers have relied greatly on hand collection and jugular vein blood sampling from mammals but there is lack of details on mammalian, amphibian and reptilian-specific traps like Sherman traps for rodents (Anthony, Ribic et al. 2005) and drift fences for amphibians. Moreover, there is lack of standardization in reporting like some studies have mentioned vague descriptions like nets and hand collection, which doesn\u0026rsquo;t reflect standardization in trapping mechanisms.\u003c/p\u003e\n\u003cp id=\"_Toc208690175\"\u003eFuture Recommendations\u003c/p\u003e\n\u003cp\u003eFuture research should focus on standardizing trapping protocols, ensuring species-specific sampling strategies, and incorporating non-invasive genetic sampling methods to improve biodiversity assessments in Pakistan. Each publishing journal should set exact trapping technique as a requirement for publication along with mesh size, lure types and collection duration. Additionally, single method may be a way to mis or underrepresentation of species therefore, incorporation of techniques should be used. Ethical consideration should be a way out in studies where animals are involved, besides eDNA should be a focus for researchers on DNA barcoding.\u003c/p\u003e\n\u003cp id=\"_Toc208690176\"\u003eAnalysis of Types of Articles\u003c/p\u003e\n\u003cp\u003eThe present review reveals that majority of the articles utilized are research articles while some are Rapid Communications, Short Communications, MITO Communications and Review Articles.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690177\"\u003eDominance of Research Articles\u003c/p\u003e\n\u003cp\u003eThe dataset is heavily skewed towards Research Articles, which are detailed studies presenting original findings based on field or laboratory investigations. These articles have presented data which lead to reproducibility, undergone peer review, are more reliable and accurate. However, these articles have focused on specific case studies and limited to few species in many cases and are limited to certain geographic area.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690178\"\u003eShort and Rapid Communications\u003c/p\u003e\n\u003cp\u003eThese are types of articles which briefly discusses and focuses on new discoveries, methodological improvements, or preliminary results. The data set shows that these articles have provided timely insights, are focused and concise. However, these articles are with limited data and lack statistical analysis. Moreover, these are not peer-reviewed extensively which may lead to compromise quality control.\u003c/p\u003e\n\u003cp id=\"_Toc208690179\"\u003eMITO Communications\u003c/p\u003e\n\u003cp\u003eIn mitochondrial genome sequencing and its applications in phylogenetics, these two types of articles play a key role. These are highly relevant to DNA barcoding and provide genome-level insights. However, these have narrow focus as ecological and taxonomic discussions are missing and are in short format, lacking extensive methodological comparisons.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690180\"\u003eReview Articles\u003c/p\u003e\n\u003cp\u003eCritical analysis has been provided by review articles on specific perspective or section of DNA barcoding. These types of studies are limited in the present dataset and discusses only specific parts or aspects of DNA barcoding (Muhammad Tahir and Akhtar 2016, Hanif, Manan et al. 2023). A lack of multiple review articles reveals lack of multiple and broader meta-analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690181\"\u003eDNA Barcoding Sampling in Pakistan\u003c/p\u003e\n\u003cp\u003eThe currently available data on COI-based DNA barcoding reveals that extensive geographic coverage has been established across Pakistan. However, a critical analysis reveals notable gaps, biases and underrepresentation of specific taxa and ecosystems.\u003c/p\u003e\n\u003cp id=\"_Toc208690182\"\u003eGeographic Bias\u003c/p\u003e\n\u003cp\u003eIn DNA barcoding studies, unequal geographic distribution is observed in sampling (\u003cstrong\u003eTable 3\u003c/strong\u003e). The dataset shows that Punjab dominated sampling efforts with significant locations from Lahore, Faisalabad, Sargodha and Multan (\u003cstrong\u003eFigure 3\u003c/strong\u003e). However, several locations like Baluchistan, Gilgit-Baltistan, Khyber Pakhtunkhwa and most of the Sindh has gained less attention despite their ecological importance. Multiple cities and river systems like Indus, Ravi and Chenab has provided more data which could be due to accessibility in Punjab, so we can assume that it is Punjab-Centric research. Baluchistan despite being the largest province with unique ecosystems covering mountains, deserts and marine, only few locations in Quetta, Ziarat and Hazarganji-Chiltan National Park have been covered in DNA barcoding. There seems a lack of surveys due to logistical challenges, political instability and limited research funding. Broader Karakoram, Himalayas and Hindu Kush regions have been underreported despite of being biodiversity hotspots (\u003cstrong\u003eTable 3\u003c/strong\u003e). Only Skardu has been given a little approach from Gilgit Baltistan. The omission of biodiversity hotspots such as distinct alpine and glacial regions is a significant research gap. Pakistan has relatively a smaller number of publications compared to neighboring countries like India, Iran, China and Bangladesh (\u003cstrong\u003eFigure 5\u003c/strong\u003e). China is leading in this scenario with more than thousand publications.\u003c/p\u003e\n\u003cp id=\"_Toc208690183\"\u003eUnderrepresentation of Certain Ecosystems\u003c/p\u003e\n\u003cp\u003eSome of the critical ecological zones have been underrepresented in DNA barcoding studies. In Punjab, rivers like Indus, Chenab, Ravi, Jhelum and Satluj have been sampled widely, however coastal and marine biodiversity is underrepresented despite their importance with respect to mangroves, coral reefs, estuaries and deep-sea ecosystems. In Sindh, few sites like Sandspit (Karachi), Fish Harbour Road (Karachi), and Sindh Coast have been sampled while the nearby marine species are still underreported, showing a biasing towards certain ecosystem due to unviability of facilities and funds. Some of the most important spots like the Cholistan desert is sampled however Thar desert is still missing, despite its unique xeric biodiversity. Moreover, Kharan and Makran deserts are also missing from the dataset. When protected areas are compared to open areas, Hazarganji-Chiltan National Park, Juniper forests in Ziarat have been discussed and sampled while northern forests, alpine meadows and temperate ecosystems are overlooked. The Margalla Hills, Deosai National Park, Fairy Meadows, and other ecologically rich areas of Gilgit-Baltistan and Khyber Pakhtunkhwa remain absent from barcoding studies (\u003cstrong\u003eFigure 3, Table 3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp id=\"_Toc208690184\"\u003eOverrepresentation of some areas\u003c/p\u003e\n\u003cp\u003eAs compared to other ecosystems, agricultural and urban ecosystems have been widely studied and explored. Pests, pollinators and invasive species have been barcoded with Ayyub Agricultural Research Institute and PARS (UAF Faisalabad) being dominated. An overrepresentation has been shown for Lahore, Karachi and Faisalabad that thrive human-modified habitats. \u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690185\"\u003eTaxonomic Bias and Underreporting\u003c/p\u003e\n\u003cp\u003eIn the current dataset, some species or ecosystem have been focused more as compared to others. For instance, there has been a more focus on economically significant species while holistic approach to biodiversity is missing. While talking taxonomically, some groups like insects, freshwater fish and agricultural pests have been reported widely while amphibians, reptiles, fungi and invertebrates have been underreported. Species of terrestrial vertebrates like birds, reptiles and mammals have been focused less as compared to occupied and protected places like national parks and zoos. Microbial DNA barcoding is nearly missing despite its importance and omnipresence in soil, water and gut that are considered crucial for ecological and health studies.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690186\"\u003eInstitutional and Logistical Constraints\u003c/p\u003e\n\u003cp\u003ePunjab and Sindh have gained more focus that suggests the research facilities, funding and role of academic institutions. Security concerns, political instability and limited infrastructure may explain less exploration of Balochistan, Gilgit-Baltistan, and interior Sindh. Moreover, cross-border and multi-geographic sampling is rare which may limit regional comparative studies on biodiversity.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690187\"\u003eConservation and Policy Implications\u003c/p\u003e\n\u003cp\u003eAs DNA barcoding is successful when two major biases are removed, the geographic and taxonomic constrains which limit its applications in conservation biology. The dataset reveals that many endemic and threatened species remain unstudied in marine, desert and high-altitude biodiversity, affecting conservation policies. Biodiversity monitoring programs are negatively affected when less species is barcoded from national parks and protected areas. A more balanced DNA barcoding efforts are need of the day in Pakistan\u0026rsquo;s like rich cultural and ecologically diverse country.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690188\"\u003eFuture Recommendations\u003c/p\u003e\n\u003cp\u003eTo have a balanced focus on rich biodiversity barcoding of Pakistan, major gaps and biases should be addressed. Sampling should be expanded to underrepresented locations of Baluchistan, Gilgit-Baltistan, Khyber Pakhtunkhwa and parts of Sindh. Marine and high-altitude ecosystems should be focused with species focus on neglected groups like reptiles and mammals. Multinational collaborative research should be the key point for future research and international partnerships should be part of the study designs for cross-border studies.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690189\"\u003eSampling Effort and Species Identified\u003c/p\u003e\n\u003cp id=\"_Toc208690190\"\u003eAnalysis of Number of Sampling Sites\u003c/p\u003e\n\u003cp\u003eSubstantial variation has been observed in the number of sampling sites across different studies utilized for this review ranging from multi-site investigations to studies with extremely limited geographical coverage (\u003cstrong\u003eTable 1\u003c/strong\u003e). The inconsistency highlights several key points regarding the comprehensiveness, representativeness, and potential limitations of DNA barcoding efforts in the country. Some studies have demonstrated extensive sampling efforts with 1858, 491 and 329 sites (\u003cstrong\u003eTable 1\u003c/strong\u003e). These studies and others like these provide robust datasets which helps in species identification and genetic diversity assessments. These types of studies are rare across 80 plus articles of the current review. In contrast to this, a high number of studies have reported few sampling sites like ten or below reaching to only one site in few studies. This can lead to underrepresentation of species and showcases biases in barcoding, reducing effectiveness of the said studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA high number of studies have not mentioned the number of sites and labelled here as \u0026ldquo;Not Mentioned\u0026rdquo; (\u003cstrong\u003eTable 1\u003c/strong\u003e). It raises concerns about scientific rigor and data reproducibility. Therefore, results can be questioned, and it is difficult to draw conclusions. Future research may be restricted due to data constrains and it will be difficult to compare, reproduce or validate data. Besides above, several studies have mentioned multiple sites which fall into the similar category of ambiguity. Missing of exact figures limits meaningful comparison and meta-analyses.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690191\"\u003eAnalysis of Number of Samples\u003c/p\u003e\n\u003cp\u003eNumber of samples were analysed across different studies on DNA barcoding in Pakistan. These numbers ranged from single-digit figures to over 60000 samples (Ashfaq, Khan et al. 2022). Some of the studies reported high number of samples such as 60,273, 53,092, 10,792, 10,653, and 8,641 (\u003cstrong\u003eTable 1\u003c/strong\u003e) that provide robust dataset that enhances species identification and reporting, biodiversity assessments and increases reliability and validity of DNA barcoding databases. These studies have been supported by external funding that is why they supported high number of samplings. Besides these, many studies reported few numbers of sampling such as 1, 3, 4 or 5. These studies contribute well to DNA barcoding studies however they are often inadequate for biodiversity assessments and conclusions. They are often coupled with poor statistical analysis and potential misidentification and incomplete representation when at species-level biodiversity is considered. In several studies from Pakistan, number of samples is missing (Not Mentioned) which limits the impact of DNA barcoding. These studies conclude that there is a lack in transparency, data validation and reproducibility. Future studies should focus on standard number of samplings in DNA barcoding while more focus should be given to Large-scale sampling.\u003c/p\u003e\n\u003cp id=\"_Toc208690192\"\u003eAnalysis of Number of Species identified\u003c/p\u003e\n\u003cp\u003eThe country\u0026rsquo;s DNA barcoding efforts are supported by studies that extended from one species to thousand number of species across different articles. These variations are associated with several factors like comprehensiveness, scope and methodological approaches.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690193\"\u003eLarge-Scale Studies: Comprehensive Biodiversity Assessment\u003c/p\u003e\n\u003cp\u003eExtensive taxonomic coverages have been demonstrated in some of the studies like 1,364 species across 1,375 (Ashfaq, Khan et al. 2022) genera in a study while another with 379 (Ashfaq, Akhtar et al. 2017) species spanning 52 families. These studies contribute efficiently in terms of importance of DNA barcoding for species delimitation, evolutionary studies and conservation strategies. Another study with 254 families which fits in 17 orders also adds to broad taxonomic range. These studies demonstrate well designed sampling strategies which is crucial for DNA barcoding. These studies have improved large-scale studies and encourages others to follow in the same range and improved Pakistan\u0026rsquo;s representation at BOLD and GenBank level.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690194\"\u003eModerate Species Coverage studies: Limited Impact\u003c/p\u003e\n\u003cp\u003eMany of the studies have reported handful of species, genera and families. Some of these have 1 to 5 species with certain studies even focusing on single species or a single genus (\u003cstrong\u003eTable 1\u003c/strong\u003e). These are valuable for specific taxonomic groups; however, they provide limited contribution to overall efforts. Small sample sizes reduce the ability to assess intraspecific variation, that is crucial for differentiating cryptic species and understanding population dynamics. Moreover, some of the studies have focus on specific taxonomic groups like one limited to 86 pheretimoid species complex (Hussain, Liaqat et al. 2022) and the great gap between morphologically and molecular identified species as 50 species identified morphologically but only 8 species identified molecularly (Khan, Kakar et al. 2024). Some of the studies have not mentioned the exact number of species which produces ambiguity for taxonomic researchers. This lack of clarity reduces the usefulness of such studies for comparative analyses and biodiversity assessments. Additionally, the use of BINs (Barcode Index Numbers) instead of species names, as seen in the study reporting 15 BINs (Ashfaq, Hebert et al. 2014), may indicate that taxonomic validation was incomplete, limiting the practical application of the data.\u003c/p\u003e\n\u003cp id=\"_Toc208690195\"\u003eNew Species: Promising but Underutilized\u003c/p\u003e\n\u003cp\u003eOut of studied articles, few studies report the discovery of new species, such as one reported 11 species including 6 new ones and another study with 2 species with 1 new species (\u003cstrong\u003eTable 4\u003c/strong\u003e)(Memon, Meier et al. 2006, Sajid, Zahid et al. 2021). This highlights the potential of DNA barcoding in uncovering previously unrecognized biodiversity in Pakistan. However, the low number of articles that reports new species can be think of as underexplained or underexplored. There also seems a lack of expertise in species validation through DNA barcoding and molecular phylogenetics. In conclusion there is a need for expansion as well standardization of sampling protocols across the country. Smaller studies should focus on poorly studied taxa to fill the gap while extensive studies should be encouraged for biodiversity assessment. Efforts should be made towards expanding taxonomic coverage and species validation.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690196\"\u003eAnalysis of Most Abundant Species\u003c/p\u003e\n\u003cp\u003eMany diversity indices like Shannon Diversity Index and Simpson\u0026rsquo;s Diversity index are extensively used to estimate species diversity and richness (\u003cstrong\u003eTable 4\u003c/strong\u003e). However, in barcoding studies it is still scarce limited to few studies (Anjum, Ilahi et al. 2025). Therefore, it is needed to carry out biodiversity estimation parallel to identification. In the current scenario, reporting of most abundant species is inconsistent, with many studies failings to provide such information. Some of the studies have mentioned explicitly the dominant species such as such as \u003cem\u003eCulex quinquefasciatus (61%)\u003c/em\u003e, \u003cem\u003eBactrocera zonata\u003c/em\u003e, \u003cem\u003eHypophthalmichthys molitrix\u003c/em\u003e, and \u003cem\u003eAnopheles stephensi.\u0026nbsp;\u003c/em\u003eMajority of list is labelled as Not Available (\u003cstrong\u003eTable 4\u003c/strong\u003e). Species that are abundant in any ecosystem play a crucial role in ecosystem stability or functioning. For example, \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e and \u003cem\u003eAedes aegypti\u003c/em\u003e are not just dominant mosquito species but also significant vectors for diseases like dengue and malaria (Rodriguez 2005). Understanding their abundance and distribution can help in vector control and public health strategies. Similarly, dominant agricultural pests such as \u003cem\u003eBactrocera zonata\u003c/em\u003e can have serious economic implications.\u003c/p\u003e\n\u003cp\u003eOn the other end of the spectrum, less abundant species may indicate rare or threatened taxa requiring conservation attention. Species such as \u003cem\u003eGazella bennettii\u003c/em\u003e and \u003cem\u003eVulpes vulpes\u003c/em\u003e are notable mentions, as their population trends could indicate habitat disturbances or conservation success. If studies do not mention the most or least abundant species, critical information about species decline or dominance is lost. When dominant species are reported, it provides insights into potential biotic homogenization, where a few species outcompete and replace native biodiversity. The presence of invasive species such as \u003cem\u003eHypophthalmichthys molitrix\u003c/em\u003e (silver carp) and \u003cem\u003eOxya hyla hyla\u003c/em\u003e (a grasshopper species) suggests ecological shifts that could affect native species and ecosystems. Without data on species abundance, it is difficult to assess the impact of invasive species or take appropriate management actions. Accurate reporting improves DNA barcoding and its utility in DNA databases. Without mentioning the true status, the databases may not reflect true ecological importance of each species.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690197\"\u003eIssues with \u0026quot;Not Available\u0026quot; Data\u003c/p\u003e\n\u003cp\u003eThe unavailability of data regarding species abundance may be coupled to several issues such as the focus of study on species identification rather than ecological significance. It may also be associated with incomplete sampling or bias in specimen collection.\u003c/p\u003e\n\u003cp id=\"_Toc208690198\"\u003eConspecific Genetic Distance: Pakistan\u0026rsquo;s Perspective\u003c/p\u003e\n\u003cp\u003eIn the current review, the intraspecific divergence varies significantly across articles (\u003cstrong\u003eTable 4\u003c/strong\u003e). Some studies have provided clear ranges or their mean values while others do not report it and labelled as \u0026ldquo;Not Available\u0026rdquo;. A broad range of genetic distances are available as low as 0.0% and as high as 67.10% (\u003cstrong\u003eTable 4\u003c/strong\u003e). This genetic distance is a crucial metric in DNA barcoding and helps to provide and establish a threshold. Generally, this value remains less than 2% but depending upon evolutionary history, geographic separation and species, it can vary.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690199\"\u003eWide Variability in Reported Values\u003c/p\u003e\n\u003cp\u003eHere, we report high inconsistency in intraspecific values from Pakistan based on COI gene. Many of the studies have reported values between 0-2.4% which aligns with standard barcoding expectations(Dinh, Ngatia et al. 2019). For instance, the studies reporting 0-1.6%, 0.0-2.26%, and mean values around 0.2%-0.7% suggest expected intraspecific variation (\u003cstrong\u003eTable 4\u003c/strong\u003e). Some of the studies from the current scenario report high values as high as 32.18% which exceed normal values which may indicate presence of cryptic species (Dinh, Ngatia et al. 2019), taxonomic errors, variable genetic markers and geographic structuring. Some of the studies have reported no variation which reflect low genetic diversity and limited sampling. Several studies have not reported conspecific genetic distances. This lack of data reduces the chance of comparability and is a gap in establishing threshold for Pakistan\u0026rsquo;s biodiversity.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690200\"\u003eImplications for Species Delimitation and Accuracy\u003c/p\u003e\n\u003cp\u003eAbsence of clear genetic distance may lead to misidentification and indicate overlooked taxonomic diversity. For species delimitation, the threshold is set to be 2%, however the variability in the dataset suggests that single universal threshold may not be sufficient therefore work on taxon-specific threshold is necessary. Future studies should adhere to standardized methods to improve the consistency and reliability of reported values.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690201\"\u003eCongeneric Genetic Distance in DNA Barcoding\u003c/p\u003e\n\u003cp\u003eIn the whole dataset, interspecific genetic divergence is inconsistent (\u003cstrong\u003eTable 4\u003c/strong\u003e). Few studies have provided well-defined ranges or their mean values from very low values of (0.0000\u0026ndash;0.0067%) to exceptionally high distances (37.02%)(Islam, Qasim et al. 2018). Congeneric distances help to define the boundary between species.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690202\"\u003eWide Variation in Reported Values\u003c/p\u003e\n\u003cp\u003eMany studies report expected values such as 2.3\u0026ndash;17.8%, 2.8\u0026ndash;23.2% (mean 8.8%), and 7.93% summarized and outlined in \u003cstrong\u003eTable 4\u003c/strong\u003e. these values are in line with global thresholds and successfully defined species boundaries.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSome of the studies have reported extreme values such as 37.02% which may be attributed to deep genetic divergence, misidentification and presence of cryptic species. Some of the values are less than 1% which may indicate recent speciation, hybridization or taxonomic issues. Many of the studies have not presented values for congeneric distances, which limits the comparison. In the current review, this gap may be due to focus on identification instead of evolutionary relationships, incomplete sampling and use of various markers instead of one universal primer.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690203\"\u003eThe Barcode Gap Challenge\u003c/p\u003e\n\u003cp\u003eThe barcode gap-the distance which separates species is crucial for distinguishing species. Congeneric distance should be more than conspecific distance. Some studies have reported overlapping values which results in blurring identification. Therefore, there is a need for taxon-specific threshold adjustment, co-validation of morphological and molecular identification and the integration of mitochondrial markers alongside nuclear markers.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690204\"\u003eFocused vs. Ignored Taxa, Limitations, and Recommendations\u003c/p\u003e\n\u003cp\u003eIn Pakistan, a wide range of taxa including insects, fishes, birds, mammals, arachnids, reptiles, and amphibians have been covered through DNA barcoding. However, this tremendous focus during the past two decades is uneven across different groups and geographies.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690205\"\u003eGroups Received More Attention\u003c/p\u003e\n\u003cp\u003eA significant portion of studies have focused on insect species including species from Diptera, Hemiptera, Lepidoptera, Hymenoptera and Orthoptera of which pests and disease vectors are dominant. Species of mosquitoes like \u003cem\u003eAedes aegypti\u003c/em\u003e, \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e, \u003cem\u003eAnopheles mosquitoes,\u0026nbsp;\u003c/em\u003epests like\u003cem\u003e\u0026nbsp;\u003c/em\u003eThrips (\u003cem\u003eThysanoptera\u003c/em\u003e), whiteflies (\u003cem\u003eHemiptera: Aleyrodidae\u003c/em\u003e), stink bugs (\u003cem\u003eHemiptera: Pentatomidae\u003c/em\u003e), fruit flies (\u003cem\u003eBactrocera spp.\u003c/em\u003e), cotton bugs (\u003cem\u003eDysdercus spp.\u003c/em\u003e, \u003cem\u003eOxycarenus hyalinipennis\u003c/em\u003e), and mealybugs (\u003cem\u003ePhenacoccus solenopsis\u003c/em\u003e) and beneficial insects like \u003cem\u003eApis mellifera\u003c/em\u003e (honeybee) and \u003cem\u003eCoccinellidae\u003c/em\u003e (ladybird beetles, important for biological control) have gained special attention. Spiders and arachnids belonging to order Araneae and have been documented well with respect to DNA barcoding. While talking about fishes, more focus has been given to economically important species like members from Cyprinidae family, marine fishes (\u003cem\u003eSillago indica\u003c/em\u003e) and endangered species like \u003cem\u003eTor putitora.\u003c/em\u003e Few studies on Doves, pigeons, pheasants and starlings, few mammals like ungulated including \u003cem\u003eGazella bennettii\u003c/em\u003e, \u003cem\u003eMoschus cupreus\u003c/em\u003e, \u003cem\u003eMuntiacus muntjac\u003c/em\u003e, \u003cem\u003eCapra hircus\u003c/em\u003e, \u003cem\u003eBos taurus\u003c/em\u003e and bats like Vespertilionidae, Emballonuridae, \u003cem\u003ePipistrellus coromandra\u003c/em\u003e have been barcoded. Some studies have examined turtles (\u003cem\u003eLissemys punctata\u003c/em\u003e) and toads (\u003cem\u003eDuttaphrynus spp.\u003c/em\u003e), however, herpetofauna remain understudies largely.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690206\"\u003eIgnored or Understudied Taxa\u003c/p\u003e\n\u003cp\u003eWhen studied and understudied groups are compared to each other, Pakistan\u0026rsquo;s coastal and deep-sea biodiversity remains largely unexplored. Corals and molluscs and crustaceans have been ignored. Studies on carnivores such as foxes, hyenas, jackals and leopards are missing. Rodents, shrews and hedgehogs have not been studies still. \u0026nbsp;Pakistan is blessed with snake and lizard diversity (Khalid, Attaullah et al. 2019), however only few studies have focused them. Molecular work on fungi and microorganisms is scarce across Pakistan.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690207\"\u003eLimitations in DNA Barcoding Studies\u003c/p\u003e\n\u003cp\u003eOne of the major limitations on DNA barcoding in Pakistan is the focus on economically important species while ignoring the ecologically significant species. Many of the studies are restricted to specific regions like Punjab. However, diverse regions like Balochistan and Khyber Pakhtunkhwa have been ignored. Moreover, many of the studies have focused on few species while other taxa are largely ignored. Finally, availability of the universal or species-specific markers is also a significant problem.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc208690208\"\u003eComparison of Pakistan\u0026rsquo;s Biodiversity and Molecular Work\u003c/p\u003e\n\u003cp\u003eCurrently over 198 fish species, 198 mammal species, 666 bird species, 177 reptile species, and 22 amphibian species have been identified in Pakistan (\u003cstrong\u003eFigure 4\u003c/strong\u003e) which is far more than the barcoded species and there is still a large gap in between the two. Moreover, approximately 6,000 species plant species but very few DNA barcoding studies exist. While microorganisms are Extremely underexplored at the molecular level.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe current study concludes that COI-based DNA barcoding in Pakistan has faced with multiple challenges. These challenges include biased geographic studies, limited resources in terms of financial as well as laboratory, fragmented studies, ignored taxa (e.g., fungi, microbes, reptiles, and marine organisms), missing barcode library, and limited availability of biodiversity data, therefore these should be the focus of next studies. Multinational collaboration should be strengthened to enhance DNA barcoding progress. Barcoding outcomes should be linked to policy and conservation strategies. Furthermore, ignored taxa can be studied if global and local collaboration between institutions and governments are established. The study further concludes the unavailability of expertise in COI-based DNA barcoding at institutional level.\u003c/p\u003e\u003cp\u003eRecommendations\u003c/p\u003e\u003cp\u003eTo address these taxonomic gaps, taxonomic coverage should be expanded to neglected taxa including carnivores, rodents, small mammals, snakes, geckos, skinks and frogs and to aquatic life across waters of Pakistan.Geographic sampling should be prioritized in underepresented region, specifically Balochistan, Khyber Pakhtunkhwa and Gilgit-Baltistan. COI gene should be coupled with other genes as well as molecular markers should be assessed alongside morphological characters. Moreover, barcoding outcomes should be linked to policy management and conservation strategies. Conservation strategies should be focused and eDNA should be the top priority of DNA barcoding researchers with the use of Artificial intelligence for species delimitation and sequencing as well. We develop and propose a standardized checklist (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e6\u003c/span\u003e) to enhance reproducibility and comparability of future DNA barcoding studies in Pakistan. Additionally, a three-tier proposal has been introduced to address the gaps in future studies (Fig.\u0026nbsp;6).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.A: Wrote the main manuscript and developed the idea.I.I and Q.Z supervised and reviewed the manuscript.M.S.K prepared figures.R.K prepared tables and edited the manuscript.I.U visualized the figures and curated the data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAkhtar T, Ali G (2016) DNA barcoding of Schizothorax species from the Neelum and Jhelum Rivers of Azad Jammu and Kashmir. 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Pakistan Journal of Zoology 49(5)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMassey AL, Bronzoni RVdM, da Silva DJF, Allen JM, de L\u0026aacute;zari PR, dos Santos-Filho M, Canale GR, Bernardo CSS, Peres CA, Levi T (2022) Invertebrates for vertebrate biodiversity monitoring: Comparisons using three insect taxa as iDNA samplers. Mol Ecol Resour 22(3):962\u0026ndash;977\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMemon N, Meier R, Manan A, SU KFY (2006) On the use of DNA sequences for determining the species limits of a polymorphic new species in the stink bug genus Halys (Heteroptera: Pentatomidae) from Pakistan. Syst Entomol 31(4):703\u0026ndash;710\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeusnier I, Singer GA, Landry J-F, Hickey DA, Hebert PD, Hajibabaei M (2008) A universal DNA mini-barcode for biodiversity analysis. 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Annals Anim Sci 23(2):363\u0026ndash;389\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRehman A, Jafar S, Raja NA, Mahar J (2015) Use of DNA barcoding to control the illegal wildlife trade: a CITES case report from Pakistan. J Bioresource Manage 2(2):3\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRodriguez MH (2005) Malaria and dengue vector biology and control in Latin America. Frontis: 129\u0026ndash;141\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRuedi M, Manzinalli J, Dietrich A, Vinciguerra L (2023) Shortcomings of DNA barcodes: a perspective from the mammal fauna of Switzerland. Hystrix, the Italian. J Mammal 34(1):54\u0026ndash;61\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSajid M, Zahid M, Shah M, Rasool M, Ullah I, Ahmad R, Habibullah P, Majeed N (2021) IDENTIFICATION OF THE ORB WEAVING SPIDER (ARANEAE: ARANEIDAE) FAUNA OF DIR LOWER (PAKISTAN) THROUGH DNA BARCODING. JAPS. J Anim Plant Sci 31(4)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSajjad A, Jabeen F, Ali M, Zafar S (2023) DNA barcoding and phylogenetics of Wallago attu using mitochondrial COI gene from the River Indus. J King Saud University-Science 35(6):102725\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSkalak SL, Sherwin RE, Brigham RM (2012) Sampling period, size and duration influence measures of bat species richness from acoustic surveys. Methods Ecol Evol 3(3):490\u0026ndash;502\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUllah S, Ahmad H and M. A. Rafi CO1 based DNA barcoding of some pentatomomorpha bugs (Hemiptera: Heteroptera) from Swat, Pakistan\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 6 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"DNA barcoding, COI gene, Biodiversity, Species identification, Pakistan fauna, Genetic divergence, Review","lastPublishedDoi":"10.21203/rs.3.rs-7936079/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7936079/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDNA barcoding using the cytochrome oxidase I (COI) gene has revolutionized species identification and biodiversity research in Pakistan since 2006. This review evaluates the progress, trends, and challenges of COI-based DNA barcoding across various taxa, focusing on taxonomic coverage, genetic divergence, sampling methodologies, publication trends, and phylogenetic analyses. A total of 120 articles were downloaded, of which 91 were retained based on strict relevancy. Research efforts are unevenly distributed, with insects, particularly mosquitoes and fruit flies, receiving the most attention, followed by economically significant freshwater fish like Cyprinidae. However, mammals, amphibians, marine organisms, fungi, and microbes remain largely underrepresented, highlighting the need for broader taxonomic studies. Geographic coverage and sample sizes vary widely, affecting statistical reliability and species representation. Methodological inconsistencies, such as unreported collection sites and varying trapping techniques, limit reproducibility and comparative analysis. Genetic divergence data reveal inconsistencies, with conspecific distances typically between 0\u0026ndash;2% but sometimes reaching extreme values (e.g., 67.10%), suggesting cryptic species or sequencing errors. Congeneric distances also vary significantly, emphasizing the need for taxon-specific barcoding thresholds instead of a universal cutoff. Phylogenetic analyses predominantly use MEGA software, with Neighbor-Joining and Maximum Likelihood methods being most common, while Bayesian inference remains underutilized. The publication trend was slow from 2006 to 2012 but showed steady growth from 2013 to 2021 and a sharp rise from 2022 onwards due to increased funding and technological advancements. Research is mainly published in international journals, with some contributions in national journals like the Pakistan Journal of Zoology. To enhance DNA barcoding in Pakistan, improvements such as expanded taxonomic and geographic coverage, standardized methodologies, increased data sharing, and integration with multigene approaches are necessary. Addressing these gaps will improve the accuracy and global relevance of COI-based DNA barcoding, supporting better conservation and sustainable management of Pakistan\u0026rsquo;s biodiversity.\u003c/p\u003e","manuscriptTitle":"COI-Based DNA Barcoding in Pakistan: Progress, Challenges, and Future Directions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-30 18:02:40","doi":"10.21203/rs.3.rs-7936079/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e618076e-00bf-4f66-87c0-6366e7390d97","owner":[],"postedDate":"October 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-30T18:02:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-30 18:02:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7936079","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7936079","identity":"rs-7936079","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}