Assembling a DNA barcode reference library for the spiders (Arachnida: Araneae) of Pakistan

RESEARCH ARTICLE Assembling a DNA barcode reference library for the spiders (Arachnida: Araneae) of Pakistan Muhammad Ashfaq ID1☯*, Gergin Blagoev1☯, Hafiz Muhammad Tahir2, Arif M. Khan3, Muhammad Khalid Mukhtar4, Saleem Akhtar5, Abida Butt6, Shahid Mansoor7, Paul D. N. Hebert1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, Canada, 2 Department of Zoology, GC University, Lahore, Pakistan, 3 Department of Biotechnology, University of Sargodha, Sargodha, Pakistan, 4 Department of Zoology, University of Sargodha, Sargodha, Pakistan, 5 Directorate of Entomology, Ayub Agricultural Research Institute, Faisalabad, Pakistan, 6 Department of Zoology, University of the Punjab, Lahore, Pakistan, 7 National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan ☯ These authors contributed equally to this work. * OPEN ACCESS Citation: Ashfaq M, Blagoev G, Tahir HM, Khan AM, Mukhtar MK, Akhtar S, et al. (2019) Assembling a DNA barcode reference library for the spiders (Arachnida: Araneae) of Pakistan. PLoS ONE 14(5): e0217086. https://doi.org/10.1371/ journal.pone.0217086 Editor: Sebastian D. Fugmann, Chang Gung University, TAIWAN Received: January 15, 2019 Accepted: May 4, 2019 Published: May 22, 2019 Copyright: © 2019 Ashfaq et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Collection data, a photograph, a taxonomic assignment, and DNA barcode (COI-5p) sequence for each specimen are available in the public dataset, "DS-MASPD DNA barcoding spiders of Pakistan" on the Barcode of Life Data System (BOLD) (www.boldsystems.org). (dx.doi.org/10.5883/DS-MASPD). Funding: This study was enabled by grant 106106001 “Engaging Developing Nations in iBOL” from the International Development Research Centre in Canada and by grant HEC No. 20-1403/R& D/09 Abstract Morphological study of 1,795 spiders from sites across Pakistan placed these specimens in 27 families and 202 putative species. COI sequences >400 bp recovered from 1,782 specimens were analyzed using neighbor-joining trees, Bayesian inference, barcode gap, and Barcode Index Numbers (BINs). Specimens of 109 morphological species were assigned to 123 BINs with ten species showing BIN splits, while 93 interim species included representatives of 98 BINs. Maximum conspecific divergences ranged from 0–5.3% while congeneric distances varied from 2.8–23.2%. Excepting one species pair (Oxyopes azhari–Oxyopes oryzae), the maximum intraspecific distance was always less than the nearest-neighbor (NN) distance. Intraspecific divergence values were not significantly correlated with geographic distance. Most (75%) BINs detected in this study were new to science, while those shared with other nations mainly derived from India. The discovery of many new, potentially endemic species and the low level of BIN overlap with other nations highlight the importance of constructing regional DNA barcode reference libraries. Introduction With nearly 48,000 known species in 117 families [1], spiders are a major component of terrestrial ecosystems with important practical applications as biocontrol agents [2] and as bio-indicators [3,4]. Prior studies have documented 4,300 spider species in Europe [5] and a similar number (3,800) in the Nearctic [6]. By contrast, just 2,300 species have been reported from South Asia [7], suggesting that many species await detection in this region. Although studies on the spider fauna of Pakistan began nearly a century ago [8], work has recently intensified, but most of these studies have produced regional checklists (S1 Table). Unfortunately, these PLOS ONE | https://doi.org/10.1371/journal.pone.0217086 May 22, 2019 1 / 21 DNA barcoding spiders of Pakistan “Sequencing DNA Barcodes of Economically Important Insect Species from Pakistan” from the Higher Education Commission of Pakistan awarded to MA. Sequence analysis was made possible by a grant from the Government of Canada through Genome Canada and Ontario Genomics in support of the International Barcode of Life (iBOL) project awarded to PDNH. This is a contribution to the Food From Thought project supported by the Canada First Research Excellence Fund awarded to PDNH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. publications often employ invalid or incorrect species names or only identify specimens to a family [9], compromising their value [10–12]. It is likely that many species reported as new discoveries from Pakistan [13] await description. For example, in her dissertation research on spiders of Punjab, Parveen [13] reported the discovery of 33 new species but only one has been formally described [9]. Examination of prior taxonomic work (S1 Table) indicates that just 400 species of spiders have been documented from Pakistan. Considering the country’s diverse ecosystems [14], this count must seriously underestimate the true diversity of its fauna given the much higher numbers reported for India (1686) [15] and Iran (528) [16]. The limited knowledge of the spider fauna of Pakistan is a particular example of the barrier to our general understanding of spider biodiversity in a global context, a factor compromising both scientific progress and conservation efforts [17]. The poor documentation of spider diversity of Pakistan reflects, in part, the paucity of taxonomic specialists working on the group [18]. Moreover, spiders pose a challenge for morphological approaches because cryptic species are common [19], and sexual dimorphism is often striking [20]. DNA barcoding [21] provides an alternate approach to identifications. It employs sequence diversity in a standard gene region (COI-50 ) to discriminate both morphologically cryptic species and all life stages, even for species with sexual dimorphism [22,23]. Although concerns about the use of single marker [24,25] or discordance between the barcode and other gene regions [26] have been voiced [27], the advantages of employing a single standard gene region for DNA barcoding is now very well established [28]. Fifteen years after its introduction, this approach has demonstrated its effectiveness in discriminating species in diverse groups, including spiders [29–34]. The use of DNA barcoding for specimen identification and species discovery is greatly facilitated by BOLD, the Barcode of Life Data System (http://www.boldsystems.org). This informatics platform assembles specimen metadata and sequences and provides tools to facilitate data analysis and publication [35]. It also enables species discrimination by assigning each COI sequence cluster to a (...truncated)