Skip to main content
NCBI home page
PLOS One logoLink to PLOS One
. 2019 May 22;14(5):e0217086. doi: 10.1371/journal.pone.0217086

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

Muhammad Ashfaq 1,*,#, Gergin Blagoev 1,#, Hafiz Muhammad Tahir 2, Arif M Khan 3, Muhammad Khalid Mukhtar 4, Saleem Akhtar 5, Abida Butt 6, Shahid Mansoor 7, Paul D N Hebert 1
Editor: Sebastian D Fugmann8
PMCID: PMC6530854  PMID: 31116764

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 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-5′) 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 Barcode Index Number (BIN) [36], which is an analogue of Operational Taxonomic Unit (OTU). Because BINs have high congruence with species recognized through morphological analysis [37–40], they are now routinely used as a species proxy [41,42]. Consequently, they have gained wide adoption [41,43] for cryptic species recognition [40,43], species discovery [44], taxonomic revisions [45], and faunal assessments [46,47]. The DNA barcode reference libraries available for diverse animal groups [48–54] are helping to identify newly collected specimens [45,54] and to speed taxonomic progress [33]. By assigning sequences from unidentified specimens to a species proxy [44], the BIN system has greatly augmented the application of barcode data in groups where taxonomic knowledge is poor. These barcode libraries are, in effect, forming the foundation for a global “DNA library of life” [55].

At present, BOLD holds 6.8 million records derived from specimens representing 587,000 BINs (accessed 13 April, 2019). This total includes 117,000 records from spiders that have been assigned to more than 10,000 BINs. Past work on spiders has had varied motivations [39,56–60], but just two prior studies have aimed to construct a comprehensive DNA barcode library for a national fauna–Canada [61] and Germany [62]. The need for similar work in other regions is evident, particularly in south Asia. For example, barcode records are only available for 73 species of spiders from India [35,63] and for 41 species from Pakistan [64–66]. The current study aimed to develop a barcode library for the spider fauna of Pakistan and investigate the spider diversity overlap with other regions using BINs. The study addresses the gap for reference data in the country by expanding DNA barcode coverage for Pakistan to 202 species.

Materials and methods

Ethics statement

No specific permissions were required for this study. The study did not involve endangered or protected species.

Spider collection

From 2010 to 2016, 1,795 spiders were collected at 225 sites in Pakistan (Fig 1). Each spider was provisionally identified by collectors in Pakistan before it was sequenced for the barcode region of the mitochondrial COI gene [21]. GB subsequently validated and refined identifications by examining (including genitalic dissections) representative specimens from each barcode cluster. Generic and species assignments generally followed taxonomic publications on Asian spiders (S1 Table), but nomenclature was updated as required to follow the World Spider Catalog [1]. Collection data, a photograph, and a taxonomic assignment for each specimen are available in the public dataset, "DS-MASPD DNA barcoding spiders of Pakistan" (dx.doi.org/10.5883/DS-MASPD) on BOLD. The 1,795 specimens are held in four repositories: Centre for Biodiversity Genomics, University of Guelph, Guelph, Canada (585); National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan (1126); University of Sargodha, Sargodha, Pakistan (84). The location of any particular specimen is reported in the dataset.

Fig 1. Map showing collection localities for the 1,795 spiders examined in this study.

Fig 1

Open in a new tab

The map was developed using www.simplemappr.net. The author of SimpleMapper has waived all copyrights and no permission is needed to use. GPS coordinates (Latitude, Longitude) for the collection localities were: 24.45, 70.8; 25.488, 67.821; 25.681, 67.781; 25.756, 67.739; 25.757, 67.732; 25.759, 67.737; 25.76, 67.732; 25.801, 67.733; 25.812, 67.739; 25.9, 69.85; 28.083, 70.283; 28.261, 70.647; 28.293, 70.115; 28.304, 70.134; 28.306, 70.128; 28.308, 70.132; 28.308, 70.134; 28.309, 70.13; 28.309, 70.131; 28.309, 70.133; 29.083, 69.083; 29.103, 70.324; 29.104, 70.324; 29.105, 70.328; 29.24, 71.415; 29.242, 71.413; 29.39, 71.68; 29.393, 71.688; 29.393, 71.684; 29.394, 71.682; 29.396, 71.683; 29.401, 71.627; 29.429, 71.548; 29.454, 71.161; 29.518, 71.645; 29.584, 71.439; 29.868, 71.291; 29.9167, 69.9667; 30, 70.6; 30.026, 71.381; 30.053, 71.385; 30.065, 71.363; 30.105, 71.417; 30.189, 71.455; 30.189, 71.458; 30.189, 71.457; 30.191, 71.457; 30.516, 72.583; 30.518, 72.624; 30.519, 72.606; 30.52, 72.624; 30.522, 72.635; 30.523, 72.629; 30.525, 72.624; 30.529, 72.63; 30.531, 72.655; 30.531, 72.632; 30.533, 72.63; 30.534, 72.633; 30.534, 72.606; 30.537, 72.638; 30.538, 72.641; 30.54, 72.608; 30.585, 72.993; 30.6, 73.0667; 30.65, 73.1; 30.66, 73.1; 30.6612, 73.1086; 30.791, 72.594; 30.8, 72.05; 30.832, 72.512; 30.85, 72.083; 30.85, 72.544; 30.854, 72.538; 30.855, 72.54; 30.855, 72.539; 30.856, 72.572; 30.857, 72.542; 30.859, 72.566; 30.862, 72.56; 30.862, 72.554; 30.866, 72.555; 30.875, 72.557; 30.959, 73.984; 31.024, 74.531; 31.033, 73; 31.0833, 73.95; 31.2167, 73.8667; 31.3333, 73.4167; 31.3833, 73.0167; 31.3833, 73; 31.393, 73.027; 31.394, 73.026; 31.4167, 73.05; 31.4167, 73.0667; 31.45, 73.7; 31.45, 73.6833; 31.45, 73.1333; 31.463, 74.436; 31.4667, 73.2; 31.496, 74.294; 31.5, 73.2667; 31.532, 73.063; 31.5333, 74.3333; 31.56, 72.54; 31.6167, 73.8667; 31.64, 74.13; 31.825, 72.541; 31.8424, 70.8952; 31.86, 73.276; 31.924, 72.863; 31.965, 72.867; 31.976, 72.328; 31.986, 72.832; 32.027, 72.653; 32.034, 72.703; 32.05, 73; 32.055, 72.946; 32.059, 73.011; 32.063, 73.042; 32.0667, 72.6667; 32.0667, 72.6833; 32.067, 73.05; 32.074, 72.684; 32.077, 72.671; 32.077, 72.67; 32.078, 72.672; 32.08, 72.9; 32.081, 72.667; 32.082, 72.675; 32.083, 73.067; 32.0837, 72.6719; 32.084, 72.68; 32.088, 72.673; 32.093, 72.684; 32.1, 73.067; 32.102, 72.957; 32.109, 72.846; 32.11, 72.655; 32.119, 72.679; 32.122, 72.681; 32.125, 72.693; 32.1333, 74.1833; 32.15, 74.1833; 32.17, 72.26; 32.19, 73.025; 32.267, 72.476; 32.275, 72.904; 32.287, 72.43; 32.3054, 72.3482; 32.5333, 69.85; 32.56, 72.02; 32.59, 72.999; 32.59, 72.008; 32.59, 73.049; 32.59, 73.999; 32.591, 73.008; 32.591, 72.999; 32.5916, 72.3446; 32.592, 73.011; 32.592, 72.999; 32.593, 72.999; 32.594, 73.02; 32.594, 72.999; 32.595, 72.999; 32.5964, 72.217; 32.597, 73.041; 32.601, 73.369; 32.601, 73.038; 32.603, 73.042; 32.624, 73; 32.629, 73.009; 32.63, 73.005; 32.632, 73.013; 32.637, 73.008; 32.637, 72.008; 32.652, 73; 32.656, 73.005; 32.657, 73.004; 32.658, 73.003; 32.6581, 73.0034; 32.659, 73.008; 32.6592, 72.2433; 32.755, 72.677; 33.686, 73.076; 33.714, 73.132; 33.714, 73.133; 33.714, 73.13; 33.715, 73.132; 33.716, 73.129; 33.7167, 73.0333; 33.7167, 73.05; 33.7667, 73.8833; 33.8, 72.9167; 33.8167, 73.8167; 33.9, 73.3833; 33.9167, 73.3833; 34.333, 73.204; 34.334, 73.201; 34.38, 73.52; 34.38, 73.54; 34.385, 73.544; 34.386, 73.546; 34.386, 73.545; 34.541, 73.348; 34.543, 73.348; 34.546, 73.349; 34.638, 73.461; 34.639, 73.461; 34.639, 73.462; 34.7333, 72.35; 34.7667, 72.35; 34.776, 73.527; 34.777, 73.526; 34.778, 73.528; 34.78, 73.53; 34.78, 73.531; 34.8167, 72.3333; 35.426, 74.098; 35.461, 72.588; 35.465, 72.584; 35.4667, 72.5833; 35.478, 72.588; 35.918, 74.29; 35.918, 74.289.

Molecular analysis

DNA extraction, PCR, and Sanger sequencing were performed at the Canadian Centre for DNA Barcoding (CCDB) (http://ccdb.ca/resources/) using standard protocols. A single leg was removed from each specimen with a sterile forceps and transferred into a well in a 96-well microplate pre-filled with 30 μl of 95% EtOH. DNA was subsequently extracted by tissue lysis at 56°C overnight followed by a column-based protocol [67]. PCR amplification of the COI-5′ barcode region employed the primer pair C_LepFolF and C_LepFolR (http://ccdb.ca/site/wp-content/uploads/2016/09/CCDB_PrimerSets.pdf). This primer cocktail includes equal volume of LepF1 [68] /LCO1490 [69] and LepR1 [68] /HCO2198 [69], respectively. The target COI region was amplified using 2 μL of DNA template in a 12.5 μL reaction containing standard PCR ingredients [30] employing the following PCR regime: 94°C (1 min), 5 cycles of 94°C (40 s), 45°C (40 s), 72°C (1 min); 35 cycles of 94°C (40 s), 51°C (40 s), 72°C (1 min) and final extension of 72°C (5 min). Amplicons were analyzed on a 2% agarose E-gel 96 system (Invitrogen Inc.) and were sequenced bidirectionally using the BigDye Terminator Cycle Sequencing Kit (v3.1) on an Applied Biosystems 3730XL DNA Analyzer. Sequences were assembled, aligned, and edited using CodonCode Aligner (CodonCode Corporation, USA) and validated in MEGA5 [70] to ensure they lacked a stop codon.

Data analysis

All sequences were submitted to BOLD (DS-MASPD) where those meeting required quality criteria (>507 bp, <1% Ns, no stop codon or contamination flag) were assigned to a BIN [36]. An accumulation curve, BIN discordance, genetic distance analysis, barcode gap analysis (BGA), and geo-distance correlation were determined using analytical tools on BOLD. The Accumulation Curve plots the rise in the number of BINs with increased sampling effort making it possible to ascertain if asymptotic diversity has been reached. The BGA determines if the maximum sequence divergence within members of a species or BIN is less than the distance to its Nearest-Neighbor (NN) species or BIN, a condition required for unambiguous identification [71,72]. The geo-distance correlation ascertains the correlation between geographic distance and genetic distance in each species or BIN employing two methods. The Mantel Test [73] examines the relationship between the geographic distance (km) and genetic divergence (K2P) matrices. The second approach compares the spread of the minimum spanning tree of collection sites and maximum intra-specific divergence [61]. The relationship between geographic and intraspecific distances was analyzed for each species with at least one individual from three or more sites. The analysis included all the conspecific records public on BOLD.

A neighbor-joining (NJ) tree was generated in MEGA5 using the Kimura-2-Parameter (K2P) [74] distance model along with pairwise deletion of missing sites. Nodal support on the NJ tree was estimated by 1000 bootstrap replicates. Bayesian inference (BI) was calculated by MrBayes v3.2.0 [75] using representative sequences of the 221 BINs and employing Phalangium opilio (Arachnida: Opiliones) and Galeodes sp. (Arachnida: Solifugae) as outgroups. The data was partitioned in two ways; i) a single partition with parameters estimated across all codon positions, ii) a codon-partition in which each codon position was allowed different parameter estimates. Sequence evolution was modelled by the GTR+Γ model independently for the two partitions using the ‘‘unlink” command in MrBayes. Analyses were run for 10 million generations using four chains with sampling every 1000 generations and the BI trees were obtained using the Markov Chain Monte Carlo (MCMC) technique. Posterior probabilities were calculated from the sample points once the MCMC algorithm converged. Convergence was determined when the standard deviation of split frequencies was less than 0.022 and the PSRF (potential scale reduction factor) approached 1, and both runs converged to a stationary distribution after the burn-in stage (the first 25% of samples were discarded by default). The resultant trees were visualized in FigTree v1.4.0. The NJ and Bayesian analyses were employed to assess support for the BINs detected in this study, not to reconstruct the phylogeny of Araneae.

Results

Coupling of the DNA sequence results with detailed morphological analysis made it possible to assign 1,574 of the 1,795 barcoded specimens to one of 109 species, but the other 221 specimens could only be placed into one of 93 interim species. Collectively, these specimens included representatives of 27 families, 113 genera, and 202 species (Table 1). Most species were only represented by a single sex, usually females. Two-thirds (1,256) of the specimens were immatures that lacked the diagnostic characters required for species assignment. However, their DNA barcodes allowed them to be linked to adults whose identification was established through morphology. Four families (Amaurobiidae, Atypidae, Ctenidae, Segestriidae), 43 genera, and 74 species identified here represent first records for Pakistan (Tables 1 and S1). As adults from 12 of the 93 interim species possessed clear morphological differences from any known species in their genus, they are likely new to science (Table 1).

Table 1. Species, maximum barcode divergence (K2P), nearest neighbor distance (NN), and BIN assignment of 1,795 spiders collected in Pakistan.

No. Taxa N K2P NN BINs
Agelenidae C. L. Koch,1837
1 Draconarius sp. 1GAB_PAK 2 0 8.8 BOLD:AAO2052
2 Draconarius sp. 2GAB_PAK 2 0 8.8 BOLD:AAO2053
*NP 3 Tegenaria domestica (Clerck, 1757) 1 N/A 19 BOLD:AAF1312
NP Amaurobiidae Thorell,1870
*NS 4 Himalmartensus cf. martensi Wang & Zhu, 2008 1 N/A 14 BOLD:ACB2928
Araneidae Clerck, 1757
NP 5 Araneus affinis Zhu, Tu & Hu, 1988 2 0.6 12 BOLD:AAV7611
6 Araneus mitificus (Simon, 1886) 20 1.7 13 BOLD:AAV1598
7 Araniella sp. 1GAB_PAK 4 0.8 10 BOLD:AAV1625
8 Argiope aemula (Walckenaer, 1841) 8 1.4 10 BOLD:ACG0732
9 Argiope anasuja Thorell, 1887 1 N/A 9.5 BOLD:ACB2926
NP 10 Argiope lobata (Pallas, 1772) 2 0.5 12 BOLD:ACI8559
NP 11 Argiope pulchella Thorell, 1881 6 0.8 9.5 BOLD:ACG0576
12 Argiope trifasciata (Forsskål, 1775) 15 1.1 10 BOLD:AAQ2634
NP 13 Chorizopes wulingensis Yin, Wang & Xie, 1994 2 0.5 12 BOLD:ABX7347
U 14 Cyclosa confraga (Thorell, 1892) 8 0.8 18 BOLD:ADF2726
15 Cyclosa hexatuberculata Tikader, 1982 4 0 11 BOLD:ADD8756
NP 16 Cyclosa moonduensis Tikader, 1963 9 1.1 11 BOLD:ACZ2455
17 Cyrtophora citricola (Forsskål, 1775) 66 1.6 13 BOLD:AAO2032
18 Eriovixia excelsa (Simon, 1889) 40 1.1 16 BOLD:AAQ0105
19 Gea subarmata Thorell, 1890 1 N/A 10 BOLD:ACG0733
*NS 20 Hypsosinga cf. alboria Yin, Wang, Xie & Peng, 1990 4 0 10 BOLD:ABX7344
*NP 21 Hypsosinga wanica Song, Qian & Gao, 1996 17 1.6 10 BOLD:AAQ0134
22 Larinia phthisica (L. Koch, 1871) 5 0.9 10 BOLD:AAO2160
23 Larinia sp. 1GAB_PAK 1 N/A 11 BOLD:ABX7407
* 24 Leviellus sp. 1GAB_PAK 2 0 14 BOLD:AAV1590
NP 25 Neoscona polyspinipes Yin, Wang, Xie & Peng, 1990 20 0.8 4.9 BOLD:AAO1983
NP 26a Neoscona scylla (Karsch, 1879) 13 1.9 7.8 BOLD:ACI8762
26b Neoscona scylla (Karsch, 1879) 16 BOLD:AAO1997
27 Neoscona sp. 1BAG_PAK 1 N/A 7.6 BOLD:ACI2573
28 Neoscona sp. 2BAG_PAK 1 N/A 9 BOLD:ADD4537
NP 29 Neoscona subfusca (C. L. Koch, 1837) 1 N/A 8.6 BOLD:AAV3851
30 Neoscona theisi (Walckenaer, 1841) 160 2.5 7.6 BOLD:ACM3489
31 Neoscona vigilans (Blackwall, 1865) 38 1.5 4.9 BOLD:AAO2202
32 Plebs himalayaensis (Tikader, 1975) 2 0 17 BOLD:ACI8675
NP Atypidae Thorell, 1870
*NS 33 Calommata sp. 1GAB_PAK 1 N/A 21 BOLD:ACP9624
Cheiracanthiidae Wagner, 1887
NP 34 Cheiracanthium inornatum O. Pickard-Cambridge, 1874 5 2.3 7.4 BOLD:ACC4872
NP 35 Cheiracanthium insulanum (Thorell, 1878) 20 3.3 6.9 BOLD:AAQ0110
36 Cheiracanthium sp. 1GAB_PAK 2 0.2 11 BOLD:ACA7676
37 Cheiracanthium sp. 2GAB_PAK 2 1.1 4.9 BOLD:ABW2880
38 Cheiracanthium sp. 3GAB_PAK 2 0.2 4.9 BOLD:AAU6055
Clubionidae Wagner, 1887
39 Clubiona drassodes O. Pickard-Cambridge, 1874 28 0.9 13 BOLD:AAV1620
40 Clubiona filicata O. Pickard-Cambridge, 1874 18 0.9 13 BOLD:AAV1603
41 Clubiona sp. 1GAB_PAK 1 N/A 8.8 BOLD:AAV1602
42 Clubiona sp. 2GAB_PAK 1 N/A 8.8 BOLD:AAO2055
Corinnidae Karsch, 1880
43 Castianeira sp. 1GAB_PAK 1 N/A 16 BOLD:ACP7698
NP Ctenidae Keyserling, 1877
* 44 Anahita sp. 1GAB_PAK 1 N/A 12 BOLD:ADF5307
* 45 Ctenus sp. 1GAB_PAK 1 N/A 9 BOLD:AAV1591
* 46 Ctenus sp. 2GAB_PAK 1 N/A 9 BOLD:ABW2888
Filistatidae Ausserer, 1867
47 Kukulcania sp. 1GAB_PAK 1 N/A 22 BOLD:ABX7408
Gnaphosidae Pocock, 1898
48 Berlandina afghana Denis, 1958 1 N/A 14 BOLD:AAV1613
49 Drassodes sp. 1GAB_PAK 1 N/A 12 BOLD:AAV1404
*NP 50 Drassyllus coreanus Paik, 1986 2 0 14 BOLD:AAV0899
51 Gnaphosa jodhpurensis Tikader & Gajbe, 1977 2 1.2 15 BOLD:ACR0656
*NP 52 Haplodrassus signifer (C. L. Koch, 1839) 1 N/A 13 BOLD:ACB2432
53 Micaria sp. 1GAB_PAK 1 N/A 13 BOLD:ACP3811
* 54 Phaeocedus sp. 1GAB_PAK 2 1.2 14 BOLD:AAV1605
*NP 55 Scopoides maitraiae (Tikader & Gajbe, 1977) 2 0 16 BOLD:ACZ1655
*NP 56 Trachyzelotes kulczynskii (Bösenberg, 1902) 1 N/A 13 BOLD:AAQ2633
NS 57 Zelotes cf. puritanus Chamberlin, 1922 2 0.8 12 BOLD:AAQ0137
NP 58 Zelotes shantae Tikader, 1982 1 N/A 12 BOLD:ADD7482
59 Zelotes sp. 1GAB_PAK 1 N/A 12 BOLD:ACZ4032
*NP 60 Zimiris diffusa Platnick & Penney, 2004 1 N/A 14 BOLD:AAV1616
Hersiliidae Thorell, 1870
61 Hersilia savignyi Lucas, 1836 16 1.1 17 BOLD:AAP4789
Linyphiidae Blackwall, 1859
62 Gnathonarium dentatum (Wider, 1834) 5 0 14 BOLD:AAQ0150
* 63 Mermessus sp. 1GAB_PAK 1 N/A 14 BOLD:ACP3810
*NP 64 Neriene emphana (Walckenaer, 1841) 3 0.8 14 BOLD:ACI8558
Lycosidae Sundevall, 1833
* 65 Alopecosa sp. 1GAB_PAK 1 N/A 9.2 BOLD:AAV1615
NS 66 Arctosa cf. serrulata Mao & Song, 1985 1 N/A 9.4 BOLD:ACB2931
67 Arctosa sp. 1GAB_PAK 1 N/A 13 BOLD:AAV1608
68 Draposa oakleyi (Gravely, 1924) 19 1.6 5.8 BOLD:ABX7398
NS 69 Evippa sp. 1GAB_PAK 5 1.4 8.3 BOLD:ABX7397
70 Evippa sp. 2GAB_PAK 1 N/A 8.3 BOLD:ABW2890
71a Hippasa pisaurina Pocock, 1900 16 4.1 5.8 BOLD:AAO2058
71b Hippasa pisaurina Pocock, 1900 1 BOLD:ADF3448
72 Hippasa sp. 1GAB_PAK 1 N/A 5.8 BOLD:ADE8277
* 73 Hogna sp. 1GAB_PAK 5 0.6 10 BOLD:AAQ0158
* 74 Hogna sp. 2GAB_PAK 1 N/A 11 BOLD:ADF5080
75 Lycosa poonaensis Tikader & Malhotra, 1980 5 0.6 10 BOLD:ABW2889
76 Lycosa sp. 1GAB_PAK 1 N/A 10 BOLD:AAO2168
E 77 Lycosa terrestris Butt, Anwar & Tahir, 2006 45 0.9 4.3 BOLD:AAO2150
NP 78 Pardosa mionebulosa Yin, Peng, Xie, Bao & Wang, 1997 3 1.6 5.3 BOLD:ACZ3882
79 Pardosa pseudoannulata (Bösenberg & Strand, 1906) 5 0.6 5.9 BOLD:AAO2149
80 Pardosa sp. 1GAB_PAK 3 0.2 5.9 BOLD:AAO2146
81 Pardosa sp. 2GAB_PAK 1 N/A 4.9 BOLD:AAO2148
82 Pardosa sp. 3GAB_PAK 1 N/A 5.2 BOLD:AAV1588
83 Pardosa sp. 4GAB_PAK 13 2.4 4.6 BOLD:AAO2147
84 Pardosa sp. 5GAB_PAK 4 0.8 5.2 BOLD:AAV1589
NP 85 Pardosa sutherlandi (Gravely, 1924) 7 0.2 4.6 BOLD:ABX7411
NP 86 Trochosa aquatica Tanaka, 1985 17 0.6 5.9 BOLD:AAV3200
87 Trochosa sp. 1GAB_PAK 3 0.3 5.9 BOLD:ADF4175
*NP 88 Wadicosa fidelis (O. Pickard-Cambridge, 1872) 75 1.9 7.2 BOLD:AAG7456
Oecobiidae Blackwall, 1862
89 Oecobius putus O. Pickard-Cambridge, 1876 10 0.4 14 BOLD:AAV1624
Oxyopidae Thorell, 1870
E 90 Oxyopes azhari Butt & Beg, 2001 112 3.6 3.6 BOLD:AAO1991
E 91 Oxyopes chenabensis Mukhtar, 2017 5 0.9 6.4 BOLD:ABX7410
NP 92 Oxyopes heterophthalmus (Latreille, 1804) 8 0.3 4.9 BOLD:AAD0599
93 Oxyopes hindostanicus Pocock, 1901 123 3 5.6 BOLD:AAO1990
NP 94 Oxyopes macilentus L. Koch, 1878 8 1.5 1.3 BOLD:AAF9665
NP 95a Oxyopes matiensis Barrion & Litsinger, 1995 3 2.1 1.3 BOLD:ACX5149
95b Oxyopes matiensis Barrion & Litsinger, 1995 5 BOLD:ABX7414
E 96 Oxyopes oryzae Mushtaq & Qadar, 1999 52 1.9 3.6 BOLD:AAO1989
97 Oxyopes sp. 1GAB_PAK 1 N/A 6.7 BOLD:ACZ2323
NS 98 Oxyopes sp. 2GAB_PAK 3 1.2 5.7 BOLD:ACZ4097
99 Oxyopes sp. 3GAB_PAK 1 N/A 11 BOLD:ACP4193
NP 100 Peucetia ranganathani Biswas & Roy, 2005 14 0.8 11 BOLD:ACB4190
101 Peucetia sp. 1GAB_PAK 1 N/A 13 BOLD:ACB4188
Philodromidae Thorell, 1870
102 Philodromus sp. 1GAB_PAK 1 N/A 12 BOLD:ADD8987
103 Philodromus sp. 2GAB_PAK 3 2 13 BOLD:ABX7412
*NP 104 Pulchellodromus mainlingensis (Hu & Li, 1987) 2 0 12 BOLD:ACB4189
NS 105 Rhysodromus cf. xinjiangensis (Tang & Song, 1987) 4 0 13 BOLD:AAO2159
NP 106 Thanatus vulgaris Simon, 1870 2 0.3 15 BOLD:AAQ0111
Pholcidae C. L. Koch, 1850
107 Artema sp. 1GAB_PAK 1 N/A 19 BOLD:ABW2886
NP 108 Artema transcaspica Spassky, 1934 2 1 19 -
109 Crossopriza lyoni (Blackwall, 1867) 2 0.3 16 BOLD:AAG2795
110a Crossopriza maculipes (Spassky, 1934) 4 5.3 16 BOLD:ACN4846
110b Crossopriza maculipes (Spassky, 1934) 7 BOLD:AAU5412
110c Crossopriza maculipes (Spassky, 1934) 1 BOLD:ACB2929
Pisauridae Simon, 1890
*NP 111 Pisaura mirabilis (Clerck, 1757) 4 0.5 10 BOLD:AAE4245
112 Pisaura sp. 1GAB_PAK 1 N/A 12 BOLD:AAO2059
Salticidae Blackwall, 1841
NP 113 Bianor albobimaculatus (Lucas, 1846) 21 0.7 12 BOLD:AAP4728
114 Bianor sp. 1GAB_PAK 1 N/A 13 BOLD:ACI8750
NP 115 Epocilla sirohi Caleb, Chatterjee, Tyagi, Kundu, Kumar, 2018 7 1.9 11 BOLD:ADD4346
116 Euophrys sp. 1GAB_PAK 1 N/A 13 BOLD:ADD1307
*NS 117 Evarcha sp. 1GAB_PAK 3 0.8 9 BOLD:AAV1614
118 Hasarius adansoni (Audouin, 1826) 2 0 13 BOLD:AAW0165
NP 119 Hyllus dotatus (Peckham & Peckham, 1903) 3 0.7 11 BOLD:AAV1597
NP 120 Menemerus brevibulbis (Thorell, 1887) 3 1.4 7.7 BOLD:AAO2155
121 Menemerus marginatus (Kroneberg, 1875) 1 N/A 11 BOLD:AAV1611
122 Menemerus nigli Wesolowska & Freudenschuss, 2012 12 1.1 7.7 BOLD:AAQ0156
*NP 123 Modunda staintoni (O. Pickard-Cambridge, 1872) 3 0.8 14 BOLD:AAV0387
NP 124 Mogrus cognatus Wesolowska & van Harten, 1994 12 1.4 8.1 BOLD:AAV1599
125 Mogrus sp. 1GAB_PAK 1 N/A 8.1 BOLD:ACZ1977
126 Mogrus sp. 2GAB_PAK 6 0.8 10 BOLD:AAQ2635
127 Myrmarachne melanocephala MacLeay, 1839 1 N/A 6.7 BOLD:AAV1609
128 Myrmarachne robusta (Peckham & Peckham, 1892) 5 1.4 6.7 BOLD:ACS0377
*NP 129 Philaeus chrysops (Poda, 1761) 1 N/A 9.8 BOLD:ACE4347
* 130 Philaeus sp. 1GAB_PAK 1 N/A 9.8 BOLD:AAV0574
131 Phintella vittata (C. L. Koch, 1846) 11 0 9.9 BOLD:ACR1776
132a Plexippus paykulli (Audouin, 1826) 34 5 8.8 BOLD:AAO2152
132b Plexippus paykulli (Audouin, 1826) 4 BOLD:AAO2151
132c Plexippus paykulli (Audouin, 1826) 1 BOLD:ACU8433
132d Plexippus paykulli (Audouin, 1826) 1 BOLD:ABX7409
132e Plexippus paykulli (Audouin, 1826) 1 BOLD:ACZ4027
133 Plexippus sp. 1GAB_PAK 2 0.2 8.8 BOLD:AAV1604
134a Pseudicius admirandus Logunov, 2007 8 1.4 9.4 BOLD:AAQ0115
134b Pseudicius admirandus Logunov, 2007 2 BOLD:ADD4534
NP 135 Rhene albigera (C. L. Koch, 1846) 1 N/A 5.9 BOLD:AAV5815
NP 136 Rhene flavigera (C. L. Koch, 1846) 4 0 5.4 BOLD:ADD7823
137 Rhene sp. 1GAB_PAK 1 N/A 5.4 BOLD:ACU6737
*NS 138 Sonoita cf. lightfooti Peckham & Peckham, 1903 1 N/A 13 BOLD:ADD9560
139 Stenaelurillus arambagensis (Biswas & Biswas, 1992) 3 0.3 11 BOLD:ABX7343
* 140 Talavera sp. 1GAB_PAK 1 N/A 12 BOLD:ACZ2472
141 Telamonia dimidiata (Simon, 1899) 17 1.4 10 BOLD:ACG1123
142 Thyene imperialis (Rossi, 1846) 56 3.5 9 BOLD:AAO2153
143 Thyene sp. 1GAB_PAK 1 N/A 11 BOLD:AAV1607
*NS 144 Trite sp. 1GAB_PAK 13 0.5 11 BOLD:AAO2154
NP Segestriidae Simon, 1893
* 145 Ariadna sp. 1GAB_PAK 1 N/A 20 BOLD:AAO2054
Sparassidae Bertkau, 1872
NP 146 Heteropoda maxima Jäger, 2001 20 0.3 4.3 BOLD:ACB5077
147a Heteropoda sp. 3GAB_PAK 1 2.3 5.4 BOLD:ABW2881
147b Heteropoda sp. 3GAB_PAK 1 BOLD:AAO2057
148 Heteropoda sp. 4GAB_PAK 1 N/A 4.3 BOLD:ACB5549
149 Olios sp. 1GAB_PAK 1 N/A 3.9 BOLD:ADD6859
150 Olios sp. 2GAB_PAK 10 0.5 3.9 BOLD:ADD7417
151 Olios sp. 3GAB_PAK 4 0.3 4.1 BOLD:ACB4191
152 Olios sp. 4GAB_PAK 4 1.1 7.2 BOLD:AAQ0159
153 Olios sp. 5GAB_PAK 15 2.2 7.2 BOLD:AAQ0157
154a Olios tener (Thorell, 1891) 4 1.9 11 BOLD:AAQ0107
154b Olios tener (Thorell, 1891) 1 BOLD:ADK3497
154c Olios tener (Thorell, 1891) 1 BOLD:ADJ7965
155 Pseudopoda prompta (O. Pickard-Cambridge, 1885) 4 0.9 13 BOLD:AAO2056
156a Spariolenus tigris Simon, 1880 1 4.1 12 BOLD:ADF5077
156b Spariolenus tigris Simon, 1880 1 BOLD:ABW2878
Tetragnathidae Menge, 1866
*NP 157 Glenognatha tangi (Zhu, Song & Zhang, 2003) 3 1.2 18 BOLD:AAQ0147
158 Guizygiella indica (Tikader & Bal, 1980) 8 1.1 14 BOLD:ABX7345
159 Leucauge celebesiana (Walckenaer, 1841) 7 0.2 11 BOLD:AAO2068
160 Leucauge decorata (Blackwall, 1864) 30 0.5 11 BOLD:AAG8516
* 161 Metleucauge sp. 1GAB_PAK 1 N/A 19 BOLD:AAV1600
NP 162 Tetragnatha boydi O. Pickard-Cambridge, 1898 3 0 15 BOLD:ACB2930
NP 163 Tetragnatha cavaleriei Schenkel, 1963 2 0.5 16 BOLD:AAT8904
164 Tetragnatha javana (Thorell, 1890) 43 2.8 17 BOLD:AAO2174
165 Tetragnatha mandibulata Walckenaer, 1841 1 N/A 15 BOLD:AAK2567
NP 166 Tetragnatha maxillosa Thorell, 1895 4 0.3 15 BOLD:AAK2560
NP 167 Tetragnatha nitens (Audouin, 1826) 6 0.8 15 BOLD:AAD3790
168 Tetragnatha sp. 1GAB_PAK 1 N/A 16 BOLD:ABW2885
Theraphosidae Thorell, 1869
* 169 Chilobrachys sp. 1GAB_PAK 1 N/A 4.3 BOLD:ADD5278
* 170 Chilobrachys sp. 2GAB_PAK 1 N/A 4.3 BOLD:AAQ0160
Theridiidae Sundevall, 1833
*NP 171 Emertonella taczanowskii (Keyserling, 1886) 1 N/A 12 BOLD:AAV1610
172 Enoplognatha sp. 1GAB_PAK 1 N/A 12 BOLD:ACI8909
173 Enoplognatha sp. 2GAB_PAK 1 N/A 15 BOLD:ACP4208
* 174 Euryopis sp. 1GAB_PAK 1 N/A 12 BOLD:AAQ0155
175 Latrodectus sp. 1GAB_PAK 1 N/A 19 BOLD:AAV1732
176 Latrodectus sp. 2GAB_PAK 1 N/A 16 BOLD:AAO3347
* 177 Meotipa sp. 1GAB_PAK 2 2 12 BOLD:AAQ0152
178 Phylloneta sp. 1GAB_PAK 11 0.3 11 BOLD:AAV3043
*NP 179 Steatoda cingulata (Thorell, 1890) 2 0 14 BOLD:ABW2877
NP 180 Theridion melanostictum O. Pickard-Cambridge, 1876 1 N/A 11 BOLD:AAV1617
181 Theridion sp. 1GAB_PAK 1 N/A 11 BOLD:ACB2932
182 Theridion sp. 3GAB_PAK 1 N/A 12 BOLD:AAV1623
Thomisidae Sundevall, 1833
*NP 183 Coriarachne melancholica Simon, 1880 1 N/A 7.9 BOLD:ACI8639
*NP 184 Ebelingia kumadai (Ono, 1985) 3 0 12 BOLD:AAV1619
185 Henriksenia hilaris (Thorell, 1877) 1 N/A 11 BOLD:AAV1618
NP 186 Lysiteles kunmingensis Song & Zhao, 1994 3 0 9.9 BOLD:ACI8899
* 187 Misumenoides sp. 1GAB_PAK 1 N/A 11 BOLD:AAV1594
* 188 Misumenops sp. 1GAB_PAK 2 1.9 11 BOLD:AAV1596
* 189 Ozyptila sp. 1GAB_PAK 1 N/A 11 BOLD:ADF5201
190a Runcinia insecta (L. Koch, 1875) 40 4.9 11 BOLD:AAI0997
190b Runcinia insecta (L. Koch, 1875) 2 BOLD:AAQ0108
*NP 191 Tharpyna indica Tikader & Biswas, 1979 1 N/A 12 BOLD:AAV1606
NP 192 Thomisus onustus Walckenaer, 1805 1 N/A 8.6 BOLD:AAD7031
E 193a Thomisus zaheeri Parveen, Khan, Mushtaq, Ahmad & Rana, 2008 30 4.3 11 BOLD:AAP4819
193b Thomisus zaheeri Parveen, Khan, Mushtaq, Ahmad & Rana, 2008 1 BOLD:AAQ0153
194 Tmarus dostinikus Barrion & Litsinger, 1995 13 0.2 11 BOLD:ABX7413
NS 195a Tmarus sp. 1GAB_PAK 3 2.9 11 BOLD:ABX7346
195b Tmarus sp. 1GAB_PAK 5 BOLD:ADJ6297
195c Tmarus sp. 1GAB_PAK 4 BOLD:ADK4624
195d Tmarus sp. 1GAB_PAK 1 BOLD:ADK4625
NP 196 Xysticus joyantius Tikader, 1966 1 N/A 13 BOLD:ADF4849
197 Xysticus sp. 1GAB_PAK 3 0.6 7.9 BOLD:ACI8898
198 Xysticus sp. 2GAB_PAK 1 N/A 12 BOLD:ADF4647
Uloboridae Thorell, 1869
* 199 Hyptiotes sp. 1GAB_PAK 1 N/A 15 BOLD:AAQ2632
200a Uloborus sp. 1GAB_PAK 4 4 14 BOLD:AAW8359
200b Uloborus sp. 1GAB_PAK 1 BOLD:ABW2879
Zodariidae Thorell, 1881
* 201 Zodarion sp. 1GAB_PAK 1 N/A 14 BOLD:AAV1621
* 202 Zodarion sp. 2GAB_PAK 1 N/A 14 BOLD:ACG0983
Total 1795 221
Open in a new tab

N = number of individuals; K2P = maximum Kimura 2-parameter distance; NN = distance to Nearest Neighbor species; BIN = Barcode Index Number; NP = new species or family to Pakistan; * = new genus to Pakistan; E = endemic species to Pakistan; U = undescribed opposite sex; NS = putative new species to science.

As the accumulation curve failed to approach an asymptote (Fig 2), it is certain that more species await detection. Although one species (Artema transcaspica) failed to qualify for a BIN assignment because its only sequence was too short, the other 108 morphological species were assigned to 123 BINs with 10 species showing a split to two or more BINs (Table 1 and Fig 3). The 93 interim species were allocated to 98 BINs with three showing BIN splits (Table 1), making the total BIN count 221 –with 94 of them singletons. NJ clustering (Fig 3) and Bayesian inference (Fig 4), supported the monophyly of all 221 BINs. Barcode distances (K2P) varied for differing taxonomic ranks with conspecific values ranging from 0.0–5.3% (mean = 0.8%), congenerics from 2.8–23.2% (mean = 8.8%), and confamilials from 4.3–26.7% (mean = 15.1%) (Table 2). Excepting 14 species, maximum intraspecific divergences did not exceed 2% in the 90 species that were represented by two or more specimens (Table 1). The barcode gap analysis showed that maximum intraspecific distance for all but one of the 90 species with two or more records was less than its NN distance (Oxyopes azhari was the exception, overlapping with Oxyopes oryzae) (Fig 5). The Mantel test was non-significant (P>0.01) for 60 of the 69 species and the regression line for all species showed a weak positive relationship (R2 = 0.08; y = 0.0003x + 2.62) (Fig 6).

Fig 2. Accumulation curve for morphological species and barcode index numbers (BINs) for 1,795 spiders from Pakistan.

Fig 2

Open in a new tab

Fig 3. NJ analysis of spider species based on the analysis of 1,782 COI sequences.

Fig 3

Open in a new tab

Bootstrap values (50% or higher; 1000 replicates) are shown above the branches. The scale bar shows K2P distances. The node for each species with multiple specimens is collapsed to a vertical line or triangle, with the horizontal depth indicating the level of intraspecific divergence. Species assigned to multiple BINs are indicated in bold. The tree is presented in two parts.

Fig 4. Bayesian phylogenetic analysis of spiders from Pakistan based on COI sequences.

Fig 4

Open in a new tab

Posterior probabilities are indicated at the nodes. Taxa are followed by the BINs. Phalangium opilio (Arachnida: Opiliones) and Galeodes sp. (Arachnida: Solifugae) were employed as outgroups. Due to its large size, the tree is presented in two parts.

Table 2. Sequence divergences (K2P) for differing levels of taxonomic affinity for the COI-5′ gene region for the spiders from Pakistan.

Analysis was restricted to sequences >400 bp.

Distance class n Taxa Comparisons Min (%) Mean (%) Max (%)
Intraspecific 1702 122 44347 0 0.8 5.3
Congeners 1338 44 56792 2.8 8.8 23.2
Confamilial 1662 15 137164 4.3 15.1 26.7
Open in a new tab

Fig 5. Barcode gap analysis for spider species represented by three or more records.

Fig 5

Open in a new tab

Points that fall above the 1:1 line (blue) indicate the presence of a local barcode gap. NN = Nearest-Neighbor species.

Fig 6. Intraspecific sequence divergence (K2P) for the COI gene (blue dots) versus geographic distance (km) for spider species from Pakistan with data from other regions.

Fig 6

Open in a new tab

The relationship between genetic and geographic distances is indicated by a regression line. P-values for the Mantel Test are indicated by red vertical lines.

The similarity between the spider fauna in Pakistan and that of other nations was calculated by examining BIN overlap. Less than a quarter (52/221) of the BINs from Pakistan were represented among the 10,229 spider BINs reported in prior studies. As expected, the highest overlap (23%) was with India, but the proportion of shared BINs was far lower for the other 43 countries (Fig 7).

Fig 7. Percentage of spider BINs shared between Pakistan and 41 other nations.

Fig 7

Open in a new tab

Discussion

Most prior work on the spider fauna of Pakistan has had a regional focus and only employed morphological approaches. For example, 157 species were reported from the province of Punjab [9], 56 from the district of Sargodha [76], 23 from Peshawar [11], and 13 from Buner [77]. A recent checklist for the spiders of Pakistan [10] included records for 239 species, but the present study has substantially increased this total by adding first records for 84 described species and another 93 that could not be assigned to a known taxon. Most importantly, this study generated a DNA barcode reference library for 202 species, facilitating their future identification.

Because the spider fauna of Pakistan has seen such limited study, the discovery of new species was not unexpected, and follows a pattern seen for spiders in other regions. For example, the analysis of 80 species of Salticidae from Papua New Guinea revealed 34 species and five genera new to the country [78]. Likewise, 6% of the 136 spider species recovered from the Northern Cape Province, South Africa were new [79]. This study employed a mix of methods for spider collection, including beating, sweeping, and pitfalls. The choice of sampling method impacts species detection [80] and extensive sampling is critical to generate comprehensive species coverage [81]. Although the present study involved collections at 225 sites, the resultant species accumulation curve did not reach an asymptote, indicating that many more species await detection.

The present study revealed a close correspondence (93%) between BINs and morphospecies as 188 of the 202 species were assigned to a unique BIN, reinforcing a pattern seen in other groups [37,38,40]. For example, the concordance between BINs and species was 78% in a study that examined 30,000 Canadian spiders representing 1,018 species [61] with most discordances reflecting BIN splits suggestive of overlooked species. Stronger species-BIN correspondence has been reported in several insect groups; 96% for Erebidae (Lepidoptera) from the Iberian Peninsula [38], 94% for tiger moths from Brazil [82] and 92% for beetles from central Europe [40]. However, some arthropod groups have shown relatively low level of species-BIN concordance; for example, orthopterans in Central Europe (76%) [83], waterstriders in Germany (82%) [84] and katydids in China (75%) [85]. Thirteen (6%) species in this study were assigned to two or more BINs (BIN splits), and one species (Plexippus paykulli) was assigned to five. BIN splits often indicate the presence of a species complex [43]. For example, 13% of 1,018 species of Canadian spiders [61], 13% of 1,541 Canadian Noctuoidea [86], 5.7% of 1,872 Finnish beetles [87], and 20% of 62 global mealybugs [88] possessed BIN splits. Although in most cases the subsequent morphological investigation has revealed overlooked species [89], other factors can cause BIN splits/mergers, such as hybridization [90], incomplete lineage sorting [83], or rapid speciation [91].

K2P divergences >2% were found in 14 of the 202 spider species from Pakistan with a maximum value of 5.3%. There was, however, no significant relationship between intraspecific divergence and the number of specimens analyzed. For example, 12 specimens of Crossopriza maculipes (3 BINs) showed 5.3% divergence and were assigned to three BINs while 160 specimens of Neoscona theisi possessed a maximum divergence of 2.5%. High COI divergence is not uncommon in spiders. For example, the maximum intraspecific divergence in 561 spider species from Germany was 10.1%, but it was below 2.5% in 95% of the cases with an arithmetic mean of 0.7% [62]. The divergence could depend on several factors such as the number of specimens analyzed, the number of localities, the geographic distance between them and the dispersal capabilities of the particular species [92,93]. With the exception of a single species (Oxyopes azhari), high conspecific distances did not impede the capacity of DNA barcodes to discriminate the species encountered in our study. However, species with BIN splits and high divergences are likely to represent a cryptic species complex. Preliminary morphological analyses including genitalic dissections of specimens from taxa with BIN splits in this study reinforced this conclusion.

Correlation analysis revealed only a weak relationship between the geographic range of the species examined in this study and their intraspecific divergence value. The Mantel test was significant for a few (13%) species, but species identification was not impeded as maximum intraspecific distances were nearly always less than NN distances. Similar results have been reported for Lepidoptera from Europe [94], Pakistan [32] and Central Asia [95]. Although a study that examined a single tribe, Agabini, of aquatic beetles in Europe [96] argued that regional divergences were so great as to obscure species assignments, this result is clearly not the rule [72].

Because BINs are generally an effective species proxy [41], we used them to assess faunal overlap. This work revealed that most (76%) BINs detected in this study were first records. Just 52 BINs have records from other nations and 13 of these were shared only with India. The BIN overlap with other nations was considerably lower for the spiders (24%) of Pakistan than for its Lepidoptera (42%) [42], but this difference almost certainly reflects the intensive barcode studies on the latter group. Although DNA barcoding has been used to assess regional biodiversity [41,47] and to ascertain species connections [42], the limited data availability complicates interpretation. Although further sampling will add new BINs, it is also likely to raise BIN overlap with other regions, improving our understanding of faunal overlap. Such efforts to better document local biodiversity are also certain to reveal new species as evidenced by the discovery of 93 taxa in this study that could not be assigned to a known species.

Supporting information

S1 Table. Taxonomic publications consulted for this study.

(DOCX)

Click here for additional data file. (51.6KB, docx)

Data Availability

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 Statement

This study was enabled by grant 106106-001 “Engaging Developing Nations in iBOL” from the International Development Research Centre in Canada and by grant HEC No. 20-1403/R& D/09 “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.

References

  • 1.World Spider Catalog. World Spider Catalog. Version 20.0. Natural History Museum Bern, online at http://wsc.nmbe.ch, accessed on 7 January 2019. doi: 10.24436/2.
  • 2.Riechert SE, Lockley T. Spiders as biological control agents. Annu Rev Entomol. 1984;29: 299–320. [Google Scholar]
  • 3.Maelfait J-P, Hendrickx F. Spiders as bio-indicators of anthropogenic stress in natural and semi-natural habitats in Flanders (Belgium): some recent developments. In: Selden PA (ed.), Proceedings of the 17th European Colloquium of Arachnology, Edinburgh; 1998. pp. 293–300.
  • 4.Rybak J. Accumulation of major and trace elements in spider webs. Water Air Soil Pollut. 2015;226: 105 10.1007/s11270-015-2369-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.van Helsdingen PJ. The merits of a European checklist of spiders (Arachnida: Araneae). Logunov DV, Penney D, (Eds). Eur Arachnology. 2003;1: 101–106. [Google Scholar]
  • 6.Ubick D, Paquin P, Cushing P, Roth V. Spiders of North America: An Identification Manual, Second Edition 2nd Edition. American Arachnological Society; 2017: 425 pp. [Google Scholar]
  • 7.Siliwal M, Molur S. Checklist of spiders (Arachnida: Araneae) of South Asia including the 2006 update of Indian spider checklist. Zoos' Print J. 2007; 22: 2551–2597. [Google Scholar]
  • 8.Dyal S. Fauna of Lahore. 4. -Spiders of Lahore. Bulletin of the Department of Zoology of the Panjab University. 1935; 1, i–ii, 119–252. [Google Scholar]
  • 9.Parveen R, Khan AA, Mushtaq S, Rana SA. A checklist of the spiders of the Punjab. Pak J Agric Sci. 2007;44: 625–626. [Google Scholar]
  • 10.Ghazanfar M, Hussain M, Hashim M, Fahid AM. Checklist of spider (Araneae) fauna of Pakistan: A review. J Entomol Zool Studies. 2016;4: 245–256. [Google Scholar]
  • 11.Perveen F, Jamal A. Checklist of spider fauna of FR Peshawar, FATA, Pakistan. Arthropods. 2012;1: 35−39. [Google Scholar]
  • 12.Sial N, Ruby T, Malik S, Mushtaq S. A checklist of the spiders of Cholistan and neighbouring areas. Pak J Agric Sci. 2012;49: 301–304. [Google Scholar]
  • 13.Parveen R. Taxonomic study on some spiders of Punjab, Pakistan. PhD Thesis. Department of Zoology and Fisheries, Agriculture University Faisalabad, Pakistan. 2003: 356 pp. http://prr.hec.gov.pk/jspui/handle/123456789//4123
  • 14.Baig MB, Al-Subaiee FS. Biodiversity in Pakistan: Key issues. Biodiversity 2009;10: 20–29. [Google Scholar]
  • 15.Keswani S, Hadole P, Rajoria A. Checklist of spiders (Arachnida: Araneae) from India-2012. Indian J Arachnol. 2012;1: 1–129. [Google Scholar]
  • 16.Mirshamsi O, Marusik YM, Zamani A, Moradmand M, Kashefi R. Annotated checklist of the spiders of Iran (Arachnida: Araneae). Iranian J Biosyst Fauna Iranica. 2015;1: 1–108. [Google Scholar]
  • 17.Borges PAV, Wunderlich J. Spider biodiversity patterns and their conservation in the Azorean archipelago, with descriptions of new species In: Systematics and Biodiversity; 2008. pp. 249–282. [Google Scholar]
  • 18.Drew LW. Are we losing the science of taxonomy? BioScience. 2011;61: 942–946. [Google Scholar]
  • 19.Zhang Y, Li S. A spider species complex revealed high cryptic diversity in South China caves. Mol Phylogenet Evol. 2014;79: 353–358. 10.1016/j.ympev.2014.05.017 [DOI] [PubMed] [Google Scholar]
  • 20.Hamilton CA, Formanowicz DR, Bond JE. Species delimitation and phylogeography of Aphonopelma hentzi (Araneae, Mygalomorphae, Theraphosidae): cryptic diversity in North American tarantulas. PLOS ONE. 2011;6: e26207 10.1371/journal.pone.0026207 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Hebert PDN, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proc Biol Sci. 2003;270: 313–321. 10.1098/rspb.2002.2218 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Dhananjeyan KJ, Paramasivan R, Tewari SC, Rajendran R, Thenmozhi V, Leo SV, et al. Molecular identification of mosquito vectors using genomic DNA isolated from eggshells, larval and pupal exuvium. Trop Biomed. 2010;27: 47–53. [PubMed] [Google Scholar]
  • 23.Iftikhar R, Ashfaq M, Rasool A, Hebert PDN. DNA barcode analysis of thrips (Thysanoptera) diversity in Pakistan reveals cryptic species complexes. PLOS ONE. 2016;11: e0146014 10.1371/journal.pone.0146014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Dupuis JR, Roe AD, Sperling FAH. Multi-locus species delimitation in closely related animals and fungi: one marker is not enough. Mol Ecol. 2012;21: 4422–36. 10.1111/j.1365-294X.2012.05642.x [DOI] [PubMed] [Google Scholar]
  • 25.Abe TA, Spence JR, Sperling FAH. Mitochondrial introgression is restricted relative to nuclear markers in a water strider (Hemiptera: Gerridae) hybrid zone. Can J Zool. 2005;83:432–444. [Google Scholar]
  • 26.Pazhenkova EA, Lukhtanov VA. Nuclear genes (but not mitochondrial DNA barcodes) reveal real species: Evidence from the Brenthis fritillary butterflies (Lepidoptera, Nymphalidae). J Zool Syst Evol Res. 2018; 10.1111/jzs.12252 [DOI] [Google Scholar]
  • 27.Hickerson MJ, Meyer CP, Moritz C. DNA Barcoding will often fail to discover new animal species over broad parameter space. Syst Biol. 2006;55: 729–739. 10.1080/10635150600969898 [DOI] [PubMed] [Google Scholar]
  • 28.Stein ED, White BP, Mazor RD, Jackson JK, Battle JM, Miller PE, et al. Does DNA barcoding improve performance of traditional stream bioassessment metrics? Freshw Sci. 2014;33: 302–311. [Google Scholar]
  • 29.Yang J, Zhang X, Zhang W, Sun J, Xie Y, Zhang Y, et al. Indigenous species barcode database improves the identification of zooplankton. PLOS ONE. 2017;12: e0185697 10.1371/journal.pone.0185697 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Hebert PDN, DeWaard JR, Zakharov EV, Prosser SW, Sones JE, McKeown JT, et al. A DNA 'barcode blitz': rapid digitization and sequencing of a natural history collection. PLOS ONE. 2013;8: e68535 10.1371/journal.pone.0068535 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Slowik J, Blagoev GA. A survey of spiders (Arachnida: Araneae) of Prince of Wales Island, Alaska; combining morphological and DNA barcode identification techniques. Insecta Mundi. 2012;251: 1–12. [Google Scholar]
  • 32.Blagoev GA, Nikolova NI, Sobel CN, Hebert PDN, Adamowicz SJ. Spiders (Araneae) of Churchill, Manitoba: DNA barcodes and morphology reveal high species diversity and new Canadian records. BMC Ecol. 2013;13: 44 10.1186/1472-6785-13-44 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Cao XW, Liu J, Chen J, Zheng G, Kuntner M, Agnarsson I. Rapid dissemination of taxonomic discoveries based on DNA barcoding and morphology. Sci Rep. 2016;6: 13 10.1038/s41598-016-0006-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Nadolny AA, Omelko MM, Marusik YM, Blagoev GA. A new species of spider belonging to the Pardosa lugubris-group (Araneae: Lycosidae) from Far East Asia. Zootaxa. 2016;4072: 263–281. 10.11646/zootaxa.4072.2.8 [DOI] [PubMed] [Google Scholar]
  • 35.Ratnasingham S, Hebert PDN. BOLD: The Barcode of Life Data System (http://www.barcodinglife.org). Mol Ecol Notes. 2007;7: 355–364. 10.1111/j.1471-8286.2007.01678.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Ratnasingham S, Hebert PDN. A DNA-based registry for all animal species: the Barcode Index Number (BIN) system. PLOS ONE. 2013;8: e66213 10.1371/journal.pone.0066213 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Ashfaq M, Akhtar S, Khan AM, Adamowicz SJ, Hebert PDN. DNA barcode analysis of butterfly species from Pakistan points towards regional endemism. Mol Ecol Resour. 2013;13: 832–43. 10.1111/1755-0998.12131 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Ortiz AS, Rubio RM, Guerrero JJ, Garre MJ, Serrano J, Hebert PDN, et al. Close congruence between Barcode Index Numbers (BINs) and species boundaries in the Erebidae (Lepidoptera: Noctuoidea) of the Iberian Peninsula. Biodivers Data J. 2017;5: e19840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Wirta H, Varkonyi G, Rasmussen C, Kaartinen R, Schmidt NM, Hebert PDN, et al. Establishing a community-wide DNA barcode library as a new tool for arctic research. Mol Ecol Resour. 2016;16: 809–822. 10.1111/1755-0998.12489 [DOI] [PubMed] [Google Scholar]
  • 40.Hendrich L, Morinière J, Haszprunar G, Hebert PDN, Hausmann A, Köhler F, et al. A comprehensive DNA barcode database for Central European beetles with a focus on Germany: adding more than 3500 identified species to BOLD. Mol Ecol Resour. 2015;15: 795–818. 10.1111/1755-0998.12354 [DOI] [PubMed] [Google Scholar]
  • 41.Hebert PDN, Ratnasingham S, Zakharov EV, Telfer AC, Levesque-Beaudin V, Milton MA, et al. Counting animal species with DNA barcodes: Canadian insects. Phil Trans R Soc B. 2016;371: 20150333 10.1098/rstb.2015.0333 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Ashfaq M, Akhtar S, Rafi MA, Mansoor S, Hebert PD. Mapping global biodiversity connections with DNA barcodes: Lepidoptera of Pakistan. PLOS ONE. 2017;12: e0174749 10.1371/journal.pone.0174749 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Ashfaq M, Hebert PDN. DNA barcodes for bio-surveillance: Regulated and economically important arthropod plant pests. Genome. 2016;59: 933–945. 10.1139/gen-2016-0024 [DOI] [PubMed] [Google Scholar]
  • 44.Mutanen M, Kekkonen M, Prosser SWJ, Hebert PDN, Kaila L. One species in eight: DNA barcodes from type specimens resolve a taxonomic quagmire. Mol Ecol Resour. 2015;15: 967–984. 10.1111/1755-0998.12361 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Miller SE, Hausmann A, Hallwachs W, Janzen DH. Advancing taxonomy and bioinventories with DNA barcodes. Philos Trans Royal Soc B. 2016;371: 20150339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Telfer AC, Young MR, Quinn J, Perez K, Sobel CN, Sones JE, et al. Biodiversity inventories in high gear: DNA barcoding facilitates a rapid biotic survey of a temperate nature reserve. Biodivers Data J. 2015;30: 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Ashfaq M, Sabir JSM, El-Ansary HO, Perez K, Levesque-Beaudin V, Khan AM, et al. Insect diversity in the Saharo-Arabian region: Revealing a little-studied fauna by DNA barcoding. PLOS ONE. 2018;13: e0199965 10.1371/journal.pone.0199965 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Kerr KC, Stoeckle MY, Dove CJ, Weigt LA, Francis CM, Hebert PDN. Comprehensive DNA barcode coverage of North American birds. Mol Ecol Notes. 2007;7: 535–543. 10.1111/j.1471-8286.2007.01670.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Hawlitschek O, Moriniere J, Dunz A, Franzen M, Rodder D, Glaw F, et al. Comprehensive DNA barcoding of the herpetofauna of Germany. Mol Ecol Resour. 2016;16: 242–253. 10.1111/1755-0998.12416 [DOI] [PubMed] [Google Scholar]
  • 50.Dinca V, Zakharov EV, Hebert PDN, Vila R. Complete DNA barcode reference library for a country's butterfly fauna reveals high performance for temperate Europe. Proc Biol Sci. 2010;278: 347–355. 10.1098/rspb.2010.1089 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Raupach MJ, Hendrich L, Kuchler SM, Deister F, Moriniere J, Gossner MM. Building-up of a DNA barcode library for true bugs (Insecta: Hemiptera: Heteroptera) of Germany reveals taxonomic uncertainties and surprises. PLOS ONE. 2014;9: e106940 10.1371/journal.pone.0106940 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Moriniere J, Hendrich L, Balke M, Beermann AJ, Konig T, Hess M, et al. A DNA barcode library for Germany’s mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera). Mol Ecol Resour. 2017;17: 1293–1307. 10.1111/1755-0998.12683 [DOI] [PubMed] [Google Scholar]
  • 53.Porco D, Chang CH, Dupont L, James S, Richard B, Decaens T. A reference library of DNA barcodes for the earthworms from Upper Normandy: Biodiversity assessment, new records, potential cases of cryptic diversity and ongoing speciation. Appl Soil Ecol. 2018;124: 362–371. [Google Scholar]
  • 54.Garcia-Robledo C, Kuprewicz EK, Staines CL, Kress WJ, Erwin TL. Using a comprehensive DNA barcode library to detect novel egg and larval host plant associations in a Cephaloleia rolled-leaf beetle (Coleoptera: Chrysomelidae). Biol J Linnean Soc. 2013;110: 189–198. [Google Scholar]
  • 55.Gall LLE, Delsuc F, Hourdez S, Lecointre G, Rasplus JY. Toward the DNA library of life. Eur J Taxon. 2017;266: 1–9. [Google Scholar]
  • 56.Robinson EA, Blagoev GA, Hebert PDN, Adamowicz SJ. Prospects for using DNA barcoding to identify spiders in species-rich genera. ZooKeys. 2009;16: 27–46. [Google Scholar]
  • 57.Blagoev GA, Dondale CD. A new species of Alopecosa (Araneae: Lycosidae) from Canada: a morphological description supported by DNA barcoding of 19 congeners. Zootaxa. 2014;3894: 152–160. 10.11646/zootaxa.3894.1.12 [DOI] [PubMed] [Google Scholar]
  • 58.Magalhaes ILF, Martins PH, Nogueira AA, Santos AJ. Finding hot singles: matching males to females in dimorphic spiders (Araneidae: Micrathena) using phylogenetic placement and DNA barcoding. Invertebr Syst. 2017;31: 8–36. [Google Scholar]
  • 59.Piacentini LN, Scioscia CL, Carbajal MN, Ott R, Brescovit AD, Ramirez MJ. A revision of the wolf spider genus Diapontia Keyserling, and the relationships of the subfamily Sosippinae (Araneae: Lycosidae). Arthropod Syst Phylo. 2017;75: 387–415. [Google Scholar]
  • 60.Ivanov V, Lee KM, Mutanen M. Mitonuclear discordance in wolf spiders: Genomic evidence for species integrity and introgression. Mol Ecol. 2018;27: 1681–1695. 10.1111/mec.14564 [DOI] [PubMed] [Google Scholar]
  • 61.Blagoev GA, deWaard JR, Ratnasingham S, deWaard SL, Lu L, Robertson J, et al. Untangling taxonomy: a DNA barcode reference library for Canadian spiders. Mol Ecol Resour. 2016;16: 325–341. 10.1111/1755-0998.12444 [DOI] [PubMed] [Google Scholar]
  • 62.Astrin JJ, Hofer H, Spelda J, Holstein J, Bayer S, Hendrich L, et al. Towards a DNA barcode reference database for spiders and harvestmen of Germany. PLOS ONE. 2016;11: e0162624 10.1371/journal.pone.0162624 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Gaikwad S, Warudkar A, Shouche Y. Efficacy of DNA barcoding for the species identification of spiders from Western Ghats of India. Mitochondrial DNA Part A. 2017;28: 638–644. [DOI] [PubMed] [Google Scholar]
  • 64.Tahir HM, Naseem S, Akhtar S, Ashfaq M, Butt A, Mukhtar MK. DNA barcode record of some common spiders from Punjab, Pakistan. Pak J Zool. 2016;48(1): 159–164. [Google Scholar]
  • 65.Naseem S, Tahir HM. Use of mitochondrial COI gene for the identification of family Salticidae and Lycosidae of spiders. Mitochondrial DNA. 2018;29: 96–101. 10.1080/24701394.2016.1248428 [DOI] [PubMed] [Google Scholar]
  • 66.Majeed N, Butt A, Krammer H-J, Astrin JJ. DNA barcoding in jumping spider communities of Pakistan reveals a new species (Araneae: Salticidae). ZooKeys. (In Press). [Google Scholar]
  • 67.Ivanova NV, deWaard JR, Hebert PDN. An inexpensive, automation-friendly protocol for recovering high quality DNA. Mol Ecol Notes. 2006;6: 998–1002. [Google Scholar]
  • 68.Hebert PDN, Penton EH, Burns JM, Janzen DH, Hallwachs W. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc Natl Acad Sci USA. 2004;101: 14812–14817. 10.1073/pnas.0406166101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994;3: 294–299. [PubMed] [Google Scholar]
  • 70.Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28: 2731‒2739. 10.1093/molbev/msr121 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Meyer CP, Paulay G. DNA barcoding: Error rates based on comprehensive sampling. PLOS Biol. 2005;3: e422 10.1371/journal.pbio.0030422 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72.Candek K, Kuntner M. DNA barcoding gap: reliable species identification over morphological and geographical scales. Mol Ecol Resour. 2015;15: 268–277. 10.1111/1755-0998.12304 [DOI] [PubMed] [Google Scholar]
  • 73.Mantel N. The detection of disease clustering and a generalized regression approach. Cancer Res. 1967;27: 209–220. [PubMed] [Google Scholar]
  • 74.Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980;16: 111–120. [DOI] [PubMed] [Google Scholar]
  • 75.Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012;61: 539–542. 10.1093/sysbio/sys029 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 76.Mukhtar MK, Khan SY, Jabeen S, Tahir HM, Qadir A, Ahmad KR, et al. A preliminary checklist of the spider fauna of Sargodha (Punjab), Pakistan. Pak J Zool. 2012;44: 1245–1254. [Google Scholar]
  • 77.Ahmad S, Akhtar N, Saeed K. Some observations on spider fauna of district Buner, Khyber Pakhtunkhwa, Pakistan. J Entomol Zool Studies. 2015;3: 47–52. [Google Scholar]
  • 78.Zhang J-X, Maddison WP. New euophryine jumping spiders from Papua New Guinea (Araneae: Salticidae: Euophryinae). Zootaxa. 2012;3491: 1–74. [Google Scholar]
  • 79.Dippenaar-Schoeman AS, Haddad CR, Lyle R, Lotz LN, Foord SH, Jocque R, et al. South African National Survey of Arachnida: A checklist of the spiders (Arachnida, Araneae) of the Tswalu Kalahari Reserve in the Northern Cape province, South Africa. KOEDOE. 2018;60: a1486. [Google Scholar]
  • 80.King JR, Porter SD. Evaluation of sampling methods and species richness estimators for ants in upland ecosystems in Florida. Environ Entomol. 2005;34: 1566–1578. [Google Scholar]
  • 81.Skalak SL, Sherwin RE, Brigham RM. Sampling period, size and duration influence measures of bat species richness from acoustic surveys. Methods Ecol Evol. 2012;3: 490–502. [Google Scholar]
  • 82.Zenker MM, Rougerie R, Teston JA, Laguerre M, Pie MR, Freitas AVL. Fast census of moth diversity in the neotropics: A comparison of field-assigned morphospecies and DNA barcoding in tiger moths. PLOS ONE. 2016;11: e0148423 10.1371/journal.pone.0148423 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 83.Hawlitschek O, Moriniere J, Lehmann GUC, Lehmann AW, Kropf M, Dunz A, et al. DNA barcoding of crickets, katydids and grasshoppers (Orthoptera) from Central Europe with focus on Austria, Germany and Switzerland. Mol Ecol Resour. 2017;17(5):1037–1053. 10.1111/1755-0998.12638 [DOI] [PubMed] [Google Scholar]
  • 84.Havemann N, Gossner MM, Hendrich L, Moriniere J, Niedringhaus R, Schafer P, et al. From water striders to water bugs: the molecular diversity of aquatic Heteroptera (Gerromorpha, Nepomorpha) of Germany based on DNA barcodes. PeerJ. 2018;6: e4577 10.7717/peerj.4577 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 85.Zhou Z, Guo H, Han L, Chai J, Che X, Shi F. Singleton molecular species delimitation based on COI-5P barcode sequences revealed high cryptic/undescribed diversity for Chinese katydids (Orthoptera: Tettigoniidae). BMC Evol Biol. 2019;19: 79 10.1186/s12862-019-1404-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 86.Zahiri R, Lafontaine JD, Schmidt BC, DeWaard JR, Zakharov EV, Hebert PDN. A transcontinental challenge–a test of DNA barcode performance for 1,541 species of Canadian Noctuoidea (Lepidoptera). PLOS ONE. 2014;9: e92797 10.1371/journal.pone.0092797 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 87.Pentinsaari M, Hebert PDN, Mutanen M. Barcoding beetles: a regional survey of 1872 species reveals high identification success and unusually deep interspecific divergences. PLOS ONE. 2014;9: e108651 10.1371/journal.pone.0108651 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 88.Ren J-M, Ashfaq M, Hu X-N, Ma J, Liang F, Hebert PDN, et al. Barcode index numbers expedite quarantine inspections and aid the interception of nonindigenous mealybugs (Pseudococcidae). Biol Invasions. 2018;20: 449–460. [Google Scholar]
  • 89.Landry J-F, Hebert PDN. Plutella australiana (Lepidoptera, Plutellidae), an overlooked diamondback moth revealed by DNA barcodes. Zookeys. 2013;327: 43–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 90.Gottsberger B, Mayer F. Behavioral sterility of hybrid males in acoustically communicating grasshoppers (Acrididae, Gomphocerinae). J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2007;193: 703–714. 10.1007/s00359-007-0225-y [DOI] [PubMed] [Google Scholar]
  • 91.Hawlitschek O, Hendrich L, Espeland M, Toussaint EFA, Genner MJ, Balke M. Pleistocene climate change promoted rapid diversification of aquatic invertebrates in Southeast Australia. BMC Evolutionary Biology. 2012;12:142 10.1186/1471-2148-12-142 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 92.Papadopoulou A, Bergsten J, Fujisawa T, Monaghan MT, Barraclough TG, Vogler AP. Speciation and DNA barcodes: testing the effects of dispersal on the formation of discrete sequence clusters. Philos Trans R Soc Lond B Biol Sci. 2008;363: 2987–2996. 10.1098/rstb.2008.0066 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 93.Huemer P, Hebert PDN, Mutanen M, Wieser C, Wiesmair B, Hausmann A, et al. Large geographic distance versus small DNA barcode divergence: Insights from a comparison of European to South Siberian Lepidoptera. PLOS ONE. 2018;13: e0206668 10.1371/journal.pone.0206668 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 94.Huemer P, Mutanen M, Sefc KM, Hebert PDN. Testing DNA barcode performance in 1000 species of European Lepidoptera: large geographic distances have small genetic impacts. PLOS ONE. 2014;9: e115774 10.1371/journal.pone.0115774 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 95.Lukhtanov VA, Sourakov A, Zakharov EV, Hebert PDN. DNA barcoding Central Asian butterflies: increasing geographical dimension does not significantly reduce the success of species identification. Mol Ecol Resour. 2009;9: 1302–1310. 10.1111/j.1755-0998.2009.02577.x [DOI] [PubMed] [Google Scholar]
  • 96.Bergsten J, Bilton DT, Fujisawa T, Elliott M, Monaghan MT, Balke M, et al. The effect of geographical scale of sampling on DNA barcoding. Syst Biol. 2012;61: 851–869. 10.1093/sysbio/sys037 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Taxonomic publications consulted for this study.

(DOCX)

Click here for additional data file. (51.6KB, docx)

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).

Articles from PLoS ONE are provided here courtesy of PLOS

RESOURCES