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

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.

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

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 3

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

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.

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

Fig 5

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

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.

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.

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.

Taxonomic publications consulted for this study.

(DOCX)

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