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

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.

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.

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.

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.

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