As DNA extraction techniques have improved, researchers in Arthropod conservation genetics have moved away from ‘non-lethal sampling’ (sampling of tissue which may impact the individual’s future life but does not kill (Vila et al. 2009)) and begun to explore ‘non-invasive sampling’ (sampling which confers minimal costs to the individual but that is targeted to a specific species (Feinstein 2004)) and environmental DNA (eDNA, genetic material from bulk environmental samples, not necessarily targeted toward a taxonomic group (Barnes and Turner 2016)). Spider webbing represents a potential source of DNA for such applications (Xu et al. 2015).

DNA isolation was tested on samples of webbing from two species (Psalmopoeus cambridgei Pocock 1895, Theraphosidae, and Pholcus phalangioides Fuesslin 1775, Pholcidae) that produce different web forms. Psalmopoeus construct vertical sheet webs in enclosed spaces in trees, which are then covered in loose material surrounding the web structure; primarily detritus and leaves (Bushell pers. obvs.). Pholcus build ‘space webs’ which are used as prey-detection structures from which the spider hunts prey (Jackson and Brassington 1987). Both species produce different web forms to species investigated previously (Latrodectus spp., Theridiidae, Xu et al. 2015).

Samples of captive Psalmopoeus cambridgei webbing were cleaned of large particles of detritus, but the majority of the fine detritus (pieces of prey, faeces from the spider, and local substrates etc.) remained stuck to the web, a potential source of non-target DNA (Xu et al. 2015). Webbing was cut into pieces to give individual sample weights of 2.3–8.5 mg.

Webs known to belong to Pholcus phalangioides were collected from a house in Wales (52.4113, −3.9897). Four samples of webbing without visible exoskeletons from either the web holder or prey were used, weighing 2.4–7.4 mg.

DNA from web samples was extracted using a standard CTAB phenol:chloroform:isoamyl alcohol (PCIA) method (Winnepenninckx et al. 1993), chosen for its relative low per-sample cost and applicability to low weight eDNA samples (Blake et al. 2015). Samples were placed in 350 μl of CTAB and 10 μl of Proteinase K and incubated overnight at 37 °C with occasional vortexing. 350 μl of PCIA was then added and the mixture shaken for 20 min. Following centrifugation at 15,000 RPM for 20 min, the upper aqueous phase was removed and subjected to an ethanol precipitation with 1 ml of absolute ethanol. Following a second precipitation using 1 ml of 70 % ethanol, eluted pellets were air dried and resuspended in 100 μl of ddH2O. Agarose gel electrophoresis of neat DNA solution revealed high molecular weight DNA in all cases. The concentration of the extracted DNA, estimated using a Nanodrop 2000 (ThermoScientific), was 15.6–23.3 ng/μL for the Psalmopoeus webbing, and 1.1–7.4 ng/μL for the Pholcus webbing, though these weights likely include DNA from prey and detritus (Xu et al. 2015).

Polymerase chain reaction (PCR) was used to amplify fragments of the cytochrome oxidase 1 (COI) gene in both species. Species-specific primers were designed for Psalmopoeus cambridgei, whilst within-genus primers were designed for Pholcus phalangioides (Table 1). The widely used universal invertebrate COI Folmer primers (Folmer et al. 1994) were also tested for both species. PCRs were performed in 20 μl volumes consisting of 10 μl of Biomix (Bioline), 1 μl of each primer (10 μM), 3 μl of DNA diluted from stock to 1/50, and 5 μl of ddH2O. Thermocycler conditions for all reactions were: 95 °C/3 min, 45 × (95 °C/30 s, 50 °C (45 °C for the Folmer primers)/45 s72 °C/45 s), 72 °C/3 min.

Table 1 Details of novel primers used in the study
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Most PCRs produced amplicons of the expected sizes, with the largest amplicon being generated by the Folmer primers (710 bp) (Fig. 1), though this reaction failed for the Psalmopheus material. Only the smallest Pholcus phalangioides sample failed consistently to produce amplicons. Sequencing of amplicons was performed using AB BigDye technology. BLASTn confirmed species identify for both Psalmopoeus cambridgei and Pholcus phalangioides, demonstrating amplification of the target region and species.

Fig. 1
figure 1

PCR success from Pholcus phalangioides webbing DNA extracts on a 3.5 % agarose TBE gel with HyperLadder 50 bp (Bioline). Lane 1: Phol-415F+Phol633R. Lane 2: Phol-459F+Phol633R. Lanes 3, 4: Folmer primers on two different samples of P. phalangioides webbing. The 300 and 700 bp ladder markers are labelled

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This work demonstrates that large fragments of COI (710 bp) can be amplified from a range of spider webs, joining Xu et al. (2015) and Sint et al. (2015) in the recent push toward advancing Araneae conservation genetics. However, caution should be used when using universal primers for species surveys due to the potential mixture of species contained within the DNA extraction, ideally taxa-specific primers should be developed and used where possible. The large fragment of mtDNA suggests that this material will also permit genotyping of a range of nuclear markers such as microsatellites, AFLP etc., as shown by other studies on low-quality environmental DNA (Nyström et al. 2012; Calvignac-Spencer et al. 2013; Blake et al. 2015; Thomsen and Willerslev 2015). Studies on a variety of web producing species should greatly benefit from this technique, including work on DNA barcoding, ecological genetic surveys, and fine-resolution population connectivity.