Spider Population Genetics

Genomic DNA Variation Between Orb, Sheet Web and Ground-Dwelling Spiders

Stephanie Sermon

Abstract.

Variation between orb, sheet web and ground-dwelling spiders were studied at the DNA level. Genomic DNA was extracted from eight species of spiders (2 orb, 2 sheet web weavers, and 4 non-orb weavers). The DNA concentration was quantified spectrographic methods and calculations from these readings indicated dilutions for running a RAPD-PCR. Samples were tested for polymorphic RAPD using six different 10-mers primers. It was found that primer three worked best with spider DNA. The resulting DNA was tested on a 2% argarose gel stained with ethidium bromide. Gel bands were measured and compared to a 100bp ladder. Differences were analyzed first by comparison in a chart based on molecular weights and second by using a dendrogram analysis.

Introduction

There are 35,000 species of named spiders worldwide and approximately 3,000 of those are in North America and in addition the majority of species have not yet been identified. Currently, spiders are classified on physical characteristics such as, coloration, spinnerets, and number and arrangement of eyes. The goal of this research was to take the current physical identifications and compare the types of spiders at the genomic level in order to detect evolutionary relationships.

The current way of classification based on physical characteristics is as follows; orb weaving spiders are typically classified by their shape, color and size. These spiders are usually 1/16 –1 1/8” long. It is common for the male to be smaller than the female. They have eight eyes arranged in two horizontal rows of four each. The chelicerae normally have a small bump in the outer portion. Orb weavers spin big webs, generally in a circular fashion that radiate out from the center. Many species actually replace the entire web daily. The spider usually rests with it head downward and near the center of the web.

Ground dwellers are in the family Lycosidae. They have 8 dark eyes of unequal size arrange in three rows. The abdomen and cephalothoraxes are usually long. They have dark mottled colors to camouflage them among dead leafs and stones. Except for one species these spiders do not spin webs. Typical size is ½”.

Sheet web builders have long slender legs with strong bristles. There are generally 8 eyes arranged in 2 parallel rows. They are typically smaller in size around 1/16- 3/8” long. Their webs are flat sheet like or dome shaped webs. They hide beneath their webs until prey are trapped. They also use this dome shape to escape from predators.

By looking at these classifications it would be expected that the sheet weaving and orb weaving spiders would be more closely related evolutionarily. This statement is only a speculation though based on the fact that they both build a type of web to live on whereas ground dwellers do not. In order to get genetic proof or disproof of their relationship a RAPD was run on the genomic DNA of all three types of these spiders.

Previous research has examined the relationship of kin in spiders. The bases for the research was to examine the evolution of cooperation and group living in spiders from sub social family groups and whether they were constrained of their cannibalistic nature. A group of social living spiders was observed when they were placed together and starved for 7-10 days that social group was then compared to a group of non kin spiders that were placed together and starved. The results showed that non-kin spiders were more likely to result to cannibalism than the kin spiders did. It has not been found that RAPD’s have been performed on spiders to look at evolutionary relationships.

Materials and Methods

Spider Material. Eight species of spiders were collected. Orb-weavers were Araneus angulatus, and Neriene radiata. Sheet web weavers included Frontinella pyramitela, and Floinda coccinea. Ground-dwelling species were Kukulcania hibernalis, Angelena labyrinthica, Tidarren sisyphoide and Lycosa rabida.

DNA extraction. For DNA extraction of the eight species the protocol E from the Wizard Genomic DNA purification kit was used (Promega- address). Although this protocol was designed for plant material, it proved to isolate genomic DNA from arachnids as well. Spiders were crushed with frozen CO₂ by mortar and pestle and the resulting powder was placed in a 1.5 microcentrifudge tube. After most of the CO₂ evaporated, 600ml of nuclei lyses solution was added and vortexed for 3 sec to wet the tissue and then incubated at 65° C for 15 min. Next, 3ml of Rnase was added and mixed by inversion followed by 15 min incubation at 37° C. Following incubation the samples cooled to room temperature before proceeding. Two hundred µl of protein precipitation solution was added and the samples were vortexed for 20 seconds to extract as much of the protein as possible. Tubes were then centrifuged at 13,000-16,000 g for 3 min and the supernatant containing the DNA was transferred to a sterile 1.5 microcentrifuge tube with 600µl of room-temperature isopropanol. Next, the solution was mixed by inversion and centrifuged again at 13,000-16,000 g for 1 min. The supernatant was decanted and 600µl of 70% ethanol was added to wash the DNA and centrifuged again for 1 minute. Then the ethanol was aspirated and the DNA was left to dry for 15 minutes. Finally 100µl of DNA rehydration solution was added and the samples were incubated at 65°C for 60 min with periodic gentle mixing. The DNA was then stored at 2-8° C.

Spectrophotomer. The DNA aliquot was diluted with sterile TE buffer and 20µl of the Genomic DNA solution was mixed thoroughly with 475µl of TE in a new tube, for a 1/20 dilution. The DNA was measured for concentration and purity at wavelengths 260nm for DNA, 280nm for RNA, and 234nm for protein using a blank of 1000µl TE.

Gel Electrophoresis. Electrophoresis was carried out in a minigel electrophoretic system. To check DNA purity an 0.8% agarose gel was run in 1X TBE Buffer and 2µl of ethidium bromide was mixed into the gel solution after it had been melted in the microwave for 5 minutes. A second gel with 2µl of ethidium bromide was run was for the RAPD product to measure the length of the bands. The gels contained 2 wells with the standard molecular weight ladder at either end with 8 samples of genomic DNA run between them.

RAPD reaction. Extracted DNA was used in a RAPD reaction to get substance that could be analyzed by gel electrophoresis. A Ready-To-Go RAPD kit was used (Promega). In this kit were beads that provide the reagents for RAPD reactions in a convenient ambient-temperature-stable bead. These beads contain thermo stable polymerases, dNTPs, BSA, and buffer. The primer for RAPD analysis consists of a single oligonucleiotide. Primer three worked best for this research (5’ –d[GTAGACCCGT]-3’). Sterile procedure was observed for this reaction. It was checked that the bead was at the bottom of the tube. Next, 25pmol of a single RAPD primer was added. Then 25ng template DNA and enough distilled water to equal 25µl was mixed with the primer. A short gentle vortex is then required to thoroughly mix the solution and then centrifuged to collect contents at the bottom of the tube. An Overlay was placed on the solutions with 50µl of mineral oil to prevent evaporation and thermo cycled at 95° C for 5 minutes for one round, then 95° C for 1 minute, 36°C for 1 minute, and 72°C for 2 minutes.

Photography. First a Nikon digital camera was used to photograph the gels on a Fotodyne UV illuminator. After many unsuccessful attempts a Fotodyne Polaroid camera was set up using film number 52.

Data Analysis. DNA fragments for each spider were measured on the agarose gels. The distance each band migrated from the well was measured with respect to a standard 100bp DNA ladder from Promega. Using Log graphing paper and the curve of the standard molecular weight ladder the base length the bands were calculated. The RAPD products are shown in Figure 1 from gel electrophoresis. Each size band that was reproducible were recorded and then scored as positive or negative for each spider species. The bands were recorded in excel spreadsheets for comparison. Table 1 shows the bands observed of the ground dwelling spiders. Table 2 compares the bands of the sheet web builders and the orb weavers. A second gel on ground dwellers were then compared again in Table 3 to check for reproducibility. Table 4 shows the results of a gel run to compare all eight species. Table 5 shows the results of a similarity test on two spiders of the same species. When a gel was run before comparisons were made the length in centimeters a band moved was measured and recorded in an excel spread sheet to calculate molecular weight of the band. Table 6 shows the measurements of the molecular weights of ground dwelling spiders. The measurements for orb weaving and sheet weaving spiders can be viewed in table 7. Table 8 shows the distances for the second gel on ground dwellers. All eight species and the movements of the bands are shown in table 9. Table 10 shows the measurements of the reproducible bands of ground dwelling spiders.

Results and Discussion.

By analysis of the molecular weight bands observed Agelena labyrinthica and Lycosa rabida were the most closely related spiders. These are both ground dwellers and by further research physical characteristic classification determined them cousins. They had 9 bands in common at 1150, 1100, 1090, 1000, 880, 460, 440, 400, and 380. Kykulcania hibernalis and Florinda coccina were the next most closely related, having 7 bands in common at 1100, 1000, 800, 700, 560, 500, and 400. The relationship here is a ground dweller vs. a sheet web weaver. The third closest relationship was Kykulcania hibernalis and Agelena labyrinthica with 6 bands in common 1100, 1000, 700, 500, 460, and 400. Again, these two spiders are both ground dwellers. A second pair also showed to have 6 bands in common and their relationship is a ground dweller vs. an orb weaver. This pair was Agelena labyrinthica and Araneus angulatus; the bands were measured at 900, 600, 460, 400, 380, 320. The next closest determine pairs had five bands in common, these pairs were Tidarren sisyphoides and Agelena labyrinthica, two ground dwellers, with bands at 700, 600, 440, 400,and 320. Tidarren sisyphoides and Araneus angulatus, a ground dweller and an orb builder, with bands at 600, 520, 480, 400, and 320. Agelena labyrinthica and Florinda coccina, a ground dweller vs. a sheet web builder, with bands at 1100, 1000, 700, 500, and 400. Lastly, Lycosa rabida and Araneus angulatus, a ground dweller vs. an orb weaver, with bands measuring 580, 480, 460, 400, and 380. There were also 3 pair of spiders with 4 bands in common; Florinda coccina and Frontinella pyramitella, 2 sheet web builders, Kykulcania hibernalis and Frontinella pyramitella, ground dweller and sheet web builder, and Tidarren sisyphoides and Kykulcania hibernalis 2 ground dwellers. The least closely related pairs were Neriene radiata and Lycosa rabida and Neriene radiata and Araneus angulatus neither of these two pairs had any bands in common.

By comparison in this research ground dwellers from one species to the other. There were also more instances when ground dwellers were more genetically related to sheet web builders than orb weavers to sheet web builders. By examination of the relationships of the bands it is possible that sheet web builders and orb weaving spiders developed from ground dwelling spiders. It was determined that this method of RAPD-PCR research would be useful in comparing different species of the different types of spiders because there are so many different combinations of possibilities.

Figure 1. Gel electrophoresis. A: Orb weavers vs. Ground dwellers. B: Orb weavers vs. Sheet web builders. C: Ground Dwellers. D: Ground Dwellers vs. Sheet web builders

Molecular weight (bp)	K. hibernalis₁	T. sisyphoids₁	A. labyrinthica₁	T. sisyphoids₂	A. labyrinthica₂	L. rabida₁	K. hibernalis₂	L. rabida₂
1150	-	-	-	-	+	+	-	-
1090	-	-	-	-	+	-	-	+
1000	-	-	-	-	+	+	-	-
910	-	-	-	-	-	-	+	-
900	-	-	-	-	+	-	-	-
890	+	-	-	-	-	-	-	-
880	-	-	-	-	+	-	-	-
740	+	-	-	-	-	-	-	-
700	+	+	-	-	-	-	-	-
660	+	-	-	-	-	-	-	-
600	-	+	-	-	+	-	-	-
520	-	+	-	-	-	-	-	-
500	+	-	-	-	-	-	-	-
480	-	+	-	-	-	+	-	-
440	-	+	-	-	-	-	-	-
420	-	+	-	-	-	+	-	-
350	-	-	-	-	-	+	-	-

Table 1. Ground dwelling spiders gel 1

Molecular weights (bp)	Florinda coccina₁	Florinda coccina₂	Frontinella pyramitella₁	Frontinella pyramitella₂	Araneus angulatus₁	Araneus angulatus₂	Neriene radiata₁	Neriene radiata₂
960	-	-	-	-	-	-	+	-
900	-	-	-	-	+	-	-	-