DOI: 10.5061/dryad.nzs7h44qj
title: ddRAD of Hawaiian Ariamnes spiders

description: The diversification of a host organism can be influenced by both the
 external environment and its assemblage of microbes. Here, we use a young
 lineage of spiders, coupled with a chronologically arranged series of
 volcanic mountains, to determine the evolutionary history of a host and
 its associated microbial communities, altogether forming the “holobiont”.
 Using the stick spider Ariamnes waikula (Araneae, Theridiidae) on the
 island of Hawaiʻi, and outgroup taxa on older islands, we tested whether
 the host spiders and their microbial constituents have responded in
 similar ways to the dynamic abiotic environment of the volcanic
 archipelago. The expectation was that each component of the holobiont (the
 spider hosts, intracellular endosymbionts, and gut microbiota) should show
 a similar pattern of sequential colonization from older to younger
 volcanoes. In order to investigate this, we generated ddRAD data for the
 host spiders and 16S rRNA gene amplicon data from their microbiota.
 Results showed that the host A. waikula is strongly structured by
 isolation, suggesting sequential colonization from older to younger
 volcanoes. The endosymbiont communities were markedly different between
 Ariamnes species on different islands, but more homogenized among A.
 waikula populations. In contrast, the gut microbiota was largely conserved
 across all populations and species, and probably mostly environmentally
 derived.  Our results highlight the different evolutionary
 trajectories of the distinct components of the holobiont, showing the
 necessity of understanding the interplay between components in order to
 assess any role of the microbial communities in host diversification.
description: To examine the population structure of the spider host, we used
 ddRAD to obtain reduced representation genome-wide SNP data. Genomic DNA
 was extracted from spider legs with several modifications to the Qiagen
 DNeasy kit protocol. Legs were first removed from each specimen using
 sterile tweezers so that the abdomen remained intact for the microbial DNA
 analysis. DNA was then extracted by placing the tissue in Proteinase K and
 lysis buffer and grinding them with a sterile pestle to break up the
 exoskeleton. We then added 4uL RNase A (100 mg/ml) and incubated the
 extractions for two minutes at room temperature. Tubes with tissue and
 extraction solution were then placed overnight in a heat block at 56°C.
 The remainder of the extraction protocol was performed following the
 manufacturer’s instructions. We built ddRAD libraries following an adapted
 protocol of Peterson et al. (2012) (Saarman & Pogson, 2015; see
 Maas et al. 2018 for protocol optimization steps). Briefly, we started the
 ddRAD protocol with a total of 100 nanograms of DNA per sample. The DNA
 was digested using SphI‐HF (rare‐cutting) and MlucI (frequent‐cutting)
 restriction enzymes. We assessed fragmentation with a Bioanalyzer High
 Sensitivity chip (Agilent). We multiplexed 15-20 individuals per library
 for a total of eight ddRAD libraries. We used a Sage Science Pippen Prep
 to size select 451-551bp (including internal adapters) fragments, and
 confirmed the sizes using a Bioanalyzer. Ten indexing polymerase chain
 reaction cycles (PCRs) were run on each library to enrich for
 double‐digested fragments and to incorporate a unique external index for
 each library pool. The eight libraries were sequenced using 100bp
 paired-end sequencing on one Illumina HiSeq 2500 lane at the Vincent J.
 Coates Genomic Sequencing Facility at UC Berkeley. To
 characterize the microbial community within the A. waikula hosts, 71
 individuals from eight populations of three Hawaiian Islands were selected
 for analysis. We focused on the mid and hindgut, both located in the
 spider’s opisthosoma. The preservation in ethanol led to considerable
 shrinkage of the opisthosoma and thus did not allow us to separately
 dissect out the gut. Instead, we used the whole opisthosoma to extract
 DNA. Specimens which did not have the opisthosoma intact were not used.
 The digestive tract comprises the majority of the opisthosoma’s cavity. In
 addition, it contains silk glands, the heart, lungs, and gonads. The
 opisthosoma was removed with a sterile razor blade and then washed in
 ethanol to remove external bacteria (Hammer et al. 2015). We considered it
 to be representative of the “gut microbiota”, even if it technically
 consists of the “opisthosoma microbiota”, but previous studies have shown
 that the gut microbiota dominates in the opisthosoma (Sheffer et al. 2020,
 Kennedy et al. 2020). The tissue was then transferred into lysis buffer
 and finely ground with a sterile pestle. DNA was extracted using the
 Gentra Puregene Tissue Kit (Qiagen, Hilden, Germany) according to the
 manufacturer’s protocol. Spider abdominal tissue can contain PCR
 inhibitors (Schrader et al. 2012), thus we cleaned the DNA extract with
 0.9X AmPure Beads XP. We next amplified a ~300 bp
 fragment of the V1-V2 region of the bacterial 16S rRNA using the Qiagen
 Multiplex PCR kit according to the manufacturer’s protocols and using the
 primer pair MS-27F (AGAGTTTGATCCTGGCTCAG) and MS-338R
 (TGCTGCCTCCCGTAGGAGT) (Gibson et al. 2014). PCRs were run with 20ng of
 template DNA and 30 cycles at an annealing temperature of 55°C. PCR
 products were separated from leftover primer by 1X AmPure Beads XP. A
 six-cycle indexing PCR was performed on the cleaned products, adding dual
 indexes to every sample using the Qiagen Multiplex PCR kit. Indexing was
 performed according to (Lange et al. 2014). The dual indexed libraries
 were isolated from leftover primer as described above, quantified using a
 Qubit fluorometer, and pooled in equal amounts into a single tube. The
 library was sequenced on an Illumina MiSeq using V3 chemistry and 300 bp
 paired reads. We also performed blank extraction controls and negative PCR
 controls (without DNA template) in order to discard contaminants from our
 final dataset.