DOI: 10.5061/dryad.4mw6m90g7
title: Infrequent oceanic long-range dispersal and evolution of a top terrestrial arthropod predator in the sub-Antarctic

description: The UNESCO world heritage sub-Antarctic terrestrial ecosystems are unique.
 They have been isolated for over 30 million years by constant circum-polar
 currents and winds, and shaped by climatic cycles that surpass the
 tolerance limits of many species. Despite this recognition, surprisingly
 little is known about how these ecosystems acquired their native
 terrestrial fauna and how it changed over deep time scales. Here the
 patterns and timing of colonization and speciation in the largest and
 dominant arthropod predators in the Eastern sub-Antarctic – spiders of the
 genus Myro – are demonstrated for the first time. Our results indicate
 that this lineage originated from Australia before the Plio-Pleistocenic
 glacial cycles and underwent an adaptive radiation on the Crozet
 archipelago. We discuss the gain and loss of pre-adaptations acting as
 filter that enabled only one of four Myro species native to the Crozet
 islands to repeatedly disperse via the Antarctic circum-polar current,
 resulting in an outstanding distribution range over 9000 kilometres. The
 results highlight the outstanding role of the volcanic Crozet archipelago
 for the evolution of arthropod life in the sub-Antarctic, and the
 potential of terrestrial macro-invertebrates to achieve rare but
 ecologically influential trans-oceanic dispersal events over thousands of
 kilometres under hostile conditions.
description: <strong><em>Collection and field observation.
 </em></strong>Fieldwork in Tasmania was done under the
 research Authority No. FA18285, for Maatsuyker Island FA19197, and for
 Macquarie Island FA15234 of the DPIPWE (Department of Primary Industries,
 Parks, Water and Environment, Tasmania). Fieldwork on Crozet and Kerguelen
 islands (French Southern territories) was done under the project
 136-SUBANTECO (French Polar Institute) with the collection permits
 A2020-89 (26/08/2020) and A-2021-115 (12/10/2021) from Terres Australes et
 Antarctique Françaises. The collection sites are indicated on the maps in
 Fig. S3. Specimens from Marion Island were obtained under the permit
 MARION-E/MA2022-6 by DFFE (Department of Forest, Fisheries and the
 Environment). Fieldwork in New Zealand was performed under the Research
 and Collection Authority 71225-RES by the DOC (Department of Conservation,
 New Zealand). Specimens of Toxopidae were hand
 collected from webs and retreats or by turning stones and sifting
 litter. <em>Myro</em> spp. from Macquarie Island and
 Maatsuyker Island were retrieved from pitfall trapping with 100% propylene
 glycol as fixative. As the Heard Island archipelago is rarely visited, we
 examined and subsampled from a historical specimen of <em>M.
 kerguelensis</em> collected during an expedition in 1985 and
 deposited with the Tasmanian Agricultural Insect Collection.
 Species were identified using taxonomic literature. We did not
 use sub-species names (e.g., <em>M. kerguelensis
 crozetensis</em>), as sub-species taxonomy is highly inconsistent in
 arachnology. <strong><em>DNA sequencing
 approach.</em></strong> For molecular phylogenetics, long
 fragments of two mitochondrial genes (Cytochrome Oxidase I (COI), and
 Cytochrome B (CytB)) and the nuclear ribosomal cluster (spanning from
 highly conserved 18S-28S, including the fast evolving Internal Transcribed
 Spacer (ITS) regions) were sequenced, providing informativeness at both
 shallow and deeper phylogenetic levels [1]. These genes are widely used in
 barcoding, metabarcoding and molecular phylogenetics of spiders, and thus
 have the benefit of an abundance of legacy data that can be included in
 the analysis [2-4]. Our approach differed from previous phylogenetic
 studies on spiders using these amplicons by using new, long
 amplicon-producing primers (Tab S1) and a Nanopore sequencing approach to
 capture longer sections of mentioned genes (~1,050 bp of COI, ~750 bp of
 CytB and ~3,700 bp of the nuclear ribosomal cluster instead of ~300 bp
 fractions) [1, 5]. <strong><em>DNA
 extraction.</em></strong> Genomic DNA was extracted from
 separated legs of specimens that were either stored in absolute ethanol at
 -70 °C (45 out of 52 specimens, collected between one and three years
 before extraction) or in 70% ethanol at room temperature (collected
 between two and ten years before DNA extraction) (S4). For the extraction,
 the Qiagen Puregene Cell kit was used following the manufacturer’s
 protocol, with the addition of Proteinase K (ThermoFisher, Waltham, MA)
 and GlycoBlue (ThermoFisher) to enhance DNA yield. DNA pellets were eluted
 in 25 µL of Qiagen DNA hydration solution.
 <strong><em>Marker
 amplification.</em></strong> For the amplification of the
 markers, the primers listed in Table S2 with attached index sequences were
 used, such that each sample received a unique combination of indexes. The
 unique indexes were generated using the Barcode Generator 
 (http://comailab.genomecenter.ucdavis.edu/index.php/Barcode_generator,
 Accessed August 2020) with a minimum distance of 10 bases between each
 index and a length of 20bp. PCR was performed using the Qiagen Multiplex
 kit for the two mitochondrial markers and the Qiagen UltraRun LongRange
 kit for the nuclear ribosomal marker (due to its length exceeding 3000
 bp). Each PCR reaction contained 5 µL PCR master mix from the
 corresponding kit, 3 µL RNase free water, 0.5 µL of each 10 µM forward and
 reverse primer and 1 µL of template DNA. Thermocycler protocols were
 chosen following the manufacturer’s recommendations for each kit, and were
 further adjusted following a preliminary round of PCR optimization. For
 the amplification of mitochondrial markers the following PCR settings were
 used: initial denaturing step at 95 °C for 15 min, 30 amplification cycles
 (denaturing at 94 °C for 30 s, annealing at 48 °C for 90 s, and extension
 at 72 °C for 90 s), final extension at 68 °C for 15 min. For the nuclear
 ribosomal marker the settings used were: initial denaturing at 93 °C for 3
 min, 40 amplification cycles (denaturing at 93 °C for 30 s and
 annealing/extension at 68 °C for 2 min), final extension at 72 °C for 10
 min. Indexed PCR products were checked on 1 % agarose gels, then pooled in
 roughly equimolar amounts based on gel band intensity.
 <strong><em>Library preparation and
 sequencing.</em></strong> Two pools were made – one for the
 mitochondrial and one for the nuclear marker – because, based on previous
 work, amplicons differing greatly in length were expected to perform
 differently in library prep. Pools were then cleaned of residual primers
 using 0.7X magnetic beads following the AMPure XP protocol (Beckman
 Coulter, Pasadena, CA). Nanopore library prep was performed separately for
 the mitochondrial and nuclear pools, using the SQK-LSK109 kit (Oxford
 Nanopore Technologies, Oxford, UK) following Pomerantz et al. [6], and
 using the Short Fragment Buffer (SFB) for the mitochondrial and the Long
 Fragment Buffer (LFB) for the nuclear library. Libraries were quantified
 using a Qubit Fluorometer with the high-sensitivity dsDNA assay
 (ThermoFisher) and then combined into a final pool containing roughly 20
 fmol of the mitochondrial and 30 fmol of the nuclear library. The
 relatively larger amount of nuclear library was chosen because the shorter
 mitochondrial fragments were expected to outperform the longer nuclear
 fragments during sequencing. This final pool was sequenced on a MinION
 using an R9.4.1 flow cell (Oxford Nanopore Technologies) according to the
 manufacturer’s protocol.
 <strong><em>Approach for historical
 specimen.</em></strong> Due to the age of the historical
 specimen of <em>M. kerguelensis </em>from Heard Island
 (collected 37 years before DNA extraction), with only small amounts of
 tissue (2 legs) available, we used a DNA extraction method more suitable
 for degraded DNA following the protocol outlined in Derkarabetian et al.
 [7], a method originally described in Tin et al. [8]. As the DNA was too
 fragmented for the long read approach, short (200-300bp) amplicons of COI
 and 18S gene fragments were amplified and sequenced on an Illumina
 platform following the protocol by Krehenwinkel et al. [3].
 <strong><em>Sequence
 assembly.</em></strong> Sequences were processed following
 Pomerantz et al. (2022). In brief, samples were first demultiplexed by
 index combination using <em>minibar</em> (Krehenwinkel et al.
 2019). They were then demultiplexed by marker (COI, CytB or ribosomal
 cluster) based on inner primer sequences, and consensus sequences were
 generated, using <em>NGSpeciesID</em> [9]. In many cases,
 multiple consensus sequences were generated for a given sample due to
 coamplification of paralogs, NUMTs [10] or contaminants such as fungi. To
 identify the correct sequence for each sample, all sequences were checked
 by BLAST search [11] against the NCBI nucleotide database (accessed
 10/2022), and erroneous sequences were removed from the data
 set. Legacy sequence data were added from Wheeler et
 al. [2] and from the Barcode of Life project [12] (fractions of COI and
 18S genes for 17 specimens; Tab. S1).
 <strong><em>Sequence
 alignment.</em></strong><em> </em>All samples of
 each individual marker sequence were aligned with ClustalW MSA with gap
 opening penalty 15.00 and gap extension penalty 6.66 in MEGA11 [13].
 Alignments were visually inspected for obvious alignment errors, which
 were corrected by hand. The ITS-region contains many homopolymers, which
 are often subject to sequencing errors, causing misalignments particularly
 in this region. In such cases, excessive sites were removed. Some
 hyper-variable sites in the ITS could only be poorly aligned, even between
 samples of the same species, and were removed. The final concatenated
 alignment of all markers contains 4487 bp with 997 parsimony-informative
 and 306 singleton sites. References
 [1] Krehenwinkel, H., Pomerantz, A., Henderson, J.B., Kennedy,
 S.R., Lim, J.Y., Swamy, V., Shoobridge, J.D., Graham, N., Patel, N.H.
 & Gillespie, R.G. 2019 Nanopore sequencing of long ribosomal DNA
 amplicons enables portable and simple biodiversity assessments with high
 phylogenetic resolution across broad taxonomic scale.
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 sets for simple and cost efficient arthropod systematics by multiplex PCR
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 Evolution</em> <strong>9</strong>, 2181-2193.
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description: All files included in this submission can be viewed with text
 editors. For viewing and analysing the files, however, programmes that can
 handle sequence alignments, such as MEGA, are recommended.
description: # Infrequent oceanic long-range dispersal and evolution of a top
 terrestrial arthropod predator in the sub-Antarctic --- Sequence data
 Myroinae ## Description of the data and file structure
 1_consensus_sequences.zip: Raw consensus sequences generated from Nanopore
 reads after demultiplexing by specimen index and marker.
 2_alignment_concatenated.zip: Final concatenated alignment (ITS reduced)
 used for phylogenetic analyses. The zip archive contains the concatenated
 alignment (fasta file) and the partition (nexus file).