DOI: 10.5061/dryad.73n5tb31c
title: Polyandry and non-random fertilisation maintain long-term genetic diversity in an isolated island population of adders (Vipera berus)

description: Conservation genetic theory suggests that small and isolated populations
 should be subjected to reduced genetic diversity i.e., heterozygosity and
 allelic diversity. Our 34 years study of an isolated island population of
 adders (Vipera berus) in southern Sweden challenges this notion. Despite a
 lack of gene flow and a yearly mean estimated reproductive adult
 population size of only 65 adult adders (range 12 to 171), the population
 has been able to maintain high levels of heterozygosity and allelic
 diversity similar to that observed in two mainland populations. Even a
 14-year major “bottleneck” i.e., a reduction in adult adder numbers,
 encompassing at least four adder generations, did not result in any
 reduction in the island adders’ heterozygosity and allelic diversity.
 Female adders are polyandrous, and fertilisation is non-random, which our
 empirical data and modelling suggest underpinning the island adders’
 ability to maintain a high level of heterozygosity. Our empirical results
 and subsequent modelling suggest that the positive genetic effects of
 polyandry in combination with non-random fertilisation, often overlooked
 in conservation genetic analyses, deserve greater consideration when
 predicting long-term survival of small and isolated populations.
description: Blood samples were stored in 70% ethanol and DNA was isolated by
 phenol–chloroform extraction. Sequencing for SNP
 genotyping was performed by DArTseqTM (DArT Pty Ltd, Canberra), after
 complexity reduction using a combination of restriction enzymes. For
 fragment size selection and next generation sequencing details, see Kilian
 et al. (2012) and Georges et al. (2018). To achieve the most appropriate
 complexity reduction (the fraction of the genome represented, controlling
 average read depth and number of polymorphic loci), four combinations of
 restriction enzymes (Pstl enzyme combined with either Hpall, Sphl, Nspl or
 Msel) were evaluated and the restriction enzyme combination of Pstl
 (recognition sequence 5'-CTGCA|G-3') and Sphl
 (5'-GCATG|C-3') was selected. Only fragments
 generated by the Psti-Sphl double digest were effectively amplified in 30
 rounds of polymerase chain reaction (PCR). Amplifications consisted of an
 initial denaturation step of 94°C for 1 min, followed by 30 cycles of PCR
 with the following temperature profile: denaturation at 94°C for 20 s,
 annealing at 58°C for 30 s and extension at 72°C for 45 s, with an
 additional final extension at 72°C for 7 min. After PCR, equimolar amounts
 of amplification products from each sample were pooled and applied to cBot
 (lllumina) bridge PCR for sequencing on the lllumina Hiseq 2500. The
 sequencing (single end) was run for 77 cycles.
 Sequences generated from each lane were processed using
 proprietary DArT Pty Ltd analytical pipelines as described by Georges et
 al. (2018). In particular, one third of samples were processed twice from
 DNA, using independent adaptors, to allelic calls as technical replicates,
 and scoring consistency (repeatability) was used as the main selection
 criterion for high quality/low error rate markers. The resultant data set
 contained the SNP genotypes and various associated metadata of which
 CloneiD (unique identity of the sequence tag for a locus), repAvg
 (proportion of technical replicate assay pairs for which the marker score
 is identical), CallRate (proportion of individuals scored at a particular
 locus) and SnpPosition (position in the sequence tag at which the defined
 SNP variant base occurs) used in our analyses. The SNP
 data and associated metadata were read into a genlight object (Jombart
 2008) to facilitate processing with package dartR (Mijangos et al., 2022).
 Only loci with 99% or greater repeatability (repAvg) were chosen for
 subsequent analysis. Further filtering was undertaken on the basis of call
 rate (95%). Finally, we filtered out secondary SNPs where they occurred in
 a single sequenced tag, retaining only one SNP at random. This additional
 filtering resulted in 7504 SNP markers for the 84 adders, and we regard
 the data remaining after filtering to be highly reliable.
 Georges A, Gruber B, Pauly GB, White DF, Adams M,
 Young MJ, Kilian A, Zhang X, Shaffer HB, Unmack PJ (2018) Genomewide SNP
 markers breathe new life into phylogeography and species delimitation for
 the problematic short-necked turtles (Chelidae, Emydura) of eastern
 Australia. Mol Ecol 27:5195–5213. Jombart T (2008)
 adegenet, a R package for the multivariate analysis of genetic markers.
 Bioinformatics 24:1403-1405. Kilian A, Wenzl P,
 Huttner E, Carling J, Xia L, Blois H, Caig V, Heller-Uszynska K, Jaccoud
 D, Hopper C, Aschenbrenner-Kilian M, Evers M, Peng P, Cayla C, Hok P,
 Uszynski G (2012) Diversity arrays technology, a generic genome profiling
 technology on open platforms. Methods Mol Biol 888:67-89.
 Mijangos, J., Gruber, B., Berry, O., Pacioni, C. and Georges, A.
 2022. dartR v2: an accessible genetic analysis platform for conservation,
 ecology, and agriculture Methods in Ecology and Evolution
 13:2150–2158.
description: # Title: Polyandry and non-random fertilisation maintain long-term genetic
 diversity in an isolated island population of adders (Vipera berus) ##
 Description of the Data and file structure Blood samples were stored in
 70% ethanol and DNA was isolated by phenolchloroform extraction.
 Sequencing for SNP genotyping was performed by DArTseqTM (DArT Pty Ltd,
 Canberra), after complexity reduction using a combination of restriction
 enzymes. For fragment size selection and next generation sequencing
 details, see Kilian et al. (2012) and Georges et al. (2018). To achieve
 the most appropriate complexity reduction (the fraction of the genome
 represented, controlling average read depth and number of polymorphic
 loci), four combinations of restriction enzymes (Pstl enzyme combined with
 either Hpall, Sphl, Nspl or Msel) were evaluated and the restriction
 enzyme combination of Pstl (recognition sequence 5'-CTGCA|G-3')
 and Sphl (5'-GCATG|C-3') was selected. Only fragments generated
 by the Psti-Sphl double digest were effectively amplified in 30 rounds of
 polymerase chain reaction (PCR). Amplifications consisted of an initial
 denaturation step of 94C for 1 min, followed by 30 cycles of PCR with the
 following temperature profile: denaturation at 94C for 20 s, annealing at
 58C for 30 s and extension at 72C for 45 s, with an additional final
 extension at 72C for 7 min. After PCR, equimolar amounts of amplification
 products from each sample were pooled and applied to cBot (lllumina)
 bridge PCR for sequencing on the lllumina Hiseq 2500. The sequencing
 (single end) was run for 77 cycles. Sequences generated from each lane
 were processed using proprietary DArT Pty Ltd analytical pipelines as
 described by Georges et al. (2018). In particular, one third of samples
 were processed twice from DNA, using independent adaptors, to allelic
 calls as technical replicates, and scoring consistency (repeatability) was
 used as the main selection criterion for high quality/low error rate
 markers. The resultant data set contained the SNP genotypes and various
 associated metadata of which CloneiD (unique identity of the sequence tag
 for a locus), repAvg (proportion of technical replicate assay pairs for
 which the marker score is identical), CallRate (proportion of individuals
 scored at a particular locus) and SnpPosition (position in the sequence
 tag at which the defined SNP variant base occurs) used in our analyses.
 The SNP data and associated metadata were read into a genlight object
 (Jombart 2008) to facilitate processing with package dartR (Gruber et al.
 2018). Only loci with 99% or greater repeatability (repAvg) were chosen
 for subsequent analysis. Further filtering was undertaken on the basis of
 call rate (95%). Finally, we filtered out secondary SNPs where they
 occurred in a single sequenced tag, retaining only one SNP at random. This
 additional filtering resulted in 7504 SNP markers for the 84 adders, and
 we regard the data remaining after filtering to be highly reliable. The
 raw data as recieved from the commercial supplier (Diversity Arrays
 Technology) are held in compact binary form of a genlight object (R
 package Adegenet available from the CRAN repository). This file has been
 compressed using adegenet: :saveRDS(). The metadata associated with each
 individual (the population to which it belongs) is held in comma delimited
 file ind_metadata.csv. The filtered data (see methods above) are also held
 in an adegenet genlight object, compressed using adegenet::saveRDS(). To
 access the data, use library(adegenet) gl. raw &lt;-
 readRDS(file="adder \_raw.RDSdata") gl. filtered &lt;- read
 RDS(file="adder_filtered_run1.RDSdata") and then work with the
 genlight object as outlined by Jombart, 2008 or Mijangos et al. 2022. *
 Jombart T (2008) adegenet, a R package for the multivariate analysis of
 genetic markers. Bioinformatics 24:1403-1405. * Mijangos, J., Gruber, B.,
 Berry, 0., Pacioni, C. and Georges, A. 2022. dartR v2: an accessible
 genetic analysis platform for conservation, ecology, and agriculture
 Methods in Ecology and Evolution 13:2150-2158 ## Sharing/access
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