DOI: 10.5061/dryad.8cz8w9gzz
title: Immigration allows population persistence and maintains genetic diversity despite an attempted experimental extinction

description: Widespread fragmentation and degradation of habitats make organisms
 increasingly vulnerable to declines in population. Immigration is a key
 process potentially affecting the rescue and persistence of populations in
 the face of such pressures. Field research addressing severe demographic
 declines in the context of immigration among interconnected local
 populations is limited due to difficulties in detecting such demographic
 events and the need for long-term monitoring of populations. In a
 17-subpopulation metapopulation of the butterfly, Parnassius smintheus,
 all adults observed in two adjacent patches were removed over eight
 consecutive generations. Despite this severe and long-term reduction in
 survival and reproduction, the targeted populations did not go extinct.
 Here, we use genetic data to assess the role of immigration versus in situ
 reproduction in allowing the persistence of these populations. We
 genotyped 471 samples collected from the targeted populations throughout
 the removal experiment at 171 single nucleotide polymorphisms. We found no
 reduction of genetic diversity of the targeted populations over time, but
 a decrease in number of loci in Hardy-Weinberg equilibrium, consistent
 with a high level of immigration from multiple, surrounding populations.
 Our results highlight the role of connectivity and movement in making
 metapopulations resilient to even severe and protracted localized
 population reductions.
description: The Jumpingpound Ridge metapopulation occurs within a network of
 17 patches of alpine meadow habitat ranging in size from 0.2 ha to 22.7
 ha. This system has been the subject of long-term population monitoring
 and genetic sample collection since 1995, with mark-recapture performed
 during the annual flight season to monitor population size and movement.
 The removal experiment on patches P and Q was conducted from 2001 to 2008
 to investigate the effects of severe and continuous population reduction
 in patches P and Q on the population dynamics in surrounding, neighbouring
 patches . Through the course of the experiment, patches P and Q were
 surveyed every one to three days during the flight season each year, and
 all observed butterflies were captured by hand netting and removed from
 the site. In total, 4,830 butterflies were removed from P and Q over the
 eight-year period. The removed individuals were labelled with the date and
 location of capture, and then stored, dried, and pinned in a collection
 housed at the University of Cincinnati. We sampled from
 this collection of pinned specimens from patches P and Q, removing one leg
 per individual across each year of the removal experiment. For years in
 which the number of captured individuals in a patch was lower than 30, we
 sampled all available individuals. Otherwise, we sampled between 30-60
 individuals per patch per year.  Samples from 2001 represent the basal or
 initial state of the populations, as they would not yet have responded to
 the experimental manipulation. Additionally, leg tissues from 42 frozen
 whole butterflies, removed during the 2008 flight season were also used;
 in combination with pinned samples from 2008 these represent the final
 year of the experimental removals. In total, we sampled a total of 471
 individuals from patches P and Q across eight years.
 Our SNP panel contained 171 unlinked SNP loci and was developed
 based on a double digest restriction site associated DNA sequencing
 (ddRADseq) dataset for the species [44]. Briefly, multiple ddRADseq
 libraries were constructed from 80-100 individuals each (for a total of
 501 individuals, sampled from Jumpingpound Ridge and the surrounding
 region and not overlapping with the individuals removed from patches P and
 Q) by digesting genomic DNA using the restriction enzymes
 <em>Nla</em>III and <em>Eco</em>RI, then selecting
 and amplifying fragments between 200 and 500 bp and sequencing the
 libraries on an Illumina HiSeq 2500 sequencer. We identified SNPs from the
 resulting sequences using the program STACKS with the following
 parameters: minimum stack depth (m) = 3, mismatches allowed between
 putative catalog loci (n) = 3, mismatches allowed between putative alleles
 (M) = 2, mismatches allowed to align secondary reads (N) = 4, and a
 maximum allowed missing data of 50%.<em> </em>This resulted in
 a ddRADSeq dataset of 8814 SNP loci. From the ddRADSeq sequences, we
 filtered SNPs with at least 40 bp upstream and downstream flanking
 sequence to allow space for designing primers for the iPlex Gold MASSarray
 assay, and used the software PLINK to identify and remove statistically
 linked loci. To identify SNPs that might putatively be functional, we
 aligned the filtered ddRADSeq sequences to a transcriptome of thorax
 tissue of adult butterflies captured during flight using Magic-BLAST. We
 defined fragments with at least a 90% sequence match as putatively
 expressed loci. Potential functions of these putatively expressed loci
 were identified using Trinotate, and 35 loci were chosen to reflect a
 variety of cellular functions such as metabolism, transcription
 regulation, transmembrane proteins, protein modification, and insect
 development. An additional 130 loci having both less than 10% sequence
 match to the transcriptome and a greater than 90% sequence match to a
 shotgun sequenced genome were chosen at random to include in the final SNP
 panel. Finally, an additional six non-synonymous SNPs were included from
 the coding region of phosphoglucose isomerase (<em>Pgi</em>) ,
 a gene associated with dispersal and flight metabolism in other insects,
 including the Glanville fritillary butterfly. Of the
 171 SNPs targeted for genotyping, assays for two SNPs failed altogether,
 seven SNPs failed across more than 70% of samples, and nine SNPs were
 monomorphic across all samples in all years from both patches P and Q.
 These 19 SNPs were removed, and we used the genotype data from the
 remaining 152 polymorphic SNPs for further analyses.  Of the 477
 individuals genotyped from P and Q, six individuals were successfully
 genotyped at less than 80% of all SNPs and were also removed from the
 dataset, with data from 471 individuals total remaining.
description: ***Parnassius smintheus*** **(Rocky Mountain Apollo) Removal Experiment
 SNP (Single Nucleotide Polymorphism) Genotype Data** This dataset consists
 of three sheets: 01-08 PQ Removal, 05 Surrounding Populations, and 08
 Surrounding Populations, each containing SNP (single nucleotide
 polymorphism) genotype data of *Parnassius smintheus* (Rocky Mountain
 Apollo), collected from a metapopulation system of this alpine butterfly
 in Jumpingpound Ridge, Kananaskis, Alberta, Canada (50° 57'N, 114°
 54'W), consisting of 17 alpine meadow habitat patches (Please refer
 to Figure 1 of
 [https://doi.org/10.1098/rsos.240557](https://doi.org/10.1098/rsos.240557)
 for the map of Jumpingpound Ridge including the name and location of each
 habitat patch). This population system has been researched since 1995,
 with population monitoring and genetic sampling being conducted during the
 summer flight season annually. From 2001 to 2008, all adult individuals
 found in two adjacent patches, P and Q, were removed annually as part of a
 long-term removal experiment meant to induce local extinction, testing its
 effects on the population dynamics of the surrounding patches.    471
 Samples included in the 01-08 PQ Removal sheet were sampled from
 individuals collected from P and Q as part of this long-term artificial
 removal experiment. 05 and 08 Surrounding Populations sheets contain
 samples from surrounding populations of P and Q (M,O,R,S) that lie within
 the maximum observed dispersal distance of the butterfly during flight
 season (2 km), from two of the years that coincide with the removal
 experiment along with 2005 and 2008 P and Q populations. All samples were
 genotyped on a SNP panel containing 171 unlinked SNPs, with P and Q
 samples being genotyped in 2021 and the M,O,R,S samples in 2019 (Please
 refer to [https://ir.lib.uwo.ca/etd/8456](https://ir.lib.uwo.ca/etd/8456)
 for more details regarding the development and design of the panel). SNPs
 which failed to genotype or were monomorphic across all individuals in the
 P,Q dataset were removed, with the final total number of loci used for the
 analysis being 152 SNPs presented in 01-08 PQ Removal sheet. SNPs
 presented in 05 and 08 Surrounding Populations contain all successfully
 genotyped SNPs that are shared among the P, Q samples and M,O,R,S samples
 in 2005 (138 SNPs) and 2008 (151 SNPs). For more details regarding the
 methodology of the removal experiment please refer to
 [https://doi.org/10.1111/j.1600-0706.2010.18666.x](https://doi.org/10.1111/j.1600-0706.2010.18666.x) **Description of the data and file structure** ·       *Year*: Year in which the sample was collected ·       *Patch*: Meadow habitat patch from which the sample was collected within the Jumpingpound Ridge metapopulation. For the full map of the system with the locations and names of each patch please refer to figure 1 of [https://doi.org/10.1098/rsos.240557](https://doi.org/10.1098/rsos.240557) ·       *Sample Name*: Unique ID designated to each individual genotyped ·       *Columns D ~*: Diploid genotype results at each single nucleotide polymorphism (SNP) of the panel.