DOI: 10.5061/dryad.d51c5b054
title: Contrasting genetic responses to habitat fragmentation for two Lycaenid butterfly species

description: Biodiversity is currently declining at the global scale. Apart from
 species declines and lowered abundances, the loss of genetic diversity is
 equally concerning as it may undermine fitness and the potential to adapt
 to future environmental change. We compared genetic diversity of
 historical and recent Alpine populations of two butterfly species, Lycaena
 helle and L. hippothoe, over a period of about 10 years. Using
 microsatellite markers, we found no changes over time in L. helle, while
 genetic diversity decreased, and differentiation increased in L.
 hippothoe. Lycaena helle inhabits peat bogs and wetland fallows with
 populations being strongly isolated, while L. hippothoe used to occur in
 population networks on hay meadows, with the latter being strongly exposed
 to agricultural intensification. We conclude that currently L. hippothoe
 populations are strongly declining due to changes in land use, resulting
 in genetic erosion potentially due to the collapse of population networks.
description: <strong>Population
 sampling and study area</strong> We studied <em>L.
 helle</em> populations in Germany (Rhineland-Palatinate, Bavaria)
 and <em>L. hippothoe</em> populations in the European Alps,
 including sampling sites in Austria (Tyrol) and Italy (South Tyrol) (Fig.
 1; Table 1). We sampled 20 individuals each in five populations of
 <em>L. helle</em> (except for population 5 with only 15
 individuals in 2005-06 and population 1 with only 14 individuals in 2017)
 in 2005-06 and in 2017. For <em>L. hippothoe</em>, eight
 populations with 18 individuals each were sampled in 2006-07 and in
 2017-19. This design allowed us to investigate changes in the genetic
 constitution of five and eight populations over time in two different
 <em>Lycaena</em> species. Butterflies were caught with an
 insect net in the field. One leg was removed and stored in 100% ethanol
 until DNA extraction. Subsequently, we released the butterflies
 immediately. To avoid the recapture of individuals, we marked all sampled
 individuals with a blue dot on the left hindwing before release.
 <strong>DNA
 extraction and microsatellite analyses</strong> DNA was extracted with the
 E.Z.N.A. ® Tissue DNA Kit (Omega bio-tek, Germany) following the
 manufacturer’s instructions. We used five microsatellite markers for
 <em>L. helle</em> and 14 for <em>L.
 hippothoe</em>, following the polymerase chain reaction (PCR)
 amplifications and thermal cycling profiles given in Habel <em>et
 al</em>. (2008) and Trense <em>et al</em>. (2019)
 (Supplementary Table S1). Genotyping of microsatellites was performed on
 an ABI 3130XL sequencer (Applied Biosystems, Germany). The allele length
 of microsatellite loci was determined using the program GENEIOUS version
 11.1.5 (Kearse <em>et al</em>., 2012;
 https://www.geneious.com). <strong>Data
 analyses</strong> For comparisons, we performed
 separate analyses for the five past (2005-06; 5_past) and recent (2017;
 5_recent) population samples of <em>L. helle</em>, and the
 eight past (2006-07; 8_past) and recent (2017-19; 8_recent) population
 samples of <em>L. hippothoe</em>. The microsatellite loci were
 checked for potential null alleles using the program MICROCHECKER version
 2.2.3 (van Oosterhout <em>et al</em>., 2004). Numbers of
 alleles, alleles per locus, polymorphic loci, gene diversity, inbreeding
 coefficient (<em>F<sub>IS</sub></em>), pairwise
 <em>F<sub>ST</sub></em> and
 <em>R<sub>ST</sub></em> values, observed and
 expected heterozygosity, and analyses of molecular variance (AMOVA) were
 computed with ARLEQUIN version 3.5 (Excoffier & Lischer, 2010).
 Numbers of private alleles,
 <em>G’’<sub>ST</sub></em> and Jost’s D values,
 Nei’s genetic distance (Nei, 1972), and unbiased Nei’s genetic distance
 were calculated using GENALEX version 6.51 (Peakall & Smouse,
 2006, 2012). FSTAT version 2.9.3 (Goudet, 1995) was used to calculate
 allelic richness. FREENA was used to control for the impact of null
 alleles on <em>F<sub>ST</sub></em> values (Chapuis
 & Estoup, 2007). Hardy-Weinberg equilibrium (HWE) and linkage
 disequilibrium were tested with GENEPOP version 4.2 (Raymond &
 Rousset, 1995; Rousset, 2008). Resulting<em> P</em>-values for
 linkage disequilibrium and pairwise
 <em>F<sub>ST</sub></em> values were corrected for
 multiple testing using the Benjamini-Hochberg method (Benjamini &
 Hochberg, 1995). To test whether results are affected by the different
 numbers of microsatellite loci across species, we additionally performed
 analyses based on random selections of five out of 14 loci for
 <em>L. hippothoe</em>, using 15 replicates (see Supplementary
 Table S2 – S17). To assess statistical significance across replicates, we
 calculated the harmonic mean p-value (HMP) for each molecular index. The
 HMP significance threshold was 0.039. Variation in molecular indices
 across past and recent populations was analysed with Mann-Whitney U and
 Wilcoxon tests using STATISTICA version 12.0 (Tulsa, StatSoft). We used
 both approaches because the individuals tested were independent (different
 individuals were sampled within the different periods) while populations
 were sampled repeatedly. Furthermore, the genetic
 population structure was examined by a Bayesian analysis, as implemented
 in STRUCTURE version 2.3.4 (Pritchard <em>et al</em>., 2000),
 using an admixture model to allocate individual microsatellite genotypes
 to K gene clusters. For the four data sets, we tested 10 iterations for
 each K = 1-5 for <em>L. helle</em> and K = 1-8 for
 <em>L. hippothoe</em> with 50 000 burn-in steps and 500 000
 Markov chain Monte Carlo (MCMC) cycles. The Delta K method (Evanno
 <em>et al</em>., 2005) was used to estimate the most likely
 number of clusters in STRUCTURE HARVESTER version 0.6.94 (Earl &
 von Holdt, 2012). Afterwards, we used CLUMPAK (Kopelman <em>et
 al</em>., 2015) to combine the cluster assignments across replicate
 analyses and to illustrate aligned cluster assignments.