Computational protocol: Trapped in the extinction vortex? Strong genetic effects in a declining vertebrate population

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Protocol publication

[…] We assessed the genetic similarity of mates using band-sharing coefficients derived from multi-locus DNA fingerprints [], following standard laboratory and scoring procedures (e.g. []). Although band-sharing does not give an exact measure of relatedness between two individuals, it provides an index that reflects their relatedness. Such an index, however, still allows statistical testing of e.g. differences in relatedness between groups (e.g. [,-]). Recent studies have often used microsatellite markers to estimate relatedness. However, indices of relatedness based on microsatellite genotyping and DNA fingerprinting frequently correlate, as documented by several previous studies (e.g. [,]) and also supported by our findings (see Results).We hybridized DNA with the multi-locus probe per [] and scored on average 28.5 bands in males (range 10-37) and 29.0 bands in females (range 15-38). Our sample consisted of 40 pairs (Table ), first formed between 1993 and 2003. We examined the influence of genetics on hatching success in a subsample of 36 pairs. For these pairs, we selected all their first clutches in which at least one egg hatched, thereby removing environmental influences such as predation on hatching rates. We then calculated each pair's total hatching success over the years as: sum of hatchlings/sum of eggs produced. The mean number of analyzed clutches per pair was 1.6 (range 1-6 clutches). In one year, three of the pairs produced a clutch that survived beyond the due hatching date and was subsequently abandoned by the parents (thus resulting in complete hatching failure). These cases may or may not represent inbreeding depression, and we conservatively excluded them from the analysis (including them yielded the same result; not shown).Individual genetic diversity of offspring was assessed by allelic heterozygosity at microsatellite loci. We genotyped a sample of 76 individuals at nine polymorphic loci (Table ). Sixty-four of these were chicks used in the subsequent analyses (Table and below), the remaining 12 individuals were only used for assessing the degree of marker polymorphism. Allele frequencies and expected frequency of heterozygotes were calculated using CERVUS v. 2.0 []. Two loci deviated from Hardy-Weinberg Equilibrium (homozygote excess; Table ), possibly indicating non-amplifying alleles (null alleles) or allelic dropouts []. We therefore excluded these loci from further analyses (including them yielded qualitatively similar results; data not shown). We used GENEPOP v. 3.1b [] to investigate potential linkage between loci. After sequential Bonferroni correction [,], however, none of the locus pairs were in significant linkage disequilibrium.Sixty-four of the genotyped individuals each represented one randomly selected offspring from 64 different parental combinations (37 of which were fingerprinted). All of these individuals were typed at all loci, and we therefore calculated multi-locus heterozygosity as the number of heterozygous loci divided by the number of loci examined (i.e. seven). Seven (10.9%) of the genotyped chicks failed to hatch (died before or during the hatching process, Figure ). Two of the hatched chicks fledged in 2004 and were excluded when analysing recruitment rate, as they might have been alive without us detecting them (our field effort was greatly reduced from 2005 onwards). Of the remaining 55 chicks (born 1993-2003), 26 (47.3%) were recruited to the breeding population.Microsatellite genotyping was based on PCR reactions carried out in 10 μl 1× Taq polymerase buffer B containing 15 ng of template DNA, 0.5 U of Taq polymerase (Promega), and final concentrations of the following components: 20 μM (Calp2) or 200 μM (all others) of each dNTP, 1 mM (Calp4) or 1.5 mM (Calp2 and Calp5) or 2 mM (all others) of MgCl2, and 0.4 μM (Calp4 and Calp5) or 1 μM (all others) of both forward and reverse primer. One primer of each pair was dye-labelled (WellRED D2-PA, D3-PA or D4-PA; Proligo) and PCR amplification was carried out on an Eppendorf Mastercycler Gradient. All thermal profiles consisted of an initial 2 min denaturation at 94°C and a final 5 min extension step at 72°C, whereas the denaturation (94°C), annealing (varying temperatures, see below) and extension (72°C) steps of each amplification cycle all lasted 30 sec. For all Ruff primers, the following annealing temperatures were used in a touch-down program: 15 cycles at 52-45°C (decreasing with 0.5°C in each subsequent cycle), followed by 25 cycles at 45°C. The PGT83 and 4A11 loci were amplified with 20 cycles at 57-47°C (decreasing with 0.5°C in each subsequent cycle), followed by 20 cycles at 47°C. For the Calp4 and Calp5 microsatellites, the annealing temperature was 61.5°C during 40 cycles (Calp4) and 56°C during 35 cycles (Calp5). The Calp2 locus was amplified at 58°C (5 cycles), followed by 57°C (15 cycles) and finally 56°C (20 cycles). The size of amplification products was determined on a CEQ™8000 Genetic Analysis System (Beckman Coulter) using the Fragment Analysis Module (software version 8.0.52). […]

Pipeline specifications

Software tools Cervus, Genepop
Application Population genetic analysis
Organisms Ilex paraguariensis