Computational protocol: Inter- and intraspecific genetic and morphological variation in a sibling pair of carabid species

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Protocol publication

[…] Body size (elytral length) and wing size were measured by means of a calibrated ocular under a binocular microscope. Generally, carabid wing size follows an allometric relationship with body size. [] developed an index that corrects for this allometry, i.e. percentage of maximal realisable relative wing size. Relative wing size is wing length × width divided by elytral length × width. Relative wing size is then expressed as a percentage of the maximal wing size for a beetle of a given size. This index was shown to be an unbiased estimator for comparing different individuals, populations and species of carabid beetles []. In ground beetles, females are generally larger than males. Therefore, we analyse male and female body sizes separately. To be complete, we analyse female and male relative wing size also separately. Body size and relative wing size are compared between species and populations with ANOVA's. Total variance is partitioned among groups (species or species and ecotypes), among populations within groups, and within populations by carrying out a nested design ANOVA using STATISTICA (version 7.1; StatSoft Inc., Tulsa, UK) on both a micro- and macroscale. [...] Data are used from five polymorphic enzymes: aldehyde oxidase (AO, E.C., glucose-6-phosphate isomerase (GPI, E.C., isocitrate dehydrogenase 1 and 2 (IDH1, IDH2, E.C., phosphoglucomutase (PGM, E.C. Protocols of electrophoresis are provided by []. Earlier work showed that one locus (IDH1) was non-neutral and we will always analyse it separately [].Departures from Hardy-Weinberg equilibrium expectation were tested with an exact test using the GENEPOP software (Version 3.2; []). Significance levels were adjusted by using sequential Bonferroni correction. Similarly as in the analyses for body and wing size, total genetic variance is partitioned among groups (species, ecotypes), among populations within groups, and within populations by carrying out a hierarchical analysis of molecular variance (AMOVA) using ARLEQUIN (version 3.000; []) on both a micro- and macroscale.PCRs for nucleotide sequencing of COI utilized primers C1-J-1718 and C1-N-2191 and for 16S we utilized primers LR-J-1307 and LR-N-13398 []. DNA amplification reactions were performed in a 25 μL final volume. Each reaction mix contained 5 μL of extract, 1× buffer (Sigma), 1.5 mM MgCl2, 200 μM of each dNTP, 0.4 μM of each primer and 0.6 U RedTaq DNA polymerase (Sigma). Initial denaturation was for 2 min at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min 30 s at 48°C (and 46°C for 16S), and 2 min at 72°C; 9 min at 72°C completed the program. The reaction was purified with columns following manufacturer's recommendations. Sequencing was done by BigDye Terminator version 3.1 kits on an ABI 3130 sequencer (Applied Biosystems). Sequences were aligned using BioEdit version 5.0.6 []. We tested for neutrality of mutations following Fu & Li's method with D* and F* test statistics using DNASP 4.0 [,]. A phylogeny of unique haplotypes was constructed from the calculated Kimura two-parameter distances using the neighbour-joining approach within MEGA ([]; 1000 bootstrap replicates). […]

Pipeline specifications

Software tools Statistica, Genepop, Arlequin, BioEdit, DnaSP
Applications Miscellaneous, Phylogenetics, Population genetic analysis
Organisms Chalceus macrolepidotus, Potomida littoralis