Computational protocol: Genetic consequences of cladogenetic vs. anagenetic speciation in endemic plants of oceanic islands

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Protocol publication

[…] The data summarized here (Table ) provide the first comprehensive genetic comparisons (with AFLPs and SSRs) in the Juan Fernández Archipelago of species that have evolved by anagenesis and cladogenesis, based on consistent samplings, laboratory methods and modes of analysis. A number of earlier studies utilizing isozymes and DNA sequences have examined genetic variation in endemic species of these islands (e.g. , ), but these investigations were not focussed on comparing modes of speciation. Genera in the present studies were selected for their representation of anagenesis and cladogenesis and for their occurrence on the two islands of different geological ages. The samples were collected during expeditions in February 2010 and 2011 from 1870 individuals in 163 populations in 15 endemic species, hence representing 14 % of the endemic angiosperms in the archipelago. The samples provide very good geographic coverage of populations over the landscape in both islands. The term population, as used here in the sense of sampling, refers to groups of individuals that were clearly delimited spatially in the field. The number of individuals analysed per population ranged from 1 to 31. The voucher data for these samples and details of data gathering and analysis are given in the respective publications. Briefly, the following approaches were used for AFLPs. Four or six selective primer combinations were chosen. Numerous (24–85) primer trials were run with each genus to determine the best combination of primers for good resolution of individuals and populations. Data were obtained on an automated DNA sequencer (ABI 3130xl, Applied Biosystems, Waltham, MA, USA). Scoring was done using GeneMarker ver. 1.85 (SoftGenetics, State College, PA, USA). For analysis of AFLP data, the programs Arlequin 3.5.1.2 (), FAMD ver. 1.25 (), R-Script AFLPdat () and SPSS ver. 15.0 (SPSS; IBM, Armonk, NY, USA) were employed to determine total number of fragments (TNB), percentage of polymorphic fragments (PPB), Shannon Diversity Index (SDI), average gene diversity over loci (AGDOL) and rarity index (RI).For SSRs, NGS methods () were used to generate 6–12 loci, selected for their repeatability and scoring convenience. Polymerase chain reaction-amplified fragments were also run on the same automated sequencer and scored with GeneMarker ver. 1.85. Data analysis involved using GENEPOP 4.0 (), Micro-Checker 2.2.3 (), FSTAT 2.9.3.2 and GENALEX 6 (). These allow analyses for observed proportion of heterozygotes (HO), expected proportion of heterozygotes (HE), number of alleles per locus (NA), inbreeding coefficient (FIS) and allelic richness standardized by five individuals (AR5).The overall pattern of higher genetic diversities in anagenetically derived species in comparison with cladogenetically derived ones was examined by a Student's t-test (average TNB, PPB, SDI, AGDOL and RI in AFLPs, and HO, HE, NA and AR5 in SSRs) and shown in Table . To improve normality of HO and HE, a square-root transformation was applied. The overall patterns of higher genetic diversities in Robinson Crusoe Island (old) than Alejandro Selkirk Island (new) were also examined in the same way. The effects of two factors (speciation mode and island) and their interaction were analysed in a two-way ANOVA in R version 3.0.0 () and shown in Table . Data from both AFLPs and microsatellites were further analysed by assessing genetic distance () with the NeighborNet algorithm () implemented by SplitsTree4 ver. 4.10 () and Population 1.2.30 (), respectively.For this article, to allow ease of visual comparisons of results among the species, emphasis has been placed on selected graphic presentations. SplitsTree NeighborNet was employed with the AFLP data, and the results are given in a series of graphs (Fig. ). Neighbour-joining based on genetic distance was used for analysis of the SSRs, and simplified networks were used to show relationships among the populations (Fig. ). For summary comparisons of genetic diversity among species, AGDOL was used with the AFLP data (Fig. ). Not all calculated values for all original populations are presented or discussed in this review. The reader is referred to the original publications for additional methods and data. Figure 3. Figure 4. Figure 5. […]

Pipeline specifications

Software tools GeneMarker, Arlequin, Genepop, GenAlEx, NeighborNet, SplitsTree
Applications Phylogenetics, Population genetic analysis
Diseases Genetic Diseases, Inborn