Computational protocol: First freshwater coralline alga and the role of local features in a major biome transition

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Protocol publication

[…] We studied 11 specimens of P. cetinaensis, recent collections of Pneophyllum taxa in the Mediterranean and Atlantic Europe, as well as type species (P. fragile, P. lobescens, P. limitatum, P. subplanum, P. zonale) and other important historical collections of Pneophyllum species collected over the last two centuries and deposited at the Natural History Museum (BM), the Muséum National d´Histoire Naturelle (PC), and at the Norwegian University of Science and Technology (TRH) (see online). Except for P. cetinaensis, the rest of the collections were collected in marine areas, intertidally or subtidally, growing as epiliths on stones or glass but also as epiphytes on seaweeds and seagrasses (see online).DNA extraction, PCR, and PCR product sequencing. Specimen surfaces without epiphytes were selected under a stereomicroscope and ground with a 2 mm drill bit for DNA extraction. Genomic DNA was extracted using a NucleoSpin® 96 Tissue kit (Macherey-Nagel, GmbH and Co. KG, Germany) following the manufacturer’s protocol. For P. cetinaensis, type specimens, and historical collections, we employed the QIAamp® DNA Micro Kit (Qiagen S.A.S., France) following the manufacturer’s protocol for tissues. The plastid gene encoding the D1 protein of photosystem II (psbA) was amplified in one reaction using the pairs of primers psbA-F1/psbA-R2 or psbA-F1/psbA600R following the thermal profile. The PCR reaction mixture followed, except for the amplification of type specimens and historical collections for which the DNA template was not diluted. PCR products were purified and sequenced by Eurofins (Eurofins Scientific, France). Voucher specimens for P. cetinaensis and recent collections of Pneophyllum were deposited in the Muséum National d´Histoire Naturelle (PC), Natural History Museum Split (NHMS), Herbarium Croaticum - University of Zagreb (ZA), and the Croatian Natural History Museum (CNHM). Sequences were submitted to the Barcode of Life Data Systems (project “NGCOR”, BOLD, http://www.boldsystems.org and GenBank (accession numbers listed in online). For the molecular analyses, publicly available sequences of Pneophyllum were included, as well as sequences from other genera of the orders Corallinales, Hapalidiales, and Sporolithales (see online).Molecular analyses. Models of sequence evolution were estimated using the Akaike Information Criterion (AIC) and the Bayesian Information Criterion (BIC) obtained in jModeltest 2.1.3. Maximum Likelihood analysis for the psbA alignment was performed under a generalized time-reversible with gamma+invariant sites heterogeneity model (GTR+G+I), and the bootstrap consisted of 1,000 replicates. The psbA alignment comprised 44 haplotype sequences ranging from 376 to 851 bp, with 294 variable sites. The alignment did not include either the holotype fragment of P. fragile or the isolectotype of P. zonale, for which DNA sequences could not be obtained. […]

Pipeline specifications

Software tools BOLD, jModelTest
Applications Phylogenetics, WGS analysis
Chemicals Calcium Carbonate