Applications of PCR-RFLPs for differentiating two freshwater sponges: Ephydatia fluviatilis and Ephydatia mülleri

Abstract

The two freshwater sponges Ephydatia fluviatilis and Ephydatia mu¨lleri belong to the widespread Spongillidae family. Their morphological tracts are very similar and can be distinguished mainly on the basis of their gemmuloscleres. However, when gemmules are absent it is essential to have an unambiguous species attribution for a population genetic study based on fresh tissues and historical collections. This article reports a simple Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, applied to DNA extracted from both gemmules and fresh tissues in order to discriminate between the two congeneric E. fluviatilis and E. mu¨lleri. Such a biomolecular method is based on the discriminative enzymes' digestion of each of the three amplified fragments 5.8S-ITS2-28S, D3 domain of the 28S subunit and COI. Two restriction enzymes were tested for a 620-642 bp fragment of 5.8S-ITS2-28S and for a 342 bp fragment of the D3 domain of the 28S, one restriction enzyme was tested for a 681 bp fragment codifying for COI. Obtained digestion patterns were diagnostic for each of the two species, providing a relatively simple, fast and cheap method for species attribution compared to sequencing.

Intragenomic Profiling Using Multicopy Genes: The rDNA Internal Transcribed Spacer Sequences of the Freshwater Sponge Ephydatia fluviatilis

“…Both of the specimens from Japan contained the same deletions we had detected and 3 additional indel and 4 substitution positions. Gigliarelli et al [20] described one substitution in ITS2, Position 263 (showing both A and G); by our annotation this position is 24 th in the beginning of 28S RNA gene. The authors did not specify the number of clones with either of those substitutions.…”

“…For ITS2, the existence of different length variants in E. fluviatilis has been found in individuals separated by a long geographical distance: from Italy [20], the length variant of 324 nt (323 nt by our annotation) and from Japan [19], the length variants of 321 and 322 nt by our annotation (289 nt by their own annotation). The specimens collected from Estonia had their ITS2 lengths from 321 to 323 nt.…”

Spatially explicit genetic structure in the freshwater sponge Ephydatia fluviatilis (Linnaeus, 1759) within the framework of the monopolisation hypothesis

“…First, using a method based on the polymerase chain reaction-restriction fragment length polymorphism (PcrrFLP) approach (Gigliarelli et al, 2008), we confirmed the taxonomic identification of all the samples, in order to exclude apparent genetic variability belonging to a closely related and very similar species -E. mülleri (Lieberkühn, 1856). then, only for samples unambiguously confirmed as E. fluviatilis, we proceeded with the genetic characterisation of rdna, mtdna and microsatellites.…”

“…the 5.8S-itS2-28S and d3/28S fragments were amplified as reported in Gigliarelli et al (2008). For coi, we amplified two overlapping regions: one was the traditional Folmer region, amplified following Gigliarelli et al (2008); the other was the i3-M11 partition (erpenbeck et al, 2006), obtained thanks to specifically designed primers.…”

“…the 5.8S-itS2-28S and d3/28S fragments were amplified as reported in Gigliarelli et al (2008). For coi, we amplified two overlapping regions: one was the traditional Folmer region, amplified following Gigliarelli et al (2008); the other was the i3-M11 partition (erpenbeck et al, 2006), obtained thanks to specifically designed primers. these were designed from the alignment of three complete poriferan coi sequences available from Genbank: E. mülleri nc010202, Lubomirskia baicalensis (Pallas, 1773) nc013760, and Tethia actinia (de Laubenfels, 1950) nc006991 (Lavrov et al, 2008, Lavrov, 2010.…”