28S rDNA C-Region:
The 28S C-DNA is universally suitable for all sponge classes (including calcareous sponges) and provides a higher resolution and easier amplification [1,2] with the current barcoding primers designed by Chombard et al. 1998 : Forward:
D2: 5′-TCCGTGTTTCAAGACGGG-3′Fig. 1: 28S rDNA secondary structure, the C-Region is highlighted in yellow. Modified after .
CO1 standard barcoding fragment
The standard barcoding fragment for the Barcoding of Life initiative is a approx. 640 bp long fragment located at the 5' site of the mitochondrial cytochrome oxidase subunit 1 (CO1). CO1 in Calcarea, however canot be amplified with standardized primers due to their high mitochondrial substitution rates . For all other sponge classes CO1 is best amplified with the degenerated (standard) CO1 barcoding primer designed by Meyer et al. :Forward:
dgLCO1490: 5′-GGT CAA CAA ATC ATA AAG AYA TYG GReverse:
dgHCO2198: 5′-TAA ACT TCA GGG TGA CCA AAR AAY CAFig. 2: Schematic view of the CO1 standard barcoding region (Folmer et al. 1994 ), and the extension region ("I3M11", Erpenbeck et al. ) with the mentioned primers .
CO1 extension region (I3M11)
The standard barcoding fragment might not display sufficient variability in sponges to species level . An additional downstream region (I3M11) exhibits a higher substitution rate suitable for population studies. The following primers for the extended fragment are designed by Misof et al.  and Erpenbeck et al. :Forward:
CO1porF1: 5′-CCN CAN TTN KCN GMN AAA AAA CA-3′ Reverse:
CO1porR1: 5′-AAN TGN TGN GGR AAR AAN G-3′ Forward:
C1Npor2760: 5′-TCT AGG TAA TCC AGC TAA ACC-3′ Reverse:
C1J2165: 5′-GAA GTT TAT ATT TTA ATT TTA CCN GG-3′ NOTE:
We suggest a nested approach in which a C1J2165/C1Npor2760 PCR-product will be reamplified by CO1porF1/CO1porR1. See  for details.
A further primer set is developed by Rot et al. , see Fig. 2. Forward:
COX1-R1: 5′-TGT TGR GGG AAA AAR GTT AAA TT-3′Reverse:
COX1-D2: 5′-AAT ACT GCT TTT TTT GAT CCT GCC GG-3′References:
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 Erpenbeck D, Voigt O, Al-Aidaroos AM, et al (2016) Molecular biodiversity of Red Sea demosponges. Mar Pollut Bull 105:507–514.
 Chombard C, Boury-Esnault N, Tillier S (1998) Reassessment of homology of morphological characters in tetractinellid sponges based on molecular data. Syst Biol 47:351–366.
 Lavrov D, Pett W, Voigt O, et al (2013) Mitochondrial DNA of Clathrina clathrus (Calcarea, Calcinea): six linear chromosomes, fragmented rRNAs, tRNA editing, and a novel genetic code. Mol Biol Evol 30:865–880.
 Meyer CP, Geller JB & Paulay G (2005) Fine scale endemism on coral reefs: Archipelagic differentiation in turbinid gastropods. Evolution 59: 113-125.
 Folmer O, Black M, Hoeh W, Lutz R & Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome C oxidase subunit I from diverse metazoan invertebrates. :. Mol. Mar. Biol. Biotechnol. 3: 294-299.
 Erpenbeck D, Hooper JNA, Wörheide G (2006) CO1 phylogenies in diploblasts and the “Barcoding of Life” - are we sequencing a suboptimal partition? Mol Ecol Notes 6:550–553.
 Misof B, Erpenbeck D & Sauer KP (2000) Mitochondrial gene fragments suggest paraphyly of the genus Panorpa (Mecoptera, Panorpidae). Molecular Phylogenetics and Evolution 17: 76-84.
 Erpenbeck D, Knowlton AL, Talbot SL, Highsmith RC & Van Soest RWM (2003 (2004)) A molecular comparison of Alaskan and North East Atlantic Halichondria panicea (Pallas 1766) (Porifera: Demospongiae) populations. Boll. Mus. Ist. Univ. Genova. 68: 319-325.
 Rot C, Goldfarb I, Ilan M & Huchon D (2006) Putative cross-kingdom horizontal gene transfer in sponge (Porifera) mitochondria. BMC Evolutionary Biology 6.