by Timothy Culwick
A new method of identifying old sponge specimens using DNA mini-barcoding.
Sponges are some of the most diverse and varied of the aquatic invertebrates with about 7,000 species described so far (Hooper 2002). Their success and morphological variability results in major problems with specimen identification. Due to similarities in hand specimen features a microscope is often needed to apply the appropriate Order let alone species separation. This difficulty is compounded in deep marine environments where much of the data come from camera drop images or broken fragments from trawling.
In Marine Biodiversity, Cardenas and Moore (2017) describe a new Geodia location on New England sea mounts and test a new way of specimen identification using mini-barcodes.
In the North Atlantic there are large sponge grounds on the slopes and shelves with most of the biomass stemming from the Geodiidea and Ancorinidae families. The grounds are both potentially ecological and environmentally important due to their size and extent. Geodiidea form the largest of the sponges in these grounds with single sponges getting up to 1m in diameter. Whilst comparatively easy to identify from large undamaged specimens due to its external morphology, broken or small specimens are much harder to separate. The damaged and diminutive species currently rely on spicule morphology which is difficult for non-specialise.
DNA Barcodes for Geodia species in the North Atlantic have been studied before (Cardenas 2013). They looked at the Folmer cytochrome oxidase subunit I (COI), 28S and 18S. of which both 28S and COI were unique for each species. Although due to slow evolution, identification of close species is limited (Schuster 2017). 18S was reported to be unique for four species but identical for two others. 18S has been previously noted to be conservative and therefore not useful for barcoding. However, this study relies on material to have no degradation of the DNA. Here we run into a problem with whole barcode sequencing. There is a limited number of specimens due to the challenge of collection at the depth and many were collected before DNA sequencing was possible. This is particularly true for most holotypes. These specimens were, and most still are, preserved using methods that degrade DNA making this type of analysis impossible.
The new specimens described by Carden and Moore (2017) were fixed in formalin which breaks down DNA so were unable to obtain a full-length barcode. They tried extracting two mini-barcodes the universal mini-barcode (=first 130bp of the Folmer barcode) and the Depressio-mini-barcode (= last 296bp of the Folmer barcode). Both barcodes hold the advantage of using an established barcoding database, so time and effort is not required to create one. The universal mini-barcode was obtained for five out of eight specimens. As with the full barcode it could not distinguish close species but importantly they all unambiguously identified the Geodia genus. This level of identification although superficially arbitrary for an organism within this phylum this is very helpful. The Depressio-mini-barcode was as sensitive as the full-length barcode with these specimens. However, it has not been used as a tool for identification on other sponges so should be treated cautiously. The potential sensitively suggested from this study is very striking and would be of great interest to look at further.
The most interesting part of this paper is what they went on to do next: they tried this mini-barcoding approach on two old specimens which would usually be considered unusable for DNA analysis. The first was a 11-14yr formalin fixed specimen from which they retrieved 100-300bp sequences. The second was a 161yr old dry holotype where two mini-barcodes were obtained. These hints of a method to open up the large proportion of collections to potential analysis which would otherwise be unusable. However, only two old specimens were tested and only one of the mini-barcodes can be considered reliable at this time. I don’t think the authors when far enough with this avenue of the paper to extrapolate the value of this technique as far as they have. There is great potential to increase the reliability of the sponge barcoding data and to look at many phylogenetic and taxonomic questions (Erpenbeck 2016). It would have been interesting to have tried with multiple samples to give an idea of the reliability and frequency of which results can be obtained. This is an interesting new avenue of DNA sequencing and could yield valuable results, but it needs further work to become the staple tool that is suggested.
Cardenas, P., & Moore, J, (2017), First records of Geodia demosponges from the New England seamounts, an opportunity to test the use of DNA mini-barcodes on museum specimens: Marine Biodiversity, pp 1–12.
Cardenas, P., H. T. Rapp, A. B. Klitgaard, M. Best, M. Thollesson, and O. S. Tendal, (2013), Taxonomy, biogeography and DNA barcodes of Geodia species (Porifera, Demospongiae, Tetractinellida) in the Atlantic boreo-arctic region: Zoological Journal of the Linnean Society, v. 169, p. 251-311.
Erpenbeck, D., M. Ekins, N. Enghuber, J. N. A. Hooper, H. Lehnert, A. Poliseno, A. Schuster, E. Setiawan, N. J. De Voogd, G. Woerheide, and R. W. M. Van Soest, (2016), Nothing in (sponge) biology makes sense – except when based on holotypes: Journal of the Marine Biological Association of the United Kingdom, v. 96, p. 305-311.
Hooper, J., & van Soest, R., (2002), Systema Porifera: A Guide to the Classification of Sponges.
Schuster, A., J. V. Lopez, L. E. Becking, M. Kelly, S. A. Pomponi, G. Woerheide, D. Erpenbeck, and P. Cardenas, (2017), Evolution of group I introns in Porifera: new evidence for intron mobility and implications for DNA barcoding: Bmc Evolutionary Biology, v. 17.