DOI: 10.5061/dryad.qfttdz0m3
title: Data from: Evaluating species richness using proteomic fingerprinting and DNA-barcoding – a case study on meiobenthic copepods from the Clarion Clipperton Fracture Zone

description: The Clarion Clipperton Fracture Zone (CCZ) is a vast deep-sea region
 harboring a highly diverse benthic fauna, which will be affected by
 potential future deep-sea mining of metal-rich polymetallic nodules.
 Despite the need for conservation plans and monitoring strategies in this
 context, the majority of taxonomic groups remains scientifically
 undescribed. However, molecular rapid assessment methods such as
 DNA-barcoding and Matrix-assisted laser desorption/ionization
 time-of-flight mass spectrometry (MALDI-TOF MS) provide the potential to
 accelerate specimen identification and biodiversity assessment
 significantly in the deep-sea areas. In this study, we successfully
 applied both methods to investigate the diversity of meiobenthic copepods
 in the eastern CCZ, including the first application of MALDI-TOF MS for
 the identification of these deep-sea organisms. Comparing several
 different species delimitation tools for both datasets, we found that
 biodiversity values were very similar, with Pielou’s Evenness varying
 between 0.97 and 0.99 in all datasets. Still, direct comparisons of
 species clusters revealed differences between all techniques and methods,
 which are likely caused by the high number of rare species being
 represented by only one specimen, despite our extensive dataset of more
 than 2000 specimens. Hence, we regard our study as a first approach toward
 setting up a reference library for mass spectrometry data of the CCZ in
 combination with DNA-barcodes. We conclude that proteome fingerprinting,
 as well as the more established DNA-barcoding, can be seen as a valuable
 tool for rapid biodiversity assessments in the future, even when no
 reference information is available.
description: Sediment sampling in the CCZ
 was conducted using a multicorer during the cruises MANGAN 2018 (SO262:,
 05/04 to 29/05/2018, Rühlemann et al., 2019), and MiningImpact2 (SO268/2:,
 30/03 to 22/05/2019, Haeckel and Linke, 2021), both on the German research
 vessel SONNE. The study area is located within the eastern part of the
 German contract area for the exploration of polymetallic nodules, which
 has been licensed by the German Federal Institute for Geosciences and
 Natural Resources (BGR) from the International Seabed Authority
 (ISA). Meiofauna was sampled using multicores with an
 (inner diameter of
 94-96 mm. Bottom water was
 sieved over a 32 µm sieve and fixed with 99.8% ethanol denatured with
 methyl ethyl ketone together with, in 2018, the upper 3 cm and in 2019 the
 upper 5 cm of sediment in a Kautex wide-neck bottle (1000 ml). All samples
 were re-fixed with the same fixative after 24 hours and stored at -20°C.
 To extract all meiofauna organisms from the sediment, samples were
 centrifuged according to the differential flotation method
 (Heip et al.,
 1985) with the colloidal
 gel Levasil®. Centrifuged samples were transferred into a Kautex wide-neck
 bottle (100 ml) and further stored at -20°C in the same fixative. All
 copepods were sorted out of the supernatant under a dissecting microscope.
 Prior to molecular processing, all individuals were photographed to
 document their basic morphology, and the ontogenetic stage was determined.
 Further processing was conducted according to
 two different protocols. In the first approach conducted on 58% of all
 available specimens, the individual was cut into two pieces. The posterior
 part was used for DNA-barcoding, while the anterior part was used for
 investigations with MALDI-TOF MS. In the second, enhanced protocol
 conducted on 42% of the specimens, the individuals were first prepared for
 MALDI-TOF MS and then washed with 10 µl molecular grade water before they
 were processed for DNA-barcoding, to increase biomass used for the MALDI
 measurements. The change of protocol only influenced the success rate of
 MALDI-TOF MS, but had no influence on the resulting DNA-barcode or the
 mass spectrum. Furthermore, the exuviae could be retained for potential
 morphological investigations in the future.
 The tissue was transferred
 with 5-µl ethanol into a 0.2 ml microcentrifuge tube. After the ethanol
 had evaporated at room temperature, 2.5-µl α-cyano-4-hydroxycinnamic acid
 (HCCA) was added and the tissue was incubated for at least 5 min.
 Thereafter, the extract with the HCCA was transferred to a metallic target
 plate and measured in a Microflex LT/SH System (Bruker Daltonics) using
 the method MBTAuto. Peak evaluation was carried out in a mass peak range
 between 2 k and 10 k Dalton (Da) using a centroid peak detection
 algorithm, a signal to noise threshold of 2 and a minimum intensity
 threshold of 600. To create a sum spectrum, 160 satisfactory shots were
 summed up. Raw spectra were imported to R and further
 processed using the R-packages MALDIquantForeign (Gibb, 2015) and MALDIquant (Gibb and Strimmer, Korbinian,
 2012). Spectra were
 square-root transformed, smoothed using the Savitzky Golay method
 (Savitzky and Golay,
 1964), baseline corrected
 using the Statistics-sensitive Non-linear Iterative Peak-clipping
 algorithm (SNIP)(Ryan et
 al., 1988) and spectra
 normalized using the Total Ion Current (TIC) method. Repeated measurements
 were averaged by using mean intensities.
description: Data was processed in R. Mass spectrometry data can be imported
 to R using the Package MaldiQuantForeign and further be processed using
 the package MaldiQuant. Gibb, S. (2015). MALDIquantForeign:
 Import/Export routines for MALDIquant. A package for R.
 https://CRAN.R-project.org/package=MALDIquantForeign Gibb, S., and Strimmer, Korbinian (2012). MALDIquant: Quantitative Analysis of Mass Spectrometry Data. <em>Bioinformatics</em> 28, 2270--2271. doi: 10.1093/bioinformatics/bts447.