Seawater samples were collected under both controlled conditions, and from natural field sites. The controlled site was a sea pen in a natural harbor basin at Fjord&Bælt (F&B) in Kerteminde, Denmark (). The pen holds four harbor porpoise in approximately 4 million liters of seawater, which is flushed daily by the tidal water movements in the harbor basin, that enter at the netted ends of the pen. Five 15 ml water samples were collected at a depth of approximately 50 cm from different points around the perimeter of the sea pen in a sterile container, which was sealed until just prior to sampling and handled using unused sterile latex gloves, which were discarded after the collection of each sample (). An additional 45 samples of 15 ml were collected at varying distances (~0–1 km) from the pen in the direction of the ebbing tide. After collection, 1.5 ml of 3 M sodium acetate and 33 ml absolute ethanol was added to the water samples to precipitate any extracellular DNA (final concentrations 0.09 M, and 66% respectively), which were then stored at −20°C until DNA extraction. As a control, a small layer of epidermis was also collected from each of the four porpoises using Scotch tape. Epidermal samples were stored in 20% dimethyl sulphoxide (DMSO) saturated with NaCl at −20°C until DNA extraction.
The sea pen at Fjord&Bælt in Kerteminde, Denmark.
Seawater samples were also collected during August 2011 at 8 locations in the western Baltic at sites where static acoustic monitoring devices, C-PODs (Cetacean and POrpoise Detector, Chelonia Ltd., U.K.), were deployed as part of the project Static Acoustic Monitoring of the Baltic Sea Harbor Porpoise (SAMBAH). The water samples were collected approximately 50 cm below the surface as above, employing the same protocols to reduce the risk of contamination. Three 50 ml samples were collected at each acoustic datalogger site and a 15 ml aliquot was taken from each of these samples and treated as above. The data recorded by the C-PODs were analyzed with the software CPOD.exe (v 2.021) with the filters ‘NBHF’ and ‘Other Cetaceans’ to search for porpoise clicks and clicks from other cetaceans, respectively. The percentage of porpoise positive days (i.e. days in which porpoise clicks were detected) were calculated for each site during the three month recording period prior to the date of water sampling (). No specific permits were required for the described field studies.
Detection of harbor porpoise DNA using qPCR at a controlled site (Fjord&Belt pen) and at natural sites.
DNA from seawater samples was extracted in a dedicated clean lab in a building separate from the location of post PCR work and extraction of DNA from the epidermis samples. Rigorous controls for preventing and monitoring contamination adopted from ancient DNA protocols were employed. Seawater samples were centrifuged at 6000 g for 10 min to pellet any precipitated DNA. One blank extraction using molecular biology grade water was included for every nine seawater samples to monitor possible contamination. Following centrifugation the supernatant was discarded and DNA was extracted from the pellet using the Qiagen DNeasy (Qiagen DNeasy, Valencia, CA, USA) kit following the manufacturer's guidelines and eluted in 100 µl of buffer. The ethanol wash step of the extraction process is expected to remove most of the Na+ monovalent ions and therefore reduce PCR inhibition due to salinity. DNA was also extracted from the epidermis samples using the Qiagen DNeasy kit.
The PrimerBlast software (NCBI, http://www.ncbi.nlm.nih.gov/tools/primer-blast/
) was used to design primers unique to the harbor porpoise that would result in amplicons ranging between 60–80 bp in size, based on records in GenBank. The primers 5′-CGCCCCATCAACACAAAGGTTTGG-3′
flanked the region corresponding to sites 82–119 of the harbor porpoise mitochondrial genome (GenBank: AJ554063; 
) and resulted in a 62 bp amplicon of the 12S region of the mitochondrial genome. This 38 bp intra-primer sequence was monomorphic in all four of the F&B harbor porpoise, as well as an additional four North Sea and Baltic harbor porpoises from the Natural History Museum of Denmark's tissue archive. Furthermore, the sequence differs by at least one base pair (either a C-A transversion or C-T transition), from the next most similar sequence present in GenBank, which was shared by several cetacean species, none of which are likely to occur at our study sites 
, and differed at 6 nucleotide sites from humans, and harbor and grey seals, which are the only other common marine mammals in the area, and thus represent possible sources of mammalian DNA in the water. To increase sensitivity and specificity of porpoise detection we performed TaqMan qPCRs detection assays. A TaqMan probe specific for the target sequence was designed (5′-TCCTGGCCTTTCTATTAGTTCTTAGCA-3′
), modified with 6-Fam dye at the 5′ end and a BHQ1 quencher at the 3′ end.
Taqman qPCRs were performed on a Stratagene Mx3000P and each 25 µl reaction contained 10 µl of DNA extract, 1× PCR buffer, 2.5 mM MgCl2, 1 mM of each primer, 0.1 mM mixed dNTPs, 2.5 µM of probe and 0.2 µl AmpliTaq Gold enzyme (Applied Biosystems) under thermocycling 50°C for 5 min and 95°C for 10 min, followed by 55 cycles of 95°C for 30 sec and 56°C for 1 min. To guard against the incorporation of erroneous data derived from contamination, and to investigate the stochasticity of successful amplifications, the PCR amplification was replicated three times for each sample. One PCR blank (containing ddH2O instead of sample) was included for every five PCRs to further monitor for contamination during PCR set up.
For initial investigation we pooled DNA extracts: DNA extracts from the five samples collected inside the F&B pen were pooled, DNA extracts from the five samples collected at less than 10 meters from the pen were pooled, and DNA extracts from thirty-eight samples collected at distances greater than 10 meters from the pen were pooled. Only if DNA was successfully amplified from at least one of the triplicate PCRs of the pooled extracts were the individual extracts included in subsequent PCRs. The amplified PCR products were purified using a Qiagen MinElute PCR purification kit. The species origin of positive PCRs were validated as authentic by cloning using the Topo TA cloning kit (Invitrogen), followed by purification and sequencing of the inserted PCR fragment (Macrogen, Europe). Additional PCRs of 43 bp of the cytochrome b gene (fwd primer: 5′-ACACACCCACTAATAAAAAT-3′; rev primer: 5′-AGCCAAAATTTCATCATGAGGA-3′) and the 53 bp of the hypervariable region of the d-loop (fwd primer: 5′-ACACATACCAATATCTAGTCTTTCCTT-3′; rev primer: 5′-CGGGCTTTAACTTATCGTATGG-3′) using the conditions above were conducted to investigate species identity in one sample, which did not match the porpoise reference sequence.