The CRMM (Centre de Recherche sur les Mammifères Marins, La Rochelle, France), presently the Joint Service Unit PELAGIS, UMS 3462, University of La Rochelle-CNRS has created the French marine mammal stranding recording program at the beginning of the 70s. The network comprises about 260 field correspondents, members of organizations or volunteers (Peltier et al. 2013).

Since 1995, the LEMM (Laboratoire d’Etude des Mammifères Marins, Océanopolis, Brest, France) has coordinated this network at a regional scale in Brittany, North West of France. Data are collected from the Brittany coastlines, analyzed, and then added to the central database maintained in La Rochelle. The Brittany coasts have been divided into 18 sections covering the whole coastline (Jung et al. 2009). In each of these areas, correspondents are trained in the analysis of stranded marine mammals. Taxonomic identification and characteristic measurements are performed following a standard procedure. The LEMM therefore compiles standardized data on a large proportion of cetaceans stranded on the Brittany coasts on a yearly basis. Whenever possible, skin, blubber, muscle and teeth samples are also collected in the field from each stranded animal. Samples are then kept in absolute ethanol or dry at -20 °C until analyses. Some harbour porpoise samples, described in the Appendix 1 and in Alfonsi et al. (2012), were stranded or by-caught in the Bay of Biscay (Atlantic coast of France).

Genomic DNA was extracted from blood samples or from muscle and skin tissues using a standardized protocol and the DNeasy Blood and Tissue kit (Qiagen), following the instructions of the manufacturer. The quality and the concentration of all the DNA extracts were estimated by agarose gel electrophoresis and by spectrophotometry using a Nanodrop 1000 (Thermo Scientific).

A 736 base-pair (bp) fragment of the 5’ region of the COI fragment (position 5352 to 6087 of the complete mitochondrial genome of the harbour porpoise, GenBank acc. no. AJ554063), was amplified using two newly designed primers, LCOIea (5’-tcggccattttacctatgttcata-3’) and HBCUem (5’-ggtggccgaagaatcagaata-3’). The 50 µl PCR final volume included approximately 50 ng of genomic DNA, and 25 pmole of each primer in the Hotgoldstar master mix x 1 (Eurogentec) with a final concentration of MgCl₂ of 2.5 mM. After an initial denaturation step of 10 min at 95 °C, the thermocycle profile consisted of 32 cycles for cetaceans or 35 cycles for pinnipeds at 95 °C for 30 s, 53 °C for 30 s and 72 °C for 60 s, with a final extension at 72 °C for 10 min.

For some animals, we also amplified and sequenced another part of the mitochondrial genome including the control region (MCR). For cetaceans, the primers and reaction conditions are described in (Alfonsi et al. 2012). For pinnipeds, two newly designed primers LMCRHgem 5’-tcatacccattgccagcattat-3’ and HMCRHgem 5’-taccaaatgcatgacaccacag-3’ amplified a 693 bp fragment from position 16160 to 55 of the Halichoerus grypus complete mitochondrial genome sequence (GenBank acc. no. X72004). PCR reaction conditions were the same as described above for pinnipeds, with the hybridization temperature set to 53 °C. PCR products were purified using the “MinElute PCR Purification Kit” and sequenced by a commercial sequence facility (Macrogen, Korea).

Electropherograms were analyzed and edited manually using the Sequence scanner software (Applied Biosystems), and alignments were produced using CLUSTAL W (Thompson et al. 1994) with default settings in Bioedit (Hall 1999). All sequences were analyzed using the Barcode of Life Data Systems (BOLD) interface (accessible at http://www.boldsystems.org), and were also compared to GenBank data using BLAST (Benson et al. 2010).

DNA sequences and specimen information have been added to two BOLD projects. The first project includes specimens for which the species had been identified without doubt using classical morphological identification, and is referred to as IMMB (Identified Marine Mammals in Brittany). The IMMB project is a part of the campaign “barcoding mammals of the world”. The second project, UMMB (Unidentified Marine Mammals in Brittany), includes specimens only identified to the genus or to higher taxonomic levels. This second project is a part of the campaign “barcoding application”.

Genetic distances (intraspecific, interspecific and minimal distance to the nearest neighbour) were calculated using the Kimura 2-parameter (K2P) model (Kimura 1980) and the MUSCLE alignment algorithm on the BOLD user interface or using the software MEGA5 (Tamura et al. 2011). Neighbour-Joining trees based on the K2P-model were built using the BOLD user interface. DnaSP v5.10 was used to calculate haplotype and nucleotide diversities (Librado and Rozas 2009). We used non-metric multidimensional scaling (nMDS) to represent MCR distances graphically and to discriminate closely related species within the Stenella-Tursiops-Delphinus complex (LeDuc et al. 1999, McGowen 2011, Perrin et al. 2013). Distance matrices were computed with the K2P-model using DNAdist (Felsenstein 1989) and were then analyzed by nMDS using Statistica (Statsoft 2005). Posterior probabilities were calculated by a LDA (linear discriminant analysis) on coordinates given by the nMDS. Phylogenetic relationships among COI sequences of harbour porpoise were depicted using a median joining network of haplotypes using Network v4.6 (www.fluxus-engineering.com).

DNA was extracted from 92 stranded animals, i.e. from dead cetaceans and pinnipeds, but also from 40 grey seals stranded alive, which were treated in the care center of Océanopolis (Brest, France) and from which a small blood sample was taken and kept at -20 °C. All the samples came from animals stranded at the coasts of Brittany, except for one grey seal (Hgc406), which stranded alive in Spain in 2009 and which was transported to the care center (Figure 2). Our sampling included 12 species of cetaceans, and three species of pinnipeds (Table 2). Two species were very common, the harbour porpoise (29 samples) and the grey seal (44 samples), thus allowing intraspecific distance analyses.

A COI amplicon was recovered from 89 samples, and good quality sequences of more than 500 bp were obtained for all samples (GenBank accession numbers KF281608–KF281697). The sequence alignment used in the analyses was 507 bp long. About 32% of the positions were polymorphic in the cetaceans and 13.1% in the pinnipeds (Table 3). The maximal intraspecific distance was 0.46% for the grey seal and 0.83% for the harbour porpoise. The COI sequences of three species of the Delphininae (Stenella frontalis, Stenella coeruleoalba and Delphinus delphis) showed very low interspecific distances (0.84% between Delphinus delphis and the nearest species Stenella frontalis, and 1.18% between the two Stenella species). All other interspecific distances were above 3.9% for pinnipeds and above 6% for cetaceans. The Neighbour-Joining (NJ) tree built on the BOLD interface using K2P-distances (Figure 3) confirms that, except for of the Delphininae, all the cetacean and pinniped species analyzed are distinguished unambiguously.

We then determined COI sequences from 10 cetacean samples whose species could not be determined accurately using morphological characters (Figure 4), either because only parts of the animal were recovered (Figure 4A) or because of the highly degraded state of the carcasses (Figure 4C). COI sequences of good qualities were obtained from all these samples, and three of them were identified unambiguously using the BOLD identification engine: Ms250511 was identified as a Balaenoptera physalus, Ds160111 as a Grampus griseus and Ds290811 as a Phocoena phocoena. The other seven samples were Delphininae, as confirmed by COI sequences. Yet, neither the BOLD identification engine, nor a BLAST search on GenBank allowed a more precise determination. We therefore sequenced MCR, which is more variable than COI, from six unidentified samples. BLAST searches on GenBank confirmed the COI results: all these samples were Delphininae, but a more precise identification could not be achieved.

We constructed a nMDS plot of the distances between MCR sequences of Stenella coeruleoalba, Stenella frontalis and Delphinus delphis taken from GenBank: for Stenella coeruleoalba, we used sequences AM498725, AM498723, AM498721, AM498719, AM498717, AM498715, AM498713, AM498711, AM498709, AM498707 (Mace et al. unpublished), for Delphinus delphis FM211560, FM211553, FM211545, FM211535, FM211527, FM211519, FM211511, FM211503, FM211495 (Mirimin et al. 2009) and DQ520121, DQ520117, DQ520113, DQ520109, DQ520105 (Hildebrandt et al. unpublished) and for Stenella frontalis GQ5041986, GQ5041987, GQ5041988, GQ5041989, GQ5041990, GQ5041991, GQ5041992, GQ5041993, GQ5041994, GQ5041995 (Kingston et al. 2009).

The three species were clearly discriminated by the nMDS (Figure 5). The posterior probabilities are given in Appendix 2. This analysis suggests that five of our unidentified samples could belong to Delphinus delphis, and one to Stenella coeruleoalba.

For the intraspecific analysis of the harbour porpoise, we included 17 additional samples of animals stranded or by-caught from the Bay of Biscay (Appendix 1, Alfonsi et al. 2012). All in all, we compared 35 sequences of grey seals, and 45 of harbour porpoises. As expected, MCR sequences were more polymorphic than COI: in harbour porpoise, 3.8% of the MCR positions were polymorphic vs. 1.30% in COI, while 4.73% of the MCR positions in the grey seal were polymorphic vs. 0.75% in COI (Table 4). Hence, MCR was 3× more polymorphic than COI in harbour porpoise and 6x in grey seals. Haplotype and nucleotide diversities were also higher for MCR than for COI.

The haplotype network of the COI sequences in harbour porpoises clearly differentiated two haplogroups (Figure 6), that correspond perfectly to those described for MCR in Alfonsi et al. (2012).

We obtained DNA sequences of good quality for almost all the samples studied, whatever their origin, their collectors, or even their state of degradation. This is consistent with the numerous molecular genetic studies that have used samples taken on stranded cetaceans or pinnipeds (e.g. Gaspari et al. 2006, Amaral et al. 2007, Fontaine et al. 2007, Mirimin et al. 2009, 2011, Alfonsi et al. 2012).

Viricel and Rosel (2012) previously demonstrated that COI sequences allowed identifying cetacean species, except for a few closely related Delphinidae species (see also Amaral et al. 2007). As expected, our NJ tree matched the overall classification, and the distance-based analysis identified correctly the sequences to the species levels for all cetaceans except within the Delphininae. The three species of pinnipeds analyzed were also unambiguously distinguished on the basis of their COI sequences.

The quality of the whole functioning and organization of the stranding network, from the field-work achieved by the correspondents to the preservation of the samples is therefore confirmed by our study. All the samples analyzed by DNA barcoding led to correct identification of the expected species with no exceptions.

We obtained COI good quality sequences for 10 unidentified animals, some of which were in a highly degraded body state. This showed that DNA barcoding can help to identify such specimens, which represent more than 16% of the stranded animals in the period 2003–2012. Hence, a routine use of DNA barcoding would noticeably decrease the proportion of unidentified animals.

Within the Delphininae, species are difficult to discriminate (Amaral et al. 2007, 2012, Viricel and Rosel 2012). In particular, Delphinus delphis, Stenella coeruleoalba and Stenella frontalis show very low interspecific COI distances, which do not allow distinguishing the species accurately. Other mitochondrial loci, such as MCR and cyt b, are neither very effective in this matter (Amaral et al. 2007, Viricel and Rosel 2012). This is attributed to recent and rapid radiation events in the subfamily, and it leads to problematic results in molecular taxonomic studies (Kingston et al. 2009, Amaral et al. 2012, Viricel and Rosel 2012, Perrin 2013). In our case, these three species produced COI and MCR sequences that did not allow to associate samples with species names, neither with the identification engine on BOLD, nor with a distance tree or a BLAST search on GenBank. nMDS of genetic distances is known to uncover sample clustering (e.g. Geffen et al. 2004, Maltagliati et al. 2006, Alfonsi et al. 2012, Weckworth et al. 2012). As such, nMDS clustering of MCR sequence distances of Delphinus delphis, Stenella coeruleoalba and Stenella frontalis, chosen randomly on GenBank among Atlantic samples, showed that individuals of the three species formed separate groups. Moreover, each individual had a high posterior probability to belong to the right group, except for one sample (i.e. 97.0% of the assignments were successful), so that all our unidentified samples could be putatively identified to the species level, based on the nMDS plot and its posterior probabilities.

DNA barcoding is informative for animals that belong to species that infrequently strand along the coasts of Brittany, which can involve either species living far off the coasts or living in deep water, but also exotic species. Such species can be more difficult to identify by the field correspondents simply because of their scarcity. Along the coast of Brittany, we observed a Stenella frontalis, a temperate to tropical Atlantic Ocean inhabitant, and three species of arctic seals (Phoca hispida, Cystophora cristata and Phoca groenlandica). It is likely that other members of such rare species are listed among the “undetermined” species, just because their morphological characteristics are less well known by field correspondents. Additionally, a species that rarely strands along the French coast may be mistakenly identified as its more common sister-species. This issue can be illustrated by the case of the two pilot whale species: Globicephala melas, the long-finned pilot whale, commonly strands along the French Atlantic coast, while only a few stranding events of Globicephala macrorhynchus, the short-finned pilot whale, have been reported (the Bay of Biscay is the northern limit of the geographical range of Globicephala macrorhynchus). The two species have overlaping morphological characters, which adds to the difficulty of detecting rare stranding events of Globicephala macrorhynchus based on morphological data only (Viricel and Sabatier unpublished data). A systematic use of DNA barcoding when morphological taxonomic characteristics are not straightforward, would clearly lower the percentage of exotic animals not listed. The existence of natural interspecific hybrids between the two Globicephala sister-species (Miralles et al. 2013), as between other cetacean species (e.g. Bérubé and Aguilar 1998, Willis et al. 2004) still reinforces the interest of such a monitoring based on molecular data.

It is important to note that a main limitation of DNA barcoding is the use of a single locus, leading to some problematic species identification such as within the Delphininae, but also to an inability to detect hybrids without complementary genetic studies. This limitation may well be removed in the near future thanks to next-generation sequencing, allowing the accumulation of large amount of DNA sequence data in a cost-effective manner. Multi-locus barcoding, including mitochondrial and nuclear polymorphic loci, will certainly represent a next step for the barcoding community.

A routine use of DNA barcoding could also allow monitoring the marine mammal biodiversity at intraspecific levels. For instance, global climate change has some effects on genetic diversity that must be studied and quantified (Pauls et al. 2012), in particular in the marine realm. Knowledge of the existence of distinct genetic groups or populations, of the history of their formation and of their movements are of a first importance to ecological understandings of natural populations, and also to the conservation efforts dedicated to them. Around the coast of Brittany, different species of marine mammals have shown variations in abundance in the last decades (Vincent et al. 2005, Jung et al. 2009). Using samples from the French Stranding Network and MCR polymorphisms, we have recently shown that two previously separated, genetically distinct, populations of harbour porpoises are now admixing along the Atlantic coast of France (Alfonsi et al. 2012). These results were unexpected according to previous work (Tolley and Rosel 2006, Fontaine et al. 2007). In this study, we show that this genetic clustering would also have been detected using COI polymorphisms, thus reinforcing the interest of a routine use of DNA barcoding in conjunction with the stranding network.

This project is part of the collaboration between the Laboratory BioGeMME of the “Université de Bretagne Occidentale” (Brest, France), Océanopolis, a public private company (http://www.oceanopolis.com), the “Parc naturel marin d’Iroise” (http://www.parc-marin-iroise.com) and the French Stranding Network, coordinated by Pelagis, Université de La Rochelle, France. All the specimens and sequence data described in this manuscript are deposited in BOLD under the institution called “Oceanopolis-BioGeMME” in two projects, UMMB and IMMB. Our mixed institution became the first contributor to BOLD for the Cetacea, as well as for the Phocidae, and these two BOLD projects will be publicly available, and all the sequences published on GenBank.