NOVEL UNIVERSAL PRIMERS for METABARCODING Edna SURVEYS of MARINE MAMMALS and OTHER MARINE VERTEBRATES

bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Elena Valsecchi1, Jonas Bylemans2, Simon J. Goodman3, Roberto Lombardi1, Ian Carr4, Laura Castellano5, Andrea Galimberti6, Paolo Galli1,7

NOVEL UNIVERSAL PRIMERS FOR METABARCODING eDNA SURVEYS OF MARINE MAMMALS AND OTHER MARINE VERTEBRATES

1 Department of Environmental and Earth Sciences, University of Milano-Bicocca, Piazza della Scienza 1, 20126 Milan, Italy 2 Fondazione Edmund Mach, Via E. Mach 1, 38010 S. Michele all'Adige (TN), Italy 3 School of Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, United Kingdom 4 Leeds Institute of Molecular Medicine, St James’s University Hospital, Leeds LS9 7TF, United Kingdom 5 Acquario di Genova, Costa Edutainment SPA, Area Porto Antico, Ponte Spinola, 16128 Genoa, Italy 6 Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy 7 MaRHE Center, Magoodhoo Island, Faafu Atoll, Republic of Maldives

Corresponding author: [email protected] ORCID ID 0000.0003.3869.6413

Key words: 12S, 16S, cetaceans, pinnipeds, fish, sea turtles

Running title: Marine Vertebrate Universal Markers for eDNA Metabarcoding bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

ACKNOWLEDGEMENTS We thank Fulvio Maffucci for providing marine turtles DNAa samples. Giudo Gnone of the Aquarium of Genoa for allowing and supporting collection of controlled environmental eDNA samples. Antonia Bruno for advises on filtering procedures of environmental samples and Anna Sandionigi for bioinformatic advice.

AUTHOR CONTRIBUTIONS E.V. designed primer sets, planned the testing approach compiled the marine mammal guideline to the primers’ use and wrote the manuscript; J.B. performed the in silico validation of the novel primer sets; R.L. carried out samples collection, wet lab tests, eDNA amplifications and analysed controlled environment HTS data; S.G. contributed to the design and implementation of the wet lab primer validation, provided support and facilities for HTS analysis at UoL, and contributed to the drafting and editing of the manuscript; I.C. provided HTS services and bioinformatics support; L.C. allowed and supported collection of water samples from tanks of the Aquarium of Genoa structure; A.G. provided useful comments on manuscript; P.G. enthusiastically supported and hosted the research study at UnMB. bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

ABSTRACT

Metabarcoding studies using environmental DNA (eDNA) and high throughput sequencing (HTS) are rapidly becoming an important tool for assessing and monitoring marine biodiversity, detecting invasive species, and supporting basic ecological research. Several barcode loci targeting teleost fish and elasmobranchs have previously been developed, but to date primer sets focusing on other marine megafauna, such as marine mammals have received less attention. Similarly, there have been few attempts to identify potentially ‘universal’ barcode loci which may be informative across multiple marine vertebrate Orders. Here we describe the design and validation of four new sets of primers targeting hypervariable regions of the vertebrate mitochondrial 12S and 16S rRNA genes, which have conserved priming sites across virtually all cetaceans, pinnipeds, elasmobranchs, boney fish, sea turtles and birds, and amplify fragments with consistently high levels of taxonomically diagnostic sequence variation. ‘In silico’ validation using the OBITOOLS software showed our new barcode loci outperformed most existing vertebrate barcode loci for taxon detection and resolution. We also evaluated sequence diversity and taxonomic resolution of the new barcode loci in 680 complete marine mammal mitochondrial genomes demonstrating that they are effective at resolving amplicons for most taxa to the species level. Finally, we evaluated the performance of the primer sets with eDNA samples from aquarium communities with known species composition. These new primers will potentially allow surveys of complete marine vertebrate communities in single HTS metabarcoding assessments, simplifying workflows, reducing costs, and increasing accessibility to a wider range of investigators. bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

INTRODUCTION

The use of DNA fragments extracted from environmental sources (e.g. soil and water samples) is becoming a well-established tool for monitoring biodiversity (Deiner et al. 2017; Jarman et al. 2018). Within the marine environment, such eDNA surveys have been used to assess the diet of marine species (Deagle et al. 2010, Peters et al.2015, McInnes et al. 2017), monitor the species diversity of marine communities (Port et al. 2016; Sigsgaard et al. 2017), determine the presence/absence of invasive species (Borrell et al. 2017) and to obtain estimates of population genetic diversity (Sigsgaard et al. 2016). Community biodiversity surveys from eDNA (i.e. eDNA metabarcoding) rely on primers targeting specific taxonomic groups and high-throughput sequencing to amplify and sequence barcoding regions from all species of interest (Creer et al. 2016). While DNA metabarcoding primers have been developed to target several individual marine taxonomic groups (e.g. elasmobranchs, teleost fish, cephalopods and crustaceans) (e.g. Jarman et al. 2016, Miya et al. 2015, Komai et al. 2019, Bylemans et al. 2018), to date no primer sets have been specifically designed to maximize the recovery and identification of marine mammals.

The few marine eDNA studies focussing on marine mammals used targeted species-specific assays (Foote et al. 2012, Baker et al. 2018) or used universal fish specific primers as a proxy to assess the total vertebrate biodiversity (e.g. Andruszkiewicz et al. 2017). All these approaches present at least one drawback when aiming to detecting the presence of cetacean and pinniped species within an eDNA sample. Primer sets designed for one group, i.e. ‘fish-specific’ primers, might amplify eDNA from other taxa, but unquantified primer mismatches risk reduced detection rates and the introduction of biases in high throughput sequencing (HTS) results (e.g. Elbrecht et al 2018). Where primers are designed for the species of interest, amplicon target size may also be a consideration. The instability and short life span of eDNA molecules (e.g. Thomsen et al. 2012), favours the use of short and informative (hypervariable) DNA regions, such as the mitochondrial 12S, 16S and cytochrome oxidase I (COI) genes, with amplicons of typically around 100-200 bp although larger mtDNA fragments have been shown to be successfully amplified from eDNA samples (Deiner et al. 2017).

There is therefore a need to develop marine mammal-specific primers suitable for metabarcoding analysis from marine eDNA samples. Mitochondrial 12S and 16S regions provide suitable targets since their sequence variation provides good taxonomic resolution for macro-eukaryotes, while also maintaining conserved sites across regions for siting primers (Deagle et al. 2014). The design criteria for such primers should be, a) primer sites which are conserved among marine mammal groups, while amplifying hypervariable DNA fragments for taxonomic resolution; b) where possible, identify marine mammal specific priming sites in order to reduce cross-amplification with human DNA, thus lessening contamination risks from investigators, swimmers, or other biological residues left by humans; c) for each primer set evaluate predicted binding efficiency and amplicon sequence diversity for other marine vertebrates such as fish and sea turtles (when necessary allowing for a single degenerate base per primer). This final point would provide to give a more accurate understanding of primer specificity and suitability for use with other vertebrate groups. Primer sets proven to have reliable affinity across multiple vertebrate Orders and could support more efficient and cost- effective eDNA biodiversity surveys. bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

In this paper we report on the development of novel “universal” marine vertebrate eDNA primers meeting the above criteria, their ‘in silico’ validation against a large marine vertebrate NCBI-Genbank dataset, and their successful initial application and validation with a high throughput sequencing analysis of environmental water samples collected from a public marine aquarium. Finally, we test for inter- and intra-specific variation over large mitogenomic data sets available for marine mammal species, presenting ready-to-use guidelines for the accurate selection of primer set of choice in specific marine mammal studies.

MATERIALS AND METHODS

Initial design of primer sets Seventy-one complete mitochondrial genome sequences, representative of most marine vertebrate groups (fish, sea turtles, birds and marine mammals), were retrieved from GenBank and used for initial primer development (Online Resource 1 in Supplementary Materials). The selected sequences represented 30 marine vertebrate families, including most marine mammal families (all 3 Pinniped families, both Sirenian and 13 Cetacean families). The selection comprised all Cetacean species occurring in the Mediterranean Sea. In addition, four human mitochondrial genomes representative of the four main human haplogroups (i.e. haplotypes 16, 31, 33 and 52 in Ingman et al. 2000) were included (Online Resource 1) in order to design primers with reduced amplification efficiency for human DNA. All sequences (n = 75) were aligned with the online tool Clustal-Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) with default parameters, and the complete ribosomal 12S and 16S genes were isolated. Potential sites for metabarcoding primers were identified by manually searching for suitable locations within alignments. Gene regions were considered suitable for designing metabarcoding primers if they encompassed a short (80-230bp) highly variable fragment, required for species discrimination, and were flanked by highly conserved sites for situating primers. Where possible, (i.e. when enough intra-mammal variation was found in proximity of the priming sites), we also tried to design, for each candidate locus, we also tried to design alternative primers which minimised the probability of amplifying human targets, by ensuring mismatches between the primers and human templates. Such variants could be preferentially used in studies specifically targeting marine mammals.

Primer evaluation and validation Three approaches were used to assess primer performance. Firstly, primers were evaluated in silico in two

steps: i) predicted primer binding and amplicon sequence diversity was assessed using the ecoPCR scripts

within the OBITOOLS software package (Ficetola et al. 2010; Boyer et al. 2016); and ii) 680 complete marine mammal mitogenome sequences deposited in Genbank, were used to quantify sequence diversity for the primer target regions within marine mammal Families, Genera and species, to provide recommendations on taxonomic resolution utility of primer sets for specific taxa. Secondly, the performance of the primers was evaluated in vitro using tissue derived DNA extracts with varying levels of degradation. Finally, eDNA samples, obtained from tanks of known species composition at the Aquarium of Genoa (Italy) as a proxy for ‘real world’ environmental samples, were used to assess the metabarcoding performance of the primers. bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

In silico primer evaluation An in silico approach was used to assess the universality of the newly designed primers against all standard nucleotide sequences in the NCBI-Genbank data repository (accessed April 2019) for three taxonomic groups: i) vertebrates (excluding Cetaceans), ii) Cetaceans only, and iii) Invertebrates. Performance of the newly designed primers were compared against the commonly used 12S-V5 vertebrate metabarcoding primers (Riaz et al. 2011) as a benchmark.

The ecoPCR script was used to simulate an in silico PCR and extract the amplifiable barcoding regions for each primer pair while allowing for a maximum of 3 base-pair (bp) mismatches between the primers and template DNA. Barcode regions shorter than 50 bp and longer than 400 bp were not considered.

Subsequently, the OBIGREP command was used to extract sequences that were reliably assigned to a species level taxonomy. Ambiguous species level identifications (i.e. sequences with ‘sp.’, ‘aff.’, etc. in the definition

of the sequence) and nuclear pseudogenes were excluded from the final sequence database. The OBIUNIQ command was then used to remove duplicate records for each species. Sequences were classified according to their higher taxonomy (i.e. Vertebrates [excluding Cetacean species], Cetaceans and Invertebrates) before summarising the data into a tabular format for further analyses using R version 3.5.2 (R Development Core Team 2010). Finally, the taxonomic resolution of the different primers was assessed by splitting the data into

their higher taxonomy and running the ECOTAXSPECIFICITY script with three different thresholds for barcode similarity (i.e. sequences were considered different if they have 1, 3 or 5 bp differences). The data obtained from the in silico analyses were imported into R and the packages tidyverse (Wickham 2016) and gridExtra (Auguie 2016) were used to construct summary figures to evaluate the taxonomic coverage, the specificity and taxonomic resolution for each primer pair.

We downloaded 680 GenBank complete marine mammal mitogenome sequences from GenBank, and evaluated levels of polymorphism within Families, Genera and species at the three proposed loci. Complete 12S and 16S genes were extracted from the retrieved sequences and aligned for each type of taxonomic comparison and the number of variable sites recorded within the three loci amplicons were reported.

In vitro primer evaluation Tissue derived DNA extracts were used to assess the performance of the newly designed primer pairs in vitro and optimise amplification conditions. DNA extracts of diverse marine vertebrate groups (cetaceans, pinnipeds, sea turtles and fish) were used as templates for PCR amplification (see Table 4). The 13 DNA templates were purposely selected to have different levels of degradation, being extracted with different techniques and spanning 1-31 years since extraction, in order to evaluate the ability of the primer to amplify low-quality DNA. High quality DNA extracts were obtained from fresh samples (i.e. muscle, skin or blood) using the Qiagen DNeasy Blood & Tissue extraction kit and following the manufacturers protocols. Low quality DNA extracts consisted of phenol-chloroform extracted DNA which was over 25 years old or DNA extracts obtained from boiling tissue samples in a buffer solution (Valsecchi 1998). For each primer pair and each of the DNA extracts a (single, duplicate, triplicate) PCR reaction was performed consisting of 25 U/µl of GoTaq G2 Flexi DNA polymerase (Promega); 1X Green GoTaq Flexi Reaction Buffer (Promega), 1.25-1.87 bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

mM MgCl2 (Promega); 0.2 mM dNTPs (Promega), 0.25-0.75 µM of each primer and dH2O to reach a final volume of 20 µl. Thermal cycling conditions followed a touch-down PCR protocol with annealing temperatures depending on primer pairs: 10/10/18 cycles at 54/55/56 °C for MarVer1; 10/10/18 cycles at 51/52/53 °C for MarVer2; 8/10/10/10 cycles at 54/55/56/57 °C for MarVer3 and finally 10/10/18 cycles at 59/60/61 °C for Ceto2. After an initial denaturation step of 4 min at 94 oC, each of the 38 cycles consisted of a 30 s at 95 oC, 30 s at the primer specific annealing temperatures as described above, followed by 40 s at 72 oC. The final extension consisted of 5 s at 72 oC. To confirm the amplification of the desired amplicon, PCR products were visually assessed using gel-electrophoresis and Sanger sequenced (GENEWIZ, UK; data not shown).

Evaluation of primer performance with environmental samples Environmental DNA samples derived from water collected from 6 tanks of the Aquarium of Genoa, Italy in June 2018, were also employed to further validate the performance of the four primer sets. The tanks contained from 1 to 14 known vertebrate species and were named after their main hosted species or typology: 1) “Manatee”, 2) “Dolphin”, 3) “Shark”, 4) “Seal”, 5) “Penguin” and 6) “Rocky shore” - a multispecies tank hosting fish and invertebrates typical of Mediterranean rocky shores. Two tanks (Dolphin and Seal) were single species, while the Shark and the Rocky shore tanks included a combination of cartilaginous and bony fish.

For each tank, a total of 13.5 litres of water was collected from the water surface using a sterile graduated 2000ml glass cylinder, while wearing sterile gloves, and were stored within a 15-litre sterile container in order to homogenise the water sample, and avoid stochastic variability due to sampling of small volumes. For each tank, 3x 1.5 litre and 3x 3 litre replicates (total 6 replicates per tank) were then aliquoted from the larger sample. To capture environmental DNA, immediately after aliquoting, each of the 6 replicates was then filtered using individual 0.45 µm pore size nitrocellulose filters, using a BioSartâ 100 filtration system (Sartorius). After filtering, membranes were placed on ice for transport to the University of Milano-Bicocca, and subsequently stored at -20oC. Two weeks later environmental DNA was extracted from the filter membranes using a DNeasy PowerSoil Kitâ (Qiagen), following the manufacturer’s protocol.

For each of the four primer sets developed in this study, PCR performance with the eDNA extractions, was initially evaluated using the same PCR conditions as the ‘in vitro’ validation. Subsequently two of the primer sets were selected for further use in a DNA metabarcoding analysis with high throughput sequencing using an Illumina MiSeq platform. For each selected primer set (metabarcoding locus), indexed forward and reverse sequencing primers were created comprising (5’ to 3’): an 8bp Illumina bar code tag - 4 random nucleotides - amplification primer sequence, and sourced from Sigma, UK. Eight forward primer indexes were combined with 12 reverse primer indexes, to allow pooling of amplicons from up to 96 uniquely identifiable samples within a single sequencing library (Taberlet et al. 2018). Trial amplifications with the Illumina barcode tagged primers suggested their yield and specificity was unchanged, and so the previously optimised PCR conditions were used to generate amplicons for MiSeq sequencing. For each locus, eDNA was amplified in triplicate in 40µl final PCR volume; 5µl of each replicate was used to check for successful amplification via agarose gel electrophoresis, and the remainder combined to yield a single pool for each sample. bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Each sample amplicon pool was first assessed for fragment size distribution using an Agilent Tapestation, and cleaned with AMPure beads (Beckman Coulter), following the manufacturer’s protocol, to remove primer dimers. The cleaned samples were quantified with a Qubit fluorometer, and then for each metabarcoding locus, separate Illumina NEBNext Ultra DNA libraries were generated, with the pooled samples in equimolar ratios. Prior to sequencing each library was further assessed for fragment size distribution and DNA quantity by Agilent Tapestation and Qubit fluorometer. The library for locus MarVer1 (see results) was sequenced in a 150bp paired-end lane, and locus MarVer3 (see results) in a 250bp paired-end lane, using an Illumina MiSeq sequencer at the University of Leeds Genomics Facility, St James’s Hospital.

Bioinformatics for environmental HTS data Paired reads were first screened for the presence of the expected primer and index sequence combinations to exclude off-target amplicons. The reads were then combined to generate the insert sequence, and the sequence of the random nucleotide region noted, such that only one instance of an insert per sample with the sample random nucleotide finger print was saved to a sample specific file (i.e. to avoid PCR duplicates and chimeric sequences). The insert data was aggregated to create a counts matrix containing the occurrence of each unique sequence in each sample. The taxonomic origin of each insert was determined by Blasting their sequence against a local instance of the Genbank NT database (Nucleotide, [Internet]). The level of homology of insert to the hit sequence was noted, as was the species name of the hit sequence. The taxonomic hierarchy for each unique insert was generated by searching a local instance of the ITIS database (ITIS, [internet]) with the annotated Genbank species name. The count matrix and taxonomic hierarchy for all annotated unique sequences were then aggregated into values for equivalent Molecular Operational Taxonomic Units (MOTUs), by combining all inserts with a set homology (>=98%) to the Genbank hit at a specified taxonomic level (i.e. ‘Order’, ‘Family’, ‘Genus’ or ‘Species’), using bespoke software (available on request). Summaries and visualisations of read counts for different taxonomic levels were generated using the R package ‘Phyloseq’ (McMurdie and Holmes, 2013).

RESULTS

Description of primer sets From the initial evaluation of aligned marine vertebrate mitochondrial sequences, four novel primer sets were identified, three within the 12S and one in the 16S genes (Table 1). The three ‘MarVer’ primer combinations (2 in 12S and 1 in 16S) were developed to amplify the target regions in any of the 71 taxa selected as representatives of marine vertebrates. To allow for variable sites between different vertebrate groups within the primer sequence, single degenerate bases were introduced (Table 1) for 4 out of the 6 MarVer primers. The fourth primer set ‘Ceto2’, was designed to preferentially amplify Cetacean DNA by minimizing base- pair mismatches for cetacean species while maximizing base-pair mismatches for other vertebrate groups. Online Resource 2 (Supplementary Material) shows variability at the priming sites across the eight marine vertebrate categories. Amplicon variability across the 71 taxa (plus the 4 human sequences) recorded in the regions targeted by the proposed markers are shown in Online Resource 3 (Supplementary Material). bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

MarVer1 MarVer1 primer set (abbr. MV1) targets a hypervariable region of the 12S gene, amplifying a fragment of about 199-212bp (Table 2). It partially overlaps with loci Tele02 (Taberlet et al. 2018), Tele03 (as named by Taberlet et al. 2018, corresponding to MiFish-U in Miya et al. 2015) and Elas01 (as named by Taberlet et al. 2018, corresponding to MiFish-E in Miya et al. 2015), targeting bony and cartilaginous fishes (Figure 1). The forward primer MarVer1F differs from the forward primers of previously described loci, in that by shifting 5- 12bp at the 5’ end, it skips variable sites distinguishing bony from cartilaginous fishes, while gaining, at the 3’ end, bases which are conserved across all surveyed marine vertebrates.

MarVer2 MarVer2 primer set (abbr. MV2) targets a variable region of the 12S gene, amplifying a smaller fragment of 85-96bp in the tested vertebrate taxa (Table 2). It is a modification of primer sets Tele01 (Valentini et al. 2016), targeting bony fishes, and Mamm01 (Taberlet et al. 2018), targeting mammals (Figure 1). The MarVer2 reverse primer partially overlaps with AcMDB01R primer designed by Bylemans et al. 2018 for freshwater fish Actinopterygii species. Both forward and reverse MarVer2 primers were designed with a degenerate base to allow amplification in all the 71 tested vertebrate taxa (Table 2). Sequence variation within MarVer2 amplicon did not resolve all 71 marine vertebrate taxa in the original alignment, with sequences shared between two or more Odontocete species within the Delphinidae family; with Sousa chinensis, Tursiops truncatus, Tursiops aduncus, Stenella coeruleoalba, Delphinus delphis and Delphinus capensis sharing one amplicon sequence, and Peponocephala electra and Feresa attenuaae sharing another.

MarVer3 MarVer3 (abbr. MV3) amplifies a variable region of the 16S gene, producing amplicons ranging in size from 232 to 274bp in the 71 marine vertebrate taxa tested (Table 2). MarVer3 is partially covered by locus Mamm02 (Taberlet et al. 2018, see Figure 1), but targets a fragment twice as long: for example, in Odontocetes MarVer3 amplifies a 233bp fragment versus a 115bp amplicon for Mamm02. The reverse primer (MarVer3R) was the only one of the presented oligonucleotides to be truly “universal”, as it was found to be fully conserved across all tested marine vertebrates (Table 2). The MarVer3 amplicon sequence resolved 69 of the 71 tested marine vertebrate species. The unresolved species also fall into the Delphinidae family: Sousa chinensis and Tursiops truncatus sharing one amplicon sequence, and Tursiops aduncus and Delphinus capensis sharing another.

Ceto2 Ceto2 primers were designed to complement the MarVer2 locus, amplifying the same 12S region, but with nucleotides specific to Cetaceans (and excluding humans) in both forward and reverse primers, and containing no degenerate bases (Table 1). Ceto2 amplicon sizes were 15bp longer than MarVer2, due to shifting of the priming sites. The Ceto2 primer set is suggested as not suitable for use with marine mammals other than Cetaceans due to primer site mismatches. The priming sites present one variable site in some Pinnipeds with the New Zealand fur seal (Arctocephalus forsteri) exhibiting a variant at the 3’ end of the bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

forward primer, while both the walrus (Odobenus rosmarus) and the harp seal (Phoca groenlandica) presented a variant towards the 5’ end of the Ceto2R primer.

Primer evaluation and validation

In silico primer evaluation For the different primer pairs and higher taxonomic groups, the total number of unique taxa for which the in silico amplification recovered target sequences is given in Figure 2. The results show that the MarVer3 primer pair amplified DNA from the most vertebrate and cetacean taxa. However, this primer pair also successfully amplifies the DNA of invertebrate species thus indicating that it has a low overall specificity to the intended taxonomic targets (Figure 2). The three other primer pairs all amplified DNA from fewer target taxa then the commonly used 12S-V5 primers.

The proportion of sequences amplified for each higher taxonomic group is a function of the bp-mismatches between the primers and template DNA is shown in Figure 3. The results of the commonly used 12S-V5 primer pair show that a high number of vertebrate sequences have very few bp-mismatches while the recovered non-vertebrate sequences generally have ≥ 5 bp-mismatches at both primer binding regions (Figure 3). A similar pattern can be observed for the MarVer2 primer pair although the bp-mismatches in the primer binding regions for non-vertebrate sequences are slightly lower (i.e. ≥ 4 bp-mismatches between both primer regions). The results of both the MarVer1 and MarVer3 primers shows that even with a low number of bp- mismatches (i.e. ≥ 2 mismatches for both the forward and reverse primers) a significant proportion of the amplified sequences belong to non-vertebrate taxa. Finally, the results of the Ceto2 primers show that this primer pair will preferentially amplify Cetacean species as the bp-mismatches between Cetacean sequences are lower compared to the number of bp-mismatches observed in the primer binding regions for most vertebrate sequences (i.e. ≤ 2 bp-mismatches for the Cetacean sequences versus ≥ 3 bp-mismatches for most vertebrate sequences).

The taxonomic resolution power of the different primer pairs was evaluated using both the Cetacean and Vertebrate sequences and the results are summarised in Figure 4. Overall the commonly used 12S-V5 metabarcoding primers have a lower resolution capacity compared to our newly designed primers (for both the Cetacean and Vertebrate taxa), with our primers assigning 20-25 % more sequences to the correct species level taxonomy (Figure 4). For the newly designed primers, differences are observed in their taxonomic assignment power, with MarVer1 and MarVer3 generally assigning a higher percentage of the sequences to the correct family, genus and species level taxonomy (Figure 4).

Primer set resolution for Marine Mammal taxonomic detection

Table 3 shows the results of the comparison performed on 680 GenBank complete marine mammal mitogenome sequences (GenBank accession numbers shown in Online Resource 4), in order to evaluate levels of polymorphism within Families, Genera and species. bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Family level. All three targeted regions contained high genetic variability within the seven analysed marine mammals Families (Pinnipeds (2), Mysticetes (1) and Odontocetes (4)). The DNA fragment amplified by MarVer3 primer set (16S region) proved to be the most diverse, highlighting 59 variable sites within the Phocoidea, and over 40 in the Otariidae, Ziphiidae and Delphinidae Families. The least variable locus of the three was MarVer2 (thus also Ceto2). Nevertheless, this marker had high discriminatory power for genera within Families, with the exception of the Monodontidae and Phocoenoidae families. Here both families contain only 2 genera, which in both cases only differ by one basepair, and therefore may not be automatically resolved as distinct MOTUs if a threshold of 98% homology is used in the annotation pipeline (Table 3).

Genus level. Nine Genera, each including 2 to 5 species, were assessed for within-genus variability, including pairwise con-generic species comparisons (Table 3). MarVer3 consistently revealed the highest levels of polymorphism (23 of the 31 inter-generic pairwise comparisons show differences of at least 1bp), while MarVer2 was least variable, showing no variation in 7 of the 31 pairwise comparisons (grey boxes in Table 3). However, MarVer2 showed good power to resolve taxon identity in the Genus Mesoplodon (Ziphiidae family). MarVer1 was the second most variable locus at this level, with the highest number of variable sites in 10 out of the 31 congeneric-species pairwise comparisons. Within all three Tursiops spp. pairwise comparisons MarVer1 was the locus showing the highest variability.

Species level. Genetic variability was investigated also below the nominal species level. This could be tested only on the few species for which large enough sample sizes were available to evaluate intraspecific variation. We assessed 14 marine mammal species for which mitogenomic data were available for a number of individuals ranging from 2 (Megaptera novaeangliae and Dugong dugong) to 152 (Balaenoptera physalus) individuals (Table 3). The 14 species were representative of seven marine mammal Families: Dugongidae (1 species), Otariidae (2 species), Phocidae (1 species), Balaenopteridae (2 species), Delphinidae (4 species), Ziphiidae (3 species) and Phocoenidae (1 species). All sequences were retrieved from GenBank (see Online Resource 4), with the exception of 22 unpublished Pusa caspica sequences (provided by SG). In only one of the 14 species (Eumetopias jubatus, fam. Otariidae) were none of the three loci polymorphic (11 individuals compared). Four of 14 species had only one informative locus: MarVer1 (12S) in two instances (Mesoplodon europaeus and Phocoena phocoena), and MarVer3 (16S) in two other cases (Tursiops truncatus and Ziphius cavirostris) (Table 3). In the Caspian seal (Pusa capsica), the killer whale (Orcinus orca) and the spinner dolphin (Stenella longirostris), two of three loci showed polymorphism. In the remaining 6 species (Dugong dugong, Arctocephalus forsteri, Balaenoptera physalus, Megaptera novaeangliae, Stenella attenuata, Mesoplodon densirostris) all 3 loci presented polymorphisms: in some instances MarVer1 was found to be the most informative locus (e.g. 12 variable sites in Balaenoptera physalus), in others MarVer3 (e.g. 6 variable sites in Stenella longirostris).

In vitro primer evaluation PCR amplicons of the expected size were generated by the four primer sets with most of the 13 DNA extracts (Table 4). The oldest DNA extract (31yo, humpback whale, Megaptera novaeangliae) was the worst performing, yielding only very faint bands for MarVer1, MarVer3 and Ceto2, and failing to produce a band bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

for locus MarVer2. All sea turtle extracts produced one strong artefact band (ca 161bp) and a fainter band in the correct size range (ca 107bp) place of the correct sized fragment for locus Ceto2. Bony fish DNA extracts also produced a band compatible in size with the Ceto2 amplicon.

Application to environmental samples Amplicons of the expected size were obtained from all 36 water samples collected at the Genoa Aquarium with the four primer sets. Locus Ceto2 amplified a faint band in the expected size range from the shark, penguin and seal tanks, in addition to the bottlenose dolphin tank, suggesting either low level amplification from non-cetacean taxa or potential transfer of dolphin eDNA between tanks (see below).

Loci MarVer1 and MarVer3 were selected for further HTS metabarcoding evaluation on the basis of the bioinformatic prediction that they would be the most informative for Mediterranean marine megafauna species (see below). For these loci after sequence quality filtering and demultiplexing, annotated read counts per sample ranged from 682 to 52,478 for MarVer1 and 1,025 to 43,003 for MarVer3, with combined reads per Tank of 10,750 to 158,950 for MarVer1, and 1,3251 to 232,015 for MarVer3 (see Table 5, Figure 5, Online Resource 5).

The percentage of resident vertebrate species with amplicons annotated to the species level within each tank ranged between 0% (Tank 6, Rocky shore) to 100% (Tank 2, Dolphin and Tank 5, Seal), mean 50.4% for MarVer1; and between 66.6% (Tank 1, Manatee) to 100% (Tanks 4, 5, 6), mean 89.7%, for MarVer3 (see Online Resource 5). Overall, for MarVer1 amplicons for 9 out of 27 resident taxa were annotated to the species level, and for 22 resident species for MarVer3, (see Table 5, Figure 5, Online Resource 5).

Amplicons for the aquarium’s four frequently used vertebrate feed species (Culpea harengus, Mallotous villosus, Merluccius productus, and Scomber scombrus), were detected in tanks 2 to 6 (in the Manatee tank, feed consists of lettuce) using both loci, with the exception of MarVer1 failing to detect Merluccius productus. Squid (unspecified species), is also supplied to the Dolphin Tank, but no cephalopod, amplicons were recovered.

Amplicons for resident species were also detected in tanks other than their host tanks at low levels (e.g. with MarVer3, dolphin and seal amplicons were detected in the manatee and shark, and penguin and rocky shore tanks respectively), suggesting possible transfer of eDNA between tanks in the aquarium, e.g. via the equipment used by staff members. Similarly, human amplicons were detected in all tanks, consistent with the practice of aquarium staff entering the water for maintenance.

Both MarVer1 and MarVer3 identified amplicons (partially overlapping between loci and tanks) that were not directly attributable to resident species or food sources (category B in Online Resource 5). These comprised 6 recurrent species, two of which were previously (but no longer) used as feed (Sardina pilchardus, Engraulis encrasicolus), and four species present in the Ligurian Sea (e.g. Auxis rochei, Auxis thazard, Belone belone, Coris julis) from which the water used to fill the tanks is drawn, after being filtered and UV irradiated. All of these unexpected species detections were at low abundance, with read counts greater than 100 to a maximum of 947 in at least 1 tank (range 0.3% to 3.7% of total reads with MarVer3 and MarVer1 respectively). Very bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

low abundance amplicons (<100 reads per tanks) for at least 20 other Mediterranean resident teleost fish species were also observed, but were not considered further as definitive detections.

Amplicons from invertebrate species (category C in Online Resource 5) were detected in two tanks by the MarVer3 locus at low abundances (read count <100), which would normally be discounted as a detection. These being, an Anthozoa species, Seriatopora hystrix, in the manatee tank, and a Sipunculid worm, of the family Phascolosomatidae, in the seal tank (Online Resource 5). Neither taxa were in the tank in which their traces were found. No invertebrate amplicons were recovered in the “rocky shore” tank which contains some Anthozoa (e.g. Anemonia viridis), some unidentified sponges growing spontaneously and hydrozoans, or from the shark tank where Aiptasia spp grows spontaneously. The other tanks (with the exception of dolphin and manatee) may also contain other spontaneously growing small invertebrates, such as copepods, amphipods and hydrozoans, but none were detected.

DISCUSSION

Comparison with previously described barcode primer sets

This study was conceived to identify cetacean specific barcode loci complementing the many already available for fish species (e.g. Miya et al. 2015, Sato et al. 2018), for use in eDNA biodiversity monitoring studies of Mediterranean marine vertebrates. However, in the primer design process, we realised that with minor adaptations, it was possible to cover the whole range of marine vertebrates in a single HTS run, potentially dramatically reducing costs for eDNA HTS biodiversity monitoring of pelagic vertebrates.

Most existing 12S/16S barcode primer sets (e.g. Taberlet et al. 2018, Miya et al. 2015, Bylemans et al. 2018) were designed for particular vertebrate groups and thus contained conserved sequence elements specific for their target taxonomic group. Most of these primer sets are partially overlapping, at least at one end, with the ones presented in this study although they are never identical (Figure 1). Our proposed primer sets were also found to be different from the universal 12S and 16S primers combinations described by Yang et al. (2014) - although MarVer3 sits within Yang et al.’s 16S target fragment - however, their amplicon sizes were too large (ca 430bp and ca 500bp for respectively the 12S and the 16S primer sets) to be easily employed in eDNA studies using current short-read HTS technology.

The 12S-V5 primer set (Riaz et al. 2011, renamed Vert01 by Taberlet et al. 2018), is the only one previously described as being specific for vertebrates. It is located adjacent to MarVer1 site (forward 12S-V5 primer partially overlaps with reverse MarVer1 sequence, see Figure 1). Within our alignments, the 12S-V5 site was not as variable as any of the three loci described in this paper. For example, looking at one of the most variable Families considered in this study, the Ziphiidae (Odontocetes), our three loci (MarVer 1, 2 and 3) recovered 37, 30 and 43 variable sites respectively (Table 3), while 12S-V5 highlighted only 13 within the same pool of sequences. Given that 12S-V5 and MarVer2 primer pairs generate similar sized amplicons (respectively 106bp and 96bp), MarVer2 detects more than twice (30) the number of variable sites found in the 12S-V5 target sequence (13). Such performance difference was also highlighted in the in silico simulation bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

(Figure 4). Moreover, within both forward and reverse 12S-V5 primer sites, polymorphisms were present within the Ziphiidae Family, suggesting that the 12S-V5 primers may not be conserved across all vertebrate classes. However, 12S-V5 performed better than MarVer1, MarVer2 and Ceto2 based on the total number of unique taxa for which DNA was amplified in the in silico simulation (Figure 2). While the lower predicted number of taxa recovered by these primers may indicate a failure to amplify DNA from some species, the completeness of the reference database used will also influence the results given that all sequences records were considered and not only the full mitochondrial genomes. Within the partial 12S sequences deposited on GenBank the fragment including locus 12S-V5 might be over-represented, as most previous studies relied on this marker.

Besides being highly conserved among Vertebrates, all three MarVer primer-sets were shown to be potentially non-exclusive to Vertebrates when 2-4bp primer/template mismatches were allowed (Figure 3). With reduced specificity, these primer pairs could potentially amplify unwanted non-vertebrate taxa. Given the high number of vertebrate taxa recovered by the MarVer3 primers in silico (Figure 2), and the observation that the majority of the non-vertebrate taxa recovered have ≥ 3 bp-mismatches in the primer binding regions (Figure 3), this primer pair should still be valuable if stringent thermal cycling conditions are used during PCR amplification. This was supported by the eDNA sequencing of aquarium samples where there was minimal recovery of invertebrate amplicons from tanks known to contain invertebrate species. This suggests the use of MarVer3 in Vertebrate biodiversity surveys would not be limited by potential homology with some invertebrate sequences.

Performance of MarVer1 and MarVer3 primer sets with environmental samples

We evaluated the performance of MarVer1 and MarVer3 primer sets with water samples collected at the Genoa aquarium, from tanks with known community compositions. Amplicons annotated to species level were recovered for 81.5% and 37% of the 27 resident taxa for MarVer3 and MarVer1 respectively. For MarVer3, the five ‘undetected’ species included Diplodus cervinus (Zebra seabream), Pterygoplichthys gibbiceps (Armored catfish), two ray species (Dasiatis americana, Teaniura grabata), and Pristis zjisron (Longcomb sawfish). In the case of D. cervinus, amplicons assigned to other non-resident Diplodus species were observed, so eDNA from the species may have been present, but annotated as a congeneric. For the four other ‘undetected’ species, there were no other incompletely (above species level) assigned reads at genus, family, or order level which could be attributed to these taxa (for Pristis zjisron no matching reference sequence for the MarVer3 region was available in Genbank). These four cases therefore appear to be genuine non-detections. These are all bottom dwelling species, whereas our water samples were collected at the surface, and therefore potentially we did not capture eDNA from these species.

For MarVer1, 9 out of 13 resident species with Genbank reference sequences covering the MarVer1 region were detected successfully. Amplicons correctly assigned to the species level were not observed for the two penguin species, Blackspot seabream (Pagellus bogaraveo), and longcomb sawfish, despite reference sequences being available. For the Magellanic penguin, amplicons assigned to the congeneric Spheniscus bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

demersus were observed, but no candidate incompletely or misattributed amplicons for the Gentoo penguin (Pygoscelis papua) or two fish species were recorded.

For 14 species (51.6% cases), no Genbank reference sequences covering the MarVer1 region were available. Reads assigned to the non-resident grouper Epinepehelus lanceolatus were observed, indicating that eDNA from the three resident groupers may have been detected but attributed to a congeneric for which a reference was available. For the remaining 11 species no other candidate amplicons attributable to related taxa were recorded.

Our demultiplexing and annotation pipeline required an amplicon sequence homology of at least 98% with other Molecular Operational Taxonomic Units (MOTUs) and with Genbank reference sequences. Therefore, the lower assignment rate for MarVer1 compared to MarVer3 likely reflects the lower Genbank coverage for the 12S region encompassed by MarVer1. In this case, reducing stringency (e.g. to 95% homology) may increase annotation rates, allowing successful attribution of amplicons to genus and/or family level, but with a requirement to check homology level for individual MOTUs before accepting species level assignments. For both primer sets, annotation success rates would be expected to increase as taxonomic coverage of Genbank reference sequences improves over time. Similarly, while MarVer3 was predicted to potentially recover invertebrate amplicons when allowing for low levels of degeneracy at priming sites, few were observed. Potentially this may also be accounted for by low coverage with reference sequences and the level of stringency applied in the annotation pipeline. The annotation of the few observed invertebrate amplicons from the aquarium samples should also be treated cautiously for the same reasons.

Optimising locus choice for different eDNA and taxon detection applications

The four loci described in this paper provide investigators with flexible options to target different barcode markers depending on priorities for their study objectives, tailored to requirements for taxonomic breadth, variation and resolution at different taxonomic levels, amplicon size where eDNA degradation is a concern (e.g. Speller et al. 2016), and sensitivity to contamination from human DNA.

Overall the 16S based MarVer3, the largest amplicon product with an expected size of approximately 245bp, has the highest potential taxonomic coverage across all vertebrates, and taxon resolution from species level upwards. Our trial eDNA HTS assay using samples from aquarium tanks demonstrated the locus performed well with environmental samples despite its relatively large amplicon size compared to the other loci. However, the MarVer3 region showed limited variability at nominal species level compared to MarVer1 and 2 amplicons (e.g. in killer whale, Orcinus orca, no variability was found among 151 individuals tested, see Table 3). This is consistent with lower rates of evolution in 16S compared to 12S genes, and suggests 12S genes may be more informative for resolving intraspecific differences (see below).

The 12S based MarVer1, Marver2, and Ceto2, offer the advantage of smaller amplicon sizes (approximately 202bp, 89bp, 104bp respectively), which may be a consideration for applications requiring work with more highly degraded eDNA templates (Nichols et al. 2018, Wei et al. 2018). bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Here we focused on evaluating taxonomic resolution with marine mammal groups, and found the relative performance of the loci varied across different families. For instance, MarVer1 was more variable than MarVer2 in most Cetacean species, but MarVer2 still had strong taxon resolution for species outside the Delphinidae family, and in particular for the Ziphiidae. In a stretch of less than 100bp, the MarVer2 region contained more polymorphic sites in the Ziphiidae (5 Genera assessed) than in the numerically and morphologically more diverse Delphinidae Family (14 Genera assessed). This observation is likely due to the older phylogenetic divergence of the Ziphiidae lineages, allowing more time for accumulation of fixed differences between species, compared to the more recently derived Delphinidae family (Nikado et al. 2001). MarVer2 was also more effective at resolving Pinnipeds of the genus Pusa compared to MarVer1. However, the incomplete resolution of MarVer2 (and thus Ceto2), within the Delphinidae would preclude its use for species level detection studies within the Mediterranean, one of our geographic areas of interest, since it cannot differentiate bottlenose, common and striped dolphins, which are sympatric in this sea. There were also notable performance differences for loci at the interspecific level within Families. For example, MarVer3 was the most polymorphic locus within the 104 spinner dolphin (Stenella longirostris) samples, but it was monomorphic within the 151 killer whale (Orcinus orca) mitogenome sequences retrieved from GenBank, see Table 3).

Resolution of intraspecific phylogeographic variation is likely to be best attempted with either more variable, or longer target amplicons (e.g. d-loop region; Kunal et al 2013). However, the large Cetacean sequence dataset we evaluated allowed us to test the potential of our loci to identify phylogeographically informative variation, which could be used for simple haplotype clade determination with eDNA for some species (Adams et al. 2019). For instance, within the MarVer1 amplicon in the 151 killer whale mitogenomes (Morin et al. 2010, 2015 and Filatova et al. 2018), some variable sites were private to either the Transient clade or to the AntB and AntC clades identified by the larger dataset.

The preliminary investigation of sequence variation in other large marine vertebrate groups (tuna and sea turtle species), suggested our loci also have potential to be informative for species identification in those taxa. While not assessed directly in this study, the MarVer loci may also prove to be useful as barcode markers for terrestrial vertebrates given taxonomic conservation of the priming sites.

Finally, the high levels of diagnostic variation seen within MarVer loci amplicons offer potential for designing additional species specific nested internal primers (Stoeckle et al. 2018). These might have utility for species focused, non-sequencing based detection applications, such as quantitative PCR, or simple agarose gel based amplicon visualisation when there is limited access to laboratory facilities, or funding. MarVer2 and Ceto2, would be most suited for this in Cetaceans (due to the clustering of variable sites in proximity of one of the priming site), while for Teleost species, MarVer3 would be the best candidate (due to many in-del mutations in fish).

Conclusions bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

This paper presents four novel primer sets targeting 12S and 16S vertebrate mitogenome regions, with a particular focus on marine mammals. Using a combination of ‘in silico’ validation, and application to eDNA samples from aquarium communities with known species composition, we show the loci to have high potential for metabarcoding and eDNA studies targeting marine vertebrates. These primer sets have broader taxonomic coverage and resolution compared to previously developed 12S and 16S primer sets, potentially allowing surveys of complete marine vertebrate communities (including fish, sea turtles, bird and mammals) in single HTS runs, simplifying workflows, reducing costs, and increasing accessibility to a wider range of investigators. They may be applied in any context focusing on resolving vertebrate taxonomic identity, from biodiversity surveys and forensics (e.g. CITES surveillance, or surveys of commercially targeted fish species), through to behavioural ecology studies and supporting conservation of rare or endangered marine vertebrate species. bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

FUNDING No funding was received for this work.

CONFLICT OF INTEREST The authors declare that they have no conflict of interest.

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Table 1 Sequences of the four described primer sets

Locus Primer ID Primer sequence (5'-3') Size Region Average amplicon size

MarVer1 MarVer1F CGTGCCAGCCACCGCG 16bp 12S ca. 202 bp MarVer1R GGGTATCTAATCCYAGTTTG 20bp

MarVer2 MarVer2F CCGCCCGTCACYCTC 15bp 12S ca. 89 bp MarVer2R CTTACCWTGTTACGACTT 18bp

MarVer3 MarVer3F AGACGAGAAGACCCTRTG 18bp 16S ca. 245 bp MarVer3R GGATTGCGCTGTTATCCC 18bp

Ceto2 Ceto2F CACGCACACACCGCCCG 17bp 12S ca. 104 bp Ceto2R GTATGCTTACCTTGTTACGAC 21bp

Table 2 Characteristics of the amplicons produced by each of the primer sets for the 71 marine vertebrate species used for primers’ design. The overall amplicon size is the mean value of the amplicon sizes recorded in the eight marine vertebrate groups analysed. Ceto2 locus is not included in this table as its characteristics are the same as MarVer2, with the only exception of having the total amplicon size increased of 15bp compared with MarVer2 due to primers positioning. The grey shading indicates, for each vertebrate group, rank of nucleotide p-distance values across the three loci (the highest shown in darker grey)

MarVer01 CODO CMYS PINN SIRE STUR SBIR TELE ELAS Overall approximate amplicon size (bp) 199 199 197 199 211 209 197 212 ca. 202 n variable sites 77 31 33 18 36 45 80 40 171 resolved over tested species 36/36 12/12 3/3 2/2 6/6 2/2 7/7 3/3 71/71 percentage of variable sites (%) 38.7 15.6 16.8 9.0 17.1 21.5 40.6 18.9 84.7 nucleotide p-distance 0.084 0.042 0.097 0.084 0.059 0.157 0.176 0.090 0.196

MarVer02 CODO CMYS PINN SIRE STUR SBIR TELE ELAS Overall approximate amplicon size (bp) 96 96 89 90 87 87 85 85 ca. 89 n variable sites 46 22 29 16 18 11 47 26 65 resolved over tested species 30/36 12/12 3/3 2/2 6/6 2/2 7/7 3/3 65/71 percentage of variable sites (%) 47.9 22.9 32.6 17.8 20.7 12.6 55.3 30.6 73.0 nucleotide p-distance 0.056 0.028 0.146 0.125 0.075 0.047 0.175 0.109 0.154

MarVer03 CODO CMYS PINN SIRE STUR SBIR TELE ELAS Overall approximate amplicon size (bp) 233 232 240 235 240 246 274 265 ca. 245 n variable sites 79 41 53 36 50 31 142 37 251 resolved over tested species 34/36 12/12 3/3 2/2 6/6 2/2 7/7 3/3 69/71 percentage of variable sites (%) 33.9 17.7 22.1 15.3 20.8 12.6 51.8 14.0 102.4 nucleotide p-distance 0.052 0.040 0.098 0.081 0.054 0.058 0.151 0.046 0.167 bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table 3 Levels of polymorphism found at the three proposed loci in a series of marine mammals Families, Genera and species for which complete mitogenomic data were available from GenBank (accession numbers listed in Appendix E, with the exception of 22 Pusa capsica entries for which data are unpublished). Numbers in the three columns on the right indicate the number of variable sites detected in the sequence targeted by the three primers sets in each given comparison: clear boxes indicate polymorphism thus usefulness of the primer set to resolve taxa case, while grey boxes show lack of diagnostic polymorphism and light grey boxes indicate lack of resolution at 98% homology threshold for MOTUs assignment

Taxonomic level Gro up Taxa (sample size) Comparisons between MV1 MV2 MV3

Family level PINN Phocidae (10 Genera) Cystophora, Erignathus, Halichoerus, Hydrurga, Lobodon, Mirounga, Monachus, Phoca, 39 29 59 Pusa, Leptonychotes Ota riida e (6 Genera ) Arctocephalus, Callorhinus, Eumetopias, Neophoca, Phocarctos, Zalophus 31 22 42 CMYS Balaenopteridae (2 Genera) Balaenoptera, Megaptera 23 14 25 CODO Ziphiidae (5 Genera) Ziphius, Mesoplodon, Indopacetus, Hyperodon, Berardius 37 30 43 Monodontidae (2 Genera) Delphinapterus, Monodon 6 1 13 Delphinidae (14 Genera) Cephalorhynchus, Sousa, Tursiops, Stenella, Delphinus, Lagenodelphis, Legenorhynchus, 41 25 42 Grampus, Peponocephala, Feresa, Pseudorca, Orcinus, Globicephala, Orcaella Phocoenidae (2 Genera) Neophocaena, Phocoena 13 1 10

Genus lev el PINN Phoca (4 species, 5 individuals) Phoca vitulina (1) vs Phoca larga (2) 0 2 2 Phoca vitulina (1) vs Phoca groenlandica (1) 3 3 11 Phoca vitulina (1) vs Phoca fasciata (1) 10 5 10 Phoca larga (2) vs Phoca groenlandica (1) 3 2 11 Phoca larga (2) vs Phoca fasciata (1) 11 4 10 Phoca groenlandica (1) vs Phoca fasciata (1) 7 4 8 Pusa (3 species, 3 individuals) Pusa caspica (1) vs Pusa hispida (1) 1 1 4 Pusa caspica (1) vs Pusa sibirica (1) 1 3 5 Pusa hispida (1) vs Pusa sibirica (1) 0 2 1 Arctocephalus (3 species, 48 individuals) Arctocephalus forsteri (46) vs Arctocephalus pusillus (1) 8 5 4 Arctocephalus forsteri (46) vs Arctocephalus townsendi (1) 4 3 5 Arctocephalus pusillus (1) vs Arctocephalus townsendi (1) 8 7 4 CODO Tursiops (3 species, 23 individuals) Tursiops truncatus (16) vs Tursiops aduncus (5) 4 0 1 Tursiops truncatus (16) vs Tursiops australis (2) 3 0 1 Tursiops aduncus (5) vs Tursiops australis (2) 3 0 2 Delphinus (2 species, 2 individuals) Delphinus delphis (1) vs Delphinus capensis (1) 1 0 2 Stenella (3 species , 176 individuals) Stenella coeruleoalba (2) vs Stenella attenuata (70) 3 1 3 Stenella coeruleoalba (2) vs Stenella longirostris (104) 1 0 4 Stenella attenuata (70) vs Stenella longirostris (104) 2 0 2 Globipephala (2 species, 2 individuals) Globicephala melas (1) vs Globicephala macrorhynchus (1) 1 1 2 Mesoplodon (5 species, 26 individuals) Mesoplodon europaeus (8) vs Mesoplodon densirostris (12) 8 9 11 Mesoplodon europaeus (8) vs Mesoplodon grayi (2) 10 9 13 Mesoplodon europaeus (8) vs Mesoplodon ginkgodens (2) 7 7 11 Mesoplodon europaeus (8) vs Mesoplodon stejnegeri (2) 12 10 12 Mesoplodon densirostris (12) vs Mesoplodon grayi (2) 6 6 9 Mesoplodon densirostris (12) vs Mesoplodon ginkgodens (2) 8 11 15 Mesoplodon densirostris (12) vs Mesoplodon stejnegeri (2) 7 6 7 Mesoplodon grayi (2) vs Mesoplodon ginkgodens (2) 10 9 12 Mesoplodon grayi (2) vs Mesoplodon stejnegeri (2) 5 8 11 Mesoplodon ginkgodens (2) vs Mesoplodon stejnegeri (2) 6 10 17 Neophocena (2 species, 12 individuals) Neophocena asiaeorientalis (4) vs Neophocena phocaenoides (8) 0 0 0

Species level SIRE Dugong dugong 2 individuals 1 2 2 PINN Arctocephalus forsteri 46 individuals 2 2 5c Eumetopias jubatus 11 individuals 0 0 0 Pusa caspica 23 individuals 2a 2b 2 CMYS Balaenoptera physalus 152 individuals 12b 1 8b Magaptera novaeangliae 2 individuals 1 1 1 CODO Orcinus orca 151 individuals 3 3b 0 Stenella longirostris 104 individuals 4a 3b 6 Stenella attenuata 70 individuals 3b 1 3 Tursiops truncatus 16 individuals 1a 0 2b Ziphius cavirostris 20 individuals 1a 1a 4 Mesoplodon densirostris 12 individuals 1 1 4 Mesoplodon europaeus 8 individuals 1 0 0 Phocoena phocoena 5 individuals 1 0 0

a all mutations present in only 1 or 2 individuals, not diagnostic to detect populations or subspecies b some of these are mutations present in only 1 or 2 individuals c further 7 variable sites were found in a single individual (NZFS8, in Emami-Khoyi et al. 2016) and were not included in this list as they might be an artifact bioRxiv preprint doi: https://doi.org/10.1101/759746; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table 4 List of tissue DNA extracts used for wet lab primer tests. “PC” indicates phenol chloroform extracts, “CK” indicates commercial kit extracts, while “BE” refers to boil extracts as described in Valsecchi 1998. The signs “✓” and “✗” specify successful amplification and PCR failure (no band or aspecific bands) respectively

Group Species Extract type Tissue Time since extraction MarVer1 MarVer2 MarVer3 Ceto2

CODO Stenella coeruleoalba PC skin 27yr Stenella frontalis PC muscle 26yr Pontoporia blainvillei BE skin 25yr CMYS Megaptera novaeangliae PC skin 31yr Eubalaena australis BE skin 24yr PINN Puca caspica CK blood 1yr STUR Lepidochelys kempii CK blood 1yr Chelonia midas CK muscle 1yr Lepidochelys olivacea CK blood 1yr Caretta caretta CK blood 1yr Caretta caretta CK muscle 1yr TELE Thunnus albacares CK muscle 1yr Merlangius merlangus CK muscle 1yr

Table 5 Species composition of the 6 tanks of the Aquarium of Genoa from which water samples were collected for extracting eDNA. The last four columns on the right show NGS outcomes for primer sets MarVer1 and MarVer3: “GB” columns indicate whether reference sequences are present in GenBank for that specific species and locus; “■” indicate successful species detection (half-full and empty square indicates a number of reads 0.001>nr>0.005 or nr<0,001 respectively); △ denotes that a congenereric was identified in place of the resident vertebrate species, “?” indicates possible detection of the resident species at a higher taxonomic group (this case together with those instances for which reference sequences were available but the species remained undetected are discussed in the text)

Tank Name Hosted species (n. individuals) Common name Vertebrate group MarVer1 MarVer3 GB detection GB detection 1 MANATEE Trichechus manatus (4) Matatee SIRE yes ■ yes ■ Piaractus brachypomus (3) Pirapitinga TELE yes ■ yes ■ Pterygoplichthys gibbiceps (2) Armored catfish TELE no X yes X 2 DOLPHIN Tursiops truncatus (10) Bottlenose dolphin CODO yes ■ yes ■ 3 SHARK Ginglymostoma cirratum (3) Nurse shark ELAS yes ■ yes ■ Stegostsoma fasciatum (1) Zebra shark ELAS yes ■ yes ■ Carcharhinus plumbeus (4) Grey shark ELAS yes ■ yes ■ Dasiatis americana (1) Southern stingray ELAS no X no X Pristis zjisron (2) Longcomb sawfish ELAS yes X yes X Teaniura grabata (2) Round stingray ELAS no X yes X Epinepehelus itajara (1) Atlantic goliath grouper TELE no yes Epinephelus costae (1) Goldblotch grouper TELE no yes ■ Epinephelus marginatus (2) Dusky grouper TELE no yes ■ Seriola dumerili (9) Greater amberjack TELE yes ■ yes Dicentrarchus labrax (7) European seabass TELE yes ■ yes ■ Diplodus sargus (4) White seabream TELE no X yes ■ Diplodus cervinus (1) Zebra sea bream TELE no X yes Pagellus bogaraveo (1) Blackspot seabream TELE yes X yes ◻ 4 PENGUIN Pygoscelis papua (12) Gentoo penguin SBIR yes X yes ■ Spheniscus magellanicus (24) Magellanic penguin SBIR yes yes ■ 5 SEAL Phoca vitulina (6) Harbour seal PINN yes ■ yes ■ 6 ROCKY SHORE Diplodus vulgaris (21) Common two-banded seabream TELE no X yes ■ Diplodus sargus (3) White seabream TELE no X yes ■ Sciaena umbra (3) Brown meagre TELE no ? yes ■ Sarpa salpa (2) Salema TELE no X yes ■ Serranus scriba (1) Painted comber TELE no X yes Scyliorhinus stellaris (2) Nursehound ELAS no X yes ■

Feed species 2,3,4,5,6 Culpea harengus Atlantic herring TELE yes ■ yes ■ 2,3,4,5,6 Mallotous villosus Capelin TELE yes ■ yes ■ 3,6 Merluccius productus North Pacific hake TELE no X yes ■ 6 Scomber scombrus Atlantic mackerel TELE yes yes ■ 2 Squid yes yes

26 Fig. 1 Map of the regions amplified by the newly presented primer sets within the 12S (light grey) and 16S (dark grey) genes. The positions of some of most commonly used barcode markers used for detecting vertebrate groups are shown for comparison. The size marker at the bottom refers to the 12S and 16S mtDNA fragment from position 72 to position 2690 in the stripe dolphin (Stenella coeruleoalba) complete mitogenome (GenBank accession number NC_012053)

4 3 7 2 6 1 5

MarVer1 MarVer2 MarVer3 Ceto2

0 bp 500 bp 1000 bp 1500 bp 2000 bp 2500 bp

27 Fig. 2 The total number of unique taxa (y-axis) recovered by the different primer pairs (x-axis). The results are shown for all three higher taxonomic groups (i.e. Vertebrates -excluding cetacean species-, Cetaceans and Invertebrates) considered during the analyses (horizontal panels)

28 Fig. 3 The proportion of sequences amplified for each higher taxonomic group as a function of the bp- mismatches between the template DNA and the forward (y-axis) and reverse (x-axis) primers. The size of the pie-charts is proportional (on a log scale) to the total number of sequences recovered for a given number of bp-mismatches between the forward and reverse primer

29 Fig. 4 The percentage of sequences correctly identified (y-axis) to the family, genus and species level taxonomy (x- axis) for the different primer pairs. Results are shown for all sequences belonging to the Cetaceans and Vertebrates (vertical panels) and using different threshold values for barcode similarity (i.e. sequences were considered different if they have 1, 3 or 5 bp differences) (horizontal panels)

Fig. 5 Taxon ‘abundance’ (read counts) for amplicons generated with the MarVer3 primer set for environmental DNA extracted from water samples collected from 6 tanks at the Genoa Aquarium. Read counts are combined across the 6 replicate samples assayed for each tank, and seqeunce demultiplexing and amplicon annotation against Genbank references was carried out using a threshold of 98% sequence similarity. Taxa presented in the figure were filtered to exclude those with read counts less than 0.005*median read count across tanks. Taxa names with *, are feed species, all others are resident

31 Online Resource 1 List of 75 taxa used for primer sets’ design. Label Family Species GenBank A.N. HOMI01 Hominidae Homo sapiens sapiens (UK) AY339432 HOMI02 Hominidae Homo sapiens sapiens (JP) AY339469 HOMI03 Hominidae Homo sapiens sapiens (AF1) AY339522.2 HOMI04 Hominidae Homo sapiens sapiens (AF2) AY339578.1 PINN01 Otariidae Arctocephalus forsteri KT693378 PINN02 Odobenidae Odobenus rosmarus NC_004029 PINN03 Phocidae Phoca vitulina NC_001325 CMYS01 Balaenidae Balaena mysticetus NC_005268 CMYS02 Balaenidae Eubalaena glacialis NC_037444 CMYS03 Cetotheriae Caperea marginata NC_005269 CMYS04 Eschrichtiidae Eschrichtius robustus NC_005270 CMYS05 Balaenopteridae Megaptara novaeangliae NC_006927 CMYS06 Balaenopteridae Balaenoptera acutorostroata NC_005271 CMYS07 Balaenopteridae Balaenoptera bonaerensis NC_006926 CMYS08 Balaenopteridae Balaenoptera edeni NC_007938 CMYS09 Balaenopteridae Balaenoptera brydei NC_006928 CMYS10 Balaenopteridae Balaenoptera borealis NC_006929 CMYS11 Balaenopteridae Balaenoptera physalus NC_001321 CMYS12 Balaenopteridae Balaenoptera musculus NC_001601 CODO01 Physeteridae Physeter catodon NC_002503 CODO02 Koogidae Kogia breviceps NC_005272 CODO03 Ziphiidae Ziphius cavirostris NC_021435 CODO04 Ziphiidae Berardius Bairdii NC_005274 CODO05 Ziphiidae Hyperoodon ampullatus NC_005273 CODO06 Ziphiidae Mesoplodon europaeus NC_021434 CODO07 Ziphiidae Mesoplodon grayi NC_023830 CODO08 Ziphiidae Mesoplodon ginkgodens NC_027593 CODO09 Ziphiidae Mesoplodon stejnegeri NC_036997 CODO10 Ziphiidae Mesoplodon densirostris NC_021974 CODO11 Iniidae Inia geofrensis NC_005276 CODO12 Lipotidae Lipotes vexillifer NC_007629 CODO13 Pontoporiidae Pontoporia blainvillei NC_005277 CODO14 Monodontidae Delphinapterus leucas NC_034236 CODO15 Monodontidae Monodon monoceros NC_005279 CODO16 Delphinidae Cephalorhynchus heavisidii NC_020696 CODO17 Delphinidae Sousa chinensis NC_012057 CODO18 Delphinidae Tursiops truncatus KF570323 CODO19 Delphinidae Tursiops aduncus NC_012058 CODO20 Delphinidae Stenella attenuata NC_012051 CODO21 Delphinidae Stenella coeruleoalba NC_012053 CODO22 Delphinidae Delphinus delphis MH000365 CODO23 Delphinidae Delphinus capensis NC_012061 CODO24 Delphinidae Lagenodelphis hosei MG599457 CODO25 Delphinidae Legenorhynchus albirostris NC_005278 CODO26 Delphinidae Lagenorhynchus obliquidens NC_035426 CODO27 Delphinidae Grampus griseus NC_012062 CODO28 Delphinidae Peponocephala electra NC_019589 CODO29 Delphinidae Feresa attenuata NC_019588 CODO30 Delphinidae Pseudorca crassidens NC_019577 CODO31 Delphinidae Orcinus orca NC_023889 CODO32 Delphinidae Globicephala melas NC_019441 CODO33 Delphinidae Globicephala macrorhynchus NC_019578 CODO34 Delphinidae Orcaella brevirostris NC_019590 CODO35 Phocoenidae Neophocaena phocaenoides NC_021461 CODO36 Phocoenidae Phocoena phocoena NC_005280 SIRE01 Trichechidae Trichecus manatus NC_010302 SIRE02 Dugongidae Dugong dugong NC_003314 STUR01 Dermochelyidae Dermochelys coriacea MF460363.1 STUR02 Cheloniidae Caretta caretta NC_016923.1 STUR03 Cheloniidae Chelonia mydas NC_000886.1 STUR04 Cheloniidae Eretmochelys imbricata DQ533485.1 STUR05 Cheloniidae Lepidochelys olivacea DQ486893.1 STUR06 Cheloniidae Natator depressa NC_018550.1 SBIR1 PhalacrocoracidaePhalacrocorax carbo NC_027267.1 SBIR2 Laridae Larus crassirostris NC_025556.1 TELE01 Scombridae Thunnus thynnus KF906720.1 TELE02 Scombridae Scomber scombrus NC_006398.1 TELE03 Moronidae Dicentrarchus labrax NC_026074.1 TELE04 Clupeidae Sardina pilchardus NC_009592.1 TELE05 Engraulinae Engraulis encrasicolus NC_009581.1 TELE06 Salmoninae Salmo salar NC_001960.1 TELE07 Xiphiidae Xiphias gladius NC_012677.1 ELAS01 Carcharhinidae Sphyrna mokarron NC_035491.1 ELAS02 Carcharhinidae Prionace glauca NC_022819.1 ELAS03 Alopiidae Carcharodon carcharias NC_022415.1

32 Online Resource 2 Variable sites at the priming sites of the described loci for each of the eight marine vertebrate groups considered in this study (71 taxa considered): CODO= Cetacean Odontocetes; CMYS= Cetacean Mysticetes; PINN= Pinnipeds; SIRE= Sirenians; STUR= Sea Turtle; SBIR= Sea Birds; TELE= Teleosts or bony fish; ELAS= Elasmobranch or cartilaginous fish. Star signs (*) indicate variable sites within the corresponding vertebrate group.

Locus Primer Primer Sequence (5'-3') vertebrate group MarVer1 T * SBIR MarVer1F CGTGCCAGCCACCGCG MarVer1R GGGTATCTAATCCYAGTTTG C CODO C CMYS C PINN C SIRE T STUR C SBIR C TELE T ELAS

MarVer2 C H. sapiens T ELAS * TELE C SBIR C STUR C SIRE C PINN C CMYS C CODO MarVer2F CCGCCCGTCACYCTC MarVer2R CTTACCWTGTTACGACTT T CODO T CMYS T PINN T SIRE T STUR T SBIR A TELE A ELAS A H. sapiens

MarVer3 A H. sapiens A ELAS A TELE G SBIR G STUR A SIRE A PINN A CMYS A CODO MarVer3F AGACGAGAAGACCCTRTG MarVer3R GGATTGCGCTGTTATCCC

Ceto2 G T H. sapiens ** ELAS * TELE T* T T STUR Ceto2F CACGCACACACCGCCCG Ceto2R GTATGCTTACCTTGTTACGAC * PINN CA A SBIR CA A TELE CA A ELAS CA H. sapiens

33 Online Resource 3 Extraction of variable sites for loci MarVer1, MarVer2, and MarVer3 for the 75 taxa used for primers design. MarVer1 111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111222222222222222 111122222223333333444444445555666666667777777777888888888899999999000000000111111112222222223333333333444444444455555555666666666777777777788888888889999999999000000111111223 1018914567891345678012345675678234567890123456789012345678901234789013456789012345670124567890123456789012345678901236789012356789012345678901234567890123456789046789123456247 HOMI01 CCATCCGATTAACAAGTCATAGAAGC-AAGATTTTAGAT------CA--CCCCCTCCC--CAAAAGCTAAACTCACCTGAGTTAAAAACTCCAGTTGACAC--AAAATAGACTACGAGTGGCTTA------ACA------TATCT----GAACAATAGCAAGACC-GC HOMI02 ...... HOMI03 ...... HOMI04 ...... PINN01 .....T...... ACT.CG.GCC.A...C.AAA...... TT..A---TC-AA....C...T....T.T.A.CA..CC....G..A.C...A.T...... T...... A...... CT...... CT...... TT.G...... PINN02 .....T...... A.T.C..GTT.A...T..AAG...... T...G---..-AA....C...T...... T.G.CA..CC....G..G.C.C.A.T...... T...... A...... TT...... AC..C...... TT.GC...... PINN03 .....T...... ACT....GCC.T...C..AA...... A---.C-.A....C...... CT.A.CA..CC....G..A.C...A...T...... C...... A...... CT...... AT...... CTGG...... T.... CODO01 .....T....G.....CT....GCATA.G....AA...G...... TT..A---TA.AA..A.-...TC.GC..TGA..A.ACC....G.CAT....A.A.T.T..GC..A..C...... A...G...... GT.C...... A...... -.CTGGC.A...... CODO02 .....T....G.....CT...AGCATA.....C.AG..A...... C..A---.A.AA..A.-....C.GCT.TGA..A..C.....G.CAT...CA.A...C..G...... A...... T.C...... AG...... CT.GC...... CODO03 .....T....G..G.A.T...... ATA...... AA...A...... TT..A---TGTAA..A.-...T...T..T.AT.A..C..G..G.CAT.A..A.A...T...C..A..C...... A...... TCT...... A...... TT.GC...... T.... CODO04 .....T....G....A.T...... ATA...... AAG..G...... TC..A---.ATAA..A.-...TC..C..T.AT.A..C.....G.CAT.A..A.A...C..GC...... A...... T.T...... A...... TT.G...... GT.... CODO05 .....T....G....A.T...... ATA...... AA...A...... TC..G---TATAA..A.-...T...T..T.AT.A..C.....GTCAT.A..A.A.T.T...C..A..C...G...A...... T.C...... A...... CT.GA.A.....T.... CODO06 .....T...... A.T...... ATA...G..AA...A...... T...G---TATAA..A.-...T...C..T.AT.A..C..G..G.CAT.A..A.A.T.T...C..A..C...... A...... T.C...... A...... A.CT.G..A...... CODO07 .....T....G....A.T...... ATA...... AA...A...... T...A---.A.AA..A.-...T...T..T.AT.A..C..G..G.CAT.A..A.A...T..GC..A...... A...... T.C...... A...... CT.G..A.....T.... CODO08 .....T...... A.T...... ATA...... AA...A...... TG..A---TGTAA..A.-G..T...C..T.AT.A..C..G..G.CAT.A..A.A...T...C..A..C...... A...... T.T...... A...... CT.GC.A...... CODO09 .....T....G....A.T...... ATA...... AA...A...... TG..A---.A.AA..A.-...T...C..T.AT.A..C..G..G.CAT.A..A.AG..T..GCC.A..C...... A...... TGT...... A...... CT.GC.A.....T.... CODO1O .....T....G....A.T...... ATA...... AA...A...... T...A---TA.AA..A.-...T...T..T.AT.A..C.....G.CAT.A..A.A...T...C..A..C...... A...... T.T...... G...... CT.G..A.....T.... CODO11 ...C.T...... G.A.T.....CATC.....CAAG..G...... TC..A---TA-TA..A.....TC..TGC..AT.A.AC.....GTCAT.A..A.T.TCT..GC.....C...... A...... TT.T...... A...... C.T.GC...... G..... CODO12 .....T...... G.A.T..G..CA.A.....AAAGA.A...... T..A---AA-TA..A...... C..TACT.AT.A..CC.G..G.CAT.A..A.A.T.T..GC..A..C...... A...... T..C...... A...C...... TT.GC.A...... CODO13 .....T...... C.A.T.....CATT...... GAG...... TC..A---TG-.A..A.....TC..TA...A....AC.....GTCAT....A.A..CT..G...AG...... A...... CT.C...... A...... CGT.GC.A.....T.... CODO14 ...C.T....G..G.A.T....GCATC.....CAAG..A...... A---TA-.A..A.....TC.GCT.T.AT.A..C.....G.CAT.A..A.ATT.T..GCCGA..C...... AA...... C..T...... A.A...... GT.GC.A...... CODO15 .....T....G..G.A.T.....CATC.....CAAG..A...... A---TA-.A..A.....TC..C..T.AT.A..C.....G.CAT.A..A.ACA.T..GCCGA..C...... AA...... C..T...... A.A...... GT.GC.A.....T.... CODO16 .....T....G..G.A.T.....CA.C.....CAAG..A...... A---TA.AA..A.....TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A...... GT.GC.A...... CODO17 .....T....G....A.T.....CATC.....CAA...A...... A---TATAA..A.....TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A...... T.GC.A...... CODO18 .....T....G....ACT.....CA.C.....CAA...A...... A---TATAA..A.....TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A...... T.GC.A...... CODO19 .....T....G...... T.....CA.C.....CAAG..A...... A---TATAA..A.....TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A...... GT.GC.A...... CODO20 .....T....G....A.T.....CA.C.....CAA...A...... A---.ATAA..A.....TC..C..TGAT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A...... GT.GC.A...... CODO21 .....T....G....A.T.....CA.C.....CAA...A...... A---TATAA..A.....TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..C...... A.A...... GT.GC.A...... CODO22 .....T....G....ACT.....CA.C.....CAA...A...... A---TATAA..A.....TC.GC..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A...... GT.GC.A...... CODO23 .....T....G....ACT.....CA.C.....CAA...A...... A---TATAA..A.....TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A...... GT.GC.A...... CODO36 .....T....G...... T.....CA.C.....CGAG..G...... T...A---.A-TA..A.....TC..T..T.GT.A..C.....G.CAT.AC.A.AC..T..GC..A..C...... AA...... T..C...... A.A.C...... GT.GC.A...... CODO24 .....T....G....A.T.....CA.C.....CAA..GA...... A---TATAA..A.....TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A...... GT.GC.A...... CODO25 .....T....G....A.T.....CATC.....CAAG..A...... T..A---TA.AA..A.....TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... T..T...... A.A...... T.GC.A...... CODO26 .....T....G..G.A.T.....CA.C.....CAAG..A...... A---TATAA..A.....TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A...... GT.GC.A...... CODO27 .....T....G....A.T.....CA.C.....CGAG..A...... A---TATAA..A.....TC.GC..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A.C...... GT.G..A...... CODO28 .....T....G....A.T...A.CA.C.....CAAG..A...... A---TATAA..A.....TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A.C...... GT.G..A...... CODO29 .....T....G....A.T...A.CA.C.....CAAG..A...... A---TATAA..A.....TC..CT.T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A.C...... GT.G..A...... CODO30 .....T....G....A.T.....CA.C.....CAAG..A...... A---TA.AA..A.....TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A.C...... GT.G..A...... CODO31 .....T....G....A.T.....CA.C.....CAA...A...... G..A---TATAA..A.....T..GC..T.AT.A..C.....GTCAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A...... GT.GC.A.....T.... CODO32 .....T....G....A.T...A.CA.C.....CGAG..A...... A---TATAA..A.....TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A.C...... T.G..A...... CODO33 .....T....G....A.T...A.CA.C.....CAAG..A...... A---TATAA..A.....TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... AA...... C..T...... A.A.C...... T.G..A...... CODO34 .T...T....G....A.T.....CATC.....CAAG..A...... C..A---TATAA..A.....TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GT..A...... A...... C..T...... A.A.C...... GT.G..A...... CODO35 .....T....G..G..CT.....CATA...G.CAAG..G...... T...G---TA-TA..G.....TC..T..T.G..A..C.....G.CAT.AC.A.GC..T..GC..A..C...... AA...... C..C...... A.A.C...... GT.GC.A...... CMYS01 .....T...... A.T...... A.A...... AAG..G...... TC..A---.GTAA..A.-...TC.GC..T.AT.A..C.....G.C.T.A..A.A...T..GCC.A...... A...... T.T...... A...... TT.GC...... CMYS02 .....T...... A.T....G.ATA...... AAG..G...... TT..A---.ATAA..A.-...TC.GC..T.AT.A..C.....G.C.T.A..A.A...T..GCC.A...... A...... T.T...... A...... TT.GC...... T.... CMYS03 .....T...... A.T...... A.A...... AA...A...... TC..G---TATA...A.-...TC..C..T.AT.A..C.....G.C.T.AC.A.A.A.T..GCC.A...... A...... T.T...... G...... T..GC...... G.T... CMYS04 .....T....G....A.T...... ATA...... AA...G...... TC..A---.ATAA..A.-...TC..C..T.AT.A..C.....G.C.T.A..A.A...T..GCC.A...... A...... T.T...... G...... A.T..GC...... CMYS05 .....T....G....A.T....G.A.A...... AAG..G...... TC..A---.ATAA..A.-...TC..C..T.AT.A..C.....G.C.T.A..A.A.T.T..GCC.A...... A...... T...... A.C...... T..GC...... CMYS06 .....T....G....A.T...... A...... AAG..A...... T..A---.ATAA..A.T...TC..T..T.AT.A..C.....G.C.T.A..A.A.T.T..GCC.A...... A...... GT.T...... A...C...... T..GC...... CMYS07 .....T....G....A.T...... A...... AAG..A...... TT..A---.ATAA..A.T...TC..T..T.AT.A..C.....G.C.T.A..A.A.T.T..GCC.A...... A...... T...... A...C....A.T..GC...... CMYS08 .....T...... A.T...... A.A...... AAG..A...... T..A---.ATAA..A.-...TC..C..T.AT.A..C.....G.CAT.A..A.A...T..GC..A...... A...... T.T...... G...C....A.T..GC...... T.... CMYS09 .....T...... A.T...... A.A...... AAG..C...... TT..A---.A.AA..A.-...TC..C..T.AT.A..C.....G.CAT.A..A.A...T..GCC.A...... A...... T.T...... G...C....A.T..GC...... T.... CMYS10 .....T...... A.T...... A.A...... AAG..G...... TC..A---.A.AG..A.-...TC..C..T.AT.A..C.....G.CAT.A..A.A...T..GCC.A...... A...... T.T...... G...C....A.T..GC...... T.... CMYS11 .....T...CG....A.T...... A...... AAG..G...... C..A---.ATGA..A.-...TC..C..T.AT.A..C.....G.C.T.A..A.A.T.T..GCC.A...... A...... T.T...... A...... T..GC...... T.... CMYS12 .....T...... A.T...... A.A...... AAG..G...... TC..T---.ATAG..A.-...TC..C..T.AT.A..C.....G.CAT.A..A.A.T.T..GCC.A...... A...... T.T...... G...... T..GC...... T.... SIRE01 ...C.T...... A.T...AGCAT.G..C...AG..G...... TC..G-A.AC-A...A.....T...TTAA.T..A..CC....G..AAG.A.T.G...A...... AC.C...... A...... C...... GC...... T.G.....G...A... SIRE02 ...... A.T...AGTA..G..C...AG..G...... A...G-A..C-AT..A.....T...T.AG.T.CA..CC.G..G..AA..A.C.G...A...... AT.C...... A...... T...... GC...... G...... A... STUR01 ....TTA.GA.G...ACT.C..CTAAT..ATGC.A.ATA...... TT..TAT.AA.AA..A.T.G.TG.CCTAATA.CC.AC.C.TCGTAAAG..AT...T..-T..C.C....T..A.AA.C...... T.CTAACGAAACA..T...... CG.C...... A.A. STUR02 ....TTA.GA.G...ACT.C.ACCAAT..ATGC.A.ACA...... GCTCTAT.AATAA..A.T.GATG.CCAAGTA.CA.AC.C.TCGTAAA...ACGG.T..-T..C.C....T..A.AA.C...... T.C.AATGAAATA..T...... CG.C...... A.A. STUR03 ....TTA.GA.G...A.T.C.ACCAAT..ATAC.A.ACA...... TTTCTAT.AATAA..A.T.G.TG.CCAAGTA..C.AC.C.TCGTAAA...ACG..T..-C..C.C.T..T..A.AA.C...... T.C.AATGAAACA..T...... TG.T...... A.A. STUR04 ....TTA.GA.G...ACT.C.ACTAAT..ATGC.A.A.A...... A..CTATTAA.AA..A.T.G.TG.CCAAGTA.CA.AC.C.TCGTAAA...ACG..T..-T..C.C....T..A.AATC...... T.C.AACAAAATA..T...... CG.C...... A.A. STUR05 ....TTA.GA.G...ACT.C.ACCAAT..ATGC.A.ACA...... ACCCTAT.AATAA..A.T.G.TG.CTAAGTA.CA.AC.C.TCGTAAA...AC.A.T..-T....C....T..A.AA.C...... T.C.AATGAAACA.CT...... CG.C...... ATAA STUR06 ....TTA.GA.GT..ACT.C.ACCAAC..ATGC.A.ACA...... TTTATAT.AATAA..A.T.G.TG.TCAAGTA.CT.AC.C.TCGTAAA...ACG..T..-C..C.C....T..A.AA.C...... T.C.AATGAAACA..T...... CG.C...... A.A. SBIR01 T.G...A.GAGG...A.T.C....ATT....G..ACATG...... T..-AT.TC.A...A.CGG..C...TA..G.....C.C.T.G..TA.AC..CAC...T..GACCC.CCTCGA..A.CC...... G..CACACGATCCAT.AA....-.C.CCA....G.G..... SBIR02 T.....A.GAG....A.T.CC.-TATA....GCACTATG...... AT..-AT.ACAGT..G.CGGAT..G.TG..A..A..C.C.TCG.C.A..G..TACT..T..GATCG.CCTTGA..ATCC...... GT.CTTTTGATTGAT.TA....-.T.CGA....G.G.A... TELE01 ....TT..GAGG.....TGC...CA.....CG..A..G....ACA..C..------AA..A.C....CG.CAC.TT.A.G.CAT.TCG.AT.C.AA.G....G..GCCCC..C...... -...... T.A...... ACC...... C.CGA....T...A... TELE02 ....TT...AGG.....TGC...CC.....CG..AG.GA...AAATTT..------AA..A.C....CG.CAC.TT.A.G.CAC.TCG..T.C.AA.G....G..GCCCCT.C...... -...... TA...... CCC.C...... C.CGA....G...A... TELE03 ....TA.GGAGGT....TG....CA.....CG..A..GACCCAACTTT..------TA....C....CG.CGCT...A...CAG.TCG.A-.C.AGAGT.A.G..G..CA.T...... A...... TTTA...... A.C...... C.CGA...... G.A... TELE04 ....TT..GAGG.T...TG.TT.-AT.....G..AT.GA...GAGTT...------AA..A..G....G.GAC.T.TC.G.CC..TCG.AT.CAGAAGTT..G..TCACA.AC...... A...... C.TT...... CT..CACCA...TCGC...GG...A... TELE05 ....TT..GAG..T...TG.T..-AG.....G..AT.GA...ATTTTT..------TA...CC....AG..AC.T.TCA.AC.T.TCG.A..CAGA.GTTA.A..CCCCTTAC...... A...... TTTT...... CGC..ACCA....CGA....G..TA... TELE06 ....TT..GAGG.T...TG..ACTA...... G..AC.GA...AAAAT...------TT..T...... CG.CAC.C...C..CCC.TCG.A..T.GG.G....G..G..CT...... CA...... TTA...... C...... CGC....C...A... TELE07 ....TT..GAGG.....TGC...CAA.....G..A..GA...AACCA...------AA..A.C....CG.CGCTCT.A...C.T.TCG.AT.C.AA.GT.T.G..GCCCA...... -...... T...... C.C...... CGA...... AA... ELAS01 ....TT..G.G.T.TA.T.C.C.TC...... A..AGAGAAAGACCT....------AACCT.T...T.TG..CTCATAA..C.T.TCG.A..CA.GA.TGG.A.T..ACA..A...... A....TAGACCTA.GGA...... ATCT...... TGTGC..TGG.....A. ELAS02 ....TT..G...T.CA.T...C.CC...... A..A.AGAATGACCTTT..------AA.CT.C...T.TG..CTTAT.A.AC.T.TCGTATTCA.AA.TGG.A.T..ACA..A...... A.....CGAATTA.GGA...... ACCT...... TGTGC..TGG.....A. ELAS03 ....TT..G...T.CA.T...CTTC...... A....AGAAGTATCTA...------.AGTA.T...T..G..CTTATCA..C.C.CCG.A..CA.AA..GG.A.TT..CA..A...... A....TCTCACT..G.A...... ATCT...... TGTGC..T.GAC...A.

34 MarVer2

11112222222222333333333344444444445555555555666666666677777777889 26790123456789012345678901234567890123456789012345678901234589346 HOMI01 CCTAAGTATAC--TTCAAA------GGACATTTAACTAAAACCCCTACGCATTTATATAGACT HOMI02 ...... HOMI03 ...... HOMI04 ...... PINN01 .....A..AT..ACC.---...... CA.A.CAAT..AT...TTAAC.TAA.A.C...-....A PINN02 .....AC.ACA.CC..---...... AA....AC.TAA.T.TGTA.A.AAATA.ATCT-...TA PINN03 .....A..A...TC.A---...... CA.GC.AC.TAA--....AAC..AA.ACATAC-...TA CODO01 ...... CA.CAG.--G.AACGCCCAAT.CAC...T.T.TG..GAGCGC-CCCC.C.-....A CODO02 ...... CCA.CAGT--..AAGCTCCAAT..A.C...CC.CG.TAAATAA-T..C.C.-....A CODO03 ...... C..TA.T.A.--C.AAGCCTAA.CT.GC....AC.TG..AAACA.-CCC.GC.-....A CODO04 ...... C.CCA.CCG.--T.GAGCCC.A.CTTGC....AC.CG.TAAGCAA-GCA.GC.-....A CODO05 .....AC..TA.CCG.--T.AAGCCCTA.CT.GC....AC.CG..AAGCAA-CCC.GC.-....A CODO06 ...... C.GTA.TCG.--C.AAGCTCTA.CT.GC....AC.CG..AAGCAA-C.C.GCG-....A CODO07 ...... C..TA.TCA.--T.AAGCTC.A.CT.GC....AC.CG.TAAGTA.-C...GCG-...TA CODO08 ...... C..TA.CCG.--T.AAGCCCTA.TT.AC....AC.TG..AAGCAA-C.C.GCG-...TA CODO09 ...... C..TA.TCA.--T.AAACTC.A.CT.AC....AC.TG..AAATAA-C...GC.-....A CODO1O ...... C..TA.TCA.--T.GAGCTC.A.CT.GC...TAC.TG..AAGTAA-C...GCG-....A CODO11 ...... CCA.CAG.--T.AGGCCCAA.TTCCC....CC.TG..AAG-.AAC.AC.CT-....A CODO12 ...... CA..AG.--..AGGCCCTA..TCAC....A..TG..AAG-.AAC.CC.C.-....A CODO13 ...... GACA.CGA.--..AAGCCCTA.CTTAC....AC.TG.TAAGT.AACCAC.CG-....A CODO14 ...... GCA.TAG.--G.AAGCCCTA..TTAC....CC.TG.TAAG-.AAG.A..C.-....A CODO15 ...... GCA.TAG.--..AAGCCCTA..TCAC....CC.TG.TAAG-.AAG.A..C.-....A CODO16 ...... GCCA.TAG.--T.GAGCCTCA..TCAC....CC.CG.TAAG-.AAGCG..C.-....A CODO17 ...... GCCA.TAG.--..AAGCCCCA..TTGC....CC.TG.TAAG-.AAGCG..C.-....A CODO18 ...... GCCA.TAG.--..AAGCCCCA..TTGC....CC.TG.TAAG-.AAGCG..C.-....A CODO19 ...... GCCA.TAG.--..AAGCCCCA..TTGC....CC.TG.TAAG-.AAGCG..C.-....A CODO20 ...... GCCA.TAG.--G.AAGCCCCA..TTGC....CC.TG.TAAG-.AAGCG..C.-....A CODO21 ...... GCCA.TAG.--..AAGCCCCA..TTGC....CC.TG.TAAG-.AAGCG..C.-....A CODO22 ...... GCCA.TAG.--..AAGCCCCA..TTGC....CC.TG.TAAG-.AAGCG..C.-....A CODO23 ...... GCCA.TAG.--..AAGCCCCA..TTGC....CC.TG.TAAG-.AAGCG..C.-....A CODO36 ...... CA.CAG.--..GAGCCTTA..TTAC...TCCCTG.TAAG-.AAGCA..C.-....A CODO24 ...... GCCA.TAG.--..AAGCCCCA..TCAC....CT.TG.TAAG-.AAGCG..C.-....A CODO25 ...... GCCA.TAG.--..AAGCCCCA..TCAC....CC.TG.TAAG-.AAGCG..C.-....A CODO26 ...... GCCA.TAG.--..GAGCCCCA..TCAC....CC.TG.TAAG-.AAGCG..C.-....A CODO27 ...... GCCA.CAG.--TAAAGCCTCA..TCAC....CCGTG.TAAG-.AAGCG..C.-....A CODO28 ...... GCCA.TAG.---AAAGCCCCA..TCAC....CC.TG.TAAG-.AAGCG..C.-....A CODO29 ...... GCCA.TAG.---AAAGCCCCA..TCAC....CC.TG.TAAG-.AAGCG..C.-....A CODO30 ...... CCA.TAG.--.TAAGCCCCA..TCAC....CC.CG.TAAG-.AAGCG..C.-....A CODO31 ..C.....CCA.CAG.--..AAGCCCCA..TCGC....CC.TG.TAAG-.AGGCG..C.-....A CODO32 ...... CCA.TAG.---AAGG.CCCA..TCAC....CC.TG.TAAG-.AAGCG..C.-.A..A CODO33 ...... CCA.TAG.---AAAG.CCCA..TCAC....CC.TG.TAAG-.AAGCG..C.-.A..A CODO34 ...... GCCA.TAG.--..AAGCCCTA..TTGC....CC.TG.TAAG-.AAGCG..C.-....A CODO35 ...... CA.CAG.--..GAGCCCTA..TCAC...TCCCTG.TAAG-.AAGCA..C.-....A CMYS01 ...... CC..TAG.--..AAGCCCCA.TTCG...... C.CG..AAGCAA-TCA..CG-....A CMYS02 ...... CC..TAG.--..AAGCCCCA.TTCG.....CCCCG.TAAGCAA-TCA..CG-....A CMYS03 ...... C...CCG.-.T.AAGCCCCA.T.CA.....CC.GG..AAGCAATCCA..CG-....A CMYS04 ...... CC..CAG.T.-.AAGCCCCA.T.CA.....CC.GG..AAGCAATTCG..C.-....A CMYS05 ...... CC..CAG.T.-.AAACCTCA.TTCA....T.C.GG..AAGCAATT.A..CG-....A CMYS06 ...... CC..TAG.T.-.GAGCCCCT.TTCA...... C.GG..AAGCAATCCA..CG-....A CMYS07 ...... CC..TAG.T.-.AAGCCCCA.T.CA...... C.GG..AAGCAATC.A..CG-...TA CMYS08 ...... CC..TAG.T.-.AAGCCCCA.TTCG...... C.GG..AAGCAATC.A..CG-....A CMYS09 ...... CC..TAG.T.-.AAGCCCCA.TTCG...... C.GG..AAGTAATC.A..CG-....A CMYS10 ...... CC..TAG.T.-.AAGCCCCA.TTCG...... C.GG..AAGCAATCCA..CG-....A CMYS11 ...... CC..CAG.T.T.AAACCCCA.TTCG...... C.GG..AAGCAA-T.A..CG-....A CMYS12 ...... CC..CAG.T.-.AAGCCACA.TTCA...... C.GG..AAGCAA.T.A..CG-....A SIRE01 .....A..CC.ATAAT---...... TAT.T.ACC..A..C.ATTT..ATA.CG...G-....A SIRE02 ...... T.ATAA.---...... CATTCCACC.TAT.TGAT....ATAGCA...G-....A STUR01 .....AA..CAAC.AA---...... AC.ACCA...A-T.....A.A..AA.CA-.AT-.TG.A STUR02 .....A---CAATC.A---...... AC.ACCA...A.T.....A.A..AA.CA..AT-.TGTA STUR03 .....AA..TAACC.A---...... AT.ACCA...A-T.....ATA..AA.CA-.AT-.TG.A STUR04 .....A--..AACC.A---...... ATCA.CAAT.AAT.....ATA..AA.CA.TAC-.TGTA STUR05 .....A--..AACC.A---...... AT.ACAA...A-T.....A.A..AA.CA..AT-.TGTA STUR06 .....AA.C.ACCCAA---...... CC.ACCA...A-T.....ATA..AA.CA-.AT-.TG.A SBIR01 ....CAAGCCA..CAA---.ATACCCCAT.AC.AAT..CCCTG..GGCCAA------.TGTA SBIR02 ....CAAGCCA..CCA---.ACA.ACCAT.AT.AAT.AACCTTATGGCTAA------.TGTA TELE01 T.C...CT...CAA.T---...... TATATA.CT.AA------A.GCTTT.AC.GCGAG.G.. TELE02 TTC...CTC..CCAC.---...... CATTCAAC.TAACT..TTA.CCATA.AC.GC.AGAG.. TELE03 T.C....C...CAA.A---...... CCGT..AACTAAT.CG.TA----AA.C..GC.AG..T. TELE04 T.C..---C.ACTAC.---...... TATA.AAATGTA.CT.A.A.A.TATTCGCCGCAG.G.. TELE05 T.C..CA.CC.TAA.T---...... CACG.G.A.TT...CC.TA.AC---..G..A.AG.G.. TELE06 T.C....TC.ATTAA.---...... CCTTC.AACTAAG...TTAACCGAA-----C.AG.G.. TELE07 ..CG..C-CCTAAACT---...... CACTTAACT.AA------A.CCTAT.A..GCGAG.GA. ELAS01 T....TA.ACAACC.A---...... CTT.TC.AT.AAC.T..TT------C..C.ACA..G.. ELAS02 T....AATA.TTTAC.---...... CTTTT.CAT.AAT.C.TTT------C.CC.ACA..G.. ELAS03 T....-A.A.AATC.A---...... CTTAT...T.A.T..TGAA------.AA.C.CA..G..

MarVer3 1111111111111111111111111111111111111111111111111111111111111111111111111111111112222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222223333333333333333333333 1122222233333333334444444444555555555566666666667777777777888888888899999999990000000000111111111122222222223333333333444444444455555555567777888888888999999990000000000111111111122222222223333333333444444444455555555666666666777777777788888888999999990000000000111111222235 5804578901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567810569013456789012467890123456789012345678901234567890123456789012345678901245679123456789012345678901234569234567890123456789012569135843 HOMI01 AGGTATTTATTAATGC--AAACAGTAC------CTAACAAACCCA---CAGGTCCTAAACTACCAAACCTGCATTAAA------AATCCTCAGCGAACCCA-ACTCGAGCAG------TA------CATGCTAAGACTTCACAGTAAGCGAACTACTAT-----ACTCAATTT---AATA-ACTT------CCCGAATTC HOMI02 ...... HOMI03 ...... HOMI04 ...... PINN01 ...C...A.C.T.CC.AA------TCAGAACCTACT.A.GTC....A.AACGG.A.AAA.T.ACT.TGC..A..GG.C.GC...... AT....AC..AA...... TGA...... T-AAA.CTAGACAT...... ATA..T.---...... CA.T...... A..A.CCT...... T...... PINN02 ...... A.C...CC.AA------T..AGAATTTATTC..TT....A.TATGG.ACAAAGT.A.T.T.C.-G..GGAC.GC...... A.....TT.G.A.....C....TGA...... T-AAA.CTAGACAT..T....A...... ---...... CA.A...... A..A.TTT...... T...... PINN03 ...... A.C...CT.AA------...AGAGCAAAT.C.GTC....A.CA.GG.AATAA.....T.T.C.-A..AG...GC...... AT....AC..AA...... TGA...... T-AAACT.AGACAA...... T.CCAC---...... CACT...... A..A.TTT...... T...... CODO01 ...C...A..C..CC.AA-T.ACCC...... AACCTC..A....CCA...GATA...AA.A.TT.TA..GG..G.C...... T.....C..AAA.CC...... TGA...... --...... TTAAACCTAGGCCT....C....AT.AC---...... CACTT...... AG.CT...... T...... CODO02 ...C...A..C.GCC.AA-..AGCC-A...... A.T.T..A....CCA...GATA...AA..TTC.TA..GGC.G.C...... TT..AAAC.C...... TGA...... -T...... T.GAGCCTAGGCCT.T..C....AT.A.---...... CACTT...... A..TA...... T...... CODO03 ...... A.CC..CC.AA--.A.C..T...... AA.T.T..A..G.CCA...GATA.C.GA..TTT.TA..GG..G.C...... TT.....C..AAA...... TGA...... -T...... T.AAACCTAGGCC...G.C...T.T.A.---...... CACTT...... A..A..TT...... T...... CODO04 ...... A..C..CT.AA--.A.C...... AA.C.T..A....CCA...GATA.C.AAG.TTT.TA..AG..G.C...... T....TC..AAA..G...... TGA...... -T...... T.AAACCTAGGC...... C...T.T.A.---...... CACTT...... A..AT.TT...... T...... CODO05 ...... A.CC..CC.AA.T.A.C..T...... AA.T.T..A....CCA...AATA.C.AA...TT.TA..GG..G.C...... TT....TC..AAA...... TGA...... -T...... T.AAACCTAGGC.T....C...TTTCA.---...... CACTT...... A..AT.TT...... T...... CODO06 ...... A.CC..CC.AA...ATA--T...... AA.TTT..A....CCA...AATA.C.AA..TTT.TA..GG..G.C...... TT....TC..AAA...... TGA...... -T...... T.AAACCTAGGCCT....C...T.TTAA---...... CACTT...... A.T.T.TT...... T...... CODO07 ...... A.CC..CC.AA...TTA--T...... AA.T.TT.A....TCA...AATA.C.AA.TTTT.TA..GG..G.C...... T....TC..AAA...... TGA...... -T...... T.AAACCTAGGC.T....C...TACTA.---...... CACTT....AAA.G.T.TT...... T...... CODO08 ...... A..C..CC.AA...TTA--...... AA.T.T..A....CCA...GATA.C.AA...TT.TA..GG..G.C...... TT.....C..AAA...... TGA...... -T...... T.AAACCTAGGC.T.T..C...TACTA.---...... CACTT...... A.T.T.TT...... T...... CODO09 ...... A.CC..CC.AA...ATC.-T...... AA.T.TT.A....CCA...AATA.C.AA..TTT.TA..GG..G.C...... T....TT..AAA...... ATGA...... -T...... T.AAACTTAGGCCT....C...TATCA.---...... CACTT....AAA.-.T.TT...... T...... CODO1O ...... A.CC..CC.AA...ATCA-T...... AA.TACT.A....CCA...AATA.C.AA.TTTT.TA..GG..G.C...... T....TC..AAA...... ATGA...... -T...... T.AAACCTAGGCCT.T..C...TATCA.---...... CA.TT....AAA.-.T.TT...... T...... CODO11 ...C...A.CC..CC....TTA.C...... AA....--AA.CACCAT..GATA.C.AA..TTT.....GG..G.T...... TT.....C.GAAA...... TGA...... T...... ATAAGCCTAGGCCT.T..C.....TGAC---...... CACTT...... GT.TT...... T...... CODO12 ...C...A.....CC...... T..CCA...... A...T..TA.CATCA...GAT-.T.AA..TTT.TA..GG....C...... T.....C..AAA..T...... TGA...... T...... A-AAGCCTAGGCCT....C....AT...---...... CACTT...... A.TTT...... T...... CODO13 ...C...A..C.GCC...... AGT..T...... AA.TTT.CA....CCA...GATA.C.AA..T.T.AT..AGC.G.C...... T....TC..AAA..C...... TGA...... T...... A-AAGCCTAGGCC..T..C....ATGAC---...... CACTT...... AG.T...... T...... CODO14 ...... A..C..CC...... A.AC...... AA....GCA....CCA...GATA.C.AA.TTTT..A..GG..G.C...... T....TC..AAA..C...... TGA...... T...... A-AAACCTAGGCCT.TG.C...T.T.A.---...... CACTT...... AT.T...... T...... CODO15 ...C...A..C...C...... AGTC.A...... AA..T..CA....CCA...GATA.C.AA.T.TT.TA..GG..G.C...... T....TC..AAG..C...... TGA...... T...... A-AAACTTAGGCCT.T..C...T.T.A.---...... CACTT...... AC.T...... T...... CODO16 ...... A..C..CT...... A..C.T...... AA..T.GTA....CCA...GATA.C.AAG.TTT.TA..AG..G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGCCT....C...TAT.G.---...... CACTT...... A..TT...... T...... CODO17 ...... A..C..CT...... A..C.T...... AA..T..TA....CCA...GATA.C.AAG.TTT.GA..AGC.G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGCCT....C...TAT.G.---...... CACTT...... A..TT...... T...... CODO18 ...... A..C..CT...... A..C.T...... AA..T..TA....CCA...GATA.C.AAG.TTT.GA..AGC.G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGCCT....C...TAT.G.---...... CACTT...... A..TT...... T...... CODO19 ...... A..C..CT...... A..C.T...... AA..T..TA....CCA...GATA.C.AAG.TTT.GA..AGC.G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGCCT.T..C...TAT.G.---...... CACTT...... A..TT...... T...... CODO20 ...... A..C..CT...... A..C.T...... AA..T..TA....CCA...GATA.C.AAG.TTT..A..AGC.G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGCCT....C...TAT.G.---...... CACTT...... A..TT...... T...... CODO21 ...... A..C..CT...... A..C.T...... AA..T..TA....CTA...GATA.C.AAG.TTT.GA..AGC.G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGCCT.T..C...TAT.G.---...... CACTT...... A..TT...... T...... CODO22 ...... A..C..CT...... A..C.T...... AA.....TA....CCA...GATA.C.AAG.TTT.AA..AGC.G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGCCT.T..C...TAT.G.---...... CACTT...... A..TT...... T...... CODO23 ...... A..C..CT...... A..C.T...... AA..T..TA....CCA...GATA.C.AAG.TTT.GA..AGC.G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGCCT.T..C...TAT.G.---...... CACTT...... A..TT...... T...... CODO36 ...... A..C..CT.AA...A.TCGT...... AA....GTA....CTA...GATA.C.AA.TTTT.TA..GG..G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACCTAGGCCT.T..C...T.T.GC---...... CACTT...... AT.TT...... T...... CODO24 ...... A..C..CT...... A..C.T...... AA..T..TA....CCA...GATA.C.AAG.TTT..A..AGC.G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGC.T..G.C...TAT.G.---...... CACTT...... A..TT...... T...... CODO25 ...... A..C..CT...... A..C.T...... AA..T..CA....CCA...AATA.C.TAG..TT.TA..AG..G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGC.T....C...TAT.AC---...... CACTT...... A..TT...... T...... CODO26 ...... A..C..CT...... A..C.T...... AA.....TA....CCA...GATA.C.AAG..TT.TA..AG..G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGCCT....C...TAT.G.---...... CACTT...... A..TT...... T...... CODO27 ...... A..C..CT...... A..C.T...... AC..T..TA....CCA...GGTA.T.AAG.TTT.TA..AGC.G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGCCT.T..C...TAT.T.---...... CACAT...... G..TT...... T...... CODO28 ...... A..C..CT...... A..C...... AA..T..TA....CCA...GATA.C.AAG.TTT.TA..AGC.G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGCCT.T..C...TAT.G.---...... CACAT...... A..TT...... T...... CODO29 ...... A..C..CT...... A..C.T...... AA..T..TA....CCA...GATA.C.AAG.TTT.TA..AGC.G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGCCT.T..C...TAT...---...... CACAT...... A..TT...... T...... CODO30 ...... A..C..CT...... A..C.T...... AA..T..TA....CCA...GATA.T.GAG.TTT..A..AGC.G.C...... T....TT..AAA..C...... TGA...... C...... A-AAACTTAGGCCT.T..C...TAT.G.---...... CACAT...... A..TT...... T...... CODO31 ...... A..C..CT...... A..C.T...... AA..T..TA....CCA...GATA.C.AAG.TTT..A..AGC.G.C...... T.....T..AAA..C...... TGA...... T...... A-AAACTTAGGCCT....C...TAT.AC---...... CACTT...... A..TT...... T...... CODO32 ...C...A..C..CT...... A..C.T...... AA.....TA....CCA...GATA.C.AAG.TTT.TA..AGC.G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGCCT.T..C...TAC.G.---...... CACAT...... A..TT...... T...... CODO33 ...... A..C..CT...... A..C.T...... AA.....TA....CCA...GATA.C.AA..TTT.TA..AGC.G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGCCT.T..C...TAC.G.---...... CACAT...... A..TT...... T...... CODO34 ...... A..C..CT...... A..C.T...... AA..T..CA....CTA...GATA.C.GAG.TTT.TA..AGC.G.C...... T....TT..AAA..C...... TGA...... T...... A-AAACTTAGGC.T.T..C...TAC.A.---...... CACTT...... A..TT...... T...... CODO35 ...... A..C..CC...... A.TC...... AA....GTA...CCTA...GATA.C.AA.TTTT.TA..GG..G.C...... T.....C..AAA..C...... TGA...... T...... A-AAACCTAGGCCT.TG.C...T.T.G.---...... CACTT...... GT.TT...... T...... CMYS01 ...C...A..C..CC.AA-..ATCA-T...... AACCTC..A....CCA...GATA.C.AA.TTTT.TA..GGC.G.C...... T.....C..AAA..C...... TGA...... -T...... T.AAGCCTAGGCC..T..C....AT.A.---...... CACTT...... C.TT...... T...... CMYS02 ...C...A..C..CC.AA-..ATCA-...... AACCTT..A....CCA...GATAGC.AA..TTT..A..GGC.G.C...... T.....C..AAA..C...... TGA...... -T...... T.AAGCCTAGGCC.....C....AT.G.---...... CACTT...... C.TT...... T...... CMYS03 ...C...A.....CC.AA-..ATCA-T...... AATC.C..A....CCA...AATA.C.AA.TTTT.TA..GG....C...... TT....TT..AAA..C...... TGA...... -T...... T.AAGCCTAGAC...T...... AC.AC---...... CACTT...... C.T...... T...... CMYS04 ...C...A..C..CC.AAT..ATCA-A...... AACCTT..A....CCA...GA.A.C.AA.TTTT.TA..GGC.G.C...... T.....C..AAA..C...... TGA...... T...... T.AAACCTAGGC...T..CG..TAT.A.---...... CACTT...... C.TA...... T...... CMYS05 ...C...A..C..CC.AAT..ATCA-...... AACCTT..A....CCA...GA.A.C.AA.TTTT.TG..GG..G.C...... T.....C..AAA..C...... TGA...... -T...... T.AAACTTAGGCC..T..C...T.T.A.---...... CA.TT...... C.TT...... T...... CMYS06 ...C...A..C.GCC.AA-.TATTA-T...... AACCTT..A....CCA...GATA.C.AA.T.TT.TA..AGC.G.C...... T....TC..AAA..C...... TGA...... -T...... T.AAACCTAGGCC..T..C...TAT.AC---...... CACTT...... C.CT...... T...... CMYS07 ...C...A..C..CC.AA-.TATCA-T...... AACCTT..A....CCA...GATA.C.AA.T.TT.TA..GGC.G.C...... T....TC..AAA..C...... TGA...... -T...... T.AAACCTAGGC...T..C...TATGAC---...... CACTT...... C.TT...... T...... CMYS08 ...C...A..C..CC..A...ATCA-T...... AACCTT..A....CCA...GATA.C.AA.TTTT.TA..GGC.G.C...... TT....TC..AAA..C...... TGA...... -T...... T.AAACTTAGGC...T..C...TAT.AC---...... CACTT...... C.TT...... T...... CMYS09 ...C...A..C..CC.AA...ATCA-T...... AACCTT..A....CCA...GATA.C.AA.TTTT.TA..GGC.G.C...... T.....C..AAA..C...... TGA...... -T...... T.AAACTTAGGC...T..C...TAT.AC---...... CACTT...... C.TT...... T...... CMYS10 ...C...A..C..CC.AA...ATCA-T...... AACCTT..A....CCA...GATA.C.AA.TTTT.TA..GGC.G.C...... T.....C..AAA..C...... TGA...... -T...... T.AAACTTAGGC...T..C...TAT.A.---...... CACTT...... C.TT...... T...... CMYS11 ...C...A..C..CC.AA-..ACCA-T...... AACCTT..A....CCA...GATA.C.AA...TT.TA..GGC.G.C...... T....TC..AAA..C...... TGA...... -T...... T.AAACTTAGGCC..T..C...TAC.A.---...... CACTT...... C.CT...... T...... CMYS12 ...C...A..C..CC.AA-..ATCA-...... AACCTT..A....CCA...GATA.C.AA.TTTT.TA..GGC.G.C...... T.....C..AAA..T...... TGA...... -T...... T.AAACTTAGGC...T..C...T.T.A.---...... CACTT...... C.T...... T...... SIRE01 ...A...A.C...CTTAA------T...... AATATAA.TATCCA..CTACGG.C.TAACCTAGCATTTTTA..AG....C...... TT....AC..GAA...... ATGATA...... C...... T-GA..C....C...... TCTG.G.--...... CACTT...C...--A.TTA...... T...... SIRE02 ...A...A....G.T.AA------...... CA....A..AC.TATACTACGG..-..ACCCAACAGTTTAA..A.C...C...... TT....AC..AAA...... ATGATA...... C...... T-AA.CC...... A.....G..TCC.TA.--...... CACTT...C...--A.TTA...... T...... STUR01 G.A..A.ACAG.TCAACT.C...TCCA...... ACCT..T.TA....GGAC.TA...C.A..T.G..T..ATCC.T...... TT...... TA...AA...... AA.AAGAACAA..C.T...... T.AACCTAGAC.T.A.T....T.CCAA----C.....GGCA..A...... T.T...... T..AC.CC. STUR02 G.A..A.ATAA.TCAACT.TT--A..T...... T..C..CC.TA....AGAC.TA...TTA..T.GTT.-TG..CC.T...... T.TT....TA...AA...... AA.AAGAACATATT.T...... T.AACCTAGA..T.A.C....T.CCAA----C.....GGCA..A...... T.T...... T..AC.CC. STUR03 G.A..A.ACAG.TCAACT.TCT.TAC...... ACT..CT.TA....GGACCTA...CTA..T.GTA..TG.CCT.T...... T.T.....TA...AA...... AA.AAGAATACA.C.T...... T.AACCTAGACC..A.T....T.CCAA----C.....GGCA..A...... T.T...... T..AC.CC. STUR04 G.A..A.ATAA.TCAACT.TTT.CAC...... ACC..CT.TA....AGAC.TA...TT...T.GTT..TG..CC.T...... T.TT....AA...AA...... -AA.AAGAACATATC.T...... T.AACCTAGACC..A.C....T.CCAA----C.....GGAA..A...... T.T...... T..AC.CC. STUR05 G.A..A.ATAA.TCAACT.TC--AC...... T..CT.CC.TA....AGAC.TA...CT...T.GTT.-TG..CC.T...... T.TT....TA...AA...... AA.AAGAACATACT.T...... T.AACCTAGA..T.A.C....T.CCGA----C.....GG.A..A...... T.T...... T..AC.CC. STUR06 G.A..A.ACAA.TCAACT.TC-.TACT...... ACC..CC..A....GGACCTA...CTA..T..TA..TG.CCT.T...... T.T.....TA...GA...... AA.AAGAACATA.C.T...... T.AACCCAGACC..A.T....T.CTAA----C.....GGCA..A...... T.T...... T..AC.CC. SBIR01 G.AA.AA.CAGC.GC.AC------.CGACACAAAAT.....CT.T.AC...AG.C..ACC.T.AC.AGGCA...GCCCGC...... TT....T..AA...AA..T...A.AA.CAAGACCTCCCCT...... T.AACC.AGAGCA..CC....TACTAA----C...... G.A.CCAC...... C.A...... T.C.CC. SBIR02 G.AA.AA.CAGCGAC.AC------.ACATACAAACCTA.C.CT.--.C...AG.C..ACT.C.CT.A..CT...GCCCGC...... TT....T..AA..TAG..T...A.AA.TAAGACCACATCT...... T..ACC.AGAGCA..CC..T.TACTAA----C...... G.A.CCAC...... C...... T.T.C.CCT TELE01 ....-GAC.CC..G..AT------...... ------...------A.CATG.C.....ACACCCCTAAACAAAGGACTAAACCAAATGAAT..CAT.ACCCCCAT.GTCT.G.G.AAT.AAA.AC.CACGTGGAATGGGAGTAC...... C..CTCCT.CAACC.AGAGCTGAGC.T.AAA.CAG.A...CTGACCAAT..--....----.GGCA...ACGCC.T...CG... TELE02 .....GAC.C.G.GC.AT------...... ------...------A.CA.G.T.....ACACCCCCAAACAAGGGACTAAACTTATTGAAATCATT.GGCCGTAT.GTC..ATG.AAC.AAA.AC.CACGTGGAATGGGAGCACA...... CTACTCCT.CAG.C.AGAGC....C.T.CAA.CAG.A.T.CTGACCAAT..C.....----.GGCA...ACGCC.T...C.... TELE03 .A..-GAC.CA-.G.GAG------...... ------...------ACTACC.T.....TTACCCCTGGATAAAGGGCAAAAGCTAAAGGTAGCCCT...CCCCAGCGTCT.G.G.TA...AA.AC.CATGTGGAATAGGAGCACCC...... CTCCTTCAACC.AGAGCTC.GC.T..GA.CAG.ACT.TTGACCAATC.A.....----.GGCA...TCGCC.T..ACG... TELE04 ....-GACGC...CCAAC------...... ------...------.CCG---...... AGCACCCTACACCAGGGCCCAAACAACGTGGTT....TTGGCACAACCGTC..G.GAGT.G.A..GCTCGAGTGGATGGGGGAAACCC...... T.AAACC.AGAGCT.AGC.G..TC.CAA.AC..TTGACCAA...-.....----.GGC..TCATGCC.....C.... TELE05 ....-GAC.C.-.GC.AA------...... ------...------...TGA...G...CGACTGAACTGAACAAGTCCTAAATACCCGCAGCCTTATGGTAATGTAGTCA.A.GAGA.GTAA.GCTCAAGCAGACCGGGAAAACCC...... TTAAGCCGAGAG.TGA.C.T...CGCAA.A.T.TTGACTGAT.T--....----.GG..GAAAAACC.TT.AC.... TELE06 ....-GAC.CC-.G..AG------...... ------...------A.CACG.C.....GTAACCTTGAATTAACAAGTAAAAACGCAGTGACCCCTAGCCCATAT.GTCT.G.G.GA...AA.GC.CATGTGGACTGGGGGCAC.G...... C..CAACC.AGAG...A.C.T..TACCAG.A.T.CTGACCAAA..-.....----.GGCA.TCACGCC.T...CG... TELE07 ....-GACG.C..A..AG------...... ------...------A.CATG.T.....ACACCCCCAAATAAGGGACCAAACTAAATGACC..CCT.GCTTTAAT.GTC..ATG.AAT.AAA.AC.CACGCGGACTGGGAGCAC..AACCCCTTCTTTACCTCTCCTCC..CAACT.AGAGCT.AGC.T.CTA.CAG.A...CTGACCAATC.-.....----.GGCA...ATGCC.T...C.... ELAS01 ...C-AAC.CAT.AATTA------...... ------...------A.TATG...CCTTATACCTCCCAGGATATAAACAAAACATACAACACTTCTAATTTAACT.GT.TTGAG.AA.G.AA.TC.CTC.T.GACTGAGTACT.--AAGTACT...... T.AAAATTAGAA.G.A.T.T.ATAGTAA.A...TTATCGAAA..-.C...----..GGA...TTACCTT.TAC.... ELAS02 ...C-AAC.C.T.AATTA------...... ------...------A.TATG...CCATCCATTTCCCAGGAAATAAACAAAATATACAACACTTCTAATTTAACT.GT.TTAAG.AA...AA.TC.CTC.T.GATTGAGTACT.--AAGTACT...... T.AAAATTAGAA.G.A.T.T.TTA.TAA.AC..TTACCGAAA..-.....----..GGA...TTTCCTT.TAC.... ELAS03 ...C-AAC.CAT.AATTA------...... ------...------A.TATG....TTAACCACTCTACGGATATAAACAGAA.ATACAATACTTTTAATTTAGCT.GT.TTAAG.AA..TTA.TC.CTT.T.GACCGAGTACTC.CAAGTACT...... T.AAAATTAGAAC..A.T.T.TTA.TAA.AC..TTATCGAAA..-.....----..GGA...TTTCCTT.TAC....

36 Online Resource 4 List of 657 GenBank entries used in this study to assess amplicons’ polymorphism at the three presented loci. NB 22 Pusa capsica sequences are not shown as unpublished yet.

CODO Ziphidae (n=50) Phocoenidae (n=18) Tursiops spp (n=24) Stenella spp (n=176) Orcinus orca (n=151)

KC776688 - KC776691 KP170488 NC_022805 EU557096 - EU557097 KR180337

KC776693 - KC776698 KC777291 KF570317 KX857264 - KX857266 GU187155 - GU187164

KC776700 - KC776712 KR108307 - KR108308 KF570323 KX857268 - KX857281 GU187166 - GU187219

KC776715 - KC776717 KT852939 KF570333 KX857284 - KX857298 HQ405752

KF032860 - KF032861 KU886000 KF570335 KX857300 - KX857301 KF164610

KF032863 KX650869 - KX650872 KF570344 - KF570345 KX857303 - KX857306 KF418372 - KF418376

KF032866 - KF032868 MF669488 - MF669489 KF570349 KX857309 - KX857315 KF418379 - KF418393

KF032870 - KF032871 MF669491 KF570353 KX857318 - KX857321 KR180299 - KR180300

KF032873 MF669493 - MF669494 KF570360 KX857323 KR180303 - KR180325

KF032876 NC_005280 KF570367 KX857325 - KX857326 KR180327

KF032878 NC_021461 KF570374 KX857330 - KX857339 KR180330 - KR180332

KF981442 NC_026456 KF570377 KX857341 - KX857342 KR180334 - KR180336

KR534596 KF570386 KX857344 KR180338 - KR180367

KY364702 KF570388 - KF570389 KX857347 - KX857349 MH062792

MG000980 KT601194 KX857351 - KX857416 NC_02388

NC_005273 - NC_005274 KT601196 KX857418 - KX857422

NC_021434 - NC_021435 KT601203 KX857424 - KX857427

NC_021974 KT601207 KX857430 - KX857438

NC_023830 MF669485 - MF669486 KX857440 - KX857445

NC_027593 NC_012058 - NC_012059 KX857447 - KX857451

NC_034348 KX857453 - KX857460

NC_036997 NC_012051

NC_012053

NC_032301

CMYS PINN SIRE Balaenoptera Phocidae (n=16) Otariidae (n=63) Odobenidae (n=2) Dugongidae (n=2) physalus (n=152) NC_008426 - NC_008441 NC_008415 - NC_008477 AJ428576 NC_003314

KC572708 - KC572860 NC_004029 AY075116

37 Online Resource 5

HTS species detection differences between loci MarVer1 and MarVer3, as inferred from controlled environment eDNA samples collected in 6 tanks of the Aquarium of Genoa (July 2018). For each tank, two sets of data are shown: one referring to the number and composition of detected species, where the species scoring more than 100 reads were considered as “detected” (top part of each section), and one showing the number of HTS reads obtained (bottom part of each section, with related pie graphs). Note that all data refer to 6 eDNA- samples replicates collected in each tank. The top part of each table section shows 7 categories: 1) “DETECTED/TOTAL vertebrate species” indicates the number of vertebrate species correctly assigned over the total number of species known to be present in the tank as main occupants (“*” indicates two instances -Shark and Rocky shore” tanks- in which one of the species detected was found but with a read count lower than 100); 2) “TANK FEED” indicates the number of molecularly detected species that were not present in the tank as living species, that however were used as food source provided to the tank hosts (thus their DNA traces originating from both hosts’ faeces and food left-overs could be found); 3) “OVERAL FEED CONTAMINATION” indicates the number of molecularly detected species that should not be in that specific sampled tank, but that are found elsewhere in the Aquarium structure, used as food source in other tanks (food buckets and/or operators’ boots or wetsuit are likely to have provided a vehicle for contamination); 4) “CONTAMINATION FROM OTHER TANKS AND HUMANS” indicates the number of molecularly detected species that matched with either species present in other tanks of the Aquarium or humans (NB: the diver in charge of the maintenance of different tanks is the same person, wearing the same wetsuit: often tank hosts -like dolphins- closely interact with him and his wetsuit); 5) “MISASSIGNED SPECIES” groups the molecularly detected species which were not those present in the tank, that however were taxonomically related to them. 6) “UNEXPECTED VERTEBRATE SPECIES” indicate the number of molecularly assigned species whose detection could not be explained. Note that all species falling in these first 6 categories were all vertebrate species (including those falling into the two “food” categories). 7) “INVERTEBRATE SPECIES” indicate the number of molecularly assigned invertebrate species. The bottom part of each table section shows the number of HTS reads scored in each of the following 3 categories: A) vertebrate species whose presence (direct or indirect) in form of eDNA traces could be justified (thus grouping together categories 1 to 5 of the above described list) [no artefact] B) vertebrate species whose presence (direct or indirect) in form of eDNA traces could not be explained [possible artefact] C) invertebrate species detection [primer aspecificity] Results are discussed in the main text.

TANKS MANATEES DOLPHINS SHARKS PRIMER SET MV1 MV3 MV1 MV3 MV1 MV3 1 DETECTED/TOTAL HOSTED VERTEBRATE SPECIES 2/3 2/3 1/1 1/1 5/14 10**/14 2 TANK FEED 0 0 2 2 2 3 3 OVERALL FEED CONTAMINATION 3 1 A 1 2 A 0 0 A 4 CONTAMINATION FROM OTHERS TANKS AND HUMAN 4 2 4 12 3 5 5 MISASSIGNED SPECIES 0 0 1 2 1 0 6 UNEXPECTED VERTEBRATE SPECIES 3 0 B 0 1 B 0 0 B 7 INVERTEBRATE SPECIES 0 1* C 0 0 C 0 0 C

HTS read count A 51608 26998 232726 196247 80565 124265 HTS read count B 877 0 0 693 0 0 HTS read count C 0 69 0 0 0 0

TANKS PENGUINS SEALS ROCKY SHORE PRIMER SET MV1 MV3 MV1 MV3 MV1 MV3 1 DETECTED/TOTAL HOSTED VERTEBRATE SPECIES 0/2 2/2 1/1 1/1 0/6 6*/6 2 TANK FEED 2 2 2 1 1 4 3 OVERALL FEED CONTAMINATION 1 1 A 2 1 A 1 0 A 4 CONTAMINATION FROM OTHERS TANKS AND HUMAN 5 8 3 9 4 10 5 MISASSIGNED SPECIES 1 0 1 0 0 3 6 UNEXPECTED VERTEBRATE SPECIES 0 2 B 0 1 B 4 3 B 7 INVERTEBRATE SPECIES 0 0 C 0 1* C 0 0 C

HTS read count A 91348 191573 158492 194057 10036 204597 HTS read count B 0 583 0 230 2627 1585 HTS read count C 0 0 0 38 0 0