The complete mitochondrial genome of a new invader fish in France, the pink salmon Oncorhynchus gorbuscha (Walbaum, 1792) (Teleostei, Salmonidae)

The complete mitochondrial genome of a new invader fish in France, the pink salmon Oncorhynchus gorbuscha (Walbaum, 1792) (Teleostei, Salmonidae) by Gaël P.J. Denys* (1, 2), Quentin Josset (3, 4), Amélie VerDe FerreirA (5) & Agnès DettAï (5) Cybium 2024, 48(2): 111-120. https://doi.org/10.26028/cybium/2024-005 IntroductIon the anadromous Pacific salmons Oncorhynchus spp. are native to the Pacific coasts of north-western America and north-eastern Asia and have a high interest for aquaculture, recreative fishing and commercial fisheries (Froese and Pauly, 2022). Many species were therefore introduced all over the world since the end of the 19th century with varying success (scott and Crossman, 1973). in France, three species were introduced (the rainbow trout O. mykiss (Walbaum, 1792), the Coho salmon O. kisutch (Walbaum, 1792) and the Chinook salmon O. tshawytscha (Walbaum, 1792)) but only O. mykiss now remains (Persat et al., 2020). since 2017, another Pacific salmon, the pink salmon Oncorhynchus gorbuscha (Walbaum, 1792), has invaded northern europe every odd year (see Baglinière et al., 2020; Beaulaton et al., 2021). this species has a very strict and simple life cycle: individuals live two years, spending 18 months in marine waters before breeding in freshwater. Adults measuring about 50 cm migrate in rivers from June to August for Asiatic populations or from July to september for American ones, and die after spawning (Heard, 1991). this species might have an impact on the predation, the competition, the disease transmission on native fish species but also on human health through tapeworm infections with Diphyllobothrium nihonkaiense yamane, Kamo, Bylund & Wikgren, 1986 (Arizono et al., 2009; Van der Veer and nentwig, 2015). Currently, its impact on native european salmonids Abstract. – the pink salmon Oncorhynchus gorbuscha (teleostei, salmonidae) is a north Pacific species. it arrived in northern europe through freshwater migration each odd year since 2017, creating management issues. in this study, we sequenced a complete mitochondrial genome for a pink salmon caught in the Bresle river (France) with a museum voucher. the sequence is 16,695 bp in length and is similar to previous mitogenomes published within this genus. Length heteroplasmy on the control region for this species was detected, as described in previous molecular studies. We tested several primers commonly used for metabarcoding studies on four markers (12s, 16s, Coi and Cytb), and all allow the discrimination of this species. this mitogenome is a reference for molecular identification, for instance in like environmental DNA studies. Résumé. – Le génome mitochondrial complet d’un nouveau poisson téléostéen invasif en France, le saumon rose Oncorhynchus gorbuscha (Walbaum, 1792) (Teleostei, Salmonidae). Le saumon rose Oncorhynchus gorbuscha (Teleostei, Salmonidae) est une espèce du Pacifique Nord arrivée en europe du nord avec une migration en eau douce chaque année impaire depuis 2017, générant des problèmes de gestion. Dans cette étude, nous avons séquencé le génome mitochondrial complet d’un saumon rose d’un spécimen enregistré en collection et capturé dans la rivière Bresle (France). La séquence a une longueur de 16 695 pb et est similaire à d’autres mitogénomes publiés chez ce genre. Cependant, nous avons mis en évidence une hétéroplasmie de longueur sur la région de contrôle pour cette espèce confirmant les études moléculaires précédentes. nous avons également testé plusieurs amorces couramment utilisées pour les études de métabarcoding sur quatre gènes (12s, 16s, Coi et Cytb). tous les marqueurs permettent la discrimination de cette espèce. notre mitogénome peut être utilisé comme référence pour l’expertise moléculaire, par exemple pour des approches d’ADn environnemental. © SFI Received: 13 Jul. 2022 Accepted: 23 Jan. 2024 Pub. online: 1 Mar. 2024 Editor: O. Otero Key words Mitogenome Pink salmon Long PCr sample multiplexing Metabarcoding Genome announcement (1) UAr Patrimoine naturel – Centre d’expertise et de données (2006 oFB – Cnrs – MnHn – irD), Muséum national d’Histoire naturelle, 36 rue Geoffroy-saint-Hilaire CP 41, 75005 Paris, France. [email protected] (2) UMr Biologie des organismes et écosystèmes aquatiques (BoreA 8067), MnHn, Cnrs, irD, sU, UCn, UA, 57 rue Cuvier CP26, 75005 Paris, France. (3) Office Français de la Biodiversité, Direction Recherche et Appui Scientifique, Service “Conservation et Gestion Durable des Espèce exploitées”, 92 chaussée de Picardie, 76260 eu, France. [email protected].fr (4) Pôle Gestion des Migrateurs Amphihalins dans leur environnement, oFB, inrAe, institut Agro, UniV Pau & Pays Adour/e2s UPPA, 65 rue de saint-Brieuc, 35000 rennes, France. (5) institut de systématique, evolution, Biodiversité, isyeB – UMr 7205 – Cnrs, MnHn, UPMC, ePHe, Muséum national d’Histoire naturelle, sorbonne Universités, 57 rue Cuvier CP26, 75005 Paris, France. [email protected], [email protected] * Corresponding author Mitogenome of oncorhynchus gorbuscha De n y s e t a l . 112 Cybium 2024, 48(2) seems to be limited (Mo et al., 2018; Beaulaton et al., 2021), except maybe on sea lamprey Petromyzon marinus Linnaeus, 1758 (Armstrong et al., 2018; Mo et al., 2018; Bonnyaud and Denys, 2021). However, it was demonstrated that the presence of fish carcasses after spawning enriches the rivers with marine nutrients that potentially impact the ecosystem (Armstrong et al., 2018; Mo et al., 2018). it can also provide new high-income feeding opportunities through egg or fry consumption by native species (rasputina et al., 2016; Dunlop et al., 2021). France recorded the southernmost pink salmon occurrences in Europe with a first observation in the Canche river (Pas-de-Calais department) and two observations in Britanny (Côtes-d’Armor and Finistère departments) in 2017, followed by six additional observations: two in the Bresle river (seine-Maritime department, normandy), one in the somme river (somme department) and in the Léguer river (Finistère department) and two others in the Manche department in 2021 (Beaulaton et al., 2021; Bonnyaud and Denys, 2021). these observations were done mainly by anglers or video counting-stations, so the number of occurrences is certainly underestimated. Molecular tools like the environmental DnA approach would be necessary to complete the range of methods used for the survey of invasive species (e.g., sepulveda et al., 2020). several studies already used this method in the native and newly invaded areas using as molecular markers sequence fragments of the mitochondrial nADH2, the cytochrome oxidase subunit 1 (Coi) and the cytochrome b markers (Mizumoto et al., 2018; Duda et al., 2021; Gargan et al., 2022). However, the popular MiFish and French environmental DNA (eDNA) studies on fishes use the 12S marker (e.g., Miya et al., 2015; Valentini et al., 2016). in the GenBank sequence repository, two mitogenomes are already available but they all lack association to a voucher or an origin other than their hatchery, and therefore cannot be validated as reference sequences (strohm et al., 2016). in this paper, we describe the mitogenome of O. gorbuscha from a voucher specimen caught in the north of France, obtained by a double-multiplexing approach. this mitogenome will be helpful to link the different molecular studies made on this taxon, especially those about the genetic variability within even and odd years populations using different molecular markers (control region, Cytb and nADH2) that are therefore not directly comparable (e.g., sato and Urawa, 2017; Podlesnykh et al., 2020) and, as molecular reference, for molecular identification using any marker from DnA barcoding sensu Hebert et al. (2003) to eDnA analyses (schroeter et al., 2020). We provide the diagnostic sites of this species on metabarcode markers already published for eDnA studies. MATERIAL AND METHODS Voucher the voucher specimen is stored in the national ichthyological Collections of the Muséum national d’Histoire naturelle (MnHn), catalogued MnHn-iC-2022-0168 (Fig. 1), and originates from the CoLisA archive (Marchand et al., 2018). the specimen (443 mm of standard length (sL) for 1178 g), a male, was caught on July 23rd, 2021 in the Bresle river at eu (normandy; Lat: 50.050628 Long: 1.418690), where the French Biodiversity Agency (oFB) operates a scientific trap since 1981 (Josset et al., 2021). identification was made according to Baglinière et al. (2020): presence of a hump on the back, black tongue and elongated black spots on the caudal fin. Brief material and method DNA extraction was carried on a fin-clip stored in 95% ethanol on an epMotion robot using a Mn Biomedical extraction kit and protocol. three long overlapping PCrs of 6-7 kbp were done following Denys et al. (2020). sequencing was performed using a double-multiplexing approach (Hinsinger et al. 2015; Denys et al., 2020). sequencing was performed on illumina Miseq at the iCM (institut du Cerveau et de la Moelle épinière, Paris V) using 250 pairedend standard V2. the mitogenome was assembled with the Geneious 11.2.2 software (Kearse et al., 2012) mapping Figure 1. – Voucher of the sequenced mitogenome, MnHniC-2022-0168, 443 mm sL, Bresle river at eu (seine-Maritime Dept.), 24th July 2021, oFB coll; credit photo: Q. Josset/oFB. Figure 2. – Coverage depending on position in the mitogenome assembly. De n y s e t a l . Mitogenome of oncorhynchus gorbuscha Cybium 2024, 48(2) 113 trimmed paired reads on a reference sequence (GenBank accession number eF455489) using the Geneious 11 sensitivity parameter set “low sensitivty/fastest” for the Geneious mapper, and carefully quality controlled by eye along their whole length in addition to the standard controls. the assembly of the control region (Cr) had a mismatch with the reference sequence (Appendix 1), so a second assembly by elongation from the assembled sequence without the Cr was performed to reconstruct the Cr without constraining it through the reference. the consensus sequence was annotated using MitoAnnotator (iwasaki et al., 2013). Sequence quality 76,484 reads (average length: 249.6) were assembled from the sequencing of the 3 long range PCrs. the read coverage throughout the entire sequence ranges from 438 to 3561, with a mean read coverage of 1147.1 fold (Fig. 2). Assembly statistics for each long fragment are given in table i. Phylogenetic reconstruction Mitogenomes from two Oncorhynchus gorbuscha CM029873 and eF455489 (= nC_010959) as well as ten other Oncorhynchus species: apache trout O. apache (Miller, 1972) MW300342, cutthroat trout O. clarkii (richarson, 1836) KP013107, gila trout O. gilae (Miller, 1950) MW300335, chum salmon O. keta (Walbaum, 1792) AP010773, coho salmon O. kisutch MF621749, masu salmon O. masou (Brevoort, 1856) KU523579, rainbow trout O. mykiss KP013084, sockeye salmon O. nerka (Walbaum, 1792) MH003639 and chinook salmon O. tshawytscha AF392054 plus a sequence of brown trout Salmo trutta Linnaeus, 1758 MF621761, arctic char Salvelinus alpinus Linnaeus, 1758 Mn530962 and european grayling Thymallus thymallus (Linnaeus, 1758) Mn852233 were retrieved from GenBank. sequences alignment was performed using the Clustal W plugin in Geneious r11.2.2, as well as nucleotide diversity and pairwise distances. the best evolutionary model was inferred in jModeltest (Darriba et al., 2012) and was Gtr+i+G for both Akaike and Bayesian information criterion. Phylogenetic analysis was inferred by Maximum Likelihood (ML) using RAxML-HPC2 Workflow on XSEDE (version 8.2.10) (stamatakis, 2014) with the inferred substitution model and 1,000 bootstrap iterations on the CiPres science Gateway (Miller et al., 2010) online platform. Comparison of metabarcode markers nine metabarcoding markers from four loci (12s, 16s, Coi and Cytb) were tested (table ii). For each locus, we table i. – statistics about the assembly of the mitogenome Oncorhynchus gorbuscha (GenBank Accession number PP272106) for the three long PCrs. Mt1 Mt2 Mt3 Length (bp) 6748 5385 5382 n reads 44685 14709 20150 Coverage min-max (average; sD) 913-3561 (1702.1; 369.4) 438-1489 (695.4; 220.8) 455-1902 (871.0; 226.4) reads lengths min-max (average; sD) 221-251 (249.2; 6.4) 224-251 (248.9; 8.1) 224-251 (248.1; 8.1) table ii. – Metabarcoding primers commonly used in the literature and tested in this study to discriminate Oncorhynchus gorbuscha from other introduced Oncorhynchus species. Locus Primers sequence 5’-> 3’ Amplicon length (bp) reference 12s MiFish Forward: GtCGGtAAAACtCGtGCCAGC reverse: CAtAGtGGGGtAtCtAAtCCCAGtttG 170 Miya et al. (2015) teleo Forward: ACACCGCCCGtCACtCt reverse: CttCCGGtACACttACCAtG 63 Valentini et al. (2016) teleo2 Forward: AAACtCGtGCCAGCCACC reverse: GGGtAtCtAAtCCCAGtttG 166 taberlet et al. (2018) 12s-V5 Forward: ttAGAtACCCCACtAtGC reverse: tAGAACAGGCtCCtCtAG 99 riaz et al. (2011) 16s Fish16sFD/16s2r Forward: GACCCtAtGGAGCtttAGAC reverse: CGCtGttAtCCCtADrGtAACt 205 Berry et al. (2017) 16sar/16sbr Forward: CGCCtGttAtCAAAAACAt reverse: CCGGtCtGAACtCAGAtCACGt 581 Duke and Burton (2020) Coi FishF1/Fishr1 Forward: ttCtCAACCAACCACAAAGACAttGG reverse: tAGACttCtGGGtGGCCAAAGAAtCA 658 Ward et al. (2005) mlCoiintF/jgHCo2198 Forward: GGWACWGGWtGAACWGtWtAyCCyCC reverse: tAiACytCiGGrtGiCCrAArAAyCA 313 Leray et al. (2013) CytB L14912-CyB/H15149CyB Forward: ttCCtAGCCAtACAytAyAC reverse: GGtGGCKCCtCAGAAGGACAtttGKCCyCA 234 Minamoto et al. (2012) Mitogenome of oncorhynchus gorbuscha De n y s e t a l . 114 Cybium 2024, 48(2) compared our sequence with those of the four Oncorhynchus species introduced in France available on GenBank and associated with a voucher (Appendix 2). Diagnostic sites characterizing species were then identified with the QUIDDiCH package (Kühn and Hasse, 2019) for r (r Core team, 2022). RESULTS AND DISCUSSION Sequence description and phylogenetic analysis the newly obtained mitogenome has a total length of 16,695 bp and follows the standard vertebrate order, similar to already published mitogenome of O. gorbuscha: 13 protein-coding genes including six coding with an incomplete codon stop (Coii, AtP6, Coiii, nADH3, nADH4 and Cytb), 22 transfer rnA genes including two trnA-Leu and 2 trnA-ser, two ribosomal rnA genes and a control region (Fig. 2; Appendix 3). intergenic spaces and overlapping sequences were found. the base composition of the entire genome was 27.9% for A, 26.4% for T, 28.7% for C and 17.0% for G. As for the two sequences of O. gorbuscha available on GenBank, the mitogenome CM029873 is reversed in the database and had to be reverse complemented to align with the dataset in standard order. the control region of the mitogenome eF455489 does not have the same length (1131 bp vs. 1041 bp). Previous genetic studies already highlighted length heteroplasmy among pink salmon populations with this marker with insertions and deletions (Brykov et al., 1999; Churikov and Gharrett, 2002; sato and Urawa, 2017). Alignment of our sequence with all Cr sequences from GenBank provided a 100% match with the haplotype OGDL-8 (sequence LC191999), one of the haplotypes widespread in Japan (sato and Urawa, 2017). the ML phylogeny (Fig. 3) is consistent with shedko et al. (2013) on mitochondrial data showing generally high support values for nodes as well as with the nuclear rAG1 marker topology (shedko et al., 2012). it differs from phylogenetic trees of Horreo (2017) and Gong et al. (2017) by the position of O. masou, which is not at a basal position within the Oncorhynchus spp. but with a weak robustness. Crespi and Fulton (2004) already demonstrated that the position of O. masou depends to the markers analysed and the phylogenetic reconstruction method. our O. gorbuscha mitogenome groups well with the two other sequences available on GenBank, corroborating its identification. Finally, we tested metabarcoding markers of four genes (12s, 16s, Coi, Cytb) in order to know if O. gorbuscha can easily be distinguished from the four other Oncorhynchus species used for restocking in non-American areas. O. gorbuscha is distinguished over these four genes by 10 diagnostic sites on the 12s (table iii), 34 on the 16s (table iV), 82 on the Coi (table V) and 61 on the Cytb (table Vi). MiFish (Miya et al., 2015) and teleo2 primers (taberlet et al., 2018) between the positions 246/250 and 416 differentiate all 4 species (table iii). However, the fragments corresponding to the 12V-5 primers (riaz et al., 2011) between the positions 444 and 543 and the teleo primers (Valentini et al., 2016) between the positions 849 and 912, while they discriminate well O. gorbuscha from other Oncorhynchus species, have Figure 3. – Maximum Likelihood phylogenetic tree of Oncorhynchus mitogenomes; bootstrap values beside the nodes. De n y s e t a l . Mitogenome of oncorhynchus gorbuscha Cybium 2024, 48(2) 115 table iii. – Diagnostic sites determined on the 12s marker for Oncorhynchus gorbuscha, O. kisutch, O. mykiss and O. tshawytscha. Diagnostic sites are in bold. the MiFish and teleo2 fragments (Miya et al., 2015; taberlet et al., 2018) between the positions 246/250 and 416, the 12V-5 fragment (riaz et al., 2011) between the positions 444 and 543 as well as the teleo fragment (Valentini et al., 2016) between the positions 849 and 912 are highlighted in grey. MiFish / teleo2 12V-5 teleo 32 38 63 118 122 128 197 307 349 355 370 390 463 490 561 592 643 662 699 730 736 738 747 788 795 864 882 936 947 O. gorbuscha tAt G t A A A tA t t A G G t C G G t C t G t CA c A t O. kisutch . G c. C G G G C . cC G A . . . A . . t C A C t G t G C O. mykiss cG . AC G . . C . . . G A . c. . A. . C A C . G t G C O. tshawytscha . G . . C G . . C G. C . t t . tA . ct C A C t G t G C table iV. – Diagnostic sites determined on the 16s marker for Oncorhynchus gorbuscha, O. kisutch, O. mykiss and O. tshawytscha. Diagnostic sites are in bold. the Fish16sFD/16s2r fragments (Berry et al., 2017) between the positions 1170 and 1357 and the 16sar/16sbr fragment (Duke and Burton, 2020) between the positions 931 and 1512 are highlighted respectively in dark and light grey. 18 19 20 29 38 40 44 52 57 59 70 98 114 115 117 118 121 132 149 162 163 164 171 176 180 189 193 O. gorbuscha CtC t A C t c G t A CG A t – t – G – – G A t – cA O. kisutch tA . . . tC A A C G . A G . – c– . – – . . . – t . O. mykiss . A t c . . C A A C G GA G . – . – . – – . . c– t G O. tshawytscha . A . . –. C A A C G . A G c A .A A A G A c .tt . 241 245 265 266 267 300 309 315 425 427 444 465 470 545 549 550 617 619 648 669 718 721 751 757 759 773 795 O. gorbuscha Ac c CGAct G A A G G AAtct–G G A t tAG A O. kisutch . t t . A . t . . C G A A . C . t . C A A . . C . A C O. mykiss ct t tA . t c. C G A A GCctcC . t.cCcA C O. tshawytscha . t t . A Gt . AC G A A . C . t . C . . G. C . A C 16sar/16sbr 16sar/16sbr + Fish16sFD/16s2r 808 816 819 823 831 844 864 881 949 1087 1131 1169 1182 1186 1194 1263 1292 1294 1308 1312 1340 1351 1352 1562 1665 O. gorbuscha t A G G C A C G GCt c A/– tAA t C t G A A GAA O. kisutch .GA A . . . . A . C A C C Gt C t.A. . A tG O. mykiss c. A A . c A . A . C A C C . t C . . . t G A . G O. tshawytscha . . A A t. . AAtC A C C . t C . c. . . A . G table V. – Diagnostic sites determined on the Coi marker for Oncorhynchus gorbuscha, O. kisutch, O. mykiss and O. tshawytscha. Diagnostic sites are in bold. the FishF1/Fishr1 fragments (Ward et al., 2005) between the positions 48 and 706 and the mlCoiintF/jgCo2198 fragment (Leray et al., 2013) between the positions 393 and 706 are highlighted respectively in light and dark grey. FishF1/Fishr1 21 99 102 108 114 117 123 132 144 148 153 156 159 162 174 210 213 228 258 267 270 276 282 285 294 315 O. gorbuscha cAc A GAAAAAC G C t tCAA G A A A t A c G O. kisutch t . t G A.GG . G t.t c C . G . . C t G C t t t O. mykiss tGt G . G. G GG . A. . C . G G A C t G C t t t O. tshawytscha ttt G . . . G . G . . . . C tG . . C t G C t t t FishF1/Fishr1 FishF1/Fishr1 + mlCoiintF/jgCo2198 318 327 339 348 354 363 366 372 378 384 402 408 423 426 429 432 438 468 480 498 507 516 519 525 546 552 O. gorbuscha c c t C/t cC t C GtG t A c c c tA G GcAAAA t O. kisutch Gt A t t G. . A . C C C A t t . C A . t GC t . . O. mykiss ttG A t . G t AACCCtt t . C A . t . t c c c O. tshawytscha At A C t A. . A cCAC A t t GC A tt . C t . . FishF1/Fishr1 + mlCoiintF/jgCo2198 561 564 568 579 586 591 594 603 615 618 627 643 651 666 669 672 675 729 738 744 747 756 783 789 792 804 O. gorbuscha t G A A CAC t t c G t t A G G c A/G ttAcC t GC O. kisutch . . G t. C t. C t A C C G A C G C GCctt. A . O. mykiss c. G C t t .cC t A C C G A C G C cC . t . cA . O. tshawytscha .AG C . C . . C t A C C G A C G C . C . t . . A t 813 819 822 831 834 843 846 861 873 885 888 891 909 915 930 957 993 1005 1017 1024 1029 1035 1044 1053 1062 1065 O. gorbuscha G A AAG c G t cC G GCc A AG G c CA c tc G c O. kisutch . . C . A A A c tt. A . t G GA A t . G t . AA t O. mykiss A G C . A G. . t A. A A/t t G . A A t tG t ct A t O. tshawytscha . . C GA A . At . AA . t G . A A t . G t . t A t 1068 1083 1098 1099 1122 1125 1146 1152 1188 1194 1208 1209 1212 1215 1224 1227 1275 1278 1287 1293 1299 1302 1305 1326 1332 1344 O. gorbuscha c t tt c c G A A c t C C t GAcCc A C A G C G G O. kisutch t C cC t t C G. G A . . . A Gttt . t t . . A A O. mykiss t C . C t t t.GG A . . cA . t . t . . . . tA A O. tshawytscha t C . C t t C . . G A t A . A . t . t G. . A. A A 1347 1359 1368 1380 1384 1443 1459 1470 1491 1500 1506 1515 1518 1524 1527 1536 1541 1546 O. gorbuscha AG C C t A A t AGC C G A GAG t O. kisutch G . . . . G t A GAt. . . A . A A O. mykiss GA.A c GcA . A . t A . A . A A O. tshawytscha G . t. . G t A . A . . . GAGA A Mitogenome of oncorhynchus gorbuscha De n y s e t a l . 116 Cybium 2024, 48(2) the exact same sequence for two (12V-5) or all three (teleo) other species making their distinction impossible. For the other markers used for 16s, Coi and Cytb genes, all species are distinguishable (tables iV-Vi). thus, this mitogenome can be used as a sequence of reference for different molecular expertise using multiple markers such as eDnA or control of mislabelling food (Wang et al., 2021). Acknowledgements. – this work was supported by the Muséum national d’Histoire naturelle (MnHn), the UAr Patrinat 2006, the UMr BoreA 8067 and the French Biodiversity Agency (oFB). We thank the team from the Long term observatory of the Bresle responsible for the capture of the specimen used in this study and also the curation staff of the INRAE fish collections (COLISA) for sending fin clip, as well as Laurent Beaulaton (OFB/INRAE) who supports this study. Laboratory access and assistance was provided by the “Service de Systématique Moléculaire” of the MNHN (UAR 2AD 2700). the authors thank two anonymous reviewers for their useful comments. 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C L14912-CyB/H15149-CyB 318 324 341 345 378 381 384 387 390 414 417 447 450 453 462 465 468 471 489 492 498 501 513 522 549 558 567 573 O. gorbuscha AA G C t A c A C G c t t c Ct G c G tc A t c t t c t O. kisutch . C A . . C t G A A . C C . A C A A A . . G C t C C t . O. mykiss GC A . . C t . . A AC C AC/A C A A A . G c C t C C t c O. tshawytscha tC A t c C t . . A . C C . GC A A A c. G C t C C t t 579 591 624 627 630 639 645 657 660 663 666 684 690 693 708 711 714 717 723 729 732 744 748 750 774 777 783 786 O. gorbuscha Ac G A G C C G t C t c c G ttt A A c A CtA C t A G O. kisutch . t A G. . . C C . . t A A . C . GC t t . C . . C C . O. mykiss Gtt. . t t A C . . t A A c A c . C t t tC . tC C A O. tshawytscha . t A . c. . C C t c t A A . C . . C t t . C G. C C . 789 795 804 816 819 825 831 840 849 852 876 891 903 906 912 915 918 927 943 945 960 970 975 978 981 982 984 987 O. gorbuscha CGC A tA t tt C C ct G C C tAtAAt t A t t A A O. kisutch . A . GCG c C . . . G . . A A CcCG.ccG C . G G O. mykiss GAt. C . . C c.tGc. . . C t C t/A c. . G C . . . O. tshawytscha . A . . C . . C . t. G . A t t C t C . G. . G C c. . 1003 1017 1026 1029 1035 1038 1044 1047 1053 1057 1059 1065 1068 1071 1083 1090 1098 1116 1119 O. gorbuscha C A C A t G C t C G t AA C tGt A A O. kisutch t G .GC A . . . . . t G.C.AC . O. mykiss . . t. C c t .t/A .ct . tC . cC . O. tshawytscha . . . . C A . c.A. t . . C A G CG De n y s e t a l . Mitogenome of oncorhynchus gorbuscha Cybium 2024, 48(2) 117 Darriba D., taboada G. L., Doallo r. & Posada D., 2012. jModeltest 2: more models, new heuristics and parallel computing. Nat. Methods, 9: 772. https://doi.org/10.1038/nmeth.2109 Denys G. P. J., secci-Petretto G. & Gomes Dos santos A., 2020. the complete mitochondrial genome of Thymallus thymallus (Linnaeus, 1758) (Actinopterygii, salmonidae) obtained by long range PCrs and double multiplexing. Cybium, 44(2): 91-94. https://doi.org/10.26028/cybium/2020-442-001 Duda J. J., Hoy M. s., Chase D. 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Mitogenome of oncorhynchus gorbuscha Cybium 2024, 48(2) 119 Appendix 2. – GenBank accession numbers for the sequences used as comparison material for the four loci used in metabarcode studies (12s, 16s, Coi, CytB) for Oncorhynchus gorbuscha, O. kisutch, O. mykiss and O. tshawytscha. O. gorbuscha: PP272106, CM029873, eF455489; O. kisutch: eF126369, MF621749, MF621751, MH003640; O. mykiss: AF392054, DQ288268 to DQ288271, HQ167664, HQ167694, HQ167682, KP013084, KP085590, Ky798500, MF621750, Mt410879, Mt667254, MZ2567214; O. tshawytscha: AF392054, HQ167665 Appendix 1. – screen captures of the problematic map to reference mapping around position 15985 of sequence eF455489. two screen captures of reads of the same area are presented (second is simply scrolled down from the first), as the high number of reads did not allow for a clear single picture. no read overlaps both sides at once without mismatch, causing coverage to drop for this mapping. Allowing free reconstruction using elongation from one of the sides recovers a different sequence with high, unambiguous coverage.