18S Metabarcoding Genetic Observations of Marine Species in the Port of Wallhamn, Sweden (2022)

Study Extent

The different sampling methods had different timings over the summer 2022: The eRAS panels were put out on the 23rd of June and taken back on the 9th of September, one was in the industrial port, three were in the marina. The artificial habitats were put out on the 23rd of June and taken back on the 4th of August, one in each area. The ARMS were put out on the 23rd of June and taken back on the 9th of September, one in the industrial port and two in the marina. In the marina, three plankton samplings occurred on the 23rd of June, the 4th of August and the 9th of September (9 plankton samples in total). Similarly, in the marina, three eDNA samplings occurred on the 23rd of June, the 4th of August and the 9th of September (9 eDNA samples in total). Three scraping/RAS samplings occured on the 9th of September in the marina.

Sampling

Artificial habitats are plastic trays filled with pottery shards, a weight at the bottom to hold the trays down and a net above to keep the shards in place. On two sides, larger holes were cut to allow fish to move freely in and out of the habitat Plankton samples were taken as vertical hauls from the bottom (5-10 m) up to the surface with a 90 µm plankton bucket. The samples were fixed in 95% ethanol directly in place with an amount of alcohol that was judged to result in a sample with at least 70% ethanol. Upon return to the lab, the plankton samples were decanted and new alcohol was added to ensure that the ethanol content was sufficiently high. The samples were then stored in a freezer (-20°C) until extraction. Water samples for eDNA were taken with Ruttner retrievers at slightly different depths on the premises and pooled in a 1 litre vessel. Water was filtered on site and fixed with 95% ethanol.The filters were stored in a freezer (-20°C) until extraction.

Quality Control

Negative controls (blank samples) were created by the same extraction procedure but without the sampled material. No DNA could be detected in these samples, with neither Nanodrop nor Qubit. The blank samples were included in the preparation of libraries (below) as a further control for contamination during the extraction of DNA. Another negative control was performed during PCR.

Method steps

1. DNA was extracted from the filters with the Nucleospin eDNA water kit (Macherey-Nagel) using the Nagel) using the technique and standard developed in the laboratory. Concentration of DNA in the extracts was measured with the Qubit® fluorometer. Note that this measures concentration of all DNA in the sample and not specific DNA from the target species. This is done to ensure that the extractions have worked.
2. The genetic analysis and comparison was based on two molecular markers (COI and 18S). These markers were amplified for each sample. Same PCR protocols were used for metabarcoding of the ARMS, eDNA and plankton samples The DNA libraries were sequenced with Illumina MiSeq, 2 x 300 bp.
3. The analysis was performed in the R-environment with a DADA2 package. After initial quality control, the sequences were filtered for low quality, as well as adapters and primers were removed. Sequencing errors were corrected by calculating an error model; and after that singletons and chimeras were removed using the “pseudo-pooling” function in DADA2.
4. The filtered and quality-controlled COI sequences were matched against the BOLD database with the python package BOLDigger. The 18S sequences were matched against the PR2 database. All subsequent analysis was performed with customized R scripts where identified species were matched against different NIS and IAS species reference lists.

N/A

1. Parmales
   rank: order
2. Verrucomonadidae
   rank: family
3. Gymnodiniaceae
   rank: family
4. Dinophyceae
   rank: class
5. Cercozoa_X
   rank: class
6. Appendicularia
   rank: family
7. Gonyaulacales
   rank: order
8. Digenea
   rank: family
9. Dino-Group-II-Clade-14
   rank: family
10. Phaeocystaceae
    rank: family
11. Bolidophyceae
    rank: class
12. Cryomonadida
    rank: order
13. Prasinodermophyceae
    rank: class
14. Chrysophyceae
    rank: class
15. Kathablepharidea
    rank: class
16. Cryptophyceae
    rank: class
17. Gymnodiniales
    rank: order
18. Pyramimonadophyceae
    rank: class
19. Prasinodermaceae
    rank: family
20. Hydrozoa
    rank: family
21. Syndiniales
    rank: class
22. Kathablepharidida_X
    rank: family
23. Cercozoa_XX
    rank: order
24. Gyrista_XX
    rank: order
25. Cryptomonadales_X
    rank: family
26. Arthropoda
    rank: class
27. Filosa-Imbricatea
    rank: class
28. Nematoda
    rank: class
29. Spirotrichea
    rank: class
30. Noelaerhabdaceae
    rank: family
31. Pycnococcaceae
    rank: family
32. Dino-Group-I-Clade-1
    rank: family
33. Dino-Group-II-Clade-1
    rank: family
34. Protoperidiniaceae
    rank: family
35. Gyrista_X
    rank: class
36. Triparmaceae
    rank: family
37. Opalozoa_X
    rank: order
38. Dino-Group-I
    rank: order
39. Pseudoscourfieldiales
    rank: order
40. MAST-1
    rank: family
41. Protaspa-lineage
    rank: family
42. Probosciales
    rank: order
43. Dino-Group-II_X
    rank: family
44. Chaetocerotaceae
    rank: family
45. Ascidiacea
    rank: family
46. Maxillopoda
    rank: family
47. Dino-Group-II-Clade-46
    rank: family
48. Thoracosphaeraceae
    rank: family
49. Heteroconchia
    rank: family
50. Choreotrichida
    rank: order
51. Litostomatea_XX
    rank: family
52. Chrysochromulinaceae
    rank: family
53. Suessiaceae
    rank: family
54. Pyrocystaceae
    rank: family
55. Urochordata_X
    rank: order
56. Peronosporales
    rank: family
57. Urochordata
    rank: class
58. Isochrysidales
    rank: order
59. Calcihaptophycidae
    rank: order
60. Prorocentrales
    rank: order
61. Chromadorea
    rank: order
62. Anthozoa
    rank: family
63. Rhabdosphaeraceae
    rank: family
64. Bivalvia
    rank: order
65. Pelagomonadaceae
    rank: family
66. Cercozoa_XXX
    rank: family
67. Crustacea
    rank: order
68. Peronosporomycetes_X
    rank: order
69. Chromadorea_X
    rank: family
70. Dino-Group-I-Clade-3
    rank: family
71. Dino-Group-II
    rank: order
72. Prasinodermales
    rank: order
73. Litostomatea_X
    rank: order
74. Mollusca
    rank: class
75. Strombidinopsidae
    rank: family
76. Pelagophyceae
    rank: class
77. Suessiales
    rank: order
78. Litostomatea
    rank: class
79. Prorocentraceae
    rank: family
80. Pelagomonadales
    rank: order
81. Peronosporomycetes
    rank: class
82. Prymnesiales
    rank: order
83. TAGIRI1-lineage
    rank: family
84. Peridiniales
    rank: order
85. Filosa-Thecofilosea
    rank: class
86. Dino-Group-II-Clade-32
    rank: family
87. Chaetocerotales
    rank: order
88. Hemiaulaceae
    rank: family
89. Dino-Group-II-Clade-3
    rank: family
90. Trematoda
    rank: order
91. Kareniaceae
    rank: family
92. Platyhelminthes
    rank: class
93. Ventricleftida
    rank: order
94. Cnidaria_X
    rank: order
95. Prymnesiophyceae
    rank: class
96. Opalozoa
    rank: class
97. Phaeocystales
    rank: order
98. Hemiaulales
    rank: order
99. Nanomonadea
    rank: order
100. Protoceratiaceae
     rank: family
101. MAST-3
     rank: family
102. Cryptomonadales
     rank: order
103. Cnidaria
     rank: class
104. Mediophyceae
     rank: class
105. MAST-12
     rank: family
106. Ebriacea
     rank: order
107. Kathablepharidida
     rank: order
108. Probosciaceae
     rank: family

This dataset covers the port of Wallhamn (Sweden) which has both a commercial/industrial part in the North and a marina in the South.