Supplementary material 1 from: Rech S, Vieira LM, Varela AI, Bastías MSR, Fuentes JP, Gordon DP, Cacciuttolo-Said M, Paschke K, Sellanes J, Thiel M (2025) Seek and you shall find: Detection of alien bryozoans along the Chilean SE Pacific coast with a simple and cost-efficient methodology. In: Anastácio P, Ribeiro F, Chainho P (Eds) Invasions in Aquatic Systems. NeoBiota 102: 269-294. https://doi.org/10.3897/neobiota.102.144725

1 Supplemental material 1 Seek and you shall find: Detection of alien bryozoans along the Chilean SE Pacific coast with 2 a simple and cost-efficient methodology 3 Running title: Detection of alien bryozoans along the Chilean SE Pacific coast 4 5 Sabine Rech1,2,3*, Leandro M. Vieira4,5, Andrea I. Varela3,6, María Soledad Romero Bastías2,7, 6 Juan Pablo Fuentes8, Dennis P. Gordon9, Martina Cacciuttolo-Said2, Kurt Paschke10, Javier 7 Sellanes2,3, Martin Thiel2,3,11 8 9 1 MARE - Marine and Environmental Sciences Centre, Regional Agency for the Development 10 of Research, Technology and Innovation (ARDITI), Edif. Madeira Tecnopolo, Caminho da 11 Penteada, Piso 2, 9020-105 Funchal, Portugal 12 2 Departamento de Biologia Marina, Facultad de Ciencias del Mar, Universidad Católica del 13 Norte, Coquimbo, Chile 14 3 Center for Ecology and Sustainable Management of Oceanic Islands (ESMOI), Facultad de 15 Ciencias del Mar, Universidad Católica del Norte, Larrondo 1281, Coquimbo, Chile 16 4 Laboratório de Estudos de Bryozoa (LAEBry), Departamento de Zoologia, Centro de 17 Biociências, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego 1235, Cidade 18 Universitária, 50670-901 Recife, PE, Brazil 19 5 Department of Sciences, Natural History Museum, Cromwell Road, London SW7 5BD, UK 20 6 Departamento de Oceanografía, Facultad de Ciencias Naturales y Oceanográficas, 21 Universidad de Concepción, Barrio Universitario S/N, Concepción, Chile 22 7 Laboratorio de Microscopía Electrónica, Dpto. de Biología Marina, Universidad Católica del 23 Norte, Larrondo 1281, Coquimbo, Chile 24 8 Estación Experimental Quempillén, Instituto de Ciencias Marinas y Limnológicas, 25 Universidad Austral de Chile, Valdivia, Chile 26 9 New Zealand Institute of Earth Science (NZIES), Private Bag 14901, Kilbirnie, Wellington 27 6241, New Zealand 28 10 Instituto de Acuicultura, Universidad Austral de Chile, Los Pinos S/N, Pelluco, Puerto Montt, 29 Chile 30 11 MarineGEO, Smithsonian Environmental Research Center, 647 Contees Wharf Road, 31 Edgewater, MD 21037, USA 32 33 * = corresponding author 34 2 Table S1. Study locations and dates. * = Results from Rapa Nui were published in Rech et 35 al. (2024a). S = austral spring/ summer, W = austral autumn/ winter. ** = in local intertidal. 36 *** = on plastic litter. ID = Site identification code. Lat = Latitude, Long = Longitude. 37 Site ID Lat Long Time period (dd.mm.yyyy) Site use Study sites (settlement plates) Rapa Nui, Hanga Piko* RN 27.15°S 109.43°W S: 20.11.2022 - 16.03.2023 Marina Coquimbo, University campus COQ 29.97°S 71.35°W W: 22.02.2022 – 22.06.2022 S: 28.09.2022 – 07.02.2023 Aquaculture/ Port Algarrobo, Cofradía Náutica del Pacífico ALG 33.36°S 71.69°W W: 07.03.2022 - 11.07.2022 S: 21.09.2022 - 31.01.2023 Marina San Antonio, International Port SAN 33.58°S 71.62°W W: 18.05.2022 - 18.09.2022 Port Puerto Montt, Yachting Club Reloncavi PMO 41.49°S 72.99°W W: 13.05.2022 - 13.09.2022 S: 13.09.2022 - 23.01.2023 Marina Ancud, Research station Quempillen ANC 41.87°S 73.78°W W: 11.05.2022 - 15.09.2022 S: 16.09.2022 - 24.01.2023 Aquaculture Additional sites (opportunistic and beach survey) Coquimbo, University campus COQ 29.97°S -71.35°W 02.09.2023** Aquaculture/ Port Ritoque, Beach RIT 32.82°S -71.52°W 22.04.2021*** Beach 3 Table S2. Specimens deposited in the biological collections SCBUCN (Sala de colecciones 38 biológicas, Universidad Católica del Norte). COQ = Coquimbo, SAN = San Antonio, PMO 39 = Puerto Montt, ANC = Ancud, RIT = Ritoque, RN = Rapa Nui. * = Results from Rapa Nui 40 were published in Rech et al. 2024a. 41 Species Substratum Site Museum code Bugulina stolonifera (Ryland, 1960) Settlement plates (sea surface) COQ SCBUCN-8176, SCBUCN -8177 SAN SCBUCN-8178 PMO SCBUCN-8179 Bugulina cf. fulva (Ryland, 1960) Settlement plates (sea surface) COQ SCBUCN-8180 Conopeum sp. Settlement plates (sea surface) ANC SCBUCN-8182 Electra monostachys (Busk, 1854) Plastic debris (beach) RIT SCBUCN-10540, SCBUCN -10541 Watersipora arcuata (Banta, 1969) Rocks (intertidal) COQ SCBUCN-8181, SCBUCN -10539 Watersipora subtorquata (d’Orbigny, 1852) Settlement plates (sea surface) RN SCBUCN-5570* 42 4 Table S3. Results of Permutational ANOVA (PERMANOVA), showing degrees of freedom 43 (df), sums of squares (SS), mean squares (M'), pseudo-F ratio (Pseudo-F) and permutation P44 value (P(perm)) and number of unique permutations (Unique perms) for each factor. Sums of 45 squares type: Type III (partial). Fixed effects sum to zero for mixed terms. Permutation method: 46 Permutation of residuals under a reduced model. Number of permutations: 9999. Please see 47 Table S1 for full site names. *Term has one or more empty cells. Bold writing = statistically 48 significant values. 49 Average species richness per sampling unit (= frame) Based on Euclidean distance. All sites and seasons included. Main test Source df SS MS Pseudo-F P(perm) Unique perms Site 5 62.889 12.578 113.2 0.0001 9960 Season 1 4 4 36 0.0003 286 SitexSeason* 3 13.5 4.5 40.5 0.0001 9073 Residuals 9 1 0.11111 Total 18 78.105 Community composition per sampling unit (= frame) Bray-Curtis similarity. All sites and seasons. except Ancud (winter). Main test Source df SS MS Pseudo-F P(perm) Unique perms Site 5 33228 6645.6 110.75 0.0001 9921 Season 1 234.59 234.59 3.9094 0.1204 9984 SitexSeason* 2 1000.9 500.45 8.34 0.0109 9972 Residuals 8 480.05 60.006 Total 16 37025 50 5 Table S4. Frequency of occurrence, indicated as the percentage of settlement plates 51 fouled, of each taxon for each combination of site and season. Visualized by a colour scale, 52 ranging from 0% (light yellow) to 100% (dark green). win = austral autumn/winter, sum = 53 austral spring/summer. Ø, St. Dev = Average frequency and standard deviation of each species 54 amongst all combinations of site and season. n (settlement plates) = 4 for San Antonio (winter); 55 n = 8 for all other combinations of site and season. NIS = non-indigenous species, cryp = 56 cryptogenic, nat = native. 57 58 6 Table S5. Measurements of Bugulina stolonifera. COQ = Coquimbo. SAN = San Antonio. 59 PMO = Puerto Montt. SCBUCN = Biological collection of Universidad Católica del Norte, 60 Coquimbo, Chile. BMNH = Natural History Museum, London, UK. MMF = Museu de História 61 Natural do Funchal, Madeira. SERC = Smithsonian Environmental Research Center specimen 62 library, Edgewater, Maryland, USA. x = information not given in source publication. n = 63 number of examined units for each feature. 64 our study Ryland 1960 Ramalhosa et al. 2017 McCann et al. 2019 Location Chile, COQ Chile, SAN Chile, PMO United Kingdom Madeira Galapagos Museum ID SCBUCN 8176 SCBUCN 8177 SCBUCN 8178 SCBUCN 8179 BMNH 1959.3.18.11959.3.18.4 MMF 4480344807 SERC 232836; 310273 Zooid length, ZL [µm] Min – Max 458 – 898 430 – 774 514 –1078 471 – 855 x 461–675 x Mean ± SD 637 ± 110 594 ± 85 761 ± 151 665 ± 93 700 ± 75 - 740 ± 43 595 560 ± 64 n 35 29 24 35 x x x Zooid width, ZW [µm] Min – Max 105 – 178 119 – 157 130 –182 112 –168 x 100 – 165 x Mean ± SD 140 ± 15 137 ± 10 160 ± 14 143 ± 14 165 ± 13 - 180 ± 15 133 145 ± 17 n 34 24 22 36 x x x ZL / ZW Mean ± SD 4.6 ± 0.9 4.3 ± 0.7 4.6 ± 0.9 4.7 ± 0.7 4.2 ± 0.4 - 4.3 ± 0.6 x x n 34 23 20 33 x x x Avicularia length, AL [µm] Min – Max 94 – 206 109 –209 123 – 217 82 – 241 x 97 – 155 x Mean ± SD 164 ± 30 173 ± 27 174 ± 32 171 ± 42 x 140 165 ± 19 n 24 20 14 29 x x x Avicularia width, AW [µm] Min – Max 72 – 144 64 – 132 76 – 131 42 – 134 x x x Mean ± SD 109 ± 19 104 ± 17 102 ± 14 95 ± 25 x x 137 ± 8 n 25 22 14 29 x x x 7 AL / AW Mean ± SD 1.5 ± 0.2 1.7 ± 0.2 1.7 ± 0.3 1.9 ± 0.4 x x x n 23 20 11 25 x x x Ovicell length, OL [µm] Min – Max 103 – 144 94 – 139 100 – 134 122 – 157 x 101 - 124 x Mean ± SD 125 ± 13 124 ± 11 116 ± 11 136 ± 9 x 122 156 ± 4 n 9 27 13 19 x x x Ovicell width, OW [µm] Min – Max 162 – 219 154 – 194 153 -208 167 – 211 x 140 - 164 x Mean ± SD 191 ± 17 177 ± 9 186 ± 18 195 ± 10 x 157 164 ± 9 n 9 26 13 19 x x x OL / OW Mean ± SD 0.7 ± 0.1 0.7 ± 0.1 0.6 ± 0.1 0.7 ± 0.0 x x x n 9 26 13 17 x x x 65 8 Table S6. Cytochrome c oxidase subunit I (COI) barcodes generated in the present 66 study. SCBUCN = Sala de colecciones biológicas de Universidad Católica del Norte. GB = 67 Genbank. 68 Bugulina stolonifera (SCBUCN #8176; GB #PP109395) >Bri4_consensus_Frame1 ACTTTATATTTTTTATTTGGCCTATGAGCTGGTATAGTTGGAAGAGGATTAAGTG CCTTAATTCGAGTAGAACTTAGTCAACCAGGAAGTTTACTAGGTAATGACCACCT TTATAACGTGATTGTTACAGCTCATGCCTTTTTAATAATTTTCTTTATAGTTATAC CCGTAATAATTGGAGGATTTGGAAATTGATTAGTCCCTCTAATACTAGGATGCCC AGATATAGCTTTTCCACGACTAAATAATATAAGATTTTGACTTTTACCACCTGCA CTAGCTCTATTACTAATATCTTCTTTAGTTGAAAGAGGAGCAGGTACAGGATGAA CAGTATACCCCCCTTTATCATCTAGTCTCGGTCATAGTGGACCATCAGTTGATTTA GCAATTTTCTCCTTACACTTAGCTGGTGTCTCATCAATTTTAGGAGCAATTAATTT TATAACATCAACAATGAATATACGAAGAGGATCAATAACTATAATACATATCCC TCTATTAGTTTGAGCAGTTTTCATTACTGCTGTGTTACTACTACTATCTCTTCCAG TTCTAGCAGGCGCAATTACTATGCTGTTAACAGATCGAAATCTTAACACATCTTT TTTTGATCCTGC Bugulina stolonifera (SCBUCN #8177; GB #PP109396) >Bri13_consensus_Frame1 AAAGATATTGGAACTTTATATTTTTTATTTGGCCTTTGAGCTGGTATAGTTGGAA GAGGATTAAGTGCCTTAATTCGAGTAGAACTTAGTCAACCAGGAAGTTTACTAG GTAATGACCACCTTTATAACGTGATTGTTACAGCTCATGCCTTTTTAATAATTTTC TTTATAGTTATACCCGTAATAATTGGAGGATTTGGAAATTGATTAGTCCCTCTAA TACTAGGATGCCCAGATATAGCTTTTCCACGACTAAATAATATAAGATTTTGACT TTTACCACCTGCACTAGCTCTATTACTAATATCTTCTTTAGTTGAAAGAGGAGCA GGTACAGGATGAACAGTATACCCCCCTTTATCATCTAGTCTCGGTCATAGTGGAC CATCAGTTGATTTAGCAATTTTCTCCTTACACTTAGCTGGTGTCTCATCAATTTTA GGAGCAATTAATTTTATAACATCAACAATGAATATACGAAGAGGATCAATAACT ATAATACATATCCCTCTATTAGTTTGAGCAGTTTTCATTACTGCTGTGTTACTACT ACTATCTCTTCCAGTTCTAGCAGGCGCAATTACTATGCTGTTAACAGATCGAAAT CTTAACACATCTTTTTTTGATCCTGCAGGAGGAGGAGATCCCATTTTATATCAAC ATCTCTTCTGATTTTTTGGTCACC Watersipora arcuata (SCBUCN #8181; GB #PP109397) 9 >Bri6_FINAL2_Frame_1 GTAGTAACAGCTCACGCATTTTTCATGATTTTTTTTATAGTAATACCAATCATAAT TGGAGGATTTGGAAACTGGTTAGTTCCTCTTATAATGGGAATCCCGGATATAGCA TTCCCACGAATAAATAACATAAGATTTTGATTACTACCCCCTGCAGCTATTTTAC TAATAATATCAGCACTAGTAGAAAGAGGTGCAGGGACAGGGTGAACAGTATACC CCCCTTTATCTTCAAACATTTCTCATAGAGGATCATCAGTAGATATAGCAATTTTC GCTCTTCACTTAGCAGGAGTGTCTTCAATTTTAGGAGCAATTAACTTTATAGTAA CAATTATCAATATACGAGGAAAAATAATAGCATTCATAAACTTACCATTATTCGT ATGAGCCGTATTTATCACTGCCCTATTATTATTACTATCTCTCCCAGTTCTAGCAG GAGCTATTACTATACTACTAACAGACCGTAATTTTAACTCATCATTCTTTGACCC AGCT 69 16 96 97 Figure S1. Examples of fouled settlement plates after retrieval. Left: Coquimbo, summer 98 2023. Right: Algarrobo, winter 2022. 99 100 101 Figure S2. Species richness per sample unit (= frame) by study site and season. n (frames) 102 = 1 for San Antonio (winter); n = 2 for all other combinations of site and season. 103 104 17 105 Figure S3. nMDS (non-metric multidimensional scaling) plot of community composition 106 based on the frequency of occurrence of each taxon on each sample unit at each combination 107 of study site and season. win = winter, sum = summer. n (sampling units) = 1 for San Antonio 108 (winter); n = 2 for all other combinations of site and season. 109 110 18 111 Figure S4. Electra monostachys, found attached to PET bottle fragment on Ritoque beach, 112 on 22nd of April 2021. Sample ID: SCBUCN #10540. a) colony overview. b) close up of 113 autozooids. 114 19 115 Figure S5. Watersipora nigra, USNM 8495, holotype, Galapagos. a) Zooids. b) Orifice 116 (close-up). c) Colony overview. 117