bioRxiv preprint doi: https://doi.org/10.1101/519041; this version posted January 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 1 Fire ! The spread of the Caribbean fire coral Millepora alcicornis in the Eastern 2 Atlantic. 3 4 Peter Wirtz & Carla Zilberberg 5 6 Peter Wirtz (email [email protected]) Centro de Ciências do Mar, Universidade do 7 Algarve, Campus de Gambelas, PT 8005-139 Faro, Portugal. Carla Zilberberg (email 8 [email protected]) Núcleo em Ecologia e Desenvolvimento Ambiental de Macaé, 9 Universidade Federal do Rio de Janeiro, Macaé, Brazil. 10 11 The Western Atlantic fire coral Millepora alcicornis is recorded from Madeira Island in the Eastern 12 Atlantic for the first time. A large colony of this species has apparently been present at a very exposed 13 site at the northern shore of Madeira Island for more than 15 years. Genetic analyses suggest that the 14 colonies of this tropical fire coral at a mid-Atlantic location (Ascension Island) and at each of three 15 Eastern Atlantic locations (Cape Verde Islands, Canary Islands, Madeira Island) originated from 16 independent long-distance dispersal events from the Caribbean area. 17 18 Key words: Long-distance dispersal, Millepora alcicornis, phylogeography, habitat change 19 20 21 22 23 24 25 ________________________________________________________________________________ 26 bioRxiv preprint doi: https://doi.org/10.1101/519041; this version posted January 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 2 27 INTRODUCTION 28 29 Long-distance dispersal events occur rarely but, lying at the heart of island biogeography theory, 30 they play a fundamental role in shaping species large-scale biogeography (Smith et al. 2018). The 31 arrival of a species into a new area can have profound impacts on local ecological communities, 32 leading altered environmental conditions and novel interactions, in particular if that species is a 33 habitat forming one and competing with local endemics. 34 Fire corals (Millepora species) are well known for being important reef builders because of their 35 large calcareous skeletons and for inflicting painful stings to humans (Lewis 2006). The genus is 36 limited to 50 m depth in tropical seas, with a clear distinction between Atlantic and Pacific species 37 (Razak & Hoeksema 2003). There are seven species in the Atlantic Ocean and until recently these 38 were only reported from the tropical western Atlantic and from the Cape Verde Islands in the Eastern 39 Atlantic (Laborel 1974, de Weerdt 1984). The species found at the Cape Verde Islands, Millepora 40 alcicornis Linnaeus, 1758, is common in the western Atlantic and has recently also been documented 41 from Ascension Island in the middle of the Atlantic Ocean (Hoeksema et al. 2017) and from the 42 Canary Islands in the Eastern Atlantic, where it apparently arrived only a short time ago (Clemente et 43 al. 2011, López et al. 2017). 44 In summer 2016, a fisherman from Madeira Island showed a large dried branch of a fire coral 45 (Millepora sp.) to the first author and claimed that it came from the north coast of this island. The 46 previous finding of fire coral at the Canary Islands (28oN) was already quite unexpected; the presence 47 of Millepora in the even colder waters of Madeira Island (32oN) would be an even greater surprise. 48 Here we report that there is indeed a large colony of fire coral at Madeira’s north coast, the type of 49 habitat it occupies, the identity of the species, and the probable origin of the colony. 50 51 52 MATERIAL AND METHODS bioRxiv preprint doi: https://doi.org/10.1101/519041; this version posted January 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 3 53 The Millepora colony was photographed under water and several branches were collected in August 54 2016. Some of these branches are now deposited in the Natural History Museum of Funchal, 55 Madeira, with the registration number MMF 46310. Fragments were preserved in 96% ethanol and 56 sent to the second author for molecular analyses. 57 Total DNA extraction followed the phenol-chlorophorm method performed by Fukami et al. (2004), 58 placing the sample in a CHAOS solution one week prior to extraction. DNA quality and 59 concentration were assessed on a 0.8% agarose gel stained with GelRed (Biotium) and visualized 60 under UV light, using the pattern Lambda DNA (125 ng/μL). 61 For species identification, the 16S rRNA gene of mitochondrial DNA (16S) was obtained from the 62 Madeira Millepora colony and compared with previously reported sequences from other Atlantic 63 Millepora specimens and species from the NCBI database (https://www.ncbi.nlm.nih.gov). A 537bp 64 fragment of the large ribosomal subunit of the mitochondrial DNA (16S) was amplified using the 65 following pair of primers: SHA 5'-ACGGAATGAACTCAAATCATGT-3; SHB 5'- 66 TCGACTGTTTACCAAAAACATA-3’ (Cunningham & Buss 1993). 67 The polymerase chain reactions (PCR) consisted of PCR buffer 1X, dNTP (2 mM), bovine serum 68 albumin (1 mg/ml), MgCl2 (1.5 mM), Taq polymerase (1U), primers (0.5 uM), ~1ng of template 69 DNA. Thermal cycling conditions started with a denaturing step at 95oC for 3 min, followed by 10 70 cycles of 94oC for 1min, 40oC for 1min and 72oC for 1min, 40 cycles at 94oC for 1min, 52oC for 71 1min and 72oC for 1min and a final extension step at 72oC for 5 min. The amplified product was 72 purified with ExoSAP-IT PCR Product Cleanup (Thermo Fisher Scientific) following manufacturer’s 73 instructions and Sanger sequencing was performed in both directions at GATC Biotech (Germany). 74 Electropherograms were edited and a consensus sequence was created with Geneious R7 75 (http://www.geneious.com, Kearse et al., 2012). Alignment was performed using the ClustalW 76 package in Geneious R7. Maximum likelihood (ML) phylogenetic reconstruction analyses and 77 substitution models’ calculations were performed with PhyML 3.0 (Guidon et al. 2010). Substitution 78 model selection was calculated using Smart Model Selection (Lefort et al. 2017) with the Akaike bioRxiv preprint doi: https://doi.org/10.1101/519041; this version posted January 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 4 79 Information Criterion and the ML reconstruction. The substitution model used for the phylogenetic 80 tree reconstruction was the HKY85 +G+I. Maximum likelihood analysis started with a neighbour 81 joining tree followed by a Nearest Neighbour Interchange searching criterion and 1000 bootstraps for 82 branch support. 83 A median-joining haplotype network was constructed using the software Networ v4.6.1.1 (Fluxus 84 Technology Ltd.). This haplotype network included all M. alcicornis sequences used in de Souza et 85 al. (2017), in addition to the sequence generated from the Millepora sample from Madeira Island in 86 this study: Forty-four Millepora alcicornis colonies from the Caribbean Province, 109 colonies from 87 the Brazilian Province, nine colonies from the Cape Verde Islands, a single colony from the Canary 88 Islands, and two colonies from Ascension Island (Hoeksema et al. 2017) were compared with the 89 colony from Madeira Island (Table 1). 90 91 RESULTS 92 93 Colony site and colony morphology 94 95 The Millepora colony of Madeira Island was found in shallow water (3m depth at low tide), in a 96 small bay at a very exposed site on the north coast of Madeira Island (approximately 32o45´N, 16 97 o43´E). Water temperature at this site varies from 16 to 23 degrees C annually (personal observations 98 by the first author). 99 The main colony had a roughly rectangular shape, approximately 4 m long and 3 m wide (figure 1). 100 There were numerous small colonies (hand-sized or smaller) scattered around the large main colony. 101 The fisherman, who guided the first author to this site, reported that there were fewer small colonies 102 previously but that the main colony was already this large size when he first encountered it more than 103 15 years ago. 104 bioRxiv preprint doi: https://doi.org/10.1101/519041; this version posted January 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 5 105 106 Figure 1: Millepora colony at the north coast of Madeira Island. The fins of the diver are 80 cm long. 107 108 The central part of the colony was characterized by erect branches up to 18 cm high, flattened 109 laterally at the tips (Figures 2-3); at the edges and at its base, the colony was encrusting. The strong 110 branches were very solid and difficult to break off, being able to resist the heavy wave action typical 111 for the north coast of Madeira Island.