Genetic diversity of Ectocarpus (Ectocarpales, Phaeophyceae) in Peru and northern Chile, the area of origin of the genome-sequenced strain Akira F. Peters, Aaron D. Mann, César A. Cordova, Juliet Brodie, Juan A. Correa, Declan C. Schroeder, J. Mark Cock To cite this version: Akira F. Peters, Aaron D. Mann, César A. Cordova, Juliet Brodie, Juan A. Correa, et al.. Genetic diversity of Ectocarpus (Ectocarpales, Phaeophyceae) in Peru and northern Chile, the area of origin of the genome-sequenced strain. New Phytologist, Wiley, 2010, 188 (1), pp.30-41. 10.1111/j.1469- 8137.2010.03303.x. hal-01806412 HAL Id: hal-01806412 https://hal.archives-ouvertes.fr/hal-01806412 Submitted on 16 Nov 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Page 1 of 32 1 Diversity of Ectocarpus (Ectocarpales, Phaeophyceae) in Peru and 2 northern Chile, the area of origin of the genome-sequenced strain 3 4 Akira F. Peters1,2,3, Aaron D. Mann4, César A. Córdova5, Juliet Brodie6, Juan A. Correa4, 5 Declan C. Schroeder1 and J. Mark Cock3 6 For Peer Review 7 1 Marine Biological Association, Citadel Hill, Plymouth PL1 2PB, UK 8 2 Bezhin Rosko, 28 route de Perharidy, 29680 Roscoff, France 9 3 UMR7139, Station Biologique, Centre National de la Recherche Scientifique et Université 10 Pierre & Marie Curie Paris VI, Place Georges Teissier, 29682 Roscoff cedex, France 11 4Departamento de Ecología and Center for Advanced Studies in Ecology and Biodiversity, 12 Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, 13 Santiago, Chile 14 5Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Ciudad 15 Universitaria, Lima, Perú 16 6Natural History Museum, Department of Botany, Cromwell Road, London SW7 5BD, UK 17 18 Author for correspondence: 19 Akira F. Peters 20 Email: [email protected] 21 Received: xx 22 Accepted: xx Manuscript submitted to New Phytologist for review Page 2 of 32 1 2 Summary 3 4 • The origin of the Ectocarpus strain used for genome sequencing (the "genome strain") 5 was Peru whence no Ectocarpus was recorded previously. To study the genetic diversity 6 in the region and to increase the number of individuals from this area available for genetic 7 experiments, 119For new Ectocarpus Peer strains wereReview isolated at eight localities along the 3000 8 km coastline from central Peru to central Chile. 9 • ITS1 genotyping revealed nine different genotypes, four of which were endemic to the 10 area studied and two were so far unknown. 11 • Individuals of the same genotype as the genome strain occurred from Peru to 12 northernmost Chile, representing 61% of the samples in this area from which five more 13 genotypes were isolated. Further south, down to central Chile, most individuals matched 14 European E. siliculosus, E. fasciculatus and E. crouaniorum. In sexual crosses, the 15 genome strain and our new isolates of the same genotype were fully compatible. 16 • Sequences from four nuclear and cytoplasmic genetic markers (ITS1, ITS2, Rubisco 17 spacer, cox3) separated the genome strain from the European species of Ectocarpus, but it 18 grouped with one of the other South American endemic genotypes. This clade may in 19 future be recognised as a separate species. 20 21 Key words: Genetic diversity, Ectocarpus, Kuckuckia, Chile, Peru 22 23 word count: summary: 197 words [200 permitted]; all text (excluding Title and Summary): 4604 24 words [6500 permitted]; tables: 1152 words; supporting material: 451 words Manuscript submitted to New Phytologist for review Page 3 of 32 1 Introduction 2 3 The Ectocarpus strain chosen for genome sequencing (henceforth referred to as the "genome 4 strain") is a male gametophyte which has the designation "Ec32" in the Ectocarpus strain 5 collection at Roscoff. It was selected because it displays an alternation of two morphologically 6 distinguishable generations (Peters et al., 2008) and produces unilocular sporangia on 7 parthenogenetic sporophytes,For both Peer of which facilitate Review studies on the complex brown algal life 8 history (for more reasons and terminology of morphology and life history see Peters et al., 2004a; 9 Coelho et al., 2007; Charrier et al., 2008). The strain was obtained from a meiospore of a field 10 sporophyte collected by the first author in November 1988 at San Juan de Marcona, Peru (Site 3 11 in Fig. 1). Identified as E. siliculosus (Dillwyn) Lyngbye in a first molecular phylogeny (Stache- 12 Crain et al., 1997, designation "SAm120h"; lineage 1c), it later showed post-zygotic 13 incompatibility (Peters et al., 2004b) when crossed with strains from Europe regarded as genuine 14 E. siliculosus (i.e. genetically close to those used by Müller (1967) for the description of the life 15 history; lineage 1a in Stache-Crain et al., 1997) or with a strain of Ectocarpus from New Zealand 16 previously used in research on virus inheritance (Müller, 1991; lineage 4 in Stache-Crain et al., 17 1997): hybrids possessed a normal morphology, but were unable to form meiospores. Further 18 crosses of the genome strain with an Ectocarpus from northern Chile, as well as with the recently 19 reinstated E. crouaniorum Thuret in Le Jolis from Europe (Peters et al., 2010), gave similar 20 results (unpublished data). Although sisters of the genome strain (female gametophytes from the 21 same field sporophyte from Peru) were available for genetic experiments, for instance to test sex- 22 linkage of a mutation (Peters et al., 2008), no female gametophyte of sufficient genetic distance 23 was available for experiments requiring outcrossing (e.g. to produce a genetic map, Heesch et al., 24 2010). Manuscript submitted to New Phytologist for review Page 4 of 32 1 2 Apart from the genome strain there was no unambiguous published record of Ectocarpus 3 from Peru. Howe (1914: p. 50) mentioned "small specimens of Ectocarpus" on Desmarestia 4 peruviana Montagne at Ancón (13 February 1907) and on Lessonia nigrescens Bory de Saint- 5 Vincent at Chincha Island (18 June 1907); however, he regarded the material as "too meager to 6 justify an attempt at determination or description". The nearest published record of Ectocarpus 7 was from Iquique, northernFor Chile, Peerat ca 800 km distanceReview from Site 3 (Ramírez & Santelices, 8 1991). 9 10 To isolate additional strains of Ectocarpus showing full compatibility with the genome 11 strain, we collected more individuals from localities along the South American Pacific coast from 12 central Peru to central Chile. They were identified based on comparisons with nuclear ribosomal 13 internal transcribed spacer 1 (ITS1) sequences available for 43 strains of Ectocarpus and seven of 14 Kuckuckia (the sister genus of Ectocarpus) from all continents except Antarctica (Stache-Crain et 15 al., 1997). In the studied area, Ectocarpus presented a surprisingly high genetic diversity 16 described in the present paper. Isolates of one genotype were genetically similar to the genome 17 strain and proved to be fully interfertile with it. Manuscript submitted to New Phytologist for review Page 5 of 32 1 Materials and Methods 2 3 Field collection and isolation of cultures. Nine localities (four in Peru, five in Chile) spanning a 4 distance of approximately 3000 km were visited for collection, most during one visit in February- 5 March 2006 (Fig. 1, Table 1). Sampling at Site 8 was done for studies on the response of 6 Ectocarpus to copper pollution (Mann et al., unpublished). From each field thallus 7 macroscopically resemblingFor Ectocarpus Peer, filaments Review were inoculated in a 2 ml Eppendorff tube 8 containing autoclaved sea water. After transfer to the Biological Station at Roscoff, isolation and 9 cultivation of clean unilalgal clonal cultures were undertaken as described previously (Peters et 10 al., 2010). A number of thalli from Site 8 were isolated by filtering nearshore surface seawater 11 and cultivating filaments that developed on filter paper, or by inoculating field macrothalli of 12 Scytosiphon and isolating all Ectocarpus thalli developing on them in culture. Only thalli 13 possessing ribbon-shaped plastids were retained for further study. 14 15 Strain designations beginning with "CCAP1310/" are from the Culture Collection of Algae 16 and Protozoa; numbers or numbers preceded by "Ec" are isolates housed in the Ectocarpus strain 17 collection at Roscoff, and are maintained by the first author. 18 19 Molecular methods. DNA was extracted from living cultures as described (Peters et al., 20 2004b). Initial identification was based on ITS1 length (see Peters et al., 2010 for rationale), 21 followed by sequencing of ITS1 or by a diagnostic PCR using a primer specific for the ITS1 22 sequence of the genome strain (for details and oligonucleotide primers used see supporting 23 information). Sequences were aligned manually using Se-Al v. 2.0a11 (Rambaut, 2002). 24 Sequences similar over the full length of ITS1 and not requiring indels of more than 20bp were Manuscript submitted to New Phytologist for review Page 6 of 32 1 considered to belong to the same ITS1 genotype (GT). For at least one member of each ITS1 2 genotype discovered, three additional markers (ITS2, 3'-rbcL+Rubisco spacer, cox3) were 3 sequenced to provide data for phylogenetic analyses. For the genome strain, the corresponding 4 data were taken from the genome. Sequences were deposited in the EMBL/Genbank/DDBJ 5 database (Table 2).