Conservation Genet Resour DOI 10.1007/s12686-013-0023-5

MICROSATELLITE LETTERS

Microsatellite loci for goreaui and other Clade C Symbiodinium

Drew C. Wham • Margaux Carmichael • Todd C. LaJeunesse

Received: 27 June 2013 / Accepted: 12 August 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract The genus Symbiodinium comprises a diverse of ‘‘Clades’’, with the most ecologically dominant and group of dinoflagellates known for their obligate relation- diverse being Clade C (LaJeunesse et al. 2004). While the ship with reef–building . Members of the sub-genus group includes phylogenetic types that display narrow and ‘clade C’ are abundant, geographically wide-spread, as broad thermal tolerances, members of clade C are often the well as genetically and ecologically diverse. colonies most severely impacted during bleaching events. For this harboring clade C are often the most exposed to physical reason, their symbiosis biology, dispersal capabilities and stressors. The genotypic diversity, dispersal and genetic genetic identities are of increasing importance to conser- connectivity exhibited by these Symbiodinium are the vation efforts as climates change globally, further affecting subjects of an increasing number of population genetic reef coral communities. studies utilizing microsatellites. Here we describe 18 new Coral are increasingly being studied with microsatellite loci and test their utility across four common multi-locus techniques because this approach offers genetic clade C types. We obtained multi-locus genotypes with resolution to the level of the individual. High-resolution individual level resolution in each of these types. Our markers, however, are not yet available for most Symbiodi- results indicate that multi-locus genotypes can be obtained nium spp. In the present study, we present 18 new loci for for many members of clade C using a subset of these Symbiodinium Clade C; 14 loci developed on a monoclonal markers. Data from these markers may assess population culture of S. goreaui (C1) and 4 loci developed from the dynamics during and recovery as well as symbiont specific to the zooanthid Zoanthus sansibaricus in resolve boundaries to genetic recombination among these the western tropical North Pacific referred to here by its closely related, yet ecologically distinct, lineages. diagnostic ITS2 sequences C3ee-ff, previously referred to as C1z (sensu Reimer 2008). In combination with previously Keywords Symbiodinium Á Microsatellites Á published markers on other clade C types (Bay et al. 2009), Population genetics Á Coral reefs these markers provide individual level resolution through multi-locus geneotypes for many members of clade C. We used 454 sequencing at the Penn State Nucleic Acids The genetically diverse dinoflagellates in the genus Sym- Facility (University Park, PA) to generate two genomic biodinium are often found in obligate symbiosis with reef libraries of [50,000 read lengths of 250–400 bp. The first building corals. This genus has been divided into a number sequence library was created from genomic DNA obtained from cultured S. goreaui (culture rt-113 from the Robert K. Trench collection). The second library was created from the Electronic supplementary material The online version of this article (doi:10.1007/s12686-013-0023-5) contains supplementary pooled DNA from the symbionts of Zoanthus sansibaricus. material, which is available to authorized users. Both libraries were prepared from 3,000 ng of double- stranded DNA, using the Nextera DNA Sample Prep Kit & D. C. Wham ( ) Á M. Carmichael Á T. C. LaJeunesse (Epicentre Biotechnologies, Madison WI) and sequenced on Department of Biology, 221 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802, USA a 454 GS-FLX sequencer (Roche Diagnostics Corporation, e-mail: [email protected] Indianapolis, IN) utilizing the Titanium Sequencing Kit 123 Conservation Genet Resour

Table 1 Size ranges of 4 clade C types, N is the number of samples genotyped and Na is the number of alleles observed Locus C1 GBR C3 GBR C1c Hawaii CZ Okinawa Range N Na Range N Na Range N Na Range N Na

Sgr_9a 254–266 2 2 Sgr_19a 169–178 3 4 Sgr_21a 132–154 3 3 125–141 10 5 128–137 42 5 Sgr_33a 190–196 3 4 Sgr_34 154–166 3 4 129–135 12 4 Sgr_37a 124–136 5 7 109–121 25 6 Sgr_40 192–215 52 6 188–209 24 6 170–176 49 4 SgrSpl_13 136–193 46 19 96 12 1 SgrSpl_22 134–140 51 5 101–107 10 3 SgrSpl_24 121–133 9 4 SgrSpl_25 110–116 55 5 107–122 10 5 105–120 24 5 94 56 1 SgrSpl_26 112–126 30 8 95–101 5 3 199–214 22 4 SgrSpl_30 208–222 44 8 173–183 11 6 206–226 22 10 201–205 58 5 SgrSpl_78 168–184 9 5 153–159 58 5 Spl_1a 122–136 12 6 215–223 52 5 Spl_16 155–167 10 5 Spl_33 192–222 10 4 209–232 58 1 Spl_89a 191–199 47 5 Data in bold included in linkage-disequalibrium test a Allele sizes include 20 bp M13 sequence used to fluorescently label PCR products with the method described in (Schuelke 2000)

(Roche). Sequences containing di-, tri- or tetranucleotide Hawaii and Spl_1 and Spl_89 in C3ee-ff from Okinawa, repeat motifs were identified following the procedures Japan. described in Wham et al. (2011). The software Primer3 These loci can be utilized in a number of genetic appli- (http://primer3.sourceforge.net/) was then used to design cations. Many of the microsatellites amplified across more primer sets for candidate microsatellites. than one of the species we tested (Table 1). Most loci were These PCR primers were screened for their ability to polymorphic across samples of a given phylogenetic type consistently amplify DNA fragments of expected allelic size and will be useful for examining diversity, gene-flow and from the monoclonal strains of S. goreaui (rt-113 and rt-152) genetic exchange. Several were polymorphic only between, and from samples obtained from animal tissues containing and hence diagnostic of, certain phylogenetic types Symbiodinium clade C. These samples were selected for their (Table 1). These may be utilized in the future as tools for diverse geographic origin (Hawaii, USA, Great Barrier Reef, identifying species within clade C. Collectively, these Australia and Okinawa, Japan) as well as their diagnostic ITS microsatellites offer additional high-resolution genetic sequence (C1, C1c, C3 and C3ee-ff). PCR reactions with markers for researchers investigating Symbiodinium ecology M13 labeled primers were preformed according to the and their use may provide new insights into species diversity, methods described in Wham et al. (2011). Loci were only mechanisms for local adaptation, as well as, population retained if they were polymorphic within or between types of dynamics during bleaching and recovery of reefs. Symbiodinium types (supplementary material 1). Dye labeled forward primers were then used to sample genetic Acknowledgments We would like to thank the Penn State diversity in populations of four sub-clade C types (Table 1). Genomics Core Facility at University Park for generating the 454 sequence data used in this study. This research was supported by the Fragment analysis returned genotypic data that was consis- Pennsylvania State University, the National Science Foundation tant with a diploid interpritation of genome copy number in grants (OCE-09287664) and by the National Science Foundation - both monoclonal cultures, as well as samples isolated from East Asia and Pacific Summer Institute (OISE-1108258). host tissue. A linkage disequilibrium test was preformed between all loci with sufficient sampling within populations References (Table 1) using the software GENEPOP (Raymond and Rousset 1995). All pairwise test were insignificant for link- Bay LK, Howells EJ, van Oppen MJ (2009) Isolation, characterisation age with the exceptions of SgrSpl_26 and Sgr_40 in C1c in and cross amplification of thirteen microsatellite loci for coral

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endo-symbiotic dinoflagellates (Symbiodinium clade C). Conserv Reimer JD (2008) Implications for different diversity levels of Genet Resour 1(1):199–203 Symbiodinium spp. (, ) within closely LaJeunesse TC, Bhagooli R, Hidaka M, Done T, deVantier L, related hosts: zoanthids (Cnidaria: Hexacorallia: Anthozoa) as a Schmidt GW, Fitt WK, Hoegh-Guldberg O (2004) Closely- case study. Galaxea 10:3–13 related Symbiodinium spp. differ in relative dominance within Wham DC, Pettay DT, LaJeunesse TC (2011) Microsatellite loci for coral reef host communities across environmental, latitudinal, the host-generalist ‘‘zooxanthella’’ Symbiodinium trenchi and and biogeographic gradients. Mar Ecol Prog Ser 284:147–161 other Clade D Symbiodinium. Conserv Genet Resour 3:541–544 Raymond M, Rousset F (1995) GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J. Heredity 86:248–249

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