7000 Years of Emiliania huxleyi Viruses in the Black Sea

7000 Years of Emiliania huxleyi Viruses in the Black Sea Marco J. L. Coolen Science 333, 451 (2011); DOI: 10.1126/science.1200072

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REPORTS

7000 Years of Emiliania huxleyi Viruses in the Black Sea Marco J. L. Coolen A 7000-year record of Coccolithovirus and its host, the calcifying haptophyte Emiliania huxleyi, was reconstructed on the basis of genetic signatures preserved in sediments underlying the Black Sea. The data show that the same virus and host populations can persist for centuries.

Major changes in virus and host populations occurred during early sapropel deposition, ~5600 years ago, and throughout the formation of the coccolith-bearing sediments of Unit I during the past 2500 years, when the Black Sea experienced dramatic changes in hydrologic and nutrient regimes.

Unit I saw a reoccurrence of the same host genotype thousands of years later in the presence of a different subset of viruses. Historical plankton virus populations can thus be included in paleoecological and paleoenvironmental studies.

Marine Chemistry and Geochemistry Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA. E-mail: mcoolen@whoi.edu

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were sources of alkenones in the underlying sapropel Unit II and that the first strains colonized the photic zone at least 7.3 ky B.P. (10), more than 4 ky earlier than the first occurrence of its preserved coccoliths (14, 15).

Presence Holocene viral PGM genotypes

iruses have a major impact on global nutrient and biogeochemical cycles in the world’s oceans (1, 2) and are also believed to play a role in regulating host communities (3).

For example, glycosphingolipids from Coccolithovirus type strain EhV-86 (4) induce lytic infection and programmed cell death in the coccolithophorid haptophyte Emiliania huxleyi (5).

In paleoecological studies, planktonic community shifts can be reconstructed from morphological and chemical signatures in sediments and provide indirect clues about past environmental conditions. Viruses of Heterosigma akashiwo (Raphidophyceae) and cyanobacteria have been recovered from sediments up to 100 years old and were found to be capable of reinfecting their planktonic hosts (6, 7), and unidentified viral particles have been observed microscopically in older marine sediment records (8). Likewise, the analysis of preserved genetic signatures in the sedimentary record by means of ancient DNA methods (9, 10) offers a promising approach to reconstruct historical viral populations and the plankton they infected in the past.

Climate shifts over Eurasia, together with global sea-level variations, modulated freshwater and saltwater inputs to the Black Sea during deglaciation and the early Holocene, leading to major hydrologic and biological changes in the basin (10–13). Geochemical and palynological evidence exists for a shoaling of the chemocline and an increased nutrient load to the photic zone during early sapropel Unit II deposition (11), and the establishment of euryhaline conditions ~5600 years before the present (5.6 ky B.P.) (12). Since ~3 thousand years ago (ka), increased riverine input of fresh water and nutrients (13) coincides with the occurrence of coccoliths (14, 15) and lipid biomarkers (i.e., long-chain alkenones) (16) of E. huxleyi in the upper laminated coccolith ooze of Unit I. Mitochondrial cytochrome oxidase subunit I (COI) genotyping using ancient DNA methods confirmed that various E. huxleyi strains

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Western Black Sea sediments (42°46.569″N, 28°40.647″E) spanning Unit II and most of Unit I (118 continuous 1-cm intervals deposited between 7.5 and 0.45 ky B.P.) that served for E. huxleyi COI genotyping (10) were used for a parallel stratigraphic analysis of Holocene E. huxleyi–infecting viruses (EhV), based on genotypic variation in their partial gene encoding the major capsid protein (MCP) (17, 18) (Fig. 1). Preserved 260–base pair (bp)–long viral MCP gene fragments were amplified by polymerase chain reaction (PCR) (19) in 83 of the 118 samples (fig. S1), and the resulting amplicons were analyzed by denaturing gradient gel electrophoresis (DGGE) (19, 20) (fig. S2). Eighty-five of 101 excised DGGE bands were successfully sequenced (19) and represented 33 unique genotypes (Fig. 1), with 95 to 98.7% nucleotide and 100% amino acid identity to coccolithoviral MCP sequences available in GenBank (fig. S3).

All genes with functional predictions identified in the EhV-86 genome (4) were aligned with GenBank coccolithoviral gene sequences

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Fig. 1. Stratigraphic analysis of Holocene GGC18 sediments and the top 2 cm of recently deposited surface sediment of MC06. (A) Relative percent abundance of preserved E. huxleyi COI genotypes (numbered 1 to 11 above the figure) (10). The presence or absence of preserved viral (B) MCP and (C) PGM genotypes in the analyzed sediment intervals is shown. See figs. S2 and S5 for corresponding similar numbered DGGE bands and figs. S3 and S6 for phylogeny. The light blue–shaded areas mark the periods when the Black Sea experienced major changes in hydrologic and nutrient regimes (11, 13).

The darker blue–shaded area marks the euryhaline period (12). The total number of retrieved host and viral genotypes during the three major environmental stages is listed below (C). All data points are plotted against calendar ages.

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REPORTS were identified together with 25 viral MCP and 12 viral PGM genotypes. During early sapropel deposition (7.4 to 5.6 ky B.P.), four unique host genotypes coincided with eight viral MCP and five viral PGM genotypes, whereas a comparable number of viral genotypes were identified with twice as many host genotypes after the establishment of euryhaline conditions at 5.6 ky B.P. (Fig. 1). A few of the same viral genotypes were recovered from sediment intervals throughout Units I and II (EhV_MCP-17, -20, and -1; and EhV_PGM-1, -15, and -2) and co-occurred with multiple host genotypes. However, most shifts in virus and host populations coincided with the Holocene environmental changes. For example, Eh-COI-2, -6, -10, and -11, and co-occurring viruses, were replaced by a different suite of genotypes with the establishment of euryhaline conditions. Viral EhV_MCP-8 and EhV_PGM-6 were only present during early sapropel and Unit I deposition. Unit I saw the return of host genotypes Eh-COI-1 and Eh-COI-2, but with a different suite of viruses (Fig. 1). In addition, both Eh-COI-3 and coincident viruses were identified only from Unit I. One E. huxleyi and one viral genotype dominated the modern photic zone and the upper 2 cm of sediment (Fig. 1 and figs. S2 and S5) deposited in recent decades. Furthermore, EhCOI-1 and the virus represented by EhV_MCP-20 and EhV_PGM-2 have co-occurred for over a century (495 to 640 years ago; Fig. 1 and fig. S1). An apparent long-term coexistence of the viral genotype EhV_MCP-17 with the host Eh-COI-1was observed during euryhaline conditions. In contrast, other viruses appeared to show limited persistence, most notably at ~4 ky B.P., when EhV_PGM-5

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Fig. 2. Quantity of preserved genetic markers of E. huxleyi and its viruses in the Holocene Black Sea sediment record as revealed by quantitative PCR. (A) E. huxleyi COI gene, (B) Coccolithovirus MCP gene, (C) Coccolithovirus PGM gene. The number of preserved gene copies is expressed per gram of total organic carbon (TOC) to compensate for variability in sedimentary TOC content. All data points are plotted against calendar ages.

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co-occurred with the unique E. huxleyi genotypes Eh-COI-8 and -9 (Fig. 1).

The analysis of genetic signatures of Coccolithovirus and its host E. huxleyi preserved in this core of Black Sea sediments provides some insights into long-term virus–phytoplankton host dynamics during the Holocene. These types of data may prove useful to paleoecologists and paleoclimatologists, and similar ancient DNA methods could be applied to other plankton species, such as the widely distributed pelagic cyanobacterium Synechococcus, which is known to be infected by DNA viruses (23).

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13. M. T. J. Van der Meer et al., Earth Planet. Sci. Lett. 267, 426 (2008).

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16. J. K. Volkman, G. Eglinton, E. D. S. Corner, T. E. V. Forsberg, Phytochemistry 19, 2619 (1980).

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20. H. Schäfer, G. Muyzer, Denaturing Gradient Gel Electrophoresis in Marine Microbial Ecology (Academic Press, San Diego, CA, 2001), pp. 425–468.

21. M. J. L. Coolen et al., Paleoceanography 21, PA1005 (2006).

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64, 4513 (1998).

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Acknowledgments: I thank C. Wuchter for suggestions for improving the manuscript, P. Dimitrov and D. Dimitrov for their expertise in Black Sea sedimentology, and T.-H. Yang and J. van de Giessen for analytical support. This research was supported by NSF grants OCE 0602423 and OCE 0825020 and a grant from the Andrew W. Mellon Foundation Endowed Fund for Innovative Research. PCR-obtained sequences encoding the coccolithoviral MCP and PGM genes have been deposited in GenBank as “environmental sequences” and are listed under accession numbers GU563596 to GU563628, GU584106 and GU584107, and HQ412623 to HQ412638.

Supporting Online Material www.sciencemag.org/cgi/content/full/333/6041/451/DC1 Materials and Methods Figs. S1 to S6 References 5 November 2010; accepted 9 June 2011 10.1126/science.1200072

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to identify an additional marker for Coccolithovirus genotyping. Fragmentation of Holocene plankton DNA in sediments leads to fewer template DNA for PCR; therefore, the targeted variable region, including conserved priming sites, should not exceed a length of ~500 bp (21, 22).

Using these criteria, a 423-bp-long region of the gene encoding phosphoglycerate mutase (PGM), known to be expressed by EhV-86 after infection (4), was selected for PCR amplification and subsequent DGGE analysis. Viral PGM gene amplicons were recovered in 81 of 118 samples (fig. S4), and all 94 excised DGGE bands (fig.

S5) were successfully sequenced. The 15 unique genotypes showed 94 to 100% nucleotide identity and 100% amino acid identity to coccolithoviral PGM sequences available in GenBank (Fig. 1 and fig S6).

The amount of host COI (425 bp) and viral PGM (423 bp) genes decreased with sediment age and depth (Fig. 2). Preservation of relatively long gene fragments was excellent until 4.9 ky B.P., as shown by the constant ratio between COI gene copy numbers and the concentration of recalcitrant alkenones found in this core (10). The COI gene and similar-sized haptophyte 18S ribosomal DNA were degraded into shorter fragments in sediments older than 4.9 ky, as indicated by the steadily decreasing gene-to-alkenone ratio (10). In contrast, the shorter, and hence better preserved (21), viral MCP gene showed greatest abundance in Unit I sediment intervals, as well as in Unit II sediments older than 5.9 ky (Fig. 2B).

Viral diversity was greatest during periods of change in the hydrologic and nutrient regimes of the Black Sea. In Unit I, four host COI genotypes