Development of DNA mismatch repair gene, MutS, as a diagnosticmarker for detection and phylogenetic analysis of algal Megaviruses

William H. Wilson a,b,n, Ilana C. Gilg a, Amy Duarte c, Hiroyuki Ogata d

a Bigelow Laboratory for Ocean Sciences, 60 Bigelow Drive, East Boothbay, ME 04544, USAb Plymouth Marine Laboratory, Prospect Place, Plymouth, PL1 3DH, UKc Humboldt State University, Arcata, CA, USAd Bioinformatics Center, Institute for Chemical Research, Kyoto University, Uji, Kyoto, Japan

a r t i c l e i n f o

Article history:Received 16 May 2014Returned to author for revisions10 June 2014Accepted 1 July 2014Available online 22 July 2014

Keywords:MegaviridaeDiagnostic markerOceanMicroalgaeMutSMismatch repair

a b s t r a c t

Megaviruses are generically defined as giant viruses with genomes up to 1.26 Mb that infect eukaryoticunicellular protists; they are clearly delineated in DNA polymerase B phylogenetic trees; in addition, commonfeatures often include an associated virophage observed during infection; the presence of an amino acyl tRNAsynthetase gene; and a nucleic acid mismatch repair protein, MutS gene. The archetypal representative of thisevolving putative family is Mimivirus, an opportunistic pathogen of Acanthamoeba spp. originally thought to bea bacterium until its genome sequence was published in 2004. Subsequent analysis of marine metagenomicdata revealed Megaviruses are likely ubiquitous on the surface ocean. Analysis of genome sequences of giantviruses isolated from naturally occurring marine protists such as microalgae and a microflagellate grazer,started the expansion of the Megaviridae. Here, we explored the possibility of developing Megavirus specificmarkers for mutS that could be used in virus molecular ecology studies. MutS is split into 15 different cladesrepresenting a wide range of cellular life, and two that contain Megaviruses, clade MutS7 and clade MutS8. Wedeveloped specific PCR primers that recognized Megavirus clade MutS8, a clade that we propose discriminatesmost of the algal Megaviruses. Analysis of seawater off the coast of Maine, US, revealed novel groups of algalMegaviruses that were present in all samples tested. The Megavirus clade MutS8 marker should be consideredas a tool to reveal new diversity and distribution of this enigmatic group of viruses.

& 2014 Elsevier Inc. All rights reserved.

Introduction

Viruses are numerically the most abundant biological agents inthe ocean and concentrations up to 108 mL�1 have been observed(Bergh et al., 1989), though typically anywhere between 1-million to100-million per mL is normal. Viruses are classically seen as havingtwo primary roles in marine ecosystems: The first is lysis of hostcells and the subsequent release of organic matter, biogenic gasesand nutrients to the water column that effect the cycling of theseelements and directly influence the community structure of ecosys-tems (Fuhrman, 1999; Suttle, 2005; Wilhelm and Suttle, 1999). Thesecond is their role in the co-evolutionary arms race with the hoststhey infect, mediating the transfer of host genes from one cell toanother by horizontal gene transfer processes such as transductionand lysogeny (latent infection), a process that can change hostfitness over evolutionary time (Martiny et al., 2014; Monier et al.,2009). The vast majority of these viruses are bacteriophages

(Breitbart, 2011), though a significant fraction (1 to 10%) of the viruspool is represented by algal viruses in the surface ocean (Brussaard,2004). Very little is known about the quantitative distribution ofgiant viruses in the ocean, despite increasing metagenomic evidencefor their presence (Monier et al., 2008a). Although, nucleo-cytoplasmic large DNA virus (NCLDV) genome abundance has beenestimated to range from 4000 to 170,000 genomes mL�1 in thesurface photic zone of the ocean (Hingamp et al., 2013).

The giant virus, or “girus” vernacular did not really take off inmarine virus-host systems in the same way as Mimivirus following itsdiscovery (Claverie et al., 2006; La Scola et al., 2003). Many of theemerging marine algal dsDNA viruses at the time were taxonomicallycharacterized as Phycodnaviridae (van Etten et al., 2002) and describedas “large viruses”, although the group did start adopting the termgiant, albeit “tiny giant” (Wilson et al., 2009). Despite this, the presenceof giant viruses in marine (and freshwater) systems was longsuspected (Bratbak et al., 1992; Garza and Suttle, 1995; Gowing,1993; Sommaruga et al., 1995; Wilson et al., 2000), and it was theexplosion of metagenomic sequences and resulting phylogeneticreconstruction that confirmed the presence of giant virus-like andNCLDV-like sequences in the ocean (Ghedin and Claverie, 2005;Hingamp et al., 2013; Monier et al., 2008a). Indeed, analysis of viral

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Virology

http://dx.doi.org/10.1016/j.virol.2014.07.0010042-6822/& 2014 Elsevier Inc. All rights reserved.

n Corresponding author at: Plymouth Marine Laboratory, Prospect Place, Plymouth,PL1 3DH, UK. Tel.: þ44 1752 633100; þ44 1752 633170;fax: þ44 1752 633101.

E-mail address: whw@pml.ac.uk (W.H. Wilson).

Virology 466-467 (2014) 123–128

DNA polymerase gene phylogeny revealed that many marine algalviruses are likely relatives of Mimivirus (Monier et al., 2008b).However, until very recently, most of the marine giant virus isolatesthat were discovered were opportunistic viral pathogens of Acantha-moeba spp. (Arslan et al., 2011; Boughalmi et al., 2013; Philippe et al.,2013), rather than viruses that infect naturally occurring eukaryoticprotists. It was the genomic characterization of giant viruses that infectthe microflagellate grazer Cafeteria roenbergensis, CroV (Fischer et al.,2010), the microalga Phaeocystis globosa, PgV-16T (Santini et al., 2013),and the brown tide microalga Aureococcus anophagefferens, AaV(Moniruzzaman et al., 2014), that removed any doubt that suchnaturally occurring Mimivirus relatives existed.

Although the phylogenetic affiliation of the giant viruses conti-nues to evolve, the family Megaviridae is emerging as a clade thatrepresents one type of giant viruses (Claverie, 2013), excluding therecently reported Pandoraviruses and Pithovirus (Legendre et al., 2014;Philippe et al., 2013). The order Megavirales has also been proposed asan alternative suggestion (Colson et al., 2013), which encompass alleukaryotic nucleocytoplasmic large DNA viruses (NCLDVs), however itappears to be used less often in the literature to date, and seemsunlikely to be used in the future. For the purpose of clarity, we havechosen to work with the taxonomic term Megaviridae in this study.Key defining features of Megaviridae typically include: an associatedvirophage (Fischer and Suttle, 2011; La Scola et al., 2008; Yau et al.,2011), a mismatch repair protein (MutS) (Ogata et al., 2011), and anamino acyl tRNA synthetase, an enzyme typically found in cellularorganisms (Claverie and Abergel, 2013). Paradoxically, Megaviridaegenomes have awide size range from as small as 371 kb for the BrownTide Virus, AaV (Moniruzzaman et al., 2014), to 1259 kb for Megaviruschilensis (Arslan et al., 2011).

With the fast evolving discovery phase of Megaviruses over thelast decade, there has been very little effort to develop molecularmarkers for molecular ecology studies, a traditional staple of a viralecology toolbox (Chen et al., 1996; Wharam et al., 2007; Wilson etal., 2006). This in part, it has been superseded by the lower cost ofmetagenomic sequencing efforts and subsequent mining of thosedata (Ghedin and Claverie, 2005; Hingamp et al., 2013). Howeverthere is still a place for the development of clade specific virusmarkers that can be used to gain high resolution temporal andspatial variability data (Martínez Martínez et al., 2012); diversitydata (Dekel-Bird et al., 2013; Larsen et al., 2008), or quantitativedata (Short et al., 2011). Of course, the advantage with abundantMegavirus sequence information is that it provides an opportunityto mine the data for useful markers. Such markers would typicallyhave the following characteristics: 1) phylogenetic relevance (uni-versal for a particular group); 2) good support (i.e. multipleexamples); 3) ability to resolve different clades; and 4) potentialto develop downstream quantitative tools (low degeneracy helps).

One of the defining features of the Megaviridae is the presence ofthe DNAmismatch repair (MMR) protein MutS (Ogata et al., 2011). It ispart of a suite of genes, typically found in cellular organisms, thatfacilitate their own DNA repair apparatus to enhance the accuracy ofgenome replication (Iyer et al., 2005). In cellular organisms, MMRrecognizes and corrects base-to-base mismatches and small insertionor deletion loops introduced during replication. This leads to 50- to1000-fold enhancement of replication fidelity. Genomic sequencing ofMimivirus revealed the presence of mutS and seven other putativegenes for DNA repair enzymes capable of correcting mismatches orerrors (Raoult et al., 2004). Previously MMRs had been the purview ofcellular organisms, until their discovery in Mimivirus. MutS wassubsequently found in Mimivirus relatives CroV, CeV, PoV, PpV,HcDNAV (Ogata et al., 2011), Megavirus chilensis (Arslan et al., 2011),PgV-16T (Santini et al., 2013) and AaV (Moniruzzaman et al., 2014),confirming the association of this gene to Megaviridae in the marineenvironment. These researchers conducted a phylogenetic analysis ofMutS, which revealed 15 distinct clades, with Megaviridae specific to

clades MutS7 and MutS8. Further analysis of global ocean sampling(GOS) expedition data (Rusch et al., 2007) revealed that mutS7 andmutS8 sequences were ubiquitous in the GOS data; Ogata et al. (2011)further speculated these sequences were of viral origin since they fellin these newly identified virus-specific clades. We used mutS7 andmutS8 sequence information as a starting point to developMegaviridae-specific PCR primers to detect and conduct a phyloge-netic reconstruction of novel giant viruses in the marine environment.

Results and discussion

PCR primer development

Nucleic acid alignments of mutS7 and mutS8 from all knownMegaviruses, plus a mutS identified from an in-progress sequencingproject of a new Chrysochromulina ericina virus isolate from the Gulfof Maine (GoM-CeV282), and a range of cellular sequences, did notidentify any obvious conserved virus-specific primer sites since thedegeneracy was too high. A more specific strategy was then adoptedto develop clademutS7- ormutS8-specific primers that would identifyMegavirus sequences and omit cellular sequences. Downstream PCRrevealed that mutS7-specific primers produced spurious results withmultiple products of the wrong size (data not shown). Hence, wefocused on clade MutS8. A degenerate forward primer was designedthat would recognize known Megaviruses in clade mutS8; and fourseparate specific reverse primers that would recognize clade mutS8sequences from GoM-CeV282; PgV-16T; PpV01B; and PoV01B (Fig. 1).This allowed a quasi-multiplex approach where the degenerateforward and four reverse primers were added to each PCR reaction.

Megavirus-specific clade mutS8 primers amplified a 550 bp pro-duct from MutS domain V (pfam00488), a domain that is structurallysimilar to the ATPase domain of ABC transporters (Junop et al., 2001).Fortuitously, the degenerate forward primer was designed over theWalker A/P-loop motif, a conserved region of the domain that impartscritical functionality (Ambudkar et al., 2006). This gave us confidencethat the primers produced a product with phylogenetic and functionalrelevance. One of the first primer sets used for marine algal viruseswas DNA polymerase (Chen and Suttle, 1995), where the internalnested primer was based on the highly conserved motif found in mostB-family (alpha-like) DNA polymerase genes. It is more of a universalmarker for algal viruses and was ideal for phylogenetic reconstruction(Chen and Suttle, 1996). However, (certainly in our hands) DNA polcould give spurious results depending on the virus or environmentsampled from. This may be caused, in part, by an intron often obser-ved in algal virus DNA pol genes (Nagasaki et al., 2005). Other algalvirus primers that have been used successfully are those amplifyingthe Major Capsid Protein (MCP) gene (Schroeder et al., 2003). Theyalso designed highly specific primers that only amplified dsDNAviruses (Coccolithoviruses) infecting one species of microalgae (Emi-liania huxleyi). These primers amplify a relatively short product(175 bp) which is less useful for phylogenetic analyses, however, theywere ideal for temporal and spatial distribution studies of Coccolitho-viruses using DGGE (Martínez Martínez et al., 2012). MCP was furtherdeveloped as a phylogenetic marker by Larsen et al. (2008) to includeother members of the Phycodnaviridae (many of which have subse-quently been putatively reclassified as Megaviruses), although theamplicons generated from degenerate primer pairs varied in sizebetween 347 to 518 bp which can make interpretation of dataproblematic.

Detection and phylogeny of MutS8 PCR products from seawater

A 550 bp PCR product was amplified in control reactions withGoM-CeV282 template DNA (results not shown), indicating theMegavirus clade mutS8 primers were ready for use with

W.H. Wilson et al. / Virology 466-467 (2014) 123–128124

environmental samples. Opportunistic seawater sampling, collected aspart of an undergraduate training program, allowed us to sample froma range of depths from coastal stations in the Damariscotta River

Estuary close to Bigelow Laboratory and off the dock of the oldBigelow Laboratory site (inset Fig. 3). DNA template that had beenextracted from cellular/viral material in 1 L of seawater collected on0.2 mm filters was used as the template for each PCR reaction. PCRproducts (550 bp) were obtained from most samples (data notshown), and sequence analysis confirmed the presence of mutS8. Toclassify the amplified sequences, they were added to a sequence align-ment containing 150 MutS reference orthologs, representing diverseMutS subfamilies (the same set as used in Ogata et al. (2011)). Phylo-genetic inference revealed 15 distinct MutS clades and all the environ-mental sequences from this study fell within the MutS8 clade, whichsignificantly expanded the MutS8 clade and changed the architectureof the final tree compared to Ogata et al. (2011), where the MutS8clade swapped positions with clades MutS3 and MutS6 (Fig. 2).

A detailed view of the MutS8 clade showed close relatives ofMegaviruses GoM-CeV282, PgV-16T and PpV 01, and there was noobvious pattern of diversity related to water depth (3 different depthswere sampled) or station number (Fig. 3). The data also revealed novel,well-supported clades of environmental sequences that likely repre-sented novel Megaviruses, although we cannot say for certain withoutcharacterized references. We did not detect any sequence orthologs toPoV-01B in this study, which fell out on a poorly supported lineage ofits own, still within the MutS8 clade.

Although we were unable to develop a universal Megaviridaemarker, we did develop a MutS8 marker that was able to differentiatewhat we believe is a subset of the Megaviridae. Despite Megavirusreference sequences being limited, our primers appear to discriminatesome of the algal Megaviruses. This is consistent with the findings ofOgata et al. (2011) who only found PoV and PpV MutS8 sequences inthe GOS database. In addition, some of the latest phylogenetic treesappear to separate algal Megaviruses from non-photosynthetic proti-stan Megaviruses (Claverie, 2013; Santini et al., 2013). One exception isthe smallest member of the Megaviridae, AaV (371 kb), that appears tocontain only MutS7, yet this virus is in an unusual phylogeneticjuncture sitting between the 2 groups after analyses of DNA pol, MCPand ATPase phylogenies (Moniruzzaman et al., 2014).

As part of this study we also investigated other markers includingMCP (Larsen et al., 2008) and DNA pol (Chen and Suttle, 1995) whichappeared to have limited utility for the development of Megavirus-specifc markers. Their main problemwas the often spurious (non-MCP

Fig. 2. Maximum Likelihood (ML) unrooted phylogenetic tree of MutS familyproteins (domain V only). The alignment and subsequent phylogeny was compiledfrom sequences generated in this study and from Ogata et al. (2011). Circles at thenodes indicate statistically significant bootstrap support for the branches. Opencircles indicate support 475%; filled circles 490%. Branch color reflects sequenceorigin: dark blue¼Bacteria, light blue¼ Archaea, green¼Eukaryota, red¼Viruses,orange¼sequences from this study. Scale bar corresponds to 0.5 substitutionsper site.(For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.)

Fig. 1. Alignment of knownmutS8 domain V (ABC ATPase domain, pfam00488) sequences. Primer binding sites are shown in boldface and colored green and red for forwardand reverse, respectively. Boxes outline the nucleotide positions of conserved motifs within the domain. Asterisks above the sequence show the codons representing theATP-binding sites of the enzyme.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

W.H. Wilson et al. / Virology 466-467 (2014) 123–128 125

or DNA pol) sequencing results or, more commonly, a size range of PCRamplicons from an environmental sample (data not shown). We alsoinvestigated A32-like ATPase, a group 1 core genes in NCLDVs (Iyer etal., 2006), however nucleic acid sequences were too divergent todesign low degeneracy primers.

Conclusions

Our Megavirus clade mutS8 primers will likely be refined asfurther sequence information becomes available from Megavirusisolates containing MutS8. Such data will also provide furthergranularity on the phylogenetic affiliation of the Megaviruses.There is additional scope to use mutS8 for quantitative studies,particularly in combination with flow cytometry, a tool that hasalready been successfully used to identify giant viruses (MartínezMartínez et al., 2013). With relatively low concentrations of giantviruses in the marine environment (Hingamp et al., 2013), flowcytometry may prove to have limited use, though methodologicalrefinements of the technology indicate it can be used to detectlower concentrations of viruses (Mojica et al., 2014). It is likely that

the Megaviridae will be split into further groups as new genomesare sequenced, preliminary investigations here and elsewhereindicate Megaviruses will be split into (at the very least) thosethat infect either non-photosynthetic protists or microalgae. Con-sequently, time will tell if mutS8 is a useful marker, however, inthis exploratory study it already appears to be revealing novelgroups of Megaviruses and should be considered as a tool to revealnew insights into diversity and distribution of this enigmaticgroup of viruses.

Materials and methods

Primer design

Megavirus mutS8 reference nucleotide sequences used forprimer design included Phaeocystis globosa virus 16T (GenBankaccession AGM15600); Phaeocystis pouchetii virus isolate 01B(FR691708.1); Pyramimonas orientalis virus isolate 01B(FR691707.1); and mutS8 sequence from a new Chrysochromulinaericina virus isolate currently being characterized by this group

Fig. 3. A. Map of sampling sites from this study. Samples were collected along a transect at the mouth of the Damariscotta River (Stations 1–3) and from the old BigelowDock at McKown Point (Station V). B. A detailed view of the MutS8 clade from the Maximum Likelihood phylogenetic tree in Fig. 2. Open circles indicate bootstrap support475%; filled circles 490%. Branch color indicates sequence origin: blue¼Bacteria, red¼Viruses, orange¼sequences from this study. Symbols at branch tips indicate thelocation and depth of sample from which the sequence was obtained. Symbol colors refer to the sample station locations (see A); black¼Station 1, green¼Station 2,blue¼Station 3, brown¼Station V. Symbol shapes reflect the depth of the sample; Δ¼surface, □¼10 m, ⬨¼17 m. Scale bar corresponds to 0.2 substitutions per site.(Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

W.H. Wilson et al. / Virology 466-467 (2014) 123–128126

(isolated on host strain Chrysochromulina ericina CCMP282from coastal waters of the Gulf of Maine, off the new BigelowLaboratory dock in July 2012, referred to as GoM-CeV282).These sequences were aligned using the ClustalX algorithm withinMEGA 5.2.1 (Tamura et al., 2011). The alignment was initiallyinterrogated for suitable primer sites using the online tool Gene-Fisher2 (http://bibiserv.techfak.uni-bielefeld.de/genefisher2/submission.html) which returned one suitable degenerate forwardprimer, but no suitable degenerate reverse primers. We thendesigned individual reverse primers from each of the four refer-ences to produce the desired amplicon length (550 bp) usingPrimerQuest

(IDT; https://www.idtdna.com/Primerquest/Home/Index). The re-verse primers were pooled to generate a mixed reverse primer stock.

Forward primer sequence was:

MutS_degen8_F1CCW AAY RMD GCN GGM AAR WCT A.

Reverse primer sequences were as follows:

MutS8_PpVR1CTT TGG GAT AGT CTA AAT C;MutS8_PgVR1CTT CCG GAT ATT CTA AAT C;MutS8_CeVR1CAT CCG GAT ATT CCA TAT C; andMutS8_PoVR2TTC GAT GGC AAC CAA TTG TG.

Study sites, sample collection and DNA extraction

Seawater samples were collected in two separate samplingtrips in coastal waters off Boothbay Harbor (off the dock on the siteof the old Bigelow Laboratory: Station V), and off East Boothbay,mid coast Maine, US (inset Fig. 3) in a transect of the DamariscottaRiver Estuary (Stations 1, 2 and 3) on 14 June 2013 aboard RV Ira C.Surface (2 m), mid (10 m), and deep (17 m) samples, coded as 1, 2,and 3 respectively were collected from Station 1 (hence coded1–1–1, 1–2–2, and 1–3–3 respectively); surface and mid sampleswere collected from Station 2 (coded 2–1–1 and 2–2–2 respec-tively); and surface and mid samples were collected from Station 3(coded 3–1–1 and 3–2–2 respectively). Only a surface sample wascollected from Station V (coded V1). This sample was processeddifferently than the others. Ten liters of seawater was concentratedby tangential flow filtration (TFF) through a 200 kDa filter. Thesample was concentrated to approximately 50 ml and subse-quently filtered onto a 0.2 μm pore size Supor-200 47 mm dia-meter filters (PALL Corp.).

Seawater at stations 1–3 was collected from depth profiles,down to approximately 17 m, using a Niskin sampler attached torope. From each depth sample, 1 l of seawater was filtered onto0.2 μm pore size Supor-200 47 mm diameter filters (PALL Corp).The filters were transferred to 2 ml cryotubes, snap frozen inliquid nitrogen and stored at �80 1C until further processing fortotal DNA preparations. Genomic DNA was isolated from theparticulate matter retained on the Supor filters using an adaptedphenol/chloroform method. The filters were cut into small easilydissolvable pieces (approx. 0.5 cm2) and placed in a 2 ml Eppen-dorff tube. Following the addition of 800 μl GTE buffer (50 mMglucose, 25 mM Tris–Cl pH 8.0 and 10 mM EDTA), 10 μl ProteinaseK (5 mg ml�1), 100 μl 0.5 M filter sterilized EDTA and 200 μl 10%SDS, samples were incubated at 65 1C for 1–2 h. DNA was thenpurified by phenol extraction as described by Schroeder et al.(2002).

PCR, sequencing and phylogeny

PCR reactions contained a final volume of 25 μl using 1 μl ofextracted DNA and using the FailSafe™ PCR System (Epicentre,

Madison, WI). Reverse primer stock was prepared by adding anequal amount of MutS8_PpVR1, MutS8_PgVR1, MutS8_CeVR1, andMutS8_PoVR2 to a 20 mM total concentration. Forward primerstock was prepared by diluting MutS_degen8_F1 to 20 mM. Eachreaction contained 1X Buffer E, 1.5 mM each primer stock (final),and 1.25 U FailSafe Enzyme Mix. PCR was carried out with atouchdown protocol; the initial denaturation was at 95 1C for4 min, followed by 20 cycles of denaturation at 95 1C for 30 s,60 1C anneal for 30 s with a 0.5 1C decrease every cycle, and a 72 1Cextension for 45 s. This was followed by 30 cycles with isothermalannealing; 95 1C for 30 s, 45 1C for 30 s, then 72 1C for 45 s. Finalextension was 72 1C for 7 min. Amplicons were visualized on a2.2% agarose DNA FlashGel™ cassette (Lonza, Rockland, ME) andpurified using the GenElute™ PCR Clean-Up kit (Sigma-Aldrich, St.Louis, MO) following the manufacturer's instructions.

The cleaned amplicons were sequenced by 454 pyrosequencing atthe Research and Testing Laboratory (Lubbock TX). Sequences weretrimmed manually based on quality, translated in frame in silico, andmerged with the alignment published by Ogata et al. (2011) in MEGA5.2.1 (Tamura et al., 2011). All sequences were then aligned withClustalX (Larkin et al., 2007) and the alignment was manually refinedin SeaView (Gouy et al., 2010). A sequence mask was applied to retainregions of unambiguous alignment. We used the LG evolutionarymodel for our Maximum Likelihood (ML) inference (Le and Gascuel,2008) as it was previously determined to be the best substitutionmodel for the data (Ogata et al., 2011) with a proportion of invariantsites incorporated into the model and the gamma shape parametercalculated from the data set. The ML inference was conducted inSeaView by invoking PhyML. The confidence of branching wasassessed using 1000 bootstrap re-samplings of the data set. Theresulting trees were visualized in MEGA 5.2.1.

Acknowledgments

We thank the skipper and REU mentor Dr. David Fields aboardRV Ira C for support during the Damariscotta River Estuarysampling trip. Sean Anderson and Dr. Pete Countway (BigelowLaboratory) collected and prepared samples at site V1. AD wassupported by an NSF Research Experience for Undergraduates(REU) internship, under grant reference OCE0755142; we thankDr. David Fields and Dr. Nicole Poulton for their mentorship duringthis program. WHW would like to acknowledge support from NSFgrant number EF0949162.

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