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<front>
<journal-meta>
<journal-id journal-id-type="nlm-ta">PLoS ONE</journal-id>
<journal-id journal-id-type="publisher-id">plos</journal-id>
<journal-id journal-id-type="pmc">plosone</journal-id>
<journal-title-group>
<journal-title>PLOS ONE</journal-title>
</journal-title-group>
<issn pub-type="epub">1932-6203</issn>
<publisher>
<publisher-name>Public Library of Science</publisher-name>
<publisher-loc>San Francisco, CA USA</publisher-loc>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.1371/journal.pone.0157011</article-id>
<article-id pub-id-type="publisher-id">PONE-D-15-55773</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Research Article</subject>
</subj-group>
<subj-group subj-group-type="Discipline-v3"><subject>Biology and life sciences</subject><subj-group><subject>Organisms</subject><subj-group><subject>Animals</subject><subj-group><subject>Vertebrates</subject><subj-group><subject>Amniotes</subject><subj-group><subject>Reptiles</subject><subj-group><subject>Testudines</subject><subj-group><subject>Turtles</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and life sciences</subject><subj-group><subject>Organisms</subject><subj-group><subject>Plants</subject><subj-group><subject>Algae</subject><subj-group><subject>Phytoplankton</subject><subj-group><subject>Diatoms</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and life sciences</subject><subj-group><subject>Organisms</subject><subj-group><subject>Animals</subject><subj-group><subject>Invertebrates</subject><subj-group><subject>Plankton</subject><subj-group><subject>Phytoplankton</subject><subj-group><subject>Diatoms</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Research and analysis methods</subject><subj-group><subject>Microscopy</subject><subj-group><subject>Electron microscopy</subject><subj-group><subject>Scanning electron microscopy</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and life sciences</subject><subj-group><subject>Ecology</subject><subj-group><subject>Marine ecology</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Ecology and environmental sciences</subject><subj-group><subject>Ecology</subject><subj-group><subject>Marine ecology</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and life sciences</subject><subj-group><subject>Marine biology</subject><subj-group><subject>Marine ecology</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Earth sciences</subject><subj-group><subject>Marine and aquatic sciences</subject><subj-group><subject>Marine biology</subject><subj-group><subject>Marine ecology</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and life sciences</subject><subj-group><subject>Ecology</subject><subj-group><subject>Community ecology</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Ecology and environmental sciences</subject><subj-group><subject>Ecology</subject><subj-group><subject>Community ecology</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Engineering and technology</subject><subj-group><subject>Equipment</subject><subj-group><subject>Imaging equipment</subject><subj-group><subject>Scanning electron microscopes</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and life sciences</subject><subj-group><subject>Organisms</subject><subj-group><subject>Animals</subject><subj-group><subject>Vertebrates</subject><subj-group><subject>Amniotes</subject><subj-group><subject>Reptiles</subject><subj-group><subject>Chelonia</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Research and analysis methods</subject><subj-group><subject>Research facilities</subject><subj-group><subject>Museum collections</subject></subj-group></subj-group></subj-group></article-categories>
<title-group>
<article-title>Epibiotic Diatoms Are Universally Present on All Sea Turtle Species</article-title>
<alt-title alt-title-type="running-head">Diatoms Are Universal Sea Turtle Epibionts</alt-title>
</title-group>
<contrib-group>
<contrib contrib-type="author" corresp="yes" xlink:type="simple">
<contrib-id authenticated="true" contrib-id-type="orcid">http://orcid.org/0000-0001-7384-3576</contrib-id>
<name name-style="western">
<surname>Robinson</surname>
<given-names>Nathan J.</given-names>
</name>
<xref ref-type="aff" rid="aff001"><sup>1</sup></xref>
<xref ref-type="aff" rid="aff002"><sup>2</sup></xref>
<xref ref-type="corresp" rid="cor001">*</xref>
</contrib>
<contrib contrib-type="author" xlink:type="simple">
<name name-style="western">
<surname>Majewska</surname>
<given-names>Roksana</given-names>
</name>
<xref ref-type="aff" rid="aff003"><sup>3</sup></xref>
</contrib>
<contrib contrib-type="author" xlink:type="simple">
<name name-style="western">
<surname>Lazo-Wasem</surname>
<given-names>Eric A.</given-names>
</name>
<xref ref-type="aff" rid="aff004"><sup>4</sup></xref>
</contrib>
<contrib contrib-type="author" xlink:type="simple">
<name name-style="western">
<surname>Nel</surname>
<given-names>Ronel</given-names>
</name>
<xref ref-type="aff" rid="aff005"><sup>5</sup></xref>
</contrib>
<contrib contrib-type="author" xlink:type="simple">
<name name-style="western">
<surname>Paladino</surname>
<given-names>Frank V.</given-names>
</name>
<xref ref-type="aff" rid="aff001"><sup>1</sup></xref>
<xref ref-type="aff" rid="aff002"><sup>2</sup></xref>
</contrib>
<contrib contrib-type="author" xlink:type="simple">
<name name-style="western">
<surname>Rojas</surname>
<given-names>Lourdes</given-names>
</name>
<xref ref-type="aff" rid="aff004"><sup>4</sup></xref>
</contrib>
<contrib contrib-type="author" xlink:type="simple">
<name name-style="western">
<surname>Zardus</surname>
<given-names>John D.</given-names>
</name>
<xref ref-type="aff" rid="aff006"><sup>6</sup></xref>
</contrib>
<contrib contrib-type="author" xlink:type="simple">
<name name-style="western">
<surname>Pinou</surname>
<given-names>Theodora</given-names>
</name>
<xref ref-type="aff" rid="aff007"><sup>7</sup></xref>
</contrib>
</contrib-group>
<aff id="aff001"><label>1</label> <addr-line>The Leatherback Trust, Goldring-Gund Marine Biology Station, Playa Grande, Guanacaste, Costa Rica</addr-line></aff>
<aff id="aff002"><label>2</label> <addr-line>Department of Biology, Indiana University-Purdue University Fort Wayne, Fort Wayne, Indiana, United States of America</addr-line></aff>
<aff id="aff003"><label>3</label> <addr-line>BioNEM Laboratory, Department of Experimental and Clinical Medicine, University “Magna Graecia” of Catanzaro, Catanzaro, Italy</addr-line></aff>
<aff id="aff004"><label>4</label> <addr-line>Division of Invertebrate Zoology, Peabody Museum of Natural History, Yale University, New Haven, Connecticut, United States of America</addr-line></aff>
<aff id="aff005"><label>5</label> <addr-line>Institute For Coastal and Marine Research, Nelson Mandela Metropolitan University, Port Elizabeth, South Africa</addr-line></aff>
<aff id="aff006"><label>6</label> <addr-line>Department of Biology, The Citadel, 171 Moultrie Street, Charleston, South Carolina, 29407, United States of America</addr-line></aff>
<aff id="aff007"><label>7</label> <addr-line>Department of Biological and Environmental Sciences, Western Connecticut State University, Danbury, Connecticut, United States of America</addr-line></aff>
<contrib-group>
<contrib contrib-type="editor" xlink:type="simple">
<name name-style="western">
<surname>Lin</surname>
<given-names>Senjie</given-names>
</name>
<role>Editor</role>
<xref ref-type="aff" rid="edit1"/>
</contrib>
</contrib-group>
<aff id="edit1"><addr-line>University of Connecticut, UNITED STATES</addr-line></aff>
<author-notes>
<fn fn-type="conflict" id="coi001">
<p>The authors have declared that no competing interests exist.</p>
</fn>
<fn fn-type="con" id="contrib001">
<p>Conceived and designed the experiments: NJR TP. Performed the experiments: NJR LR. Analyzed the data: NJR RM. Contributed reagents/materials/analysis tools: EAL-W JDZ. Wrote the paper: NJR RM JZ. Facilitated permitting and field work in South Africa: RN FVP.</p>
</fn>
<corresp id="cor001">* E-mail: <email xlink:type="simple">nathan@leatherback.org</email></corresp>
</author-notes>
<pub-date pub-type="epub">
<day>3</day>
<month>6</month>
<year>2016</year>
</pub-date>
<pub-date pub-type="collection">
<year>2016</year>
</pub-date>
<volume>11</volume>
<issue>6</issue>
<elocation-id>e0157011</elocation-id>
<history>
<date date-type="received">
<day>4</day>
<month>1</month>
<year>2016</year>
</date>
<date date-type="accepted">
<day>22</day>
<month>5</month>
<year>2016</year>
</date>
</history>
<permissions>
<copyright-year>2016</copyright-year>
<copyright-holder>Robinson et al</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/" xlink:type="simple">
<license-p>This is an open access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/" xlink:type="simple">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p>
</license>
</permissions>
<self-uri content-type="pdf" xlink:href="info:doi/10.1371/journal.pone.0157011"/>
<abstract>
<p>The macro-epibiotic communities of sea turtles have been subject to growing interest in recent years, yet their micro-epibiotic counterparts are almost entirely unknown. Here, we provide the first evidence that diatoms are epibionts for all seven extant species of sea turtle. Using Scanning Electron Microscopy, we inspected superficial carapace or skin samples from a single representative of each turtle species. We distinguished 18 diatom taxa from these seven individuals, with each sea turtle species hosting at least two diatom taxa. We recommend that future research is undertaken to confirm whether diatom communities vary between sea turtle species and whether these diatom taxa are facultative or obligate commensals.</p>
</abstract>
<funding-group>
<award-group id="award001">
<funding-source>
<institution>WCSU-AAUP</institution>
</funding-source>
<award-id>241290</award-id>
<principal-award-recipient>
<name name-style="western">
<surname>Pinou</surname>
<given-names>Theodora</given-names>
</name>
</principal-award-recipient>
</award-group>
<funding-statement>This study was funded by a WCSU-AAUP grant (#241290) to Theodora Pinou. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</funding-statement>
</funding-group>
<counts>
<fig-count count="3"/>
<table-count count="2"/>
<page-count count="8"/>
</counts>
<custom-meta-group>
<custom-meta id="data-availability">
<meta-name>Data Availability</meta-name>
<meta-value>All relevant data are within the paper.</meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
</front>
<body>
<sec id="sec001" sec-type="intro">
<title>Introduction</title>
<p>Sea turtles often harbour complex communities of epibionts [<xref ref-type="bibr" rid="pone.0157011.ref001">1</xref>, <xref ref-type="bibr" rid="pone.0157011.ref002">2</xref>, <xref ref-type="bibr" rid="pone.0157011.ref003">3</xref>]. These epibiont communities can provide valuable insights into the hosts’ behaviour [<xref ref-type="bibr" rid="pone.0157011.ref004">4</xref>, <xref ref-type="bibr" rid="pone.0157011.ref005">5</xref>] and health [<xref ref-type="bibr" rid="pone.0157011.ref006">6</xref>, <xref ref-type="bibr" rid="pone.0157011.ref007">7</xref>]; however, most studies on sea turtle epibiosis have focused exclusively on macro-epibiota. To date, little is known about the prevalence, and potential ecological function, of sea turtles’ micro-epibiota.</p>
<p>Diatoms are often some of the earliest colonizers on any marine substrate [<xref ref-type="bibr" rid="pone.0157011.ref008">8</xref>] and it has been suggested that sea turtles should harbour epibiotic diatom communities [<xref ref-type="bibr" rid="pone.0157011.ref009">9</xref>]. Moreover, numerous other studies have reported large clumps of periphytic algae growing on the carapace of several sea turtle species [<xref ref-type="bibr" rid="pone.0157011.ref002">2</xref>, <xref ref-type="bibr" rid="pone.0157011.ref010">10</xref>]. Nevertheless, direct evidence of epibiotic diatoms on sea turtles has only recently been provided on loggerhead turtles <italic>Caretta caretta</italic> [<xref ref-type="bibr" rid="pone.0157011.ref011">11</xref>] and olive ridley <italic>Lepidochelys olivacea</italic> turtles [<xref ref-type="bibr" rid="pone.0157011.ref012">12</xref>]. Consequently, we predict that epibiotic diatoms are likely present on each of the world’s seven extant sea turtle species.</p>
<p>In this study, we used a Scanning Electron Microscopy (SEM) to examine the carapace scutes or skin of flatback <italic>Natator depressus</italic>, green <italic>Chelonia mydas</italic>, hawksbill <italic>Eretmochelys imbricata</italic>, Kemp’s ridley <italic>Lepidochelys kempii</italic>, leatherback <italic>Dermochelys coriacea</italic>, loggerhead <italic>Caretta caretta</italic>, and olive ridley <italic>Lepidochelys olivacea</italic> turtles in search of epibiotic diatoms. Knowledge of the prevalence, characteristic, and diversity of epibiotic diatoms of sea turtles could provide the impetus for more detailed studies into the micro-epibiota of sea turtles.</p>
</sec>
<sec id="sec002" sec-type="materials|methods">
<title>Materials and Methods</title>
<sec id="sec003">
<title>Sample Collection</title>
<p>Carapace scutes were opportunistically collected from a single flatback, green, hawksbill, Kemp’s ridley, loggerhead, and olive ridley turtle. Samples were collected from deceased animals that had been stored in either museum or research collections. As leatherback turtles do not have an external shell like the hard-shelled Cheloniidae, we did not collect carapace samples from leatherback turtles. Instead, we collected skin samples from the flippers of nesting turtles using at 6 mm biopsy punches. Full details on sample collection and storage see <xref ref-type="table" rid="pone.0157011.t001">Table 1</xref>.</p>
<table-wrap id="pone.0157011.t001" position="float">
<object-id pub-id-type="doi">10.1371/journal.pone.0157011.t001</object-id>
<label>Table 1</label> <caption><title>Collection and storage details of sea turtle carapace and skin samples.</title></caption>
<alternatives>
<graphic id="pone.0157011.t001g" mimetype="image" position="float" xlink:href="info:doi/10.1371/journal.pone.0157011.t001" xlink:type="simple"/>
<table>
<colgroup>
<col align="left" valign="middle"/>
<col align="left" valign="middle"/>
<col align="left" valign="middle"/>
<col align="left" valign="middle"/>
</colgroup>
<thead>
<tr>
<th align="left">Species</th>
<th align="left">Scute or Skin</th>
<th align="left">Sampling Location</th>
<th align="left">Collection date and circumstance</th>
</tr>
</thead>
<tbody>
<tr>
<td align="left">Green <italic>Chelonia mydas</italic></td>
<td align="left">Carapace scute</td>
<td align="left">Hawaii, USA</td>
<td align="left">Collected from a dead-stranded turtle some time prior to 2003. Stored dry at room temperature.<xref ref-type="table-fn" rid="t001fn001"><sup>1</sup></xref></td>
</tr>
<tr>
<td align="left">Hawksbill <italic>Eretmochelys imbricata</italic></td>
<td align="left">Carapace scute</td>
<td align="left">Hawaii, USA</td>
<td align="left">Material confiscated by U.S. Customs perhaps a decade prior to 2002. Stored dry at room temperature. <xref ref-type="table-fn" rid="t001fn001"><sup>1</sup></xref></td>
</tr>
<tr>
<td align="left">Flatback <italic>Natator depressus</italic></td>
<td align="left">Carapace scute</td>
<td align="left">Northern Territory, Australia</td>
<td align="left">Collected from a dead-stranded turtle in 1981. Stored dry at room temperature.<xref ref-type="table-fn" rid="t001fn002"><sup>2</sup></xref></td>
</tr>
<tr>
<td align="left">Kemp’s ridley <italic>Lepidochelys kempii</italic></td>
<td align="left">Carapace scute</td>
<td align="left">Texas, USA</td>
<td align="left">Collected from a recently dead-stranded turtle in 2003. Stored frozen for an indeterminate period of time before being stored dry and at room temperature. <xref ref-type="table-fn" rid="t001fn001"><sup>1</sup></xref></td>
</tr>
<tr>
<td align="left">Leatherback <italic>Dermochelys coriacea</italic></td>
<td align="left">Flipper skin</td>
<td align="left">iSimangaliso Wetland Park, South Africa</td>
<td align="left">Collected from a live nesting leatherback turtle in 2013. Stored in 95% non-denatured ethanol at room temperature.<xref ref-type="table-fn" rid="t001fn003"><sup>3</sup></xref></td>
</tr>
<tr>
<td align="left">Loggerhead <italic>Caretta caretta</italic></td>
<td align="left">Carapace scute</td>
<td align="left">Florida, USA</td>
<td align="left">Collected from a dead stranded turtle some time prior to 2009. Stored dry at room temperature. <xref ref-type="table-fn" rid="t001fn001"><sup>1</sup></xref></td>
</tr>
<tr>
<td align="left">Olive ridley <italic>Lepidochelys olivacea</italic></td>
<td align="left">Carapace scute</td>
<td align="left">Hawaii, USA</td>
<td align="left">Collected from a dead animal that had been caught on a long-line in 2003. Stored dry at room temperature. <xref ref-type="table-fn" rid="t001fn001"><sup>1</sup></xref></td>
</tr>
</tbody>
</table>
</alternatives>
<table-wrap-foot>
<fn id="t001fn001"><p><sup>1</sup>Sample loaned to the Yale Peabody Museum of Natural History by John D. Zardus.</p></fn>
<fn id="t001fn002"><p><sup>2</sup>Sample loaned to the Yale Peabody Museum of Natural History by The Bishop Museum. Specimen #8294.</p></fn>
<fn id="t001fn003"><p><sup>3</sup>Sample loaned to the Yale Peabody Museum of Natural History by Nathan J. Robinson.</p></fn>
</table-wrap-foot>
</table-wrap>
<p>Prior to imaging, the leatherback skin samples were dehydrated in a graded series of hexamethyldisilazane (HMDS) of increasing concentrations until 100% of the latter. HMDS drying for SEM is generally preferred to critical point drying as it is cheaper and it less likely to distort the shape of any microbes of interest [<xref ref-type="bibr" rid="pone.0157011.ref013">13</xref>]. The carapace samples were stored dry and did not need further drying for SEM.</p>
<p>All samples were mounted on aluminium specimen mounts and sputter-coated with carbon. SEM images were collected using a FEI XL-30 field emission gun environmental scanning electron microscope at an accelerating voltage of 10kV, and a Zeiss EM900 transmission electron microscope at 80kV with an objective aperture of 90 μm diameter. Each sample was inspected haphazardly at various magnifications to search for micro-epibionts. We attempted to identify each unique diatom to the lowest taxonomic level by consulting appropriate literature [<xref ref-type="bibr" rid="pone.0157011.ref014">14</xref>, <xref ref-type="bibr" rid="pone.0157011.ref015">15</xref>, <xref ref-type="bibr" rid="pone.0157011.ref016">16</xref>, <xref ref-type="bibr" rid="pone.0157011.ref017">17</xref>, <xref ref-type="bibr" rid="pone.0157011.ref018">18</xref>, <xref ref-type="bibr" rid="pone.0157011.ref019">19</xref>, <xref ref-type="bibr" rid="pone.0157011.ref020">20</xref>, <xref ref-type="bibr" rid="pone.0157011.ref021">21</xref>, <xref ref-type="bibr" rid="pone.0157011.ref022">22</xref>,<xref ref-type="bibr" rid="pone.0157011.ref023">23</xref>, <xref ref-type="bibr" rid="pone.0157011.ref024">24</xref>, <xref ref-type="bibr" rid="pone.0157011.ref025">25</xref>]. When it was not clear that two diatoms were different taxa, they were considered as one so as not to over-estimate the number of species recorded. As each sample had been stored for various lengths of times and under varying conditions, we only attempted to determine whether micro-epibionts were present or absent—we did not attempt to quantitatively assess the abundance of micro-epibionts. Samples were not cleaned or sonicated prior to imaging as it is expected that these processes would remove the micro-epibionts of interest. SEM work was conducted at the Department of Geology and Geophysics, Yale University.</p>
</sec>
</sec>
<sec id="sec004" sec-type="conclusions">
<title>Results and Discussion</title>
<p>Diatoms were present on every sea turtle species (Figs <xref ref-type="fig" rid="pone.0157011.g001">1</xref> &amp; <xref ref-type="fig" rid="pone.0157011.g002">2</xref>) and from sea turtles from distinct ocean basins (Atlantic, Pacific, and Indian Ocean). We were able to divide all observed diatoms into 18 unique taxa (<xref ref-type="table" rid="pone.0157011.t002">Table 2</xref>). We were only able to identify a single diatom taxon to the species level (<italic>Melosira sol</italic>), all others were only identified to genus level. All diatoms were pennate, with the exception of <italic>Melosira sol</italic>. Adnate forms (<italic>Amphora</italic> spp., <italic>Cocconeis</italic> sp., <italic>Diploneis</italic> sp.) constituted 56% of all identified taxa and erect (<italic>Achnanthes</italic> sp., <italic>Poulinea</italic> spp.) and motile diatoms (<italic>Navicula</italic> sp., <italic>Nitzschia</italic> sp.) constituted 22% and 11%, respectively. The growth form of <italic>Tursiocola</italic> should be considered as uncertain. According to [<xref ref-type="bibr" rid="pone.0157011.ref020">20</xref>, <xref ref-type="bibr" rid="pone.0157011.ref026">26</xref>], <italic>Tursiocola</italic> spp. has been observed in cetaceans with one end embedded in the epidermis. However, recent observations of live diatoms collected from manatee skin suggest that some <italic>Tursiocola</italic> spp. are highly motile (TA Frankovich, personal communication).</p>
<table-wrap id="pone.0157011.t002" position="float">
<object-id pub-id-type="doi">10.1371/journal.pone.0157011.t002</object-id>
<label>Table 2</label> <caption><title>Diatom taxa found on the seven different sea turtle species.</title></caption>
<alternatives>
<graphic id="pone.0157011.t002g" mimetype="image" position="float" xlink:href="info:doi/10.1371/journal.pone.0157011.t002" xlink:type="simple"/>
<table>
<colgroup>
<col align="left" valign="middle"/>
<col align="left" valign="middle"/>
</colgroup>
<thead>
<tr>
<th align="left">Species</th>
<th align="left">Diatom species</th>
</tr>
</thead>
<tbody>
<tr>
<td align="left">Green <italic>Chelonia mydas</italic></td>
<td align="left"><italic>Cocconeis</italic> sp., <italic>Amphora</italic> sp. 1, and possibly fragments from a <italic>Navicula</italic> sp.</td>
</tr>
<tr>
<td align="left">Hawksbill <italic>Eretmochelys imbricata</italic></td>
<td align="left"><italic>Amphora</italic> sp. 2, <italic>Amphora</italic> sp. 3, and <italic>Poulinea</italic> sp. 2</td>
</tr>
<tr>
<td align="left">Flatback <italic>Natator depressus</italic></td>
<td align="left"><italic>Achnanthes</italic> sp., and <italic>Poulinea</italic> sp. 1</td>
</tr>
<tr>
<td align="left">Kemp’s ridley <italic>Lepidochelys kempii</italic></td>
<td align="left"><italic>Melosira sol</italic>, <italic>Poulinea</italic> sp. 1, and <italic>Achnanthes</italic> sp.</td>
</tr>
<tr>
<td align="left">Leatherback <italic>Dermochelys coriacea</italic></td>
<td align="left"><italic>Navicula</italic> sp., and <italic>Tursiocola</italic> sp.</td>
</tr>
<tr>
<td align="left">Loggerhead <italic>Caretta caretta</italic></td>
<td align="left"><italic>Amphora</italic> sp. 4, <italic>Amphora</italic> sp. 5, <italic>Amphora</italic> sp. 6, <italic>Amphora</italic> sp. 7, <italic>Diploneis</italic> sp., and an unknown adnate species</td>
</tr>
<tr>
<td align="left">Olive ridley <italic>Lepidochelys olivacea</italic></td>
<td align="left"><italic>Nitzchia</italic> sp., <italic>Achnanthes</italic> sp., <italic>Poulinea</italic> sp. 3, and <italic>Amphora</italic> sp. 5</td>
</tr>
</tbody>
</table>
</alternatives>
</table-wrap>
<fig id="pone.0157011.g001" position="float">
<object-id pub-id-type="doi">10.1371/journal.pone.0157011.g001</object-id>
<label>Fig 1</label>
<caption>
<title>Scanning Electron Microscope images of epibiotic diatoms found on flatback, green, hawksbill, and Kemp’s ridley sea turtles.</title>
<p>Flatback turtle: A = <italic>Achnanthes</italic> sp., B &amp; C = <italic>Poulinea</italic> sp. 1; Green turtle: D = <italic>Cocconeis</italic> sp., E = <italic>Amphora</italic> sp. 1, F = Broken pieces of <italic>Amphora</italic> sp. and <italic>Navicula</italic> sp., G = broken pieces of <italic>Amphora</italic> sp.; Hawksbill turtle: H = <italic>Amphora</italic> sp. 2, I = <italic>Amphora</italic> sp. 3, J &amp; K = <italic>Poulinea</italic> sp. 2; Kemp’s ridley turtle: L = <italic>Melosira sol</italic>, M = <italic>Poulinea</italic> sp. 1, N = (i) <italic>Achnanthes</italic> sp. &amp; (ii) <italic>Poulinea</italic> sp. 1, O = <italic>Poulinea</italic> sp. 1. All scale bars are 10 μm.</p>
</caption>
<graphic mimetype="image" position="float" xlink:href="info:doi/10.1371/journal.pone.0157011.g001" xlink:type="simple"/>
</fig>
<fig id="pone.0157011.g002" position="float">
<object-id pub-id-type="doi">10.1371/journal.pone.0157011.g002</object-id>
<label>Fig 2</label>
<caption>
<title>Scanning Electron Microscope images of epibiotic diatoms found on leatherback, loggerhead, and olive ridley sea turtles.</title>
<p>Leatherback turtle: A = <italic>Navicula</italic> sp., B, C, and D = <italic>Tursiocola</italic> sp.; Loggerhead turtle: E: <italic>Amphora</italic> sp. 4, F = <italic>Amphora</italic> sp. 5, G = <italic>Amphora</italic> sp. 6, H = <italic>Amphora</italic> sp. 7, I = <italic>Diploneis</italic> sp. and other adnate unknown diatom; Olive ridley turtle: J = Nitzschia sp., K = broken pieces of <italic>Achnanthes</italic> sp (upper left) and possibly other diatom species (arrow), L = <italic>Achnanthes</italic> sp., M = <italic>Poulinea</italic> sp. 3, N = <italic>Amphora</italic> sp. 5, O = <italic>Achnanthes</italic> sp. All scale bars are 10 μm.</p>
</caption>
<graphic mimetype="image" position="float" xlink:href="info:doi/10.1371/journal.pone.0157011.g002" xlink:type="simple"/>
</fig>
<p>When compared to descriptions of known diatom taxa, many of the 18 diatom taxa seen in this study differed in important aspects of their morphology. For example, the diatom shown in <xref ref-type="fig" rid="pone.0157011.g002">Fig 2I</xref> could not be satisfactorily assigned to any existing genus. So far, only one <italic>Poulinea</italic> sp. has been described [<xref ref-type="bibr" rid="pone.0157011.ref025">25</xref>]) and, due to differences in the central area, shape, and number of areolae, we believe that the taxa in this study to do not belong to this species. A detailed taxonomic analysis of sea turtle diatoms would therefore be a productive avenue for future research.</p>
<p>Many diatom taxa were only observed on a single host; however, three diatom species were found on multiple host species. <italic>Achnanthes</italic> sp. was found on flatback, Kemp’s ridley, and olive ridley turtles. <italic>Amphora</italic> sp. 5 was found on loggerhead and olive ridley turtles, and <italic>Poulinea</italic> sp. 1 was found on flatback and Kemp’s ridley turtles. Even with the limited sample size used in this study, the presence of comparable diatom taxa on different host species from different localities suggests that that diatom assemblages on sea turtles may be very similar in structure and composition regardless of the hosts’ species or geographic location.</p>
<p>Epibiosis in the marine environment is primarily facultative in nature [<xref ref-type="bibr" rid="pone.0157011.ref008">8</xref>] and this is probably the case with the majority of diatoms documented here. As such the survival of these epibiotic diatoms may not depend upon settling on a sea turtle host and they may be present in the micro-plankton or on a variety of substrates. Indeed, taxa such as <italic>Melirosa sol</italic> and <italic>Nitzchia</italic> sp. are often living in the plankton and may have been present in the surrounding water at the time that the sea turtle tissue sample was collected. Nevertheless, there is also evidence that some diatom taxa observed in our study are obligate sea turtle epibionts. The diatom genera, <italic>Poulinea</italic> and <italic>Chelonicola</italic>, have only recently been described from olive ridley carapaces and have not been observed elsewhere [<xref ref-type="bibr" rid="pone.0157011.ref025">25</xref>]. <italic>Tursiocola</italic> spp. may also be an obligate epibiont, even though it is not exclusive to sea turtles and has been observed on cetaceans [<xref ref-type="bibr" rid="pone.0157011.ref021">21</xref>], freshwater and marine turtles [<xref ref-type="bibr" rid="pone.0157011.ref011">11</xref>, <xref ref-type="bibr" rid="pone.0157011.ref027">27</xref> respectively] and the West Indian manatee <italic>Trichechus manatus</italic> [<xref ref-type="bibr" rid="pone.0157011.ref024">24</xref>].</p>
<p>In addition to epibiotic diatoms, other micro-organisms were also discovered by SEM. Although we could not unequivocally identify these organisms we presume them to be a hystrichosphere from a dinoflagellate (<xref ref-type="fig" rid="pone.0157011.g003">Fig 3A</xref>) and an encrusting foraminifera (<xref ref-type="fig" rid="pone.0157011.g003">Fig 3B</xref>). Furthermore, evidence of a diatom valve (<xref ref-type="fig" rid="pone.0157011.g003">Fig 3C</xref>) and a coccolithophore (<xref ref-type="fig" rid="pone.0157011.g003">Fig 3D</xref>) were also found on the foraminifera in <xref ref-type="fig" rid="pone.0157011.g003">Fig 3B</xref>. Interestingly, these micro-organisms were all found on the carapace sample from a loggerhead turtle. Loggerhead turtles generally host the most diverse macro-epibiont communities [<xref ref-type="bibr" rid="pone.0157011.ref001">1</xref>] and thus it stands to reason that loggerhead turtles also host the most diverse micro-epibiont communities.</p>
<fig id="pone.0157011.g003" position="float">
<object-id pub-id-type="doi">10.1371/journal.pone.0157011.g003</object-id>
<label>Fig 3</label>
<caption>
<title>Scanning Electron Microscope images of other epibiotic organisms found on loggerhead turtles (A and B).</title>
<p>C and D are epibionts found on the formanifera in image B. Scale bars are 10, 500, 1, and 10 μm in images A, B, C, and D respectively.</p>
</caption>
<graphic mimetype="image" position="float" xlink:href="info:doi/10.1371/journal.pone.0157011.g003" xlink:type="simple"/>
</fig>
</sec>
</body>
<back>
<ack>
<p>The samples used in this study were either donated by colleagues (green, hawksbill, Kemp’s ridley, loggerhead, and olive ridley samples), borrowed from The Bishop Museum, Honolulu, Hawaii (flatback samples), or collected directly (leatherback samples). Flatback samples were provided by Molly Hagemann from the Bishop Museum. Greg Watkins-Colwell facilitated the shipment of flatback samples to the Peabody Museum of Natural History. Green, hawksbill, and olive ridley scute samples were provided to JDZ by George Balazs, NOAA Pacific Islands Fisheries Science Center, Honolulu, Hawaii. Kemp’s ridley turtle scute samples were provided by Thane Wibbels, University of Alabama. Loggerhead material from Florida was provided to JDZ through the assistance of DuBose Griffin, Sea Turtle Stranding Coordinator for the South Carolina Department of Natural Resources. Permits for collecting skin samples from leatherback turtles were granted by the Department of Environmental Affairs, South Africa (#RES2013/10). Leatherback turtles were imported into the US under CITES permits (#12U589757A/9). We would like to thank Zhenting Jiang for providing essential assistance in the use of the SEM.</p>
</ack>
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