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The microbiome of gorgonian octocorals, Muricea, with a description of a novel, photosynthetic protistan symbiont

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The microbiome of gorgonian octocorals, Muricea, with a description of a novel, photosynthetic protistan symbiont

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THE MICROBIOME OF GORGONIAN OCTOCORALS, MURICEA, WITH A
DESCRIPTION OF A NOVEL, PHOTOSYNTHETIC PROTISTAN SYMBIONT

BY
Johanna B. Holm

A Dissertation Presented to the

FACULTY OF THE USC GRADUATE SCHOOL

UNIVERSITY OF SOUTHERN CALIFORNIA

In Partial Fulfillment of the

Requirements for the Degree

DOCTOR OF PHILOSOPHY

(BIOLOGICAL SCIENCES)

Defended: May 2015
Degree Conferral: August 2015

THESIS ADVISOR:
Dr. Karla B. Heidelberg

DEFENSE COMMITTEE:
Dr. David A. Caron (Chair), Department of Biological Sciences
Dr. Wiebke Ziebis, Department of Biological Sciences
Dr. David Bottjer, Department of Earth Sciences

Dedications

To my parents, Bruce & Eileen Holm, who were the first people in my life to tell me I
could do and be anything that I wanted. I thank them for this and their constant support
through my many endeavors.

Dr. Jean Boal of Millersville University was my first mentor. Before I met her, I didn’t
know that I could make a career out of scientific inquiry. Her mentorship directly
influenced my professional path, and I will always be grateful to her for this. She is still
an inspiration to me today.

I would like to dedicate this work to two individuals. One that I’ll never meet in person,
and one that was taken away from my life much too soon.

Dr. Rick Grigg’s doctoral dissertation was an important source for my dissertation, as no
one has studied the ecology of Muricea like he did in his 1970 doctoral dissertation. I’d
had only a single email correspondence with him before he passed away, but hoped he
might be happy to see the influence of his Ph. D. work 45 years later.

Mr. Harry Currey was a dear family friend that has been a part of my life as long as I can
remember. Like my own parents, he and his wife, Adela, were, to me, beacons of love,
happiness, hope, and family. “Brooklyn Harry” lost his life to cancer halfway through my
doctoral work, and has since been an inspiration to me to continue with scientific
research.

And to Hal, who was my boyfriend when I started this work, then my fiancé, and now my
husband. Your confidence in me is overwhelming and I love you for it. Thank you.
MICROB. OF GORG. OCTOC. MURICEA, WITH A DESCR. OF A NOV. PHOTOSYN. PROTIST. SYMBIONT

TABLE OF CONTENTS

Abstract 1
Ch. 1 Microscopic observations and description of a novel algae endozoic in the polyps of M. californica 2
Introduction...........................................................................................................................................2
Methods & Materials............................................................................................................................4
Results...................................................................................................................................................8
Discussion............................................................................................................................................10
References............................................................................................................................................13
Tables...................................................................................................................................................17
Figures..................................................................................................................................................18
Ch. 2 Prokaryotic associates of Muricea californica & Muricea fruticosa 27
Introduction...........................................................................................................................................27
Methods & Materials............................................................................................................................29
Results...................................................................................................................................................33
Discussion.............................................................................................................................................35
References.............................................................................................................................................39
Figures...................................................................................................................................................42
Ch. 3 Photosynthetic measurements of the temperate gorgonian octocoral, Muricea 51
Introduction...........................................................................................................................................51
Methods & Materials............................................................................................................................53
Results...................................................................................................................................................56
Discussion.............................................................................................................................................57
References.............................................................................................................................................60
Tables....................................................................................................................................................62
Figures...................................................................................................................................................63
Conclusion 69

MICROB. OF GORG. OCTOC. MURICEA, WITH A DESCR. OF A NOV. PHOTOSYN. PROTIST.
SYMBIONT

ABSTRACT
Coral microbial interactions have been studied for some time, primarily in response to anthropogenic
climate change and its impacts on tropical reefs. These studies focused on hermatypic, scleractinian species
(reef-building, hard corals), as coral reefs support the greatest amount of biodiversity found in one
ecosystem. Thus, temperate soft corals, such as gorgonian octocorals, have historically received far less
attention, though species from the Mediterranean Sea have begun to experience bleaching events in
response to increased water temperatures, and this has sparked examinations into gorgonian microbiomes.
More recently, gorgonian octocorals have been named as a top source of marine natural products. In some
cases, a specifically associating microbe synthesizes these natural products, rather than the gorgonian
octocoral. Eastern Pacific species of the gorgonian octocoral Muricea have been regarded as
azooxanthellate, and this was the extent of explorations into their microbiomes. They are abundant along
the southern California coasts, as important contributors of benthic heterogeneity, and considered
ecological indicators of diversity within kelp forests. This study is the first to examine the microbiome of
Muricea californica and M. fruticosa, two species with overlapping geographical ranges, but distinct
morphological characters, primarily, polyp color. M. californica maintains orange-gold polyps while M.
fruticosa polyps are white. The source of this differentiation is pigmented cells, reported and described here
for the first time by in-depth pigment and microanalyses. These cells are eukaryotic, contain chlorophyll
and the accessory carotenoid 19-hexanoyloxyfucoxanthin, and 2 membranes surround the chloroplasts,
suggesting primary endosymbiosis as is seen in the Chlorophyta, Glaucophyta, and Rhododphyta algae.
This is the first report of a coral associated with a non-dinoflagellate, endozoic algae. We show here that
each species of Muricea maintains a specific bacterial community as well, mainly composed of separate
strains of Mycoplasma, and Spirochaetes, and an enormous amount of novel bacterial diversity. The
bacterial microbiomes are also representative of photosymbiont-containing versus bleached corals,
suggesting an impact of the pigmented cells. Finally, the first measurements of photosynthesis and the
oxygen diffusive boundary layer at the Muricea polyp surfaces are reported. M. californica produced more
oxygen in the presence of light > 150 µmol photons m
-2
s
-1
than M. fruticosa, but oxygen production was
observed in these species as well, explained by the observed relative densities of the endozoic, pigmented
algae.
1
CH. 1: MICROS. OBS. AND DESCR. OF NOV. ALG. ENDOZ. IN M. CALIFORNICA
CHAPTER 1:
Microscopic observations and description of a novel algae endozoic in the polyps of M. californica

AUTHORS:
Johanna B. Holm, David A. Caron, Paul Webster, Karla B. Heidelberg

ABSTRACT
The California Golden Gorgonian, Muricea californica, and the Brown Gorgonian, M. fruticosa are octocorals that
exist in overlapping habitats in the coastal waters of southern California. They are distinguished by the color of their
polyps; M. californica polyps are bright orange-gold, while M. fruticosa polyps are white. This study explores the
source of this morphological discrepancy. These species of Muricea do not harbor the dinoflagellate photosymbionts,
Symbiodinium, but instead appear to contain non-dinoflagellate algae. These entities contain autofluorescent
chloroplasts that are connected via extensions in a network through the cell, and 19’ hexanoyloxyfucoxanthin as the
main carotenoid accessory pigment. Two membranes, implying primary endosymbiosis, surround the plastids and
sequence data from the cp23S gene suggests a Rhodophyte origin. More data are needed to resolve the genetic
identity of these organisms, but this study provides evidence that this may be the first reported non-dinoflagellate
photosymbiont present in coral polyps.

INTRODUCTION
Cnidarian-algal symbioses are common and, specifically regarding coral and anemone polyps, involve the
dinoflagellate genus Symbiodinium. Photosynthetic products, namely, fixed carbon, are transferred to the host
cnidarian, and the algae are maintained in the protective polyp environment exposed to sunlight, with access to
nitrogen and phosphorous from the host. Species of both hermatypic (reef-building) corals and non-hermatypic
gorgonian octocorals maintain zooxanthellae symbionts, though the contribution of the algae to gorgonian
octocorals is comparatively smaller (Fabricius & Klumpp 1995). Multiple clades of Symbiodinium exist and can be
observed in a single coral colony (Baker 2003). The presence, absence, and clade identification of Symbiodinium in
a coral host primarily occurs via genetic investigation of the large ribosomal subunit variable regions, specifically
the internal transcribed spacer which includes the ITS1, 5.8S, and ITS2 regions (LaJeunesse 2001).
2
CH. 1: MICROS. OBS. AND DESCR. OF NOV. ALG. ENDOZ. IN M. CALIFORNICA
Corals can also host other protists, though the nature of such associations may vary from mutualism to
parasitism. Heterotrophic Stramenopiles, such as Thraustochytrids, have been recently examined in the mucus of
many scleractinian corals (Arotsker et al. 2012), cultured and identified via 18S ribosomal RNA sequences (Harel et
al. 2008). Chromera velia, an Alveolate relative of Apicomplexans, was isolated from within the tissue of the
scleractinian coral Plesiastrea versispora in Australia (Moore et al. 2008). Taxonomic placement of C. velia was
determined via nuclear and plastid genetic markers. The ecology of these protists with the host is currently under
investigation.
Morphological descriptions via microscopic and culturing methods of algal symbionts are also important,
especially when a genetic identity is unresolved. The first coral zooxanthellae life cycle and taxonomic data came
from a microscopic study, and resulted in the establishment of the genus Symbiodinium (Freudenthal 2007) within
the Dinophyceae. Useful tools to morphologically describe protists include structural descriptions by light, epi-
fluorescence, confocal, scanning electron microscopy, and transmission electron microscopy. The observation of
particular ultrastructures can provide insights into the taxonomic origin of an organism. For example, thylakoid
membranes are indicative of photosynthetic organisms (Gibbs 1970), condensed chromatin in a nucleus is
suggestive of dinoflagellates, and the number of membranes around a plasmid provides insight into endosymbiosis
events (Gibbs 1962).
Photosynthetic pigments can also provide taxonomic inferences (Mackey et al. 1996), . Coral polyp
pigments originate from a variety of sources, and can be colored by autofluorescent, photo-protective or photo-
enhancing proteins (Salih et al. 2000, Dove et al. 1995, Dove et al. 2001), melanin in times of stress (Mydlarz &
Palmer 2011), and algal-derived photosynthetic pigments from symbionts (Jeffrey & Haxo 1968), including
astaxanthin, which may also be derived from their diet (Maia et al. 2013). Zooxanthellate corals maintain
dinoflagellate algae, Symbiodinium, that provide a distinct golden-brown coloration to polyps; a result of
photosynthetic peridinin, and photoprotective dinoxanthin, which overlay the photosynthetic chlorophylls a and c
(Jeffrey & Haxo 1968). Coral tissues can appear white, or translucent (Stoletzki & Schierwater 2005), but may also
be pigmented as a result of accumulation from their diet, especially with the xanthophyll carotenoid, astaxanthin, as
has been reported in deep-sea corals (Elde et al. 2012), and octocorals near the coast of Brazil (Maia et al. 2013).
Accumulation of carotenoid pigments in exoskeleton can also occur via non-covalent bonds with proteins
(carotenoproteins), as in many marine organisms such as copepods (Van Nieuwerburgh et al. 2005), and lobsters
3
CH. 1: MICROS. OBS. AND DESCR. OF NOV. ALG. ENDOZ. IN M. CALIFORNICA
(Bandaranayake 2006). Beyond photosynthesis, carotenoids have a range of functions from photoprotection, to
camouflage (Matsuno 2001), to antioxidation (Lesser et al. 2004).
Muricea californica are gorgonian octocorals from coastal waters of southern California, and exist in
overlapping habitats with a sister species, M. fruticosa. Polyps of M. californica are distinct from M. fruticosa due to
a golden-orange coloration (Figure 1). The source of this pigmentation has never been examined, but in 2005 van
Oppen and others determined the geographic patterns of Symbiodinium in 69 octocoral species from the Caribbean,
Great Barrier Reef, and the eastern Pacific Ocean using the ITS1 marker. It was concluded that none of the eastern
Pacific Ocean octocoral species contained Symbiodinium, including four from the Plexaurid gorgonian genus
Muricea (Van Oppen et al. 2005).
Herein, the various attributes of a pigmented algal entity observed in the polyps of the California gorgonian
octocoral Muricea californica will be described. Morphological descriptions of the symbiont in-hospite via multiple
forms of microscopy, as well as pigment profile analyses and attempts to culture and genetically identify the
symbionts are detailed providing lines of evidence for identification.

MATERIALS & METHODS
Sample Collection
Fresh samples of Muricea californica and Muricea fruticosa were collected from Big Fisherman’s Cove at
the USC-Wrigley Institute for Environmental Sciences via SCUBA or free-diving, in accordance with the California
Department of Fish and Wildlife Scientific Collection Permit #12734 during the course of the experiment (Aug-Sept
2011, 2012, 2013). Five to ten centimeter branches were snipped from healthy colonies, and placed in sterile conical
tubes. Branches were immediately brought to the lab and maintained in aquaria containing 0.2 µm filtered seawater.
Submersible pumps provided ample current and oxygen to keep branch colonies healthy.
Microscopy
For live imaging, coral polyps were examined with a BX51 microscope (Olympus) with transmitted and
epi-fluorescence light. For DAPI staining, a live polyp was removed from a colony, placed into 0.3 µM DAPI in 1X
PBS for 5 min, and mounted onto a standard microscope slide with coverslip. The same tentacle was imaged with
transmitted light and epi-fluorescent light using the chlorophyll (excitation: 480 nm, emission: 660 nm) and DAPI
filter sets (excitation: 350 nm, emission: 470 nm). Individual polyps were also mounted on a depression slide with
4
CH. 1: MICROS. OBS. AND DESCR. OF NOV. ALG. ENDOZ. IN M. CALIFORNICA
0.2 µm-filtered seawater and imaged in super-resolution with a DeltaVision OMX 3D-Structure Illumination
Microscope, with a 60x 4.2 NA oil immersion objective lens, using an excitation wavelength of 488 nm and
emission spectra at 528 (FITC, green) and 683 nm (Cy5, red). Polyp image stacks were reconstructed, deconvolved,
and aligned with softWoRx. All images and movies were false colored with red to indicate chlorophyll, and green
representing the green autofluorescence. Organelle measurements were made on the DAPI fluorescence image
(nuclei) and the 3D-SIM fluorescence images (all other structures) using ImageJ (Abramoff et al. 2004).
Sample preparation and imaging for transmission electron microscopy was done at the House Research
Institute, Los Angeles, CA. The starting material was a coral branch collected in Sep 2012 and maintained in an
aquarium containing natural sea water at 18°C with air pumps and a 12:12 h light:dark cycle at an irradiance of 300
µE m
-2
s
-1
approximately 2 weeks after collection. Living tissue was carefully removed from a colony branch,
dissected to remove calcified regions, and placed in 20% BSA solution with gold-coated specimen holders. The
sample was immediately frozen using an EMPact2 High-Pressure Freezer (Leica, Buffalo Grove, IL) and held in
liquid nitrogen for all subsequent processing. The specimen was place on frozen ethanol containing 1% osmium
tetroxide and slowly warmed to -80°C and held at this temperature for 2 h using an EM AFS2 Freeze Substation and
Embedding System (Leica, Buffalo Grove, IL). Subsequently, the specimen was gradually warmed to -60°C over 16
h and held for an additional 2 h. Finally, the sample was warmed to ambient temperature overnight and the
ethanol/osmium tetroxide solution washed with multiple rinses of fresh ethanol.
The polyp was next infiltrated with epoxy resin without catalyst, using ethanol as a carrier and embedded in
fresh ethanol containing BDMA as a hardening catalyst, and polymerized at 60°C overnight. Thin sections (50-70
nm) were prepared using an EM UC6 Ultramicrotome (Leica, Buffalo Grove, IL) and a 45° diamond knife (Diatome,
Hatfield, PA), and mounted onto Formvar/carbon coated metal specimen grids. Sections were contrasted with
solutions of uranyl and lead salts, and images were collected using a Tecnai G2 20 TEM operating at 80 kV. Digital
images were only minimally modified to adjust brightness and contrast, and cropped to suitable sizes for
presentation using ImageJ (Abramoff et al. 2004).
Coral Pigment Characterization
To compare pigment compositions, frozen, stored branches from M. californica and M. fruticosa were
shipped on dry ice to the University of Maryland Center for Environmental Sciences Horn Point Laboratory for
pigment analyses by high-performance liquid chromatography (HPLC). Thawed polyp tissue as well as whole
5
CH. 1: MICROS. OBS. AND DESCR. OF NOV. ALG. ENDOZ. IN M. CALIFORNICA
branches (tissue, sclerites, and gorgonin) were used for extractions and HPLC analysis as described in (Van
Heukelem & Thomas 2001). Samples were filtered on GF/F filters prior to extraction in cold 90%. Pigment
quantities were standardized to the tissue surface area of the original sample.
Isolation & Culturing
Attempts to isolate the pigmented cells from the coral polyps were made from Jan-Dec 2013. A few cells
(rare because tissue was very sticky), small clumps of ca. 10-20 pigmented cells, or full polyps were separated with
micropipettes and a dissecting microscope, washed with 0.2 µm-filtered seawater, and placed into a variety of media
types made with aged 0.2 µm-filtered seawater. Culture media used included L1 media (Guillard & Hargraves 1993),
with the recipe diluted either 1:2, 1:4, or 1:10 resulting in L1/2 , L1/4, or L1/10, respectively, with and without
NH
4
Cl, (0.15 mM) and the Na
2
SiO
3
9H2O omitted. Liquid, solid (with 0.8% agar), and semisolid (0.4% agar)
versions of the L1/2 and L1/4 (without and without NH
4
Cl) were produced and aseptically poured into sterile culture
vials (liquid and semi-solid), or plates (solid). Agar-based media were to simulate the polyp tissue environment.
Culturing attempts were also made with liquid LKS media (Koid et al. 2014) with and without Na
2
SiO
3
9H2O, and
with 10-fold less soil extract. Cultures were incubated at 18°C with a 12:12 h light:dark cycle at an irradiance of 300
µE m
-2
s
-1
.
Genetic Identity
Multiple primers described in coral literature were used to query samples for symbiont identification
specifically referring to M. californica. Table 1 summarizes primers used, the targeted region, the reference source
for that primer pair, and the starting material. PCR amplifications were performed following the primer source
protocols, but denaturation and annealing temperatures were altered as per polymerase-specific manufacturer
protocols. Primer Anti-coralR was designed for this study using nuclear 18S rRNA gene sequences previously
obtained from Muricea samples, and specifically designed to have a high annealing temperature when paired with
primer 570F (Weekers et al. 1994) to increase amplification specificity. The final concentrations of the PCR
reaction performed for this 570F/Anti-CoralR was as follows: Green Flexi Buffer (1X), MgCl
2
(25 mM), dNTPs
(0.2 mM each), primers (0.4 µM each), GoTaq Flexi Polymerase (1.25 units), and 10 or 20 ng template DNA. The
reactions were performed with the following cycle conditions: 1 cycle 95°C for 2 min, 30 cycles 95°C for 1 min,
65°C for 1 min, and 72°C 1.5 min, followed by 5 min extension at 72°C and a 4°C hold.
Total genomic DNA was extracted from freshly-collected M. californica, M. fruticosa, and Anthopleura
6
CH. 1: MICROS. OBS. AND DESCR. OF NOV. ALG. ENDOZ. IN M. CALIFORNICA
elegantissima (anemone), flash frozen in liquid nitrogen and stored at -80°C until DNA extraction. A. elegantissima
hosts Symbiodinium clade C ((Sanders & Palumbi 2011) and confirmed from Sep 2011 PCR) and was a positive
control for all reactions. M. fruticosa was assumed to be a negative control, though water was also used as a negative
control in all cases. Gorgonian polyps were individually removed from each frozen sample, and tentacles from A.
elegantissimia, and each sample placed into 100 µL lysis buffer (0.01 M Tris-HCl, pH 8.0, 0.25 M EDTA, pH 8.0,
0.02% SDS) and homogenized with sterile, disposable pestles (Fisher Scientific). DNA was extracted from
homogenates with a PowerWater DNA Isolation Kit (MO BIO Laboratories) following the manufacturer protocol
with the following alterations: 1) no filter was used; the homogenates were directly applied to the bead-containing
tubes, 2) samples were incubated at 4°C in solution P2 for 40 min instead of 5 min, and 3) DNA was eluted off the
column into 30 µL nuclease-free water (Thermo Scientific). DNA concentrations were measured using the Qubit BR
assay and fluorometer (Life Technologies). In some cases, DNA was amplified from specific tissue fractions to
reduce the probability of coral DNA amplification. Zooxanthellae were isolated from freshly collected M.
californica tissue following a previously described protocol (Rowan & Powers 1991). In each step, in tact
pigmented cells were confirmed via light microscopy. The DNA was then extracted from the pellet using the
protocol described above, and amplification of the 18S rDNA was performed with universal primers.
Pigmented cells isolated with a micropipette and dissecting scope were also used as source material for
PCR amplifications. Small clumps of ca. 10-20 pigmented cells were separated and washed in 0.2 µm-filtered sea
water and 3, 5, 8, and 14 clumps were placed into separate, sterile microcentrifuge tubes containing 500 µL 2X
Lysis Buffer (Schnetzer et al. 2011) and100 µL 0.5 mm zirconia/silica beads (provided by D. Caron, BioSpace
Products). Tubes were exposed to 3 rounds of heating to 70°C for 20s, vortexing for 20s, and incubation on ice for
20s. Lysates were stored at -80°C until use in PCR amplifications, in which 1:100 dilutions of lysates were used as
template DNA.
DNA was also amplified from cells lysed with Proteinase K, which has been shown to successfully extract
PCR-ready DNA from dinoflagellate cells (Ki et al. 2004). Proteinase K (200 ng/µL) was added to tubes containing
cells separated from coral polyp tissue via micropipette and dissecting scope, and incubated at 55°C for 50 mins,
95°C for 10 min, and cooled to 4°C in a thermal cycler. Additionally, amplification of pigmented cell DNA was also
attempted from fresh, raw polyp homogenate in 0.2 µm filtered seawater.
PCR products were separated in 1% agarose gels and visualized with SYBR Gold Nucleic Acid Stain (Life
7
CH. 1: MICROS. OBS. AND DESCR. OF NOV. ALG. ENDOZ. IN M. CALIFORNICA
Technologies) and UV light. If a band was observed, the PCR products were cloned using the TOPO TA Cloning
Kit (Invitrogen) as per the manufacturer’s protocol, screened using blue/white colony screening, and the insert of
positive plasmids sequenced via T7 Promotor and M13R primers at Genewiz Sequencing Facility (San Diego, CA).
Insert sequences were aligned via BLASTN to NCBI GenBank Nucleotide Collection, RefSeq, and BioProject
Databases. Reference sequences with the lowest e-value are reported, along with the coverage of the query sequence
and percent identity in Table 1. In the case of the rbcL and cp23S primers, the sequenced inserts related to algae
were aligned with ClustalW (Thompson et al. 2002) to sequences from the studies containing the original primers,
the alignment ends were manually made flush and a maximum likelihood phylogenetic tree constructed with 1,000
bootstrap replicates in Geneious v. 5.6.4 (Kearse et al. 2012). The cp23S tree includes sequences from Sherwood &
Presting (2007) (Supplemental Table 1), which include multiple lineages, and (Pochon et al. 2006), which
specifically examined Symbiodinium clade diversity, in addition to the best blast hits.

RESULTS

Microscopy
Pigmented cells were located throughout the polyps of M. californica, and exhibited chlorophyll
autofluorescence (Figure 2). The cell shapes were not consistent; some cells appeared more condensed, though not
perfectly spherical, while others were elongated in a teardrop shape. The cells ranged in size from 5-15 µm
depending on orientation and each contained a single nucleus approximately 3.2 µm in diameter (Figure 3). Aside
from a nucleus and, in some cases, a vacuole, each pigmented cell was filled with chlorophyll spheres (red auto
fluorescence with 488 nm excitation), and each sphere was approximately 0.4 µm in diameter as measured by light
microscopy (range 0.3-0.5 µm; Figure 4). Interestingly, green auto fluorescence was also present (excitation 488 nm,
and emission 528 nm), and the fluorescence pattern enveloped the spherical, red chlorophyll spheres. Three-
dimensional optical sectioning of the structures indicated that both the red and green structures were connected like
networks through the depth of a cell (Figure 4 & Movie 1).
Ultrastructural analyses of in-hospite pigmented cells confirmed that the entire cell had a membrane
separate from the surrounding coral membranes. Within the cell, the chloroplast structures and the nucleus each had
two membranes surrounding them. The symbiont cell included a nucleus (3.2 µm), and chloroplasts (mean diameter
0.99 µm, range 0.5-1.7 µm, Figure 5). This sample showed some evidence of freeze-damage, so important details
8
CH. 1: MICROS. OBS. AND DESCR. OF NOV. ALG. ENDOZ. IN M. CALIFORNICA
about organelle ultrastructures, such as the presence of thylakoid membranes within chloroplasts, or the arrangement
of chromatin in the nucleus, could not be confirmed. Additional damage included shrinkage of the organelles, as
vacuoles were clear between organelles and their surrounding membranes. However, the presence of the observable
membranes was not an artifact of the freeze-damage.
Pigment Analyses
In both species, the concentration of Chl a was an average 6-fold greater than Chl’s c1, c2, or c3, and (2.9 ±
0.8 µg cm
-2
Chl a in M. californica samples, 0.5 ± 0.1 µg cm
-2
in M. fruticosa samples, Figure 6). Chl b was not
observed. The most abundant pigment measured in M. californica samples was the light-harvesting accessory
carotenoid 19’ hexanoyloxyfucoxanthin, or Hex-fuco (5.9 ± 1.2 µg cm
-2
). M. fruticosa samples contained 0.5 ± 0.1
µg cm
-2
Hex-fuco. Relatively smaller concentrations of other accessory carotenoids were detected, including light
harvesting pigments 19’-butanyloxyfucoxanthin and fuxocanthin (0.8 ± 0.2 µg But-fuco cm
-2
and 1.5 ± 0.5 µg Fuco
cm
-2
in M. californica, and 0.1 ± 0.02 µg But-fuco cm
-2
and 0.1 ± 0.02 µg Fuco cm
-2
in M. fruticosa), and
photoprotective carotenoids alloxanthin, diatoxanthin, and zeaxanthin (1.5-1.6 ± 0.6 µg each cm
-2
in M. californica,
0.1 ± 0.04 µg each diatoxanthin and zeaxanthin cm
-2
in M. fruticosa, alloxanthin was not detected, Figure 7).
Dinoflagellate-specific pigments, peridinin and diadinoxanthin were minimally observed in M. californica (0.3-0.5 ±
0.1 µg cm
-2
), though peridinin was found at similar concentrations as 19’-butanyloxyfucoxanthin and fuxocanthin in
M. fruticosa (ca. 0.2 ± 0.05 µg cm
-2
).

Isolation and Culturing
Pigmented cells proliferated only in F/4 + NH
4
in 0.9% agar inoculated with a torn polyp bacteria were
observed in semi-solid and solid media. A reddish/orange mass approximately 3 mm wide about the polyp was
observed after 1 week. Four weeks post-inoculation, the same mass was observed at 2 mm wide and 5 mm below the
original inoculated polyp location. Samples observed via light and epifluorescence microscopy contained brown-
orange spherical cells (8-15 µm) and, when excited at 488 nm, emitted green autofluorescence (DIC, ca. 520 nm) but
no red autofluorescence (chlorophyll, ca. 660 nm). One of these autofluorescent cells burst under the blue light (the
intense, spherical fluorescence dissipated into scattering, smaller autofluorescent spheres and photobleached).
Subcultures of this sample in the same medium were made using sterile pipette tips, and removing the agar around
the cells. No subcultures were successful in proliferating any cells, nor were any other media types.
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CH. 1: MICROS. OBS. AND DESCR. OF NOV. ALG. ENDOZ. IN M. CALIFORNICA

Genetic Identity
No DNA was amplified from Muricea template starting materials using Symbiodinium-, Thraustochytrid-,
or dinoflagellate–specific primers, targeting nuclear rRNA genes, co1, or cob (Table 1), however, amplification of
the expected size was observed in all cases using the A. elegantissima (anemone) total genomic DNA. An expected
620 bp amplicon was obtained using the 570F/Anti-coral R primers, and the produced sequence was most similar to
the octocoral KF856053, Calcigorgia spiculifera 18S rRNA gene sequence (e-value=0, 100% identical to 99%
query sequence coverage).
A 294 bp fragment was amplified using primers targeting rbcL Form ID. Four clone inserts were sequenced
and were 95-99% identical. The sequences did not significantly align to any sequences in the NCBI GenBank
databases, or the sequences from a previous study characterizing marine phytoplankton populations ((Paul et al.
2000), Figure 8).
The primer sets ITSintfor2/ITS2rev amplified a ca. 200 bp fragment in each tested sample that, after
cloning and sequencing, was 84% identical to the ITS2 region of AF262339, the octocoral Alcyonium palmatum (e-
value=1e
-12
).
The cp23S Domain V region was successfully amplified from M. californica and M. fruticosa, and
produced an expected band size of 700-800 bp. Four clones were sequenced for each species. Three clones from M.
fruticosa contained identical sequences and 98% of the sequence was 89% identical to NR121960, the Tenericute
bacterium Mesoplasma florum. The fourth clone from M. fruticosa aligned most closely with NZ_JOKH01000001.1,
the γ-Proteobacterium Endozoicomonas numazuensis, isolated from sponge tissue (82% identical to 91% of the
query cp23S sequence, e-value=5e
-176
). The two clones from M. californica contained identical sequences that were
96.2% identical to Rhodophyte algae EF426657, Galaxaura rugosa, and 95.1% similar to Z18289, Palmaria
palmata (Figure 9).

DISCUSSION
Polyp color is the most conspicuous character that distinguishes southern Californica gorgonian octocorals
M. californica from M. fruticosa. The source of this orange-gold color has never been previously examined, but a
genetic survey of eastern Pacific octocorals confirmed these species do not contain dinoflagellate photosymbionts,
10
CH. 1: MICROS. OBS. AND DESCR. OF NOV. ALG. ENDOZ. IN M. CALIFORNICA
Symbiodinium (Van Oppen et al. 2005). This study determined the source of this coloration, which appears to be a
non-dinoflagellate microbial eukaryote that is capable of photosynthesis.
The pigmented entities in the polyps are ca. 5-15 µm in diameter, and contain autofluorescent chloroplasts
each a mean of 0.5 µm in diameter, as well as a source of green autofluoresence which envelopes the chloroplasts.
The chloroplasts are connected by a matrix of thin chlorophyll extensions throughout the entity of the pigmented
cells. The green autofluorescence may be from photoprotective or photoenhancing pigments, as these have been
observed in association with Symbiodinium (Matz et al. 1999, Salih et al. 2000). Host-derived pigments are
localized either above or below zooxanthellae in the polyp, and protect photosynthetic machinery from oxidative
stress, or enhance photosynthesis by either wavelength transformation or back-scattering of light, respectively (Salih
et al. 2000). Interestingly, the green autofluorescence observed in Muricea clearly localized to the photosynthetic
cells, and was not seen in the coral tissue, thus suggesting that the fluorescence is algal-derived.
Pigment profiles from both species of Muricea were obtained to define the specific pigments in M.
californica polyps, and to confirm a lack of pigments in M. fruticosa. Aside from chlorophyll, 19’-
hexanoyloxyfucoxanthin (hex-fuco) and astaxanthin were the most abundant photosynthetic accessory pigments
detected. The concentrations of astaxanthin observed were similar between both species of Muricea, while at least 6-
fold more hex-fuco was observed in the M. californica samples. Astaxanthin has been measured in octocorals before,
and was detected in extracts of zooxanthellate (M. atlantica) and azooxanthellate (Carijoa riisei) specimen (Maia et
al. 2013), both of which have reddish-brown sclerites like M. californica and M. fruticosa. Therefore, astaxanthin is
probably protein-bound and a constituent of the red sclerites of Muricea, as is observed in some crustaceans (Van
Nieuwerburgh et al. 2005, Bandaranayake 2006), and not derived from the endozoic algae. Alternatively, hex-fuco,
a derivative of fucoxanthin, is most likely the pigment responsible for the color discrepancy between the two species.
Interestingly, photosynthetic pigments were not absent in the M. fruticosa samples, as was expected. Though the
pigmented cells have not been observed via fluorescence microscopy in the polyps of M. fruticosa, they have been
quantified via transmitted light microscopy and further details on the photosynthetic capabilities of both species can
be found in Chapter 3.
To my knowledge, there are no reports of corals containing the pigment hex-fuco, whereas peridinin, the
major carotenoid of Symbiodinium, is readily detected from zooxanthellate coral polyp extracts (for example,
Apprill et al. 2007) (Jeffrey & Haxo 1968). Hex-fuco is traditionally identified with haptophyte algae, especially E.
11
CH. 1: MICROS. OBS. AND DESCR. OF NOV. ALG. ENDOZ. IN M. CALIFORNICA
huxleyi and Phaeocystis (Wright & Jeffrey 1987, Zapata et al. 2004). However, dinoflagellates have also been
observed containing hex-fuco as the major carotenoid pigment, suggesting tertiary endosymbiosis, in which a
dinoflagellate engulfed a haptophyte, and retained the plastid (Tengs et al. 2000). Haptophyte plastids have four
membranes and the plastids of the hex-fuco containing dinoflagellates maintain three membranes around their
plastids, due to the loss of one membrane (Yoon et al. 2002). The TEM data clearly show only 2 membranes
surrounding the Muricea pigmented cell plastids. Additionally, the hex-fuco containing dinoflagellate lineages were
confirmed using eukaryotic 18S and plastid 16S SSU rDNA and these sequences were easily aligned to other
dinoflagellate sequences (Tengs et al. 2000). In the case of the Muricea pigmented cells, dinoflagellate-specific 18S
primers were unsuccessful at amplifying any DNA. Thus it is unlikely that the pigmented cells in Muricea are
dinoflagellates.
The ultrastructural information provides possibilities about the identity of the pigmented cells. The two
membranes surrounding the plastids in these cells suggest a primary endosymbiosis event, as is seen in Rhodophyta,
Chlorophyta, and Glaucophyta (see Vesteg et al. 2009 for review), each of which are ancestral groups that
experienced primary endosymbiosis events. Here, a heterotrophic protist engulfed but did not digest a
cyanobacterium, resulting in a primary plastid surrounded by three membranes: two from the cyanobacterium and
one from the food vacuole. It is suggested that the cyanobacterium escaped this vacuole (and lost the third
membrane), resulting in a double-membrane bound primary plastid. It appears from the TEM data that the Muricea
algal symbionts contain primary plastids, and have since been engulfed by Muricea.
Information from plastid genes encoding for the chloroplast 23S ribosomal RNA and Ribulose-1,5-
bisphosphate carboxylase/oxygenase (RuBisCo) would help to decipher the suggestions put forth by the TEM image.
The cp23S gene successfully produced an amplicon, and the sequence was most related to red algae Galaxaura
rugosa and Palmaria palmata. The resulting information from cp23S would corroborate the observation of the
double-membrane plastids, but localization of this sequence to the pigmented cells (via fluorescence in-situ
hybridization, for example) is necessary to confirm that the sequence is not from any other contaminating sources
(such as algae located to the surface of Muricea). Amplification of the RuBisCo large subunit gene, rbcL, produced
an amplicon of expected length, but did not closely align to any available reference sequences. Multiple alignments
and phylogenetic trees were attempted (including those containing rhodophyta representatives, data not shown), but
were not successful at confirming any significant relationships.
12
CH. 1: MICROS. OBS. AND DESCR. OF NOV. ALG. ENDOZ. IN M. CALIFORNICA
Together, these data strongly suggest that the pigmented entities observed in the polyps of Muricea
californica are not closely related to the dinoflagellates. Instead, it seems that the cells are representatives of a more
ancient lineage such as the Rhodophyta. Further TEM imaging, pigment analyses including phycobilins as standards,
and genetic confirmation via localization could resolve the identification. This observation alone is a novel finding,
and expands our current knowledge of cnidarian-algal symbioses.

ACKNOWLEDGMENTS
We are grateful to Christopher Suffridge, Sarah Hu, Kellie Spafford, Lorraine Sadler, Eric Castillo, Tom
Carr and Wrigley Marine Science Center Staff for assistance in sample collections and maintain organisms.
Undergraduate summer fellows, Nicole McNabb and Samantha Wright Leigh, were integral to gorgonian data
collection. We thank the Center for Electron Microscopy and Microanalysis, specifically John Curulli and Marc
Green for microscopy assistance. Victoria Campbell and Alyssa Gellene aided in culture media preparation and
usage of the BX-51. Meg Maddox of the UMCES Horn Point Laboratory was integral to obtaining the HPLC
pigment analyses. Support was provided by a USC Wrigley Institute for Environmental Studies and the Center for
Electron Microscopy and Microanalysis. The authors declare no conflict of interest.

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CH. 1: MICROSC. OBS. AND DESCR. OF A NOV. ALGAE ENDOZ. IN THE POLYPS OF M. CALIFORNICA
Table 1. PCR primers, gene targets, starting material, polymerase type, and results for genetic identification of M.
californica pigmented cells.

a
(Correa et al. 2009)
b
(Medlin et al. 1988)
c
(Rowan & Powers 1991)
d
(Harel et al. 2008)
e
(Lin et al. 2006)
f
(Zhang
et al. 2005)
g
(Paul et al. 2000)
h
(Weekers et al. 1994)
I
J. Holm
j
(LaJeunesse 2002) & (Coleman et al. 1994)
k
(Stat et
al. 2009)

PRIMER
NAME/GENE
TARGET
SAMPLE
TYPE
POLYMERASE RESULT
SymA
a

Symbiodinium
ITS1-5.8S-ITS2 Nuclear
rDNA
DNA
GoTaq Flexi Polymerase
(Promega)
Negative
SymB
a

Symbiodinium
28S Nuclear rDNA
DNA
GoTaq Flexi Polymerase
(Promega)
Negative
SymC
a

Symbiodinium
28S Nuclear rDNA
DNA
GoTaq Flexi Polymerase
(Promega)
Negative
SymD
a

Symbiodinium
28S Nuclear rDNA
DNA
GoTaq Flexi Polymerase
(Promega)
Negative

EukA/EukB
b

18S Nuclear rDNA
Zooxanthella
e Isolation
Pellet DNA
GoTaq Flexi Polymerase
(Promega)
~1.8 kb
EukA/EukB
b
18S Nuclear rDNA Mucus
GoTaq Flexi Polymerase
(Promega)
Negative
ss3Z/ss5
c

Symbiodinium 18S Nuclear
rDNA
Lysed cell
dilutions
GoTaq Flexi Polymerase
(Promega)
Negative
Thr404f/Thr1017r
d

Thraustochytridae 18S
rDNA
DNA
Platinum Taq HiFi
Polymerase (Invitrogen)
Negative
Thr404f/Thr1017r
d

Thraustochytridae 18S
rDNA
Lysed cell
dilutions
Platinum Taq HiFi
Polymerase (Invitrogen)
Negative
Dino18SF2/18ScomR1
e
Dinoflagellate 18S rDNA
Lysed cell
dilutions
Platinum Taq HiFi
Polymerase (Invitrogen)
Negative
Dino18SF3/18ScomR1
e
Dinoflagellate 18S rDNA
Lysed cell
dilutions
Platinum Taq HiFi
Polymerase (Invitrogen)
Negative
Dinocox1f/Dinocox1R
e
Dinoflagellate cox1
Lysed cell
dilutions
GoTaq Green Master Mix
(Promega)
Negative
Dinocob1F/Dinocob1R
f
Dinoflagellate cob
Lysed cell
dilutions
GoTaq Green Master Mix
(Promega)
Negative
rbcLF/rbcLR
g

Chromophytic algal Form
ID rbcL
DNA, 4
clones
sequenced
GoTaq Green Master Mix
(Promega)
294 bp
570F
h
/Anti-Coral R
i

18S Nuclear rDNA bp
570-1050
DNA
GoTaq Flexi Polymerase
(Promega)
620bp
ITSintFor2
ITS2rev
j

Ca. 75 bp into the 150 bp
5.8S rRNA gene - the 5’
end of the 28S (nrDNA
LSU)
DNA
GoTaq Flexi Polymerase
(Promega)
200 bp
Plastid23S1f/plastid23S2
r
k

cp23S Domain V region,
Symbiodinium specific
(Chloroplast)
DNA
Q5 High Fidelity 2X Master
Mix (Qiagen)
700 bp
17
CH. 1: MICROSC. OBS. AND DESCR. OF A NOV. ALGAE ENDOZ. IN THE POLYPS OF M. CALIFORNICA

Figure 1. Muricea californica and M. fruticosa colonies co-exist in the waters of Santa Catalina Island, Los Angeles,
CA. Photo credit: Mark Steele Inset: A single polyp of M. californica. Scale Bar: 1.5 mm.
18
CH. 1: MICROSC. OBS. AND DESCR. OF A NOV. ALGAE ENDOZ. IN THE POLYPS OF M. CALIFORNICA

Figure 2. A polyp of M. californica contains brown algae containing autofluorescent chlorophyll. Top: Transmitted
light, Bottom: fluorescence light (bottom) excitation λ: 488 nm, emission λ: 660 nm. Scale bars: 100 µm.
19
CH. 1: MICROSC. OBS. AND DESCR. OF A NOV. ALGAE ENDOZ. IN THE POLYPS OF M. CALIFORNICA

Figure 3. The pigmented cells of M. californica in-hospite visualized with transmitted light (left), to observe
chlorophyll autofluorescence with excitation λ: 488 nm, emission λ: 660 nm (middle), and to observe DAPI
staining with excitation λ: 350 nm, emission λ: 470 nm. Scale bars: 10 µm.
20
CH. 1: MICROSC. OBS. AND DESCR. OF A NOV. ALGAE ENDOZ. IN THE POLYPS OF M. CALIFORNICA

Figure 4 0RYLH. 2D visualization of the 3D super-resolution images of M. californica pigmented cells in-hospite. to observe
chlorophyll (left, excitation λ: 488 nm, emission λ: 683 nm), green autofluorescence (middle, excitation λ: 488
nm, emission λ: 528 nm), and the two emission spectra overlaid. Scale bars: 15 µm.
21
CH. 1: MICROSC. OBS. AND DESCR. OF A NOV. ALGAE ENDOZ. IN THE POLYPS OF M. CALIFORNICA

Figure 5: Tranmission electron micrograph of pigmented cell in-hospite. n=nucleus, c=chloroplast, arrows indicate
membranes. Scale bar: 2 µm
22
CH. 1: MICROSC. OBS. AND DESCR. OF A NOV. ALGAE ENDOZ. IN THE POLYPS OF M. CALIFORNICA

Figure 6: Major photosynthetic pigments detected via HPLC analysis in M. californica and M. fruticosa.
23
CH. 1: MICROSC. OBS. AND DESCR. OF A NOV. ALGAE ENDOZ. IN THE POLYPS OF M. CALIFORNICA

Figure 7: Minor photosynthetic pigments detected via HPLC analysis in M. californica and M. fruticosa.
24
CH. 1: MICROSC. OBS. AND DESCR. OF A NOV. ALGAE ENDOZ. IN THE POLYPS OF M. CALIFORNICA

Figure 8: Maximum likelihood phylogenetic tree of the large subunit gene of Ribulose-1,5-bisphosphate
carboxylase/oxygenase (rbcL). Amplicons from M. californica are bold. Bootstrap proportions >50% are shown.
25
CH. 1: MICROSC. OBS. AND DESCR. OF A NOV. ALGAE ENDOZ. IN THE POLYPS OF M. CALIFORNICA

Figure 9: Maximum likelihood phylogenetic tree of the chloroplast 23S rDNA Domain V gene (cp23S). Amplicons
from M. californica are bold. Black: Cyanobacteria, Red: Rhodophyta, Orange: Stramenopiles, Green: Chlorophyta.
Bootstrap proportions >50% are shown.
26
CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

CHAPTER 2
Prokaryotic associates of Muricea californica & Muricea fruticosa

AUTHORS
Johanna B. Holm, Karla B. Heidelberg

ABSTRACT
Gorgonian octocorals are sources of novel, but understudied microbial diversity. Alternatively,
scleractinian or reef-building coral micro biomes have been thoroughly examined for some time in light of the
threats of climate change. Muricea californica and Muricea fruticosa are two species of gorgonian octocoral found
in the temperate waters of southern California. Both species survive in overlapping habitats, providing an exciting
foundation to question the species-specificity of the gorgonian microbial diversity. Here, that diversity was
compared between replicate samples collected from multiple Muricea colonies at different depths. Overlying
seawater and nearby zoanthid colonies were also sampled and confirmed gorgonian-specific associations. While
differences in microbial diversity existed between each sample, the communities showed clear Muricea species
specificity; separate strains of Mycoplasma species associated with either M. californica or M. fruticosa, and
Spirochaetes were observed moreso on M. californica. These and other differences between the observed
associations may be due to the photosynthetic potential of each species, a byproduct of the novel endozoic algae.

INTRODUCTION
Corals and their microbiomes (including microbial eukaryotes, bacteria, archaea, and viruses) together
comprise the entire metaorganism (Bosch & McFall-Ngai 2011), and these symbiotic associations are critical to host
survival (Rosenberg et al. 2007, Bourne & Webster 2013). Members of these microbiomes contribute to shared
metabolic functions such as nutrient acquisition, environmental sensing, and protection from disease (Rohwer et al.
2002, Reshef et al. 2006, Vega Thurber et al. 2009, Thomas et al. 2010). Despite the perceived importance, clear
factors shaping a coral’s microbial composition have yet to be fully discerned (Bourne & Webster 2013).
Most coral microbiome studies have focused on reef-building scleractinian corals. Few studies have
examined the diversity and composition of gorgonian-associated microbial communities (Webster & Bourne 2007,
27
CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

Brück et al. 2007, Gray et al. 2011, Bourne et al. 2013, Correa et al. 2013) and fewer have examined those of
temperate gorgonians (Vezzulli et al. 2013, La Rivière et al. 2013). The gorgonian microbiomes described to date
were targeted using culture-based, fingerprinting, and/or clone library analyses (except (Bourne et al. 2013), which
uses 454 pyrosequencing). Such strategies are capable of providing taxonomic resolution but capture only a small
portion of total microbial diversity. Additionally, there are no studies, to our knowledge, that statistically compare
gorgonian-associated prokaryotic diversity to the surrounding seawater or other benthic organisms to gorgonians,
limiting our perspective of how microbiome diversity varies between hosts and the environment.
The previous studies described above show gorgonian-associated microbial communities from various
environments, including the Great Barrier Reef, the deep-sea, and the Mediterranean Sea, were dominated by
Gammaproteobacteria (i.e. Endozoicomonas and Oceanospirillaes) or Tenericutes (especially Mycoplasma).
However, it is intriguing that similar bacterial classes dominate the observed microbiomes of these gorgonian genera
located in vastly different (and, in some cases, extreme) marine environments, and raises the question of how much
the environment influences microbiome composition. This highlights a need to more-deeply examine the gorgonian-
associated prokaryotic community with high-sampling effort and biological replication, in order to more fully
characterize prokaryotic diversity. In addition, direct comparisons between related gorgonian species, nearby benthic
fauna, and surrounding seawater from the same environment would identify the niches uniquely provided by the
gorgonian host.
Here, we examined the microbiomes of two species of Muricea, a genus of azooxanthellate (Van
Oppen et al. 2005) gorgonian octocorals found throughout the tropical and temperate eastern Pacific and western
Atlantic oceans. Muricea californica and M. fruticosa co-exist in the temperate kelp forests of southern California
and are easily distinguished from each other by the colors of their polyps, golden-orange or white, respectively
(Grigg 1970). Their overlapping habitats, similar abundances, colony structures, and general life histories make
these species of Muricea suitable for comparison. Multiple colonies of both species were sampled with biological
replication to determine the mean microbiome compositions for each colony. To test the specificity of microbial
associations, we attempted to maximize intra-, and inter-colony microbiome variations by purposely sampling
colonies from different depths. To further evaluate the degree of gorgonian-specific associations exist, we also
examined the microbiomes of nearby Parazoanthus lucificum (zoanthid, suborder Macrocnemia) colonies, and
Saccharina japonica (kelp) in addition to the surrounding seawater. Azooxanthellate P. lucificum, named for the
28
CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

brilliant bioluminescence it emits, was specifically chosen as a comparative organism because it occupies similar
space as Muricea colonies in both the water column and the benthos due to its life-history trait of infecting and
overgrowing M. californica colonies (Cutress & Pequegnat 1960). To our knowledge, only one other zoanthid
(suborder: Brachycnemina) microbiome has been described (Sun et al. 2014). This is the first study to characterize
and compare deeply sequenced microbiomes of Muricea californica, M. fruticosa, P. lucificum, and S. japonica.
0$7(5,$/6 METHODS
Sample Collection
All sample collections were made in accordance with CA-DFW Scientific Collecting Permit #12734,
issued to J. Holm. Muricea californica (Mc) and M. fruticosa (Mf) samples were collected in replicate in addition to
nearby samples of the zoanthid, Parazoanthus lucificum (Pl), and the kelp, Saccharina japonica (Sj). Samples were
collected from a rocky wall of Santa Catalina Island, CA (33º 26’ 53.9” N, 118º 28’ 42.3” W) midday on 14 October
2013.
For each Mc and Mf species, subsamples from 3 colonies from different depths were sampled in-situ (range
8-16 m). Collection depth is indicated in the sample’s name, following the replicate branch number. Sampling
techniques to reduce contamination were employed; samples were captured in 50 mL conical tubes (Falcon) without
handling. Additionally, single branches from 2 Pl colonies from depths 9 and 16 m, and one sample of kelp, Sj,
growing adjacent to the 11-12 m gorgonian colonies, were collected. The zoanthid samples were cut and collected in
a similar manner as Muricea samples. The kelp sample was collected with cleaned forceps and placed into a conical
tube. Upon returning to the boat, samples were immediately processed as follows: ethanol-wiped forceps were used
to remove a collected sample from the conical tube and the sample was dipped multiple times in 0.02 µm-filtered
seawater to remove unattached debris and contaminating overlying seawater. Samples were immediately placed in
RNAlater as per manufacturer’s instructions (Ambion) and stored at 4ºC for 3 weeks until DNA extraction.

Total DNA Extraction and PCR Amplification of 16S rRNA
Prior to extraction, samples were processed to remove RNAlater as per the manufacturer instructions. Briefly, each
sample was aseptically removed from RNAlater, weighed, and a 50 mg subsample was placed in 450-500 mL 1X
PBS, pH 8.0. Samples were centrifuged for 1 min at 4000 x g, and the supernatent containing residual RNAlater was
carefully removed. Pellets were subsequently processed using the PowerPlant Pro DNA Isolation kit (MO BIO
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

Laboratories, CA, USA) according to the manufacturer instructions using a Qiagen TissueLyser II at 30 Hz for 10
min. Due to large amounts of mucous, the Sj sample and 50 mg of each Pl sample were first ground using liquid
nitrogen and a sterilized mortar and pestle prior to DNA isolation.
Bacterial and Archaeal V4-V6 regions of the 16S rRNA gene were amplified using primers A519F
(CAGCMGCCGCGGTAA, (Wang & Qian 2009)) and 1061R (CRRCACGAGCTGACGAC, (Andersson et al.
2008)) from probeBase (Loy et al. 2007), which were predicted to amplify 76.9% bacteria and 1.2% archaeal
sequences (Klindworth et al. 2012). Final amplification reaction volumes were 25 µL and contained 1X Q5 High-
Fidelity 2X Master Mix (New England Biolabs), 100 ng template, and 1 µM of each primer. Reactions were run
with a single denaturation step at 98ºC for 30 s followed by 30 cycles at 98ºC for 30 s, 59ºC for 15 s, and 72ºC for
30 s and completed with a final extension step of 72ºC for 2 min. DNA from a previously collected, typical water
sample was also amplified using this protocol and running the PCR for 35 cycles. Amplified DNA was visualized on
a 1% agarose gel using SYBR Gold Nucleic Acid Stain, purified using DNA Clean & Concentrator-5 kits (Zymo),
and eluted with 6 µL nuclease-free water (Thermo Scientific). Amplicons were first quantified on a Nanodrop to
ensure the 260/280 absorbance ratio was near 1.8 and then quantified again using the Quant-iT dsDNA HS Assay kit
(Invitrogen, Carlsbad, CA) and measured on a Qubit fluorometer (Invitrogen).
Illumina Library Preparation and High Throughput Sequencing
Amplicon libraries were prepared for Illumina MiSeq multiplex paired-end sequencing using NEBNext Ultra DNA
Library Prep kit and the NEBNext Multiplex Oligos for Illumina Index Primers Sets 1 and 2 (New England
Biolabs). Twenty nanograms of amplicon DNA were used for each library preparation reaction. Ampure Beads
(Beckman Coulter) were used for all DNA purification steps following the manufacturer instructions. Adaptor-
ligated and indexed samples were visualized for purity and quantification using an Agilent 2100 Bioanalyzer.
Twenty samples with final molar concentrations >1 nM were submitted to the UC Davis Genome Center (Davis,
CA) for paired-end, multiplex sequencing on the Illumina MiSeq platform using the MiSeq Reagent Kit v3
(Illumina). Cluster generation, sequencing (600 cycles), image processing, demultiplexing, and quality score
calculations were all performed on the MiSeq 500 platform (Illumina). Raw read data have been submitted to the
NCBI Sequence Read Archive under BioSample Accession numbers SAMN03203155-SAMN03203174 within
BioProject Accession number PRJNA268033. We further filtered reads for quality using the IlluminaClip (default
settings), and Sliding Window (4 bases, average quality score of >25) options in Trimmomatic (Bolger et al. 2014).
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

Sequence Processing
Sequence assembly and quality control described here were performed using Mothur (v 1.32.1-v.1.33.0)
(Schloss et al. 2009). The MiSeq v3 reagent kit (Illumina) produced read lengths ca. 300 bp. Paired-end reads that
included the V4-V6 variable regions and overlapped in the 16S ribosomal RNA gene C45 region were processed.
Sequence contigs shorter than 501 bp with >10 ambiguous bases and homopolymers >10 bases were removed.
Sequences were aligned to the SILVA SSU Ref Nonredundant (NR) 119 database (Quast et al. 2013) and trimmed
to equal alignment length (608 bp with gaps, 552 bp mean sequence length). Chimeric sequences were removed
(39,076 sequences or 6.4%, UCHIME (Edgar et al. 2011)), and the remaining sequences (606,679 unique) were
taxonomically classified as described in (Wang & Qian 2009) using the SILVA SSU Ref NR 119 formatted for
Mothur.
Sequence Binning & Phylogenetic Analyses
Sequencing and assembly generated an average of 22,685 assembled 16S rRNA gene fragments per sample
(range: 11,063 – 33,481). Sequences were binned into operational taxonomic units (OTUs) of 98% identity using the
Average Neighbor method (Schloss & Westcott 2011) and resulted in 19,955 OTUs (the same number of OTUs
were observed using a 97% cutoff). Of these, 6,753 were singletons and an additional 258 OTUs were characterized
as chloroplasts, and were culled from further analyses, resulting in an average of 21,757 sequences per sample
(range: 1,785-32,715). For each sample, alpha-diversity statistics including Good’s Coverage Estimator and the
number of observed OTUs were calculated for the non-normalized dataset using Mothur (v. 1.33.3) and compared
using Student’s t-test. Sj contained the fewest number of sequences due to a high proportion of removed chloroplast
sequences, and was removed from comparative beta-diversity analyses.
Remaining samples were randomly rarified to contain an equal number of sequences for comparative
purposes (determined by the sample containing the fewest sequences, Mc_1_12m, 10,448 sequences). Bray-Curtis
Dissimilarity Indices of the abundances of remaining OTUs were calculated to assess beta-diversity, and visualized
by hierarchical clustering (hclust) using the average neighbor method and nMDS analyses (metaMDS), with R and
the Vegan package (Oksanen et al. 2013). Statistical differences in microbial community composition between
different samples (gorgonian vs. non-gorgonian), species (Mc vs. Mf), and colonies, were tested for using the adonis
function (PERMANOVA test) with 999 permutations, also in Vegan. The OTUs contributing the most to the
observed clusters were determined using the Vegan function simper, and verified using the same function on Primer-
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

E (Clarke 1993). OTU consensus taxonomies were obtained using the SILVA SSU Ref NR 119 database, and OTU
sequence representatives were extracted in Mothur. OTUs that were unclassifiable beyond Phylum or Class level,
were examined more closely using the NCBI GenBank nonredundant (NR) and 16S ribosomal RNA reference
(Bacteria and Archaea) databases (October 2014) and the BLASTN algorithm (Altschul et al. 1990). Reference
sequences with the highest percent identity and lowest e-values were used to construct a phylogenetic tree (the
number of top matches, highest percent identities, and e-values varied across OTU representatives). Maximum-
likelihood phylogenetic relationships of the OTU representative sequences were assessed using ClustalW
(Thompson et al. 2002) to align sequences, and maximum-likelihood trees calculated using the Jukes-Cantor model
of substitution (Jukes & Cantor 1969) with 1000 bootstrap replicates (Geneious v.5.6.4).
Relative abundances of rarefied microbiomes were compared across samples bacterial classes using a
Bubble Plot in MS Excel. Pie charts of the same data were composed excluding major OTUs (determined from the
SIMPER analyses) for each species of Muricea to examine underlying diversity. The OTU representative sequence
data have been submitted to the GenBank database under accession numbers KP174126-KP174134.
Quantification of pigmented cells from coral polyps
Endozoic cells are responsible for the characteristic orange-yellow pigment of Mc polyps, the identity of
which is the topic of another manuscript in preparation. While we have yet to confirm these cells as photosymbionts,
zooxanthellae are common in other species of coral, and correlate with differences in microbiome diversity (Bourne
et al. 2013). Counts of pigmented cells from Muricea were collected on two separate dates in Aug 2013 (ca. 100
polyps counted per species) and Nov 2014 (ca. 50 polyps counted per species). Colony branches were rinsed well
with 0.2 µm-filtered seawater and individual polyps were plucked with forceps immediately before counting. Polyps
were placed in 20 µL 0.2 µm-filtered seawater and homogenized via vigorous pipetting until no tissue could be
observed. Pigmented cells were counted in duplicate using a Neubauer hemocytometer and compared with a 2-
sample t-test.
Mucus Production and Microscopic Observations
Four colonies of Mc and 2 colonies of Mf were collected and maintained in tanks with unfiltered, flow
through seawater, for four weeks during June 2012. Colonies were exposed to natural light conditions. All colonies
were examined twice per day (approx. 9:00 and 18:00) for the percentage of the total colony that was visibly
covered with mucus. When visible, samples of mucus were collected with a sterile syringe, incubated with SYBR
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

Green nucleic acid stain as per the manufacturer’s instructions (Bio-Rad), and examined using an Olympus BX5100
epi-fluorescent microscope.
RESULTS
Comparative community composition analyses by 16S rDNA amplicon sequencing
Good’s coverage estimated a mean of 98.9% for all samples (range: 98.2%-99.6%), indicating adequate
sampling effort. SW had the greatest number of observed OTUs, while all organism-associated microbiomes
exhibited less diversity (Figure 1).
The microbiomes produced distinct sample-specific clusters at 20-25% (Figure 2). OTUs primarily
contributing to the observed clusters in Figure 2 were OTUs 1, 3, 4, and 7 according to the SIMPER analysis, all of
which were not taxonomically classifiable.
The seawater microbiome was different from all organism-associated microbiomes, dominated by α-
Proteobacteria, namely Candidatus Pelagibacter (53% of microbiome) and ϒ-Proteobacteria SAR86 clade (12% of
microbiome) (Figure 3). These specific taxa minimally contributed (< 0.13%) to all other microbiomes.
Pl microbiomes were also distinct. The relative abundance of OTU7 primarily distinguished the Pl samples
from all other samples (21% of Pl_16m, 84% in Pl_9m, and less than 0.03% of any other sample communities), and
most closely related to NR_044756, Spirochaeta halophila (85% identical, Figure 4). Pl_16m harbored a large
number of Flavobacteriaceae sequences (Figure 3, 48% of community), specifically OTU40 (47.8% of community),
which was 97% identical to NR_116269, Maritimimonas rapanae (Figure 4). OTU40 contributed < 0.04% to any
other microbiome, including Pl_9m.
Muricea microbiomes all contained an average of 15% unclassified Bacteria compared to the SW and Pl
samples where 2-10% of sequences were unclassifiable. Muricea were also defined by a large number of
Mycoplasma (Tenericutes) and Spirochaetes sequences.
The Muricea microbiomes were species-specific which explained 58.2% of the variability in their
microbiome compositions (PERMANOVA F
1,15
=19.5 p=0.001). Mf samples consistently had more observed OTUs
than Mc, though the difference in the means was not significant (Figure 1; t
16
=1.71, p=0.082). Reads clustered into
OTU1 composed 6-64% of Mc microbiomes, compared to < 0.001% of any other sampled microbiome, and were
noticeably abundant in the Mc_12m and Mc_3_16m microbiomes. Phylogenetic analyses indicated OTU1 was at
least 96% identical to OTU7, and also 85% identical to to NR_044756, Spirochaeta halophila (Figure 4). OTU4
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

represented 6-61% of Mc microbiome sequences, versus < 0.3% in all other samples, while OTU3 composed 12-
62% of Mf microbial communities, compared to < 0.05% of all other sample microbiomes (Figure 3). OTUs 3 & 4
were 87.5% identical to each other, and representative sequences formed a monophyletic group with multiple
Mycoplasma sequences isolated from Muricea elongata, a sister gorgonian species found in the coastal Gulf of
Mexico and the Caribbean (NCBI PopSet: 134140623, Ranzer, Restrepo, & Kerr, unpublished). OTU4, found in the
Mc samples, formed a monophyletic group with Mycoplasma sequences isolated from healthy M. elongata colonies,
while OTU3 grouped with those from bleached, diseased M. elongata colonies (Figure 4).
Other taxa showing Muricea spp. specificity included α-Proteobacteria such as Thalassospira (observed in
5 of 7 Mc samples, and no Mf samples), Nitratireductor (all Mc samples, no Mf sample), and γ-Proteobacteria like
Endozoicomonas, Caedibacter and Francisella (observed in all Muricea samples, but higher abundances in Mc
samples; Caedibacter and Francisella observed in 6 of 7 Mc samples) (Figure 5). Also, Vibrio spp. (γ-
Proteobacteria) sequences were observed in all Muricea samples, but more so in Mf samples (1-7% of Mf
communities, < 0.4% of Mc communities). Candidatus Nitrosopumilus and Sulfuricurvum were observed in all Mf
samples and no Mc samples (1-2% of mean Mf community composition). Overall, Muricea microbiomes had on
average more γ-Proteobacteria sequences than α-Proteobacteria (t= p=; Figure 5), with the exception of
Mf_3_11m, and Mf_2_10m, due to a large number of sequences from α-Proteobacteria OTU75. OTU75 was 98%
identical to uncultured Sinorhizobium, a clonal sequence isolated from bleached Muricea elongata (Figure 4, NCBI
PopSet: 134140623, Ranzer, Restrepo, & Kerr, unpublished).
Variation between Mc colony microbiomes was significant (PERMANOVA: F
1,6
=7.45, R
2
=0.60, p=0.025).
Colony Mc_9m was distinct from other Mc colonies because more Francisella (γ-Proteobacteria) sequences were
observed in each of the replicate branch communities (Figure 3). As previously stated, the large number of OTU1
sequences defined the Mc_12m microbiomes.
Mf colony microbiomes were also significantly different from each other(PERMANOVA: F
1,6
=4.33,
R
2
=0.38, p=0.007). Colonies from 8 m and 11 m clustered together and separate from the Mf_10m colony.
Approximately 80% of the 8 m and 11 m replicate branch microbiomes were dominated by OTU9 (6-17% of colony
replicate branch microbiomes) and previously described OTU4 (Figure 3). OTU9 was 94% identical to OTU1, and
>80% identical to NR_044576, Spirochaetes halophila. OTU4 contributed to only 37% of Mf_10m microbiomes;
OTU9 was minimally observed. This, and the relatively high abundance of Family NB1d sequences (γ-
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

Proteobacteria OTU54, 99% identical to AB930131, Psychrobium conchae, Figure 4), and Tenacibaculum
(Flavobacteria) sequences produced the clustering of branch replicate samples 1 & 2 from colony Mf_10m. Colony
Mf_8m replicate branch microbiomes were distinct from Mf_11m because of the relatively high abundance of
Sphingobacteria Saprospiracea (Bacteroidetes) and the Thaumarchaea Candidatus Nitrosopumilus (Marine Group
I).
Because of the life history and geographical range similarities between Mc and Mf colonies, we quantified
the endozoic, pigmented cells from their polyps (the most conspicuous morphological character) as an attempt to
explain observed differences in gorgonian species-related microbiome diversity. Comparison via cell counts in two
separate years confirmed that Mc consistently maintained more pigmented cells than Mf (Figure 6, t
2013
=30.08,
df=213, p<0.0001; t
2014
=15.262, df=90, p<0.0001).
DISCUSSION
This study provides a detailed characterization of the microbiomes of two ecologically important temperate
gorgonian corals, M. californica and M. fruticosa. We compared these microbiomes to overlying seawater and
nearby zoanthid associated microbiomes and confirm that host-associated microbial assemblages exist which are
different from the surrounding sea water (Figure 3). Using inter- and intra-colony replication, we showed that two
species of the gorgonian coral, Muricea, have specific and predictable microbial assemblages, and have distinct
members of the associated microbial community that may occupy specific niches. P. lucificum was specifically
chosen as a comparative organism because it occupies similar space, in both the water column and the benthos, as
Muricea colonies, due to its life-history trait of infecting and overgrowing M. californica colonies (Cutress &
Pequegnat 1960).
Our results revealed specific relationships between hosts and members of the microbial communities. Both
species of Muricea had an abundance of sequences from the bacterial phylum, Tenericutes, specifically from the
genus, Mycoplasma. This relationship has been observed in deep-sea gorgonians (Gray et al. 2011), the cold water
coral, Lophelia pertusa (Kellogg et al. 2009), and a species of gorgonian from the Atlantic Ocean, Muricea elongata
(Ranzer, Restrepo, & Kerr, unpublished). The data of Ranzer et. al. also showed two strains of Mycoplasma
associating separately with different colonies of M. elongata, but the distinction between those colonies was healthy
and bleached or diseased. We saw a similar relationship; Mycoplasma strains associated with healthy M. elongata
were also found on algal-dense M. californica, and Mycoplasma strains associated with bleached M. elongata were
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

also found on white-polyped M. fruticosa. The species definition of gorgonian octocorals is not clearly resolved
(Vargas et al. 2014), and we have observed M. californica exuding the pigmented cells after artificial induction
(Supplemental Figure 3). Thus, it is possible that M. fruticosa is not an entirely different species from M.
californica, but rather a “bleached”, possibly diseased, morphotype.
Members of the phylum Tenericutes such as Mycoplasma lack a cell wall and, specifically, those from the
class Mollicutes are typically considered parasites of plants, insects, and animals. Within this class, the Mycoplasma
are either facultative or obligate anaerobes, and cultured strains require secondary metabolite sterols and fatty acids
(FA) for growth (Ludwig et al. 2010). Such compounds are produced by Muricea (Popov et al. 1983, Gutiérrez et
al. 2006), and the composition of FA from soft corals is highly dependent on the coral diet: FA produced by and
transferred from symbiotic photosynthetic algae to the host coral are compositionally different than those produced
by the coral’s own biosynthesis pathways (Imbs et al. 2007). Future research could examine what types of FA M.
californica and M. fruticosa generate, respectively. M. californica polyps contain a significantly greater density of
pigmented and potentially photosynthetic, alga-like cells compared to M. fruticosa (Figure 6), and if this impacts the
types of FA present in the coral (for example, the ratio of FA from algal versus coral sources), this may provide
specific niches for the different strains of Mycoplasma we observed, but more research is needed to examine this
hypothesis.
Another major microbial distinction between the two species of Muricea was the greater abundance of
sequences from the phylum Spirochaetes in M. californica samples. The genera Spirochaeta are chemoheterotrophic
and can thrive in a variety of environments (Ludwig et al. 2010). Spirochaetes have been observed in other
cnidarian groups including the cold-water coral Lophelia (Kellogg et al. 2009), hydra Hydra attenuata (Hufnagel &
Myhal 1977), and the mucous of sea pens Pennatula phospherea and Pteroides spinosum (Porporato et al. 2013).
We observed Spirochaetes-like bacteria in the mucus of M. californica (Suppl. Fig. 1) and hypothesize that the
greater abundance of sequences in M. californica samples may be due to the daily mucus production and sloughing
performed by this species, which has not been observed in M. fruticosa (Suppl. Fig. 2).
Muricea samples consisted of relatively more γ-Proteobacteria sequences than α-Proteobacteria. Recently,
Bourne et al. (2013) showed that photosymbiont-containing marine invertebrates had a greater abundance of γ-
Proteobacteria. Those without photosymbionts generally maintained relatively more α-Proteobacteria. M.
californica and M. fruticosa contain pigmented and potentially photosynthetic algal-like cells within their polyps
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

and this may be a contributing factor to the abundances of γ-Proteobacteria sequences observed. The γ-
Proteobacteria genera from M. californica samples were limited to Endozoicomonas, Caedibacter, and Francisella.
Endozoicomonas have been observed on other corals and gorgonians (Bayer et al. 2013, Bourne et al. 2013) and
have been shown to metabolize the organic sulfur compound, dimethylsulfoniopropionate (DMSP), a byproduct of
photosynthetic algae (Raina et al. 2009). Perhaps the higher density of photosynthetically-active algal cells observed
in the polyps of M. californica may be indirectly influencing both the lower diversity of γ-Proteobacteria genera and
the presence of Endozoicomonas, specifically, if DMSP is being produced by the algal cells.
M. fruticosa samples had more observed OTUs than M. californica (Figure 1). Additionally, M. fruticosa
microbiomes contained substantially more Vibrio sequences than any of the other sample microbiomes. Members of
the genus Vibrio are amongst the known pathogens of corals (Bally & Garrabou 2007, Vezzulli et al. 2010,
Mouchka et al. 2010), and diseased corals tend to harbor greater microbial diversity compared to healthy
conspecifics (Bourne et al. 2008, Sunagawa et al. 2009). While M. fruticosa colonies may simply harbor more
microbial diversity naturally, the greater OTU diversity and number of Vibrio sequences, in addition to the particular
strain of Mycoplasma observed could be a sign of microbial community instability and possibly an indication of a
diseased state, though further study, including temporal sampling, is necessary to further elucidate this possibility.
The sequences of nitrifying microbes found uniquely amongst the host-associated microbiomes of the
Muricea were of particular interest. M. fruticosa samples contained the Archaeon Nitrosopumilus, a known
ammonia oxidizer (Francis et al. 2007), and Sulfuricurvum, a known nitrate-reducing, sulfur oxidizer. M. californica
had an abundance of Nitratireductor, another nitrate-reducing bacteria. Nitrogen cycling in corals has been observed
(Shashar et al. 1994), and a model of the role Nitrosopumilus may play in ammonia oxidation has been presented
(Siboni et al. 2008). Nitrogenous waste (dissolved inorganic nitrogen, DIN) from coral colonies is either released
into the surrounding seawater, in the case of azooxanthellate corals, or transferred to photosymbionts, if present
(Dubinsky & Jokiel 1994). In fact, the density, chlorophyll a content, and rate of photosynthesis of coral
photosymbionts is tightly coupled to the availability of DIN (Hoegh-Guldberg & Smith 1989). Thus, Muricea
metabolic waste products may offer a distinct niche for the observed nitrifying microbes, and the presence of the
potentially novel pigmented algal cells may impact this diversity by altering the amounts and types of DIN released.
Interestingly, the gorgonian samples both contained a high number of novel sequences, those that were
unclassifiable beyond the domain level, because there were no reference sequences greater than 80% identical. This
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

proportion of sequences implies that these taxa may be important sources of novel biological diversity. Gorgonians
have been targeted as promising sources of novel secondary metabolites or marine natural products (MNPs) (Rocha
et al. 2011). But in most cases, an associated microbe, and not the invertebrate host, is producing the compound(s)
of interest and our study offers novel information about the microbial organisms associated with gorgonians that
may be relevant targets for bio-discovery.
Gorgonian microbiomes, in general, are extremely understudied, especially compared to their scleractinian
relatives. While a 16S rRNA gene diversity study is not definitive of functional or metabolic interactions in a
metaorganism, a comparative approach to microbial diversity, as we have described here, can highlight and
substantiate significant differences between the available niches provided by various host organisms.

ACKNOWLEDGMENTS
We are grateful to Kellie Spafford and Lorraine Sadler and Wrigley Marine Science Center Staff for assistance in
sample collections. Undergraduate summer fellows, Nicole McNabb and Samantha Wright Leigh, were integral to
gorgonian data collection. We thank Dr. Benjamin Tully, Rohan Sachdeva, Dr. Jay Liu, Dr. Jacob Cram, Ella
Sieradzki and Dr. David Caron for their invaluable advice on sequence analysis and manuscript review. Support was
provided by a USC Wrigley Institute for Environmental Studies Rose Hills Summer Fellowship. The authors declare
no conflict of interest.

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Figure 1. The mean observed number of OTUs per sample from the non-normalized dataset. N for samples are as
follows: Mc=7, Mf=9, Pl=2. Error bars are standard error of the mean.
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

Figure 2. NMDS of Bray-Curtis Dissimilarity Indices for each sample microbiome. Orange: Mc 9m (u), 12 m ( ),
16 m(!). Purple: Mf 8 m(!), 10 m(y), 11 m("). Blue: SW ("), and Green: Pl (stars). Dotted circles indicate
significant clusters at 20% similarity. Mf: Muricea fruticosa, Mc: Muricea californica, SW: seawater, Pl:
Parazoanthus lucificum, Sj: Saccharina japonica.
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

Figure 3. Bray-Curtis Dissimilarity Indices as hierarchical clustering dendrogram. Microbiomes summarized as taxa
at the Class level per sample. Bubble size indicates relative abundance of taxa within a sample. Mf: Muricea
fruticosa, Mc: Muricea californica, SW: seawater, Pl: Parazoanthus lucificum. Sample names are
“species_biological replicate_depth of collection”.
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

Figure 4. Maximum-likelihood phylogenetic analyses for OTU representative sequences using the Jukes-Cantor
model of substitution with 1000 bootstrap replicates (bootstrap values > 0.5 displayed). Reference sequences
obtained from both NCBI NR and Reference 16S ribosomal RNA databases. See text for specific sequence percent
identities. Mf: Muricea fruticosa, Mc: Muricea californica, SW: seawater, Pl: Parazoanthus lucificum.
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

Figure 5. Mean relative abundances of microbial taxa from M. californica and M. fruticosa, with largest OTUs
removed (Mycoplasma, Spirochaetes, Unclassifiable).
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

Figure 6. Pigmented cell counts per species from 2 different seasons (Spring 2013 and Fall 2014). Sample sizes are
from ca. 100 polyps (from 10 different colonies total) for Spring 2013, and ca. 50 polyps (from 5 different colonies
total) for Fall 2014.
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

Supplemental Figure 1. Muricea californica mucous contains Spirochaetes-like cells. Mucous from M. californica
stained with SYBR Gold and examined using fluorescence microscopy.
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

Supplemental Figure 2. Mucous coverage was almost continuously observed on Muricea californica (n=4 colonies)
and little to not at all on Muricea fruticosa (n=2 colonies). Colonies were observed daily from 6/21/2013-7/19/2013.
Means were calculated from 2-24 total observations, depending on the time point.
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CH. 2: PROK. ASSOC. OF M. CALIFORNICA & M. FRUTICOSA

Supplemental Figure 3. Muricea californica branch displaying white and golden polyps (left). Scale bar: 1.5 mm. M.
californica exuding the pigmented cells following 12 h in 4-8°C (right). Scale bar: 1 mm.

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CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

CHAPTER 3:
Photosynthetic measurements of the temperate gorgonian octocoral, Muricea

AUTHORS:
Johanna B. Holm, Wiebke Ziebis, Karla B. Heidelberg

ABSTRACT
Muricea californica and M. fruticosa are gorgonian octocorals that co-exist in the temperate eastern Pacific Ocean,
and lack photosynthetic symbionts of the genus Symbiodinium. However, M. californica polyps are distinctly
colored orange-gold compared to the white polyps of M. fruticosa. M. fruticosa is noticeably more abundant in dark
caves in deeper water, leading to the question of photosynthetic capabilities by M. californica. This study
demonstrates the photosynthetic potential of these species based on the pigments present in their polyps, and
confirms photosynthetic activity at the polyp surfaces of both species using oxygen micro sensors. We produced the
first reported oxygen diffusive boundary layer measurements for gorgonian corals, and conclude that M. californica
polyps produce more oxygen than M. fruticosa, and this may be correlated to the differences in the densities of
pigmented cells contained in the polyps.

INTRODUCTION
Coral polyp pigments originate from a variety of sources. Healthy coral tissue can be colored by
autofluorescent, photo-protective or photo-enhancing proteins (Salih et al. 2000, Dove et al. 1995, Dove et al.
2001). Coral polyps containing photosynthetic symbionts, Symbiodinium, are a distinct golden-brown in color due
to the photosynthetic accessory carotenoids peridinin and dinoxanthin in addition to (Gibbs 1962) chlorophylls a and
c (Jeffrey & Haxo 1968). Species of both hermatypic (reef-building) corals and ahermatypic gorgonian octocorals
maintain zooxanthellae symbionts (Fabricius & Klumpp 1995), though azooxanthellate species exist, too. Tissues of
azooxanthellate corals can appear white, or translucent (Stoletzki & Schierwater 2005), but may also be pigmented
as a result of accumulation from their diet, especially with the xanthophyll carotenoid, astaxanthin, as has been
reported in deep-sea corals (Elde et al. 2012), and octocorals near the coast of Brazil (Maia et al. 2013).
In the coastal northeastern Pacific Ocean, gorgonian octocorals are the most common species observed with
ranges from 1-8 colonies m
-2
from Point Conception south to Baja (pers. observation via SCUBA and see Grigg
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CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

1970). The Golden Gorgonian, Muricea californica, and the Brown Gorgonian, M. fruticosa, are co-existing sister
species of gorgonian octocoral distinguished by morphological and ecological characters (sum. by Grigg 1970). The
bright orange-gold polyp color in M. californica is arguably the most conspicuous morphological character,
contrasting the white polyps of M. fruticosa. We observed during dives at ca. 25 m along the coast of Santa Catalina
Island, in caves that receive little to no sunlight, colonies with only white polyps were present, whereas M.
Californica were missing. This led to the hypothesis that the golden pigments in the polyps of M. californica may
play a role in photosynthesis by endozoic algae. Zooxanthellate sister species of Muricea located near Panama and
Belize contain clade B Symbiodinium (Goulet & Coffroth 2004), while M. californica and M. fruticosa do not
contain Symbiodinium (Van Oppen et al. 2005, and see Chapter 2). Both species of Muricea are filter feeders, but
observations have shown M. californica polyps to be passive, capturing only dead particles that stick to the polyps,
while M. fruticosa are active predators, capturing live and dead food particles as they pass through the colony (Grigg
1970).
The oxygen produced by photosynthesis and used by respiration can be measured in corals. The diffusive
boundary layer (DBL) is a result of diffusive limitation through the momentum boundary layer, or drag across a
surface in an aqueous solution, where flow is nearly zero at a surface and increases with distance from that surface.
A DBL is present at all surfaces in the marine environment, including coral colonies and its thickness depends on
the flow velocity that the surface is exposed to. The DBL represents a boundary between the organisms and the
surrounding environment, as in this thin layer the transport of dissolved substances and gases such as carbon
dioxide, dissolved organic matter, and oxygen is solely governed by molecular diffusion. DBL thickness is inversely
proportional to flow velocity, and, in the case of corals, metabolic processes like respiration and, in zooxanthellate
corals, photosynthesis, direct the diffusive flux (Patterson 1992). Molecular diffusion is very fast over very short
spatial scales (µm), but the time for diffusive transport increases with the square of the distance (Jorgensen &
Marais 1990). At night, the coral surface within the DBL is hypoxic or near anoxic, and the direction of the diffusive
flux of oxygen is toward the polyp surface. During the day, in zooxanthellate corals, oxygen concentrations within
the DBL increase due to photosynthesis and the direction of diffusive oxygen flux is away from the coral, (Shashar
et al. 1993). Direct measurements within the DBL are quite difficult because the DBL is usually less than 1 mm
thick and coral behavior can disrupt measurements but, measurements with live corals are preferred because they are
representative of in-situ conditions, yet complicate fine scale measurements because of potential movements of the
52
CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

coral (usually contraction of a polyp). Detection of oxygen production near coral surfaces via photosynthesis or
consumption by respiration can be performed with microsensors in two ways: in very small volumes with respiration
chambers, or with microsensors in the DBL.
Here, the pigments from the polyps of both M. californica and M. fruticosa are measured via HPLC
pigment analyses to confirm the presence of chlorophyll and photosynthetic carotenoids. Next, micro sensors are
used to define the diffusive boundary layer for both species of Muricea, and finally, to determine the photosynthetic
capabilities of Muricea polyps, the production of oxygen across a spectrum of PAR intensities is measured at the
coral polyp surface. These microsensors measurements are the first to be reported for gorgonian octocorals, and are
also the first description of photosynthesis from M. californica and M. fruticosa.

METHODS & MATERIALS
Sample Collection
Fresh samples of Muricea californica and Muricea fruticosa were collected from Big Fisherman’s Cove at
the USC-Wrigley Marine Science Center (WMSC) on Catalina Island via SCUBA or free-diving, in accordance
with the California Department of Fish and Wildlife Scientific Collection Permit #12734 during the course of the
experiment (Aug 2013). Five to ten centimeter branches were snipped from healthy colonies (6-7 m depth), and
placed in sterile conical tubes. Branches were immediately brought to the lab and maintained in aquaria containing
0.2 µm filtered seawater. Submersible pumps circulating the seawater provided ample current and helped to mix in
oxygen to keep branch colonies healthy. Branches were used for experiments within 1-2 d of sample collections, and
following experiments, were placed in aluminum foil and stored at -80°C.
A second set of fresh samples was collected in the same manner in Jan 2015 to confirm pigment
measurements. Four colonies each of M. californica and M. fruticosa were sampled in triplicate at depth and
transported to the USC-WMSC. Samples were processed within 1 h of sample collection. The polyps were removed
from triplicate branches, pooled together, flash frozen in liquid nitrogen, and stored in cryovials at -80°C (60-100
polyps each vial).
Photosynthetic Pigment Measurements
Frozen, stored branches were shipped on dry ice to the University of Maryland Center for Environmental
Sciences Horn Point Laboratory for pigment analyses by HPLC. Thawed polyp tissue as well as whole branches
53
CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

(tissue, sclerites, and gorgonin) were used for extractions and HPLC analysis as described in (Van Heukelem &
Thomas 2001). Samples were filtered on GF/F filters prior to extraction in cold 90% acetone. Pigment
concentrations were summed into functional groups, Total Chlorophyll, Photoprotective Carotenoids, Photosynthetic
Carotenoids, and Total Photosynthetic Pigments (defined in Table 1), and standardized to the mean surface area of
the triplicate branches from which the polyps came (ca. 12.5 cm
2
). Pigment types were analzyed, and compared
between Muricea species with 2-sample F-tests.
Oxygen Sensors
Amperometric O
2
needle sensors (OX-N, Unisense, tip diameter of 1.1 mm) as well oxygen glass
microelectrodes were used (Revsbech et al. 1980, OX-50, Unisense, tip diameter of 40-60 µm). The response of
these electrodes to increasing oxygen concentrations is linear, thus a 2-point calibration was performed, using the
same water as was used in the experimental aquaria. The seawater in the calibration chambers was either purged
with nitrogen or bubbled with air for the 0 % and 100 % saturation points, respectively. The temperature and the
salinity were recorded, and using a table for oxygen solubility in seawater (García & Gordon 1992, Millero &
Poisson 1981), and specifically the table published by the sensor manufacturer (Unisense,
http://www.unisense.com/files/PDF/Manualer/Oxygen%20Sensor%20Manual.pdf), the corresponding concentration
of oxygen in µM was looked up for the 100% saturation. Signals were amplified and converted to millivolts using a
picoammeter (PA2000 Unisense). Signals were transferred to a computer via an A/D converter and recorded using
the software Profix.
Diffusive Boundary Layer Measurements
Prior to the light-dark experiments, the diffusive boundary layer thickness was defined. Test branches (ca.
0.4 cm diameter x 5-7 cm length) were placed in a 10 L aquarium containing 0.2 µm filtered seawater. A glass
pipette blowing air across the water surface stirred the water column. The microsensor was attached to a motorized
micromanipulator that was also connected to the computer via an RS232 interface, thus allowing vertical
movements of the sensor using the software Profix. Data recording of the microsensor pA signal together with the
positioning of the sensor allows the immediate visualization of a vertical oxygen profile. The microsensor tip
remained submerged during the entire experiment. To measure the diffusive boundary layer, the branch of the
gorgonian was left in the aquarium under light conditions similar to the daylight ambient light intensity (measured
in-situ, ca. 90 µmol photons m
-2
s
-1
) until a polyp was fully extended and visibly still, as observed using a mounted
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CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

dissecting scope. Oxygen concentration was measured for 5s every 50 µm beginning 1500 µm above the polyp
surface and completing at the polyp surface. Oxygen concentrations profiles were measure to determine the DBL for
four polyps of M. californica and three polyps of M. fruticosa. . The upper boundary of the diffusive boundary layer
was determined as the distance where oxygen concentration differed from the oxygen concentration values in the
overlying sea water by > 10% (Jorgensen & Marais 1990).
Light/Dark Experimental Procedures
Prior to experiments, gorgonian skeleton and tissue were removed from the bottom 0.5 cm of a branch,
closest to the end that was cut during sample collection, to expose the gorgonin axial skeleton. The exposed tip was
secured with a branch holder, designed for this experiment, which could be attached inside of a plexiglass chamber
submerged in a 20 L aquarium. The aquarium was filled with 0.2 µm filtered seawater and was wrapped with
aluminum foil to keep the seawater in the dark and to reduce temperature fluctuations. Sample handling was
minimized as much as possible by using forceps, and samples remained submerged. A silicone-filled hole in the
chamber, located directly above the secured branch, allowed a needle sensor to be inserted and held in place relative
to the coral polyp subject. The tip of the needle sensor was carefully placed within the DBL of a single, open polyp,
as evidence by a decrease in stark decrease in oxygen compared to the overlying seawater (Figure 1). Data recording
commenced immediately after correct placement of the microsensor was confirmed. Oxygen concentration was
recorded every 10 s.
The entire setup, including the 20 L aquarium and the arms of the light source (Olympus LG-PS2-5 with a
12V 100W halogen lamp), was then carefully covered with 2 layers of black plastic to block light exposure. The
gorgonian branch was allowed to acclimate in the dark (ca. 0.5 µmol photons m
-2
s
-1
) for 1 h. The system was then
exposed to a series of step-wise, manual increases in illumination from 5-550 µmol photons m
-2
s
-1
). Advancement
to the next light step occurred after oxygen concentration plateaued in response to the previous step, which usually
occurred within 1-2 min. After exposure to the maximum light intensity (550 µmol photons m
-2
s
-1
), the light was
turned off and another dark incubation took place for 30 min. Oxygen concentration was also monitored in the
ambient seawater as a control.
Data analysis
The mean of the 6 data points from the second minute following each step-wise increase in illumination
was computed and represents the mean oxygen concentration during exposure for that sample. A Student’s T-test
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CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

was used to compare the mean oxygen concentrations in the dark to those at each light interval for each species, as
well to compare the mean oxygen concentrations between M. californica and M. fruticosa at the light levels that
produced a significant response in both species compared to darkness. To better visualize the difference in
responses, the difference in mean oxygen concentrations between each irradiance level and the dark was calculated
for every data point and averaged. For the dark measurements following the light experiment, the data from the 2
nd
,
12
th
, and 22
nd
, minutes in darkness were each averaged and plotted.
Quantification of pigmented cells from coral polyps
Counts of the pigmented cells from Muricea were collected on two separate dates to confirm the trend: 1)
polyps from branches tested in the light dark experiments (Aug 2013, ca. 100 polyps counted per species), and 2)
Nov 2014 (ca. 50 polyps counted per species). Colony branches were rinsed well with 0.2 µm-filtered seawater and
individual polyps were plucked with forceps immediately before counting. Polyps were placed in 20 µL 0.2 µm-
filtered seawater and homogenized via vigorous pipetting until no tissue could be observed. Pigmented cells were
counted in duplicate using a Neubauer hemocytometer and compared with a 2-sample t-test.

RESULTS
Photosynthetic pigments were measured in both species of Muricea. The majority of these pigments were
chlorophylls and photosynthetic carotenoids (11-14 µg cm
-2
in M. californica, 2-3 µg cm
-2
in M. fruticosa) (Figure
2), though photoprotective carotenoids were observed, as well (8-9 and 1-2 µg cm
-2
, respectively). Significantly
more photosynthetic pigments were observed in M. californica (t
PC
=3.98, df=8, p=0.001; t
PP
=2.85, p<0.01; t
TP
=2.75,
p<0.01).
At low flow velocity, and ambient light corresponding to field conditions, the diffusive boundary layer
thickness was approximately 0.7 mm for polyps M. fruticosa, and 0.5 mm for M. californica (Figure 3). Oxygen
concentration was greater at the surface of M. californica polyps than M. fruticosa.
Oxygen measurements for each sampled look similar to the experiment in (Figure 1). A sharp decrease in
oxygen was initially observed ensuring correct placement of the needle sensor, followed by a steady decrease during
the 1 h incubation period. At the onset of illumination, oxygen concentrations increase.
Oxygen production was not detected in filtered seawater alone. For both species of Muricea, mean oxygen
concentrations ranged from 20-70 µM at the surface of the gorgonian polyps in the dark (0.5 µmol photons m
-2
s
-1
),
56
CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

through irradiance levels ca. 80 µmol photons m
-2
s
-1
(Figure 4). At irradiance levels >150 µmol photons m
-2
s
-1
,
increases in mean oxygen concentrations at M. californica polyp surfaces were observed (80-100 µM oxygen),
equating to 30-70 µM increases in oxygen concentrations compared to the dark (Figure 5). Mean oxygen
concentrations at the polyp surfaces of M. fruticosa were not greater than in the dark until irradiance levels were >
230 µmol photons m
-2
s
-1
, where 10-20 µM increases were observed. The mean oxygen concentrations at M.
californica polyp surfaces were greater than M. fruticosa at irradiance levels of 130-420 µmol photons m
-2
s
-1
.
Following the light exposures, oxygen concentrations quickly decreased when samples were returned to the dark.
Quantification of the pigmented cells yielded different per polyp values over two seasons. The cell counts
from Aug 13 indicated M. californica polyps each contain ca. 3.7 x 10
4
cells, and ca. 2 x 10
4
cells per M. fruticosa
polyp. Considerably fewer pigmented cells per polyp were observed in Nov 2014. Comparison of the cell counts in
each year confirmed that Mc consistently maintained more pigmented cells than Mf (Figure 6), t
2013
=30.08, df=213,
p<0.0001; t
2014
=15.262, df=90, p<0.0001).

DISCUSSION
Southern California species of Muricea are capable of photosynthesis. The HPLC analyses of
photosynthetic pigments indicated that both chlorophyll and accessory photosynthetic carotenoids were present in
the polyps of both species of Muricea, and M. californica polyps contain more photosynthetic carotenoids than M.
fruticosa. Photosynthetic carotenoids are accessory light-harvesting pigments, that transfer energy to the
photosynthetic electron transport chain via chl a (reviewed by Mulders et al. 2014). Details regarding specific
carotenoids and taxonomic inferences can be found in Ch. 1. The obtained values of chlorophyll from this study, 1-4
µg cm
-2
,

are below the range of 5-30 µg chl a cm
-2
measured in both scleractinian (Ferrier-Pagès et al. 2003, Apprill
et al. 2007, Rodolfo-Metalpa et al. 2008, Dove et al. 2008) and gorgonian corals (Ferrier-Pagès et al. 2009).
Chlorophyll content of polyps increases with coral nutrition (Titlyanov, Titlyanova, Yamazato, & van Woesik
2001a) and darkening light regimes (shade, depth) (Titlyanov, Titlyanova, Yamazato, & van Woesik 2001b), while
it decreases with water temperature (Warner et al. 1996, 1999, Ferrier-Pagès et al. 2009). Yet, it also varies
seasonally (Fitt et al. 2000) and within a colony based on whether zooxanthellae are located in upper or lower-facing
surfaces (Jones et al. 1998). The presence of chl a observed in Muricea polyps confirms the photosynthetic potential
57
CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

of the pigmented cells, but further studies are needed to explain the low levels observed and how this might translate
into efficiency of the algal photosynthetic machinery.
To experimentally confirm the photosynthetic capabilities of Muricea polyps, the thickness of the diffusive
boundary layer was first determined at a light intensity similar to in-situ conditions. Both species of Muricea
maintained similarly thick DBLs of ca 0.6 mm. Shashar et al. (1993) measured the thickness of DBL’s of four
massive coral species and one branching coral, Styolophora, and determined that DBL thickness increased with
increasing calyx sizes (the skeletal area directly below a polyp) in the massive (non-branching) corals. The
Styolophora DBL was 3.09 mm in the dark with minimal water flow, but was reduced significantly at high flow
velocity (5 cm/s) to about 1.8 mm. Muricea polyp DBL’s were considerably smaller than the Styolophora DBL
under low-flow conditions, though calcyx sizes are comparable. These differences may be due to gorgonian colony
morphology, which is much different than even scleractinian branching colonies. Gorgonian octocorals secrete their
calcium carbonate sclerites external to the tissue which overlays the gorgonin (protein) axial skeleton. This produces
thin branches (our samples were ca. 0.4 mm in branch diameter), and large, bush-like colonies, which slow water
flow for filter feeding (Wainwright & Dillon 1969). Alternatively, scleractinian corals grow tissue overtop of the
large, calcium carbonate skeletons that are the foundation of coral reefs. This difference in colony topography
probably impacts the DBL surrounding any given gorgonian polyp. To my knowledge, there are no other reports of
the DBL’s of gorgonians or octocorals.
Photosynthesis was detected in M. californica and M. fruticosa. Oxygen at polyp surfaces increased with
irradiance, and greater concentrations of oxygen were measured from M. californica samples. Oxygen
concentrations never reached super-saturating concentrations, which is observed in other coral species at irradiances
>100 µE m
-2
s
-1
(Kühl et al. 1995), however these experiments were performed with a different methodology that
placed the micro sensors at the source of photosynthesis: the endozoic algae. This was not done in our experiments
because the coral behavior of polyp tissue contraction and complete coverage by sclerites made these types of
measurements impossible. Therefore, it is likely that produced oxygen was rapidly used inside of the polyp by the
algae or coral for respiration and thus unavailable for measurement by the micro sensors at the polyp surface.
Measuring gross photosynthetic rates of the isolated algal cells and composing a PI curve could further validate
these data, as could the use of pulse-amplitude-modulated fluorometry (Schreiber 2004), where the effective
quantum yield of Photosystem II and actual rates of electron transport can be detected. These measurements taken
58
CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

together will undoubtedly produce interesting insights regarding the nutrition modes (autotrophy versus
heterotrophy) of a temperate gorgonian from a high-nutrient environment. Gut content analyses and feeding
experiments in M. californica and M. fruticosa, concluded that only 1% of the species’ metabolic needs were met by
filter feeding of larval bivalves (Grigg 1970). While DOM and phytoplankton may significantly contribute to
fulfilling this deficit, it leaves open the possibility that autotrophy via endozoic algae may as well.
Observing photosynthesis from M. fruticosa was especially surprising, because these species are known for
their white, and presumably azooxanthellate, polyps. This prompted the need for further investigation via cell
counts. M. fruticosa also contained cells capable of photosynthesis, but the density of cells was small enough
(almost 2-fold less than in M. californica) that the polyp color is not noticeably altered from white. The difference in
cell counts between Jul and Nov may be a seasonal or depth-dependent variation, but a specific study examining this
trend in Muricea is required. Fitt (2000) examined stony coral zooxanthellae densities from the Bahamas over 4
years and observed increased densities in the winter months, and 2-6 fold changes in densities between seasons,
helping to explain the observed patterns in this study (Figure 6). Thus, the stark differences observed in Muricea
polyp cell densities are reasonable.
This is the first study to conclude that Muricea californica and M. fruticosa photosynthesize, and my data
suggests that a higher density of the endozoic algae produces more oxygen in the presence of light. However, we
note that high variability exists in these systems, and such correlations must be confirmed through more studies. It
should also be noted that the identity of these pigmented cells is still unconfirmed (but see Ch. 1), and caution must
be taken when making any comparisons between these cells and dinoflagellate zooxanthellae found in symbiotic
corals.

ACKNOWLEDGMENTS
We would like to thank Christopher Suffridge, Sarah Hu, and Helena Janulis for aid in sample collection,
and Drs. David Caron and Kenneth Sebens, and Meg Maddox for help with data interpretations. Kellie Spafford and
Lauren Czarnecki were also integral to completing these experiments. Funding was provided by the USC-Wrigley
Summer Graduate Fellowship.

59
CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

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CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

Table 1. Definitions of the summary groups representing photosynthetic pigments.
Variable Pigment Sums
Total Chlorophyll [Chl a] + [Chl b] + [Chl c]
Photoprotective
Carotenoids
[Alloxanthin] + [Diadinoxanthin] + [Diatoxanthin] + [Zeaxanthin] + [β-Carotene]
Photosynthetic
Carotenoids
[19'-Butanoyloxyfucoxanthin] + [Fucoxanthin] + [19'-Hexanoyloxyfucoxanthin] +
[Peridinin]
Total Photosynthetic
Pigments
Total Chlorophyll + Photosynthetic Carotenoids
62
CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

Figure 1. Example trace of real-time oxygen measurements of M. californica polyp surfaces (gold) in response to
increasing irradiance (blue) over time.

63
CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

Figure 2. Photosynthesis-related pigment mean concentrations in M. californica (gold) and M. fruticosa (red) ± SE.
See Table 1 for definitions of each pigment category.
64
CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

Figure 3. Mean O
2
diffusive boundary layers ± SE, beginning at 1500 µm above the surface of polyps (0) from M.
californica (gold) and M. fruticosa (red) in the presence of 90 µmol photons m
-2
s
-1
.
65
CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

Figure 4. Mean O
2
concentrations ± SE with increasing levels of irradiance at the surface of M. californica (gold),
M. fruticosa (red), and in 0.2 µm filtered sea water (blue).
66
CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

Figure 5. Difference in mean O
2
concentrations at indicated irradiance from O
2
concentrations in the dark.
Gold: M. californica, Red: M. fruticosa.
67
CH.3: PHOTOSYN. MEASUR. OF THE TEMP. GORG. OCTOC. MURICEA

Figure 6. Mean pigmented cell densities ± SE as measured via hemacytometer for M. californica (dark gray) and M.
fruticosa (light gray).
68
MICROB. OF GORG. OCTOC. MURICEA, WITH A DESCR. OF A NOV. PHOTOSYN. PROTIST.
SYMBIONT

CONCLUSION
Here, a non-dinoflagellate algae endozoic in coral polyps is described for the first time. Found in the polyps
of eastern Pacific species of Muricea, the algae are 5-15 µm in diameter, are surrounded by two membranes
in-hospite, and contain a matrix of chloroplasts enveloped with a matrix of green-autofluorescent pigment.
The major accessory pigment in these organisms is 19’hexanoylfucoxanthin, and the chloroplasts of these
cells have two membranes, which suggests a primary endosymbiosis event as is seen in ancestral algal
lineages Chlorophyta, Rhodophyta, and Glaucophyta. Preliminary genetic marker analyses showed the
presence of a Rhodophyta plastid gene, but the genetic identity of the cells is as yet unresolved. The cells
are also photosynthetic, as oxygen was measured at the surface of polyps, and microscopic analyses have
suggested that no other photosynthetic entities are present at these surfaces. M. fruticosa surprisingly
contained photosynthetic pigments and produced oxygen, though to a lesser extent than M. californica,
which was explained via comparative measurements of cell densities; M. fruticosa polyps contain half as
many of the algal cells as M. californica. The presence of these cells may contribute to the separate and
specific prokaryotic communities associated with M. californica and M. fruticosa. Major community
constituents included Mycoplasma and Spirochaetes strains, in addition to α- and γ- Proteobacteria and
uncharacterized sequences. M. fruticosa hosted more overall prokaryotic diversity and the associated strain
of Mycoplasma was related to one detected from bleached colonies of Muricea elongata, a sister species
from the Gulf of Mexico. These data along with the observation of a colony of M. californica exuding their
pigmented cells, and maintaining both white and golden polyps is suggestive that M. californica and M.
fruticosa are not separate species, but rather morphotypes of the same species.
69

Abstract (if available)

Abstract Coral microbial interactions have been studied for some time, primarily in response to anthropogenic climate change and its impacts on tropical reefs. These studies focused on hermatypic, scleractinian species (reef-building, hard corals), as coral reefs support the greatest amount of biodiversity found in one ecosystem. Thus, temperate soft corals, such as gorgonian octocorals, have historically received far less attention, though species from the Mediterranean Sea have begun to experience bleaching events in response to increased water temperatures, and this has sparked examinations into gorgonian microbiomes. ❧ More recently, gorgonian octocorals have been named as a top source of marine natural products. In some cases, a specifically associating microbe synthesizes these natural products, rather than the gorgonian octocoral. Eastern Pacific species of the gorgonian octocoral Muricea have been regarded as azooxanthellate, and this was the extent of explorations into their microbiomes. They are abundant along the southern California coasts, as important contributors of benthic heterogeneity, and considered ecological indicators of diversity within kelp forests. This study is the first to examine the microbiome of Muricea californica and M. fruticosa, two species with overlapping geographical ranges, but distinct morphological characters, primarily, polyp color. M. californica maintains orange-gold polyps while M. fruticosa polyps are white. The source of this differentiation is pigmented cells, reported and described here for the first time by in-depth pigment and microanalyses. These cells are eukaryotic, contain chlorophyll and the accessory carotenoid 19-hexanoyloxyfucoxanthin, and 2 membranes surround the chloroplasts, suggesting primary endosymbiosis as is seen in the Chlorophyta, Glaucophyta, and Rhododphyta algae. ❧ This is the first report of a coral associated with a non-dinoflagellate, endozoic algae. We show here that each species of Muricea maintains a specific bacterial community as well, mainly composed of separate strains of Mycoplasma, and Spirochaetes, and an enormous amount of novel bacterial diversity. The bacterial microbiomes are also representative of photosymbiont-containing versus bleached corals, suggesting an impact of the pigmented cells. Finally, the first measurements of photosynthesis and the oxygen diffusive boundary layer at the Muricea polyp surfaces are reported. M. californica produced more oxygen in the presence of light > 150 μmol photons m⁻² s⁻¹ than M. fruticosa, but oxygen production was observed in these species as well, explained by the observed relative densities of the endozoic, pigmented algae.

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Asset Metadata

Creator Holm, Johanna B. (author)

Core Title The microbiome of gorgonian octocorals, Muricea, with a description of a novel, photosynthetic protistan symbiont

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Marine and Environmental Biology

Publication Date 07/30/2015

Defense Date 05/12/2015

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag algae,Ecology,endosymbiosis,gorgonians,microbiology,microbiome,OAI-PMH Harvest,octocorals,photosynthesis,symbiosis

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Advisor Caron, David A. (committee chair), Bottjer, David J. (committee member), Heidelberg, Karla B. (committee member), Ziebis, Wiebke (committee member)

Creator Email holmjb@gmail.com,jholm@usc.edu

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