Marine Biology
© Springer-Verlag 2005
DOI 10.1007/s00227-005-1620-y
Research Article
Genetic and color morph differentiation in the
Caribbean sea anemone Condylactis gigantea
Nina Stoletzki (✉) · Bernd Schierwater
N. Stoletzki · B. Schierwater
ITZ, Ecology and Evolution, Tieraerztliche Hochschule Hannover, Buenteweg 17d, 30559
Hannover, Germany
N. Stoletzki
Present address: Ludwig-Maximilian Universitaet, Grosshaderner Str. 2, 82152
Planegg-Martinsried, Germany
✉ N. Stoletzki
Phone: +49-89-218074234
Fax: +49-89-218074104
E-mail: [email protected]
Received: 1 October 2004 / Accepted: 13 March 2005
Abstract The distribution of phenotypic and genetic variation across environments can provide
insights into local adaptation. The tropical sea anemone Condylactis gigantea inhabits a broad
spectrum of coral-reef habitats and displays a variety of phenotypes, particularly with respect to
color. At the coast of Discovery Bay, Jamaica, individuals with either pink or green tentacle tips
show distinct distributions. Pink morphs are more abundant in the lagoon and in deeper areas,
while green morphs are more abundant in the forereef and in shallower areas. We use DNA
sequence data (ITS1-5.8S) to investigate if variation in color is associated with genetic
differentiation in lagoon and forereef habitats about 5 km apart. Population genetic analyses reveal
two distinct ITS1-5.8S variants, which differ in relative frequency. The two variants are present
in both habitats, but a dearth of intermediates suggests reduced gene flow. In the lagoon, but not
the forereef, ITS variants show an association with color. In order to address the potential ecological
significance of color, we study UV absorbance and UV acclimatization capacities of pink and
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green color morphs in the lagoon. Color morphs differed significantly in UV-B absorbance. These
results suggest genetic and ecological differentiation in the face of gene flow over short distances.
Communicated by O. Kinne, Oldendorf/Luhe
Introduction
Gene flow and natural selection both influence the degree of population differentiation in nature
(Endler 1977; Slatkin 1973). Natural selection can drive differentiation even in the face of gene
flow (Schluter 2000). In the sea, few obvious extrinsic barriers to gene flow exist and many marine
organisms are characterized by high dispersal capabilities and wide distribution ranges (Bohonak
1999; Palumbi 1994). Not surprisingly, marine organisms with high dispersal often show little or
no genetic substructuring between populations separated by up to hundreds of kilometers (reviewed
in Palumbi 1992). There are, however, a growing number of counterexamples (e.g. Ayre and
Hughes 2000; Benzie 1994; Carlon and Budd 2002; Williams and Benzie 1998).
Coral reefs are heterogeneous environments with spatially varying selection regimes that offer
many possibilities for the evolution of niche-specific adaptations. Coral-reef anthozoans are
typically sessile and show considerable phenotypic variation in response to environmental factors
like light. Such phenotypic variation across different reef environments might be due to genetic
polymorphism or phenotypic plasticity (see, for example, Bruno and Edmunds 1997).
Light is one of the most important spatially varying factors in coral reefs (Brown 1997), especially
for the coral-reef anthozoans that rely on photosynthetic endosymbionts. The amount and proportion
of transmitted sunlight in water depends mainly on depth and turbidity. Ecological traits, like color
and UV protection, apparently differ with radiation level and might enable the organism to adapt
to the environment.
Congruent depth distributions of color phenotypes in distantly related coral-reef
anthozoans—green in shallower, lighter areas and pink in deeper, darker ones—suggest that color
is direct or indirectly important with respect to radiation (Gleason 1993; Takabayashi and
Hoegh-Guldberg 1995). The direct ecological significance, however, remains unclear. The striking
colors found in coral-reef anthozoans are host-specific, overlaying the golden-brown color of their
endosymbionts (Dove et al. 2001). Molecular analysis of the endosymbionts of Condylactis gigantea
revealed high diversity, but no correlation of symbiont type with host color (N. Stoletzki and B.
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Schierwater, unpublished work). To facilitate photosynthesis of their endosymbionts, anthozoan
endodermal tissue must be translucent. At the same time, however, translucent endoderm provides
little protection against harmful UV radiation, which is particularly high in the tropics (Whitehead
et al. 2000). Some sort of protection is necessary. UV-absorbing mycosporine-like amino acids
(MAAs) offer one potential form of protection. MAAs are present in a variety of aquatic organisms,
including anthozoans. Content and composition of MAAs often differ with depth (Dunlap and
Shick 1998; Shick and Dunlap 2002).
The Caribbean sea anemone C. gigantea shows a distinct color distribution at Discovery Bay,
Jamaica. Although a variety of color morphs are found at all sites and depths, as in other anthozoans,
green morphs dominate in clear and bright forereef water, while pink morphs dominate in turbid
and less bright lagoon water. This depth distribution suggests that the two color morphs of C.
gigantea are adapted to different radiation levels. Here, we test if the two color morphs of C.
gigantea: (1) correspond to distinct genetic variants, and (2) differ in their UV absorption. We
show that an association exists between color and genetic variants in the lagoon, but not in the
forereef habitat. We also report an association of differently colored genetic variants with UV
absorption in the lagoon.
Materials and methods
Study organism
C. gigantea is found at depths ranging from 1 to 30 m, and inhabits a variety of different habitats
from lagoon to forereef (Humann 1992). Like corals, it hosts endosymbiotic dinoflagellates of the
genus Symbiodinium (Banaszak et al. 1993). Polyp longevity and planktonic long-distance dispersal
are important life-cycle features of sea anemones (Hughes 1989). For C. gigantea, only sexual
reproduction has been reported (Shick 1991).
We conducted fieldwork at the Discovery Bay Marine Laboratory (DBML) on the north coast
of Jamaica. From January to March 1998, we surveyed two forereef, one backreef and one lagoon
site in a depth range of 1–18 m. We laid transect lines haphazardly parallel to the shore line over
a total length of 1,640 m. We found a total of 320 C. gigantea individuals within 1 m at either side
of the transect, and recorded depth and coloration of the tentacle tips for each of them.
From January to April 2001, we studied C. gigantea in two habitats about 5 km apart: (1) an
exposed forereef site where the water is clear, the wave action is strong, and light levels are high;
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(2) a protected lagoon site, where the water is turbid, the wave action is limited, and light levels
are relatively low (D’Elia et al. 1981; Fleischmann 1989; Gayle and Woodley 1998).
We determined individual anemone color using a color chart and dive light to account for
increasing light absorption with depth. We categorized sea anemones as either pink or green. Pink
morphs were defined by pink tentacle tips, and green ones by the lack of pink color. In addition
to the Jamaican C. gigantea samples, samples were taken from the geographically and
phylogenetically more distant Bermudan C. gigantea and from Mediterranean C. aurantiaca
(Giglio, Italy).
Genetic variation
At a depth of 15 m, we sampled at least ten representatives of each color morph from both the
lagoon and forereef sites for genetic analyses. We removed three to five tentacle tips (5 cm each)
per sea anemone with scissors and kept them in seawater until transference within 1 h into HOM
buffer (100 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 100 nM NaCl pH 8.0, 0.5%SDS, 50 mM
DTT). We extracted DNA following Sambrook et al. (1989). Storage until and after extraction
was at −20°C.
For molecular analyses, we sequenced the ribosomal ITS1–5.8S-ITS2 region, as it provides
high resolution at and below the species level in a variety of plant and animal species (Vogler and
DeSalle 1994), including anthozoans (Odorico and Miller 1997; Van Oppen et al. 2000, 2002a,
2002b). PCR reactions of 50 µl contained 5–50 ng of DNA template, 1×PCR buffer (Gibco BRL),
2 mM MgCl2, 5 pM of each primer, 0.1 mM total dNTP (Roth), and 0.5 units Taq Polymerase
(Gibco BRL). Primer sequences were 18SUniv.fw
5′-GGTTTCCGTAGGTGAACCTGCGGAAGGATC-3′, 28SAct.rev.
5′-GTTCCCGCTTCATTCGCCATTAC-3′ (designed for this study). PCR conditions were: 93°C
for 5 min+35× (92°C for 30 s, 60°C for 1 min, 72°C for 1 min)+72°C for 6 min (Perkin Elmer
Thermal Cycler 9600). PCR-products were cloned prior to sequencing. Direct ligation of PCR
products into a T-Vector (pGEM-T Vector System, Promega) followed standard procedures
(Sambrook et al. 1989). Transformations (in DH5 GIBCO BRL Life Technologies) followed the
manufacturer’s protocols. We re-amplified inserts from two to five positive colonies per sample
by Bac-PCR. For colony picking, we used a sterile disposable pipette tip and rinsed it in 50 µl
distilled water. After heating to 99°C for 10 min, we amplified the Bac-PCR with T7 and SP6
vector primers (Promega): 93°C for 3 min+35× (92°C for 30 s, 51°C for 30 s, 72°C for 1 min)+72°C
for 3 min.
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After purification (Geneclean III-kit, Q BIOgene), we cycle-sequenced Bac-PCR products [25×
(96°C for 10 s, 50°/52°C for 5 s*, 60°C for 4 min)] using BigDyeTM Terminator Cycle Sequencing
Ready Reaction Kit (Applied Biosystems). The 20 µl total reaction volume contained 1×Big Dye
Premix, 100 ng DNA template, and 10 pM primer. We purified PCR products with Sephadex
columns (Sigma, following Sambrook et al. 1989) and sequenced (ABI 377) both strands. We
aligned the sequences manually and compared them to GenBank sequences of other anthozoans.
We determined boundaries of the two ITS regions and 5.8S using Genbank sequences of the sea
anemone Heteractis magnifica (AF050211). We analyzed genetic data with Arlequin 2.0 (Schneider
et al. 2000) and PAUP* 4.0 (Swofford 2000). We deposited the most frequent ITS1-5.8S sequence
into GenBank (Accession number AY948135). Other copies can be inferred from Table 1.
[Table 1 will appear here. See end of document.]
UV protection
To compare the UV-absorbing capacities of the 2 color morphs “under natural conditions”, i.e. at
a location where both morphs naturally occur together, we sampled 20 individuals of each color
morph at a depth of 15 m in the lagoon habitat. To test whether the color morphs differ in their
ability to adapt to a change in the light environment, we transplanted sea anemones to different
depths. At a depth of 15 m at the lagoon habitat, we detached 20 pink and 20 green specimens of
medium size (diameter of tentacle crown 15–20 cm) with a spatula from the substrate and transferred
them to the wet laboratory. Here, we arranged sea anemones in labeled plastic cups for
transplantation, and returned them to the lagoon within 24 h. We transplanted ten specimens of
each color morph to 1 m and 18 m depths, respectively, and took samples after 10 weeks.
We lyophilized samples with liquid nitrogen, transported them on dry ice and stored them at
−80°C. For UV-absorbing assays, we followed the protocols for MAAs (see, for example, Hoyer
et al. 2002; Shick et al. 1992). We injected 10- to 40-µl aliquots of solutions in the isocratic
reverse-phase High Performance Liquid Chromatography (HPLC) system (ODS-column, mobile
phase: 0.1% acetic acid+5% methanol in water, flow rate: 0.7 ml/min). We analyzed the
chromatographies spectrophotometrically at 330 nm, using MAA standards (Palithene, Shinorine,
Porphyra 334, Asterina, and Mycosporine-Glycine) for comparisons. We quantified the relative
amount of UV absorption by the area of HPLC chromatogram peak per mg dry weight considering
the injection volume.
For statistical analyses, we grouped the UV-absorbing properties into two subdivisions:
absorption within the range 280–320 nm (UV-B), and absorption within the range 321–340 nm
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(UV-A). We used the nonparametric Mann-Whitney U-test because not all data were normally
distributed. All P-values are two-tailed and corrected for ties (P<0.05).
Results
Genetic variation
All amplified sequences of C. gigantea and C. aurantiaca are 302–307 bp long and lack the entire
ITS2 region. Three nucleotide changes distinguish the multiple C. gigantea sequences from its
mediterranean relative, C. aurantiaca (Table 1). Given this level of inter-specific differentiation,
intra-specific variation within C. gigantea is surprisingly high: we detected two major ITS variants
that also differ by three nucleotide changes (positions 91, 106, 205; Table 1). Based on these
nucleotides, the variants are named “TAC” and “CGT” (Fig. 1). Variation within the TAC variant
consists of ten nucleotide and five indel singletons, while that within the CGT variant consists of
five nucleotide and three indel singletons (Table 1).
Fig. 1 Unrooted genealogy of Condylactis gigantea ITS1-5.8S sequences and one C. aurantiaca ITS1-5.8S
sequence based on MP analyses (heuristic search). Filled circles indicate the frequent copies of the two
variants. Deletions were considered as a fifth character state.
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By sequencing multiple clones from the same individual, we detected intra-individual variation
(Table 2). Many individuals harbor different copies. However, these different copies tend to belong
to one of the two variants. Of 20 individuals, with 2–7 cloned sequences each, only 3 harbor copies
of both ITS variants (Table 2: individuals 3, 4 and 5) and 3 harbor recombinant sequences (Table 2:
individuals 2, 16 and 18). Table 2 also shows the number of individuals that harbor the common
variants TAC or CGT (see Table 1, frequent copies) and the number of individuals that harbor
singletons. We define singletons as those variants that we found once or multiple times only in a
single individual (singleton) or in several individuals (dominant singleton). Sixteen individuals
carry the TAC variant: 10 of them share the common TAC variant, and 11 harbor singletons. Ten
individuals carry the CGT variant: seven of them share the common CGT variant, and seven harbor
unique CGT singletons (Table 2).
[Table 2 will appear here. See end of document.]
We detected: (1) significant genetic structuring between the lagoon and forereef populations
(FST=0.143, P=0.0120); (2) genetic divergence among color morphs combining both habitats
(FST=0.110, P=0.013). Considering both populations separately reveals the following observation:
at the forereef, we found no association between color and ITS variant (FST=−0.030; G=0.011,
P=0.999). Both ITS variants occur in pink and green morphs with similar probabilities. In the
lagoon, however, we found a strong association between color and ITS variant (FST=0.27617,
P=0.00293; G=10.447, P=0.006). Approximately 85% (12/14) of TAC variant-harboring individuals
were pink, while approximately 75% (9/12) of CGT variant-harboring individuals were green
(Table 3).
[Table 3 will appear here. See end of document.]
To test whether the relative frequencies of the two ITS variants differ in the two habitats, we
had to account for the color-biased sampling strategy in which we picked equal numbers of both
color morphs. For the frequency of the genetic variants per habitat, we multiplied the color morph
proportion in the habitat (found in the previous survey, Table 3) with the proportion of the genetic
variant in the respective color morph in this habitat (found by genetic analyses, Table 3): e.g.
freq.TAC=prop.pink*prop.TACpink+prop.green*prop.TACgreen. The relative frequencies of the variants
differed in both habitats: in the lagoon, TAC (~60%) dominates CGT (~40%). In the forereef,
TAC (~77%) dominated CGT (~23%) even more. Both variants were present in the Bermudan
samples.
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UV absorption
Crude methanolic extracts of all C. gigantea isolates showed broad absorption in the UV region.
The absorption maxima ranged from 310 to 340 nm, suggesting a complex mixture of metabolites,
likely MAAs. We could not distinguish particular MAAs by comparison to the standards. Varying
absorption among individuals indicates differences in both the quality and quantity of UV-absorbing
substances. Only 4 out of 20 polyps at 15 m and 6 out of 13 polyps at 18 m showed UV-A
absorption.
We compared the relative absorbance of the color morphs under different conditions (Table 4).
We first tested for color morph differences in UV absorption in the lagoon habitat, at a depth of
15 m, where we found the sea anemones naturally co-occur. Green morphs absorb significantly
less UV-B (P=0.0189), and more UV-A (not significant, P=0.2955) than pink morphs. We then
tested acclimatization capacities by transplanting anemones to 1-m and 18-m depths. Ten weeks
after transplantation, both color morphs transplanted to 1 m showed significantly higher absorption
for both UV-A and UV-B radiation, compared to those transplanted to 18 m depth (UV-A pink:
P=0.0543, green: P=0.0250, UV-B pink: P=0.0126, green P=0.0094). Also, we found that the two
color morphs differed in acclimatization with respect to their relative UV-A and UV-B absorption.
After transplantation to 1 m, green morphs absorb more UV-B (50%) and less UV-A (33%) than
pink morphs, while at a depth of 15 m, it is reversed. At 18 m depth, the two color morphs show
similar low absorption of UV-A and UV-B radiation.
[Table 4 will appear here. See end of document.]
Discussion
We found two distinct ITS1-5.8S rDNA variants in C. gigantea. The divergence between them is
striking: they are as different from each other as they are from a different, related Mediterranean
species, C. aurantiaca. We also found some evidence for reduced gene flow and ecological
differentiation among the variants, and for a strong habitat effect on the association between ITS
variants and color.
The degree of intra-specific variation within C. gigantea ITS regions is comparable to the degree
of variability found by others for this marker (e.g. Vogler and DeSalle 1994). Our results suggest
reduced gene flow between ITS variants. If mating was random, we would expect to find copies
of both TAC and CGT variants within individuals of C. gigantea, present as recombinant rDNA
arrays or as heterozygotes. Although we detected some variation within individuals, copies generally
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belonged to 1 of the 2 variants: 17/20 individuals for which 2 or more sequences were sampled
harbored copies of only 1 variant. In addition, we find few recombinant sequences. This apparent
paucity of heterozygotes and recombinants could be caused by: (1) rapid concerted evolution, or
(2) assortative mating. However, concerted evolution was apparently too slow to homogenize all
recombination and mutation events. Several factors can slow concerted evolution (see Baldwin et
al. 1995; Liston et al. 1996; Nagylaki and Petes 1982; Nabeyama et al. 2000; Quijada et al. 1997).
For example, concerted evolution between ITS variants should be slower than within ITS variants.
As little as 1% sequence divergence—as is seen between the two variants of C. gigantea—can
slow concerted evolution of a multigene array (Modrich and Lahue 1996). Slow concerted evolution
should result in a higher proportion of heterozygotes and recombinants than found in our samples.
Nevertheless, we detected few. More likely, reduced gene flow between the two variants of C.
gigantea is the reason for the paucity of heterozygotes and recombinants. The apparent low level
of gene flow is surprising given that the two variants have overlapping distributions and that gene
flow within each variant occurs over large geographic ranges, as indicated by identical variants
from Jamaica and the Bermudas. Since no obvious extrinsic barriers to gene flow exist, it would
be interesting to test for plausible intrinsic barriers, e.g. synchronization of reproduction (Richmond
and Jokiel 1984) or gamete recognition patterns (Palumbi 1994, 1998) that would allow genetically
isolated populations to co-exist in sympatry.
Our data also suggest ecological differentiation in color and UV protection. In the lagoon, the
two ITS variants of C. gigantea are associated with different colors. Despite recent studies on
color polymorphisms in coral-reef animals (Dove et al. 2001, 1995; Gleason 1993, 1998; Gurskaya
et al. 2001; Kelmanson and Matz 2003; Matz et al. 1999; Takabayashi and Hoegh-Guldberg 1995;
Wiedenmann 2000), the adaptive significance of color remains unknown. Biochemical assays in
Acroporid and Pocilloporid corals show that pigments do not function as photoprotectants,
fluorescent coupling agents or UV-screens (Dove et al. 1995). Nevertheless, the distinct
(non-random) color distributions of C. gigantea and other anthozoans, with pink in darker/deeper
areas and green in brighter/shallower ones, suggest that color may be of adaptive significance.
Color has been linked to physiological performance, including UV protection. Gleason (1993),
for example, found green morphs of the coral Porites asteroides to harbor higher MAA
concentrations than brown morphs. Similarly, in our lagoon study, UV-B protection of C. gigantea
color morphs differed significantly, suggesting underlying differences in MAAs.
We detected a strong habitat effect on the two variants and their association with color. In the
lagoon, the two genetic variants are associated with different colors. In the forereef, however, the
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two variants are not associated with different colors. If color is a phenotypically plastic trait, the
difference between habitats could suggest different reaction norms of the two variants. Although
no color change was observed in our transplantation experiments, color may be set by irreversible
responses to environmental cues earlier in development, as indicated in the coral Montastrea
cavernosa (Kelmanson and Matz 2003). If, however, color is determined by a genetic polymorphism,
the habitat difference has to be explained by sea anemones (with certain combinations of ITS
variants and color alleles) that settled in the lagoon—by drift or selection—and subsequently came
to dominate this habitat.
Besides environmental factors, demographic processes affect the habitats differently. The
potential for local adaptation appears higher in the lagoon than on the forereef. On the forereef,
exposure to stronger currents and wave action could increase the rate of long-range dispersal and
recruitment. In addition, the forereef is not protected from hurricanes (see, e.g., Woodley et al.
1981), and high extinction and re-colonization rates could limit local adaptation, as populations
do not have sufficient time to adapt. In contrast, in the lagoon, protection from stronger currents,
wave action, and hurricane disturbance might facilitate larva retention and, hence, local adaptation.
It would be interesting to collect comparable data from additional forereef and lagoon sites and
to expand the geographic scope of the study.
In marine species that are characterized by potential for high gene flow, one expects little or
low neutral population differentiation. Low genetic divergence between samples of C. gigantea
over large geographic distances indicates gene flow. However, at a smaller scale, we find striking
genetic and ecological differentiation of two distinct C. gigantea variants. No matter how they
originally arose, the two variants with overlapping distributions in the face of gene flow, indicate
past or present non-random mating and/or natural selection.
Acknowledgements Very many thanks are due to Daven Presgraves who continually helped to
improve the manuscript, for advice and many helpful comments and discussions. We are also very
grateful for discussions with Allen Collins, and for many helpful comments of A. Collins, J.
Hermisson and B. Nuernberger on earlier versions of the manuscript, and of two anonymous
reviewers on the final version. Thanks are due to each of the East/West students helping in the
field, the staff members at Discovery Bay Marine Laboratories, Jamaica, and the East/West Marine
Biology Program of Northeastern University, Boston, and all the members of the ITZ. We are also
grateful to Ken Sebens for early discussions. U. Karsten kindly helped with the MAA analyses
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and allowed the use of his facilities. We acknowledge financial support from the German Science
Foundation (DFG Schi-277/10-2) to B. Schierwater and scholarship from DAAD to N. Stoletzki.
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13
14
.
.
Nucleotide ITS1
positions
0
0
8
Variant
TAC
Frequent A
copy
(n=10)
Doubleton .
Singletons .
G
.
.
.
.
.
.
.
.
.
.
.
Recombn
ian
tst .
0
1
5
G
.
.
*
.
.
.
.
.
.
.
.
.
.
.
.
.
.
0
1
2
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
G
0
4
0
changes to C. aurantiaca
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
G
0
4
7
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
G
0
4
8
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
G
0
4
9
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
T
0
5
7
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
0
6
9
C
C
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
0
9
1
.
.
.
.
.
.
T
.
.
.
.
.
.
.
.
.
.
C
0
9
6
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
1
0
6
.
.
.
.
.
.
.
.
.
*
.
.
.
.
.
.
.
A
1
2
3
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
1
2
8
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
.
*
A
1
3
8
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
*
G
1
3
9
.
.
.
.
*
.
.
.
.
.
.
.
.
.
.
.
.
A
1
4
4
5.8S
.
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
T
1
4
6
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
1
5
1
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
.
T
1
6
4
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
.
.
T
1
7
5
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
1
9
3
T
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
2
0
5
.
.
.
.
.
.
.
.
.
.
G
.
.
.
.
.
.
A
2
1
2
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
2
1
4
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
.
.
A
2
2
0
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
2
3
8
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
2
3
9
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
2
5
0
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A
2
5
1
.
.
.
.
.
.
.
*
.
.
.
.
.
.
.
.
.
A
2
5
2
.
.
.
.
.
.
.
.
.
.
.
G
.
.
.
.
.
A
2
9
4
.
.
.
.
.
.
.
.
.
.
.
.
.
T
.
.
.
C
3
0
4
Points represent identical nucleotide positions; asterisks represent indels. Highlighted in bold type are the three nucleotide sites that distinguish the two variants, as well as the nucleotide
Table 1 Table 1 Variable nucleotide positions of the ITS1-5.8S region of C. gigantea. Nucleotides of the most frequent TAC variant found in n=10 individuals serve as a reference.
15
.
Nucleotide ITS1
positions
Variant
CGT
Frequent .
copy
(n=7)
Singletons .
.
.
.
.
.
.
Ca.urantiaca .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
.
.
.
.
.
.
.
.
.
T
.
.
.
.
.
.
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
*
.
.
.
C
C
C
C
C
C
C
.
C
.
.
.
.
.
.
.
.
.
.
.
G
G
G
G
G
G
G
.
G
.
.
.
.
.
.
.
.
.
.
.
.
.
.
*
.
.
.
.
.
.
.
*
.
.
.
.
.
.
.
.
.
*
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
5.8S
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T
T
T
T
T
T
T
.
T
T
.
.
.
.
.
.
.
.
.
.
.
.
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
.
G
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
16
Individual 1
sea
anemone
No.
sequencesni/dvidiual
Common
TAC
variant
Singletons
Dominant
singletons
Recombinants
Common
CGT
variant
Singletons
5
3
1
.
1
.
.
7
4
.
3
.
.
.
2
3
.
.
2
.
.
2
4
4
1
.
2
1
.
.
4
5
.
.
2
.
2
.
4
6
.
.
.
.
2
2
4
7
.
.
.
2
.
2
4
8
.
.
.
.
2
2
4
9
1
.
3
.
.
.
4
10
1
.
2
.
.
.
3
11
1
.
2
.
.
.
3
12
.
.
.
.
.
3
3
13
.
.
.
.
.
3
3
14
.
.
.
3
.
.
3
15
.
.
.
.
2
.
2
16
.
2
.
.
.
.
2
17
.
.
.
.
.
2
2
18
.
1
.
.
.
1
2
19
.
.
2
.
.
.
2
20
2
.
.
.
.
.
2
21
or the recombinant. As opposed to a singleton, a copy found only within one individual, a dominant singleton refers to a copy found in different individuals
.
.
.
1
.
.
1
22
1
.
.
.
.
.
1
23
.
.
.
1
.
.
1
24
1
.
.
.
.
.
1
Table 2 Intra-individual ITS1-5.8S heterogeneity of 24 anemones. Given are the total number of sequenced copies and the frequency of each copy, belonging to variant TAC, GCT
17
Forereef
Lagoon
Habitat
Morph
Pink
Green
Pink
Green
60
29
28
53
67.5
32.5
35
65
Observed numbers and frequency
Color-biased sampling Genetic variants
TAC
15
12
11
2
12
8
11
9
CGT
3
9
2
2
Table 3 Color morph proportion and proportion of genetic variants within each color morph of C. gigantea at lagoon and forereef habitat
77%
23%
Estimated frequency of genetic variants
TAC
CGT
60%
40%
18
Pink
Green
Pink
Green
UV-A
UV-B
Morph
Range
Absorbance
1 m
153 ± 223.92
105 ± 138.98
191 ± 223.92
281 ± 160.47
15 m
4.3 ± 13.5
78.2 ± 149.6
254.5 ± 200.6
96 ± 27.5
Table 4 Relative UV absorbance (mean±SD) of unmanipulated sea anemonmes at 15 m, and transplants to 1 m and 18 m of depth
18 m
2.9 ± 5.04
2.56 ± 5.57
100 ± 49.85
107 ± 20.99