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2021 Coded in Colors: Morphological Differentiation Associated with Color Suggests Further Speciation of virgulata Chandler Michaele Wright

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THE FLORIDA STATE UNIVERSITY

COLLEGE OF ARTS & SCIENCES

CODED IN COLORS: MORPHOLOGICAL DIFFERENTIATION ASSOCIATED WITH

COLOR SUGGESTS FURTHER SPECIATION OF

By

CHANDLER WRIGHT

A Thesis submitted to the Department of Biological Sciences in partial fulfillment of the requirements for graduation with Honors in the Major

Degree Awarded: Spring, 2021

The members of the Defense Committee approve the thesis of Chandler Wright defended on April 12, 2021. Signatures are on file with the Honors Program office.

Dr. Don Levitan

Thesis Director

Dr. Markus Huettel

Outside Committee Member

Dr. Sophie McCoy

Committee Member

.

Abstract Octocorals are an integral taxon, with a significance to benthic communities, by providing multidimensional habitats to marine organisms and now taking over space previously occupied by hard . In the northeastern Gulf of Mexico, we have made observations leading to the question of whether the colors magenta, orange, and yellow of Leptogorgia virgulata represent different . A comparative phenotypic analysis of morphological characteristics was performed to assess whether color varies independently, or color is associated with other morphological differences, suggesting the possibility of the colors representing different species. Thirty colonies of L. virgulata were extracted from a shallow reef (3 m) at Turkey Point Shoal East in the Gulf of Mexico. Ten were yellow (wavelength 570-590 nm), 10 were orange (wavelength 590-620 nm), and 10 were magenta (wavelength 380-450 nm). Samples of L. virgulata colonies were analyzed for six quantitative morphological traits: branch thickness (BT), count (PC), polyp density (PD), calyx length (CL), sclerite length (SL) and sclerite width (SW) along with two qualitative characteristics: sclerite color composition and bicoloration. The yellow, orange, and magenta colors of L. virgulata exhibited statistically significant morphological differences for three of six variables (SW, BT, PD), more strongly between yellow and orange. These differences appear to be meaningful enough to suggest possible speciation between colors that may warrant further investigation.

Introduction In the Northeastern Gulf of Mexico, gorgonian octocorals inhabit shallow nearshore reefs (~2m), off-shore reefs (~15m), and the deep sea (Bayer, 1961; Etnoyer, 2014). They frequently are the dominant taxa of dynamic communities on hard strata and limestone (McFadden et al., 2010). Gorgonian octocorals are colonial cnidarians consisting of a soft structure, internal skeleton, and heterotrophic polyps with eight tentacles (Daly et al., 1668; Lau et.al, 2020). Their structures create a complex environment that provides habitats for many invertebrates, fish, and microbes (Lenz, et al., 2015). Octocorals demonstrate a variety in structural differences and may appear in different forms such as sea whips, sea fans, and sea plumes (Johnson & Hallock, 2020). They are an extremely diverse group that contains over 3,000 species, roughly 200 of which appear in the Gulf of Mexico (Bayer, 1981). The Leptogorgia is known to be extremely challenging to distinguish taxonomically due to the lack of a singular diagnostic characteristic within the genus to determine the species (Breedy & Guzman, 2007). Currently, the genus includes 103 named species, and at least seven considered nomen dubium across the globe (Cordeiro et al., 2020). Leptogorgia is highly morphologically diverse, however differentiating species within this genus based on is challenging because many of the species display a wide variety of intraspecific variation. One study in the Gulf of Mexico calls for a reevaluation of the classification of the species in Leptogorgia entirely. Upon conducting a mitochondrial genome analysis of Leptogorgia in the Eastern Pacific and Western Atlantic, a need for reexamination of current phylogenetic and taxonomic dogma was suggested (Silvestri et al., 2019). In recent years, several new species of Leptogorgia have also been identified. For example, in 2011, three new species of Leptogorgia were discovered in the Gulf of California, Mexico by analyzing morphologic properties including terminal branch diameter, sclerite dimensions, and color composition (Hernandez et al., 2021). Octocorals can exhibit a wide range of colors, including the gorgonian octocoral, Leptogorgia virgulata (Bayer, 1961). Leptogorgia virgulata express a variety of colors including yellow, orange, and magenta (Adams, 1981). L. virgulata thrive on hard substrates in North

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America from the Atlantic Coast, to the northern Gulf of Mexico, and extending south to Belize (Bayer, 1951), however, the geographical distribution of L. virgulata color variants is not uniform and has yet to be explained (Adams, 1981). For example, orange, magenta, and pale yellow-white are seen in Beaufort, NC. Meanwhile, magenta colored L. virgulata dominates reefs in Chesapeake Bay and Alligator Harbor in Florida. Also, in ecosystems between Panama City and Pensacola, only magenta variants have been documented (Patton, 1972). Scientists have noted these differences; however, little research has been done to investigate the cause of this diverse color distribution of L. virgulata. Some proposed reasons for geographic differences in octocoral color include phenotypic plasticity, lack of gene flow, or being different species. Studies on other octocoral species, such as Euincea flexuosa, have revealed the plastic capabilities of the sclerites through reciprocal transplant experiments (Prada et al., 2008). The original opinion on L. virgulata was that the variety is due to phenotypic plasticity or lack of gene flow among populations of the same species (Adams, 1980). However, these alternative hypotheses have not been examined in L. virgulata. There remains a question of whether color is a trait that varies independently or is associated with other morphological differences, possibly delineating species. Moreover, there is also an absence of any notable effort to perform a comparative phenotypic analysis of morphological characteristics between colors. Therefore, there is a need to determine if the color varies independently as simply intraspecific morphotypes or is paired with other morphologic differences. This could suggest different species and encourage perhaps the need for genetic analysis to more confidently delineate whether different colors of L. virgulata are gnomically the same species. In the northeastern Gulf of Mexico, FSU researchers noticed that L. virgulata in the near shore habitats at 3 meters deep are typically a collection of magenta, yellow, and orange colonies. However, on the offshore reefs, magenta-colored colonies are the dominant color present at 15 meters (Rachael Best, personal communication). The deficit of yellow and orange colonies in deep water raises the question if these colors are absent due to a lack of gene flow, plasticity in favor of magenta, or are of different species entirely. Therefore, an analysis of selected morphological characteristics was conducted on inshore colonies of the three different colors to better discern the classification of L. virgulata. If the colored colonies exhibit different morphological differences associated with color, then this could suggest further differentiation of L. virgulata. The goal of the study was to perform a comparative phenotypic analysis to determine whether colors vary independently, or if color is associated with other morphological differences, suggesting the possibility the colors represent different species.

Methods Sample Collection and Analyses Samples of Leptogorgia virgulata colonies were analyzed for six quantitative morphological traits: branch thickness (BT) polyp count (PC), polyp density (PD), calyx length (CL), sclerite length (SL) and sclerite width (SW). Also, two qualitative characteristics: sclerite color composition and bicoloration were observed. Sclerite color composition describes the mixture of solid sclerites in the colony, and the term bicoloration means two colors are observed in a single sclerite.

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Figure 1. Study site: map of Turkey Point Shoal East in relation to the Gulf of Mexico. Image from Google Earth.

With the help of Florida State University scientific diving volunteers, Rachael Best and Ashley Dawdy, 30 colonies of L. virgulata were carefully collected from an in-shore reef named Turkey Point Shoal East (N 29 53.718°, W 84 28.494°) at a depth of three meters. The colonies were brought to the surface in mesh bags and delivered to the FSU Coastal & Marine Lab the same day, November 16th, 2021. Of the 30 colonies, 10 were yellow, 10 were orange, and 10 were magenta. Each colony was placed into separate buckets containing natural seawater in the lab to increase probability of survival during the fragmentation process. Four branches of 10 cm long were severed from the apical tips of each colony, creating four replicates of each genet. Although octocorals are colonial , the colonies were fragmented to increase random sampling and more accurately account for the unknown variability. Using a digital camera, an individual photo of each branch was taken alongside a ruler for scale. Branch thickness (BT) was collected by uploading the photos into Java ImageJ and measuring the diameter at five randomized points. All diameters were measured 3 cm proximal to the apical tips, or growing ends, to avoid irregularity and improve uniformity. The remaining seven characteristics of polyp count (PC), polyp density (PD), calyx length (CL), sclerite length (SL), sclerite width (SW), color composition and sclerite bicoloration required microscopic analysis. A 2 cm branch sample was taken directly below the severed 10 cm sample and placed into labeled centrifuge tubes for preservation and transportation to the lab on FSU main campus. This 2 cm fragment was taken from the severed end of the branches to avoid collecting the smaller, underdeveloped tissues and sclerites, which populate the apical tips of octocorals (Prada, 2007). Polyp density was calculated by counting the number of calyx holes around the circumference of the fragment in a centimeter section, then standardizing for the branch thickness, assuming a cylindrical shape. For all the quantitative microscopic morphologies, except polyp density, 10 measurements were taken from each fragment, meaning totaling 40 per colony and 400 per color. For calyx length (CL), ten randomly selected calyces were measured vertically on fragments with an ocular eyepiece, then standardized with a micrometer. Calyces that exhibited degradation, disruption, or damage from preservation were avoided. Next, Bayer’s (1961) method was followed to observe the sclerites. A small piece of the specimen (approximately 2mm) was placed on a glass slide and a drop of Clorox Bleach (5%) was added. Within a minute, the sclerites separated and distributed

5 freely across the droplet. If needed, a teasing needle was gently used on the sample to facilitate further separation. After applying the coverslip, it was pushed around so the sclerites would occupy one layer, instead of stacking, which would skew measurements. Sclerites were randomly selected for length (SL) and sclerite width (SW) measurements by moving the stereomicroscope stage blindly into a new view. The sclerite located closest to the front of the ocular scale was selected for measurement, and this was repeated until 10 sclerites were analyzed per fragment. Between each fragment, all materials were carefully triple rinsed to prevent sclerites from entering other samples.

Statistical Analyses Two statistical analyses were performed to investigate the accuracy of the taxonomic classification of the color variations within L. virgulata. To analyze whether variation in color morph was associated with other morphological features, independent One-Way ANOVAs were performed on each morphological trait (BT, PC, PD, CL, SL, SW). A Principal Component Analysis (PCA) was used as a multivariate data approach to compile each of the morphologies into one visual representation and examine which traits are influential factors categorizing color variants. For both the ANOVAs and the PCA, all measures were averaged for each of the colonies, therefore N=10.

Results In this section, the characteristics of each of the three colors will be reported first, followed by comparative morphological analysis organized by trait for statistically significant differences, then the supporting principal component analysis.

Characteristics by Color Table 1. Descriptive statistics for three color variants of Leptogorgia virgulata in the Gulf of Mexico. Eight morphological characteristics were measured for each colony color. Maximums and minimums were calculated from the entire pool of data for each colony (N=40). Averages were then calculated with the result of N=10. Solid Sclerite Chromotypes: Y = yellow, W = white, M = magenta, O = orange, “&” = two or more solid colored sclerites present. Bicoloration: “ / ” = both colors exist on a single sclerite. Polyp Density: the standardized number of polyps per square centimeter of the cylinder. Means values are in parenthesis.

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Yellow

A B C

a b c

Figure 2. Yellow Leptogorgia virgulata (wavelength 570-790). Uppercase letters indicate branch; lowercase letters indicate sclerites. Aa: Yellow branch with yellow sclerites (Y). Bb: Yellow branch with yellow and white sclerites (Y&W). Cc: Yellow branch with yellow, white, and magenta sclerites (Y&W&M). Photos not to scale.

Colony Description. Branch thickness ranged from 0.159 to 0.253 cm wide ( = 0.202 SD = 0.0109, SE = 0.003). The mean polyp density was 58 polyps per centimeter squared (min = 42, max = 56, SD = 7.776, SE = 2.459, Table 1, Fig. 1). Length of the calyx varied from 0.033 to 0.967 mm long ( = 0.671, SD = 0.022, SE = 0.007, Fig. 1). Sclerite Morphology. Sclerites in yellow colonies ranged from .10 – 0.26 mm long ( = 0.169, SD = 0.009, SE = 0.003, SV = 0.001) and 0.04-0.09 wide ( = 0.070, SD = 0.002, SV = 4.9113E-05, Fig. 1). Under the microscope, all ten colonies exhibited solid yellow sclerites, varying in intensity. 100% of colonies were bicolored with white (Y/W), and 20% of colonies had magenta bicoloration (Y/M). In these, solid white and solid magenta sclerites were also present (Y&W&M, Fig. 2c). In two colonies, which had many solid white sclerites, a few rouge magenta sclerites were present as well. All colonies that had white bicoloration also had solid white sclerites (Y&W, Fig. 2b). No solid magentas were in a colony unless solid whites were also present.

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Orange

A B C

a b c

Figure 3. Orange Leptogorgia virgulata (wavelength 590-620 nm). Uppercase letters indicate branch; lowercase letters indicate sclerites. Aa: Orange branch with orange sclerites (O). Bb: Orange branch with orange, white, and magenta sclerites (O&W&M). Cc: Orange branch with orange and magenta sclerites (O&M). Photos not to scale.

Colony Description. Branches ranged in thickness from 0.135 to 0.219 cm wide ( = 0.202, SD = 0.0109, SE = 0.003). The mean polyp density per centimeter squared ranged from 92 polyps to 47 polyps, ( = 69, SD = 6.279, SE = 2.459). Calyx length ranged from 0.033 to 0.96 7 mm long ( = 0.671, SD = 0.022, SE = 0.008, Fig. 1). Sclerite Morphology. Sclerite size ranged from .010-0.26mm long ( = 0.171, SD = 0.295) and 0.04-0.09 wide ( = 0.169, SE = 0.0003, SD = 0.007, SV = 4.9113E-05). All colonies, besides two, displayed magenta bicoloration in the orange sclerites (O/M). Furthermore, amongst these eight, three colonies had solid magenta chromotypes as well (O&M, Fig. 3c). The two colonies that did not have magenta coloration were a paler shade of orange than the others and did not have solid white sclerites either. These only displayed faint white bicoloration (O/W). The only colonies which had solid white chromotypes were two colonies that also had solid magenta (O&W&M, Table 1, Fig. 3b).

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Magenta

A B

a b

Figure 4. Magenta Leptogorgia virgulata (wavelength 380 - 450 nm). Uppercase letters indicate branch; lowercase letters indicate sclerites. Aa: Magenta branch with magenta sclerites (M). Bb: Magenta branch with magenta and white sclerites (M&W). Photos not to scale.

Colony Description. Branch thickness ranged in thickness from 0.104 to 0.224 cm wide ( = 0.190, SD = 0.019, SE = 0.006, Fig. 2). The mean polyp density was 59 polyps per centimeter squared (min = 43, max = 93, SD = 12.967, SE = 4.102). Length of the calyx varied from 0.5 to 0.867 mm long ( = 0.667, SD = 0.033, SE = 0.010, Fig. 2). Sclerite Morphology. Sclerite size ranged from 0.09-0.24 mm long ( = 0.166, SD =0.007, SE = 0.002) and 0.04-0.09 mm wide ( = 0.076, SD = 0.005, SE = 0.002, Fig. 2). Coloration in magenta colonies was fairly consistent. 100% of colonies exhibited a degree of white bicoloration (M/W). The bicoloration was distinct in some, and others, it made the sclerites appear less vibrant. Only two of the ten colonies had solid white sclerites present (M&W, Fig. 4b).

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Comparative Morphological Analysis Organized by Trait

a. b.

*

*

c. d.

*

*

e f.

* *

Figure 5. Extent of variation of the means of phenotypic features of Leptogorgia virgulata in three colors, yellow, orange, and magenta (N=10) compared morphologically: a Sclerite Length (mm), b Sclerite Width (mm), c Calyx Length (mm), d Branch Thickness (cm), e Polyp Count, f Polyp Density (polyp/cm^2). Values are shown as mean values +/- standard error. Statistically significant comparisons (p < 0.05) indicated with a boldface letter. An “ * ” indicates colors that are statistically different by post hoc analysis.

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The six morphological traits (SL, SW, CL, BT, PC, PD) described in table l were evaluated across three colors of L. virgulata for statistically significant differences. When taking the approach of organizing by trait, three trait variables were found to have statistically significant differences amongst colors, and three did not (Fig. 5). First, although sclerite length did not vary between colonies, sclerite width did display a statistically significant difference (F = 6.632, p = 0.00454, Table 2). Post hoc comparisons using a Tukey HSD test revealed the difference was most noticeable between magenta and yellow. Magenta colonies had the widest sclerites ( = 0.074, +/- 0.0017), and yellow colonies had the thinnest sclerites ( = 0.0699, +/- 0.00074, Fig. 5). Next, calyx length and polyp count both did not display significant differences (Table 2). However, when the number of polyps was standardized for branch thickness, the means were significantly different (F = 3.354, p = 0.01652, Table 2). Tukey HSD post-hoc tests revealed significant difference in polyp density between yellow and orange. Orange colonies had a high density of polyps ( = 69, +/- 1.98) and the thinnest branches ( = 0.1781, +/- 0.0016, Fig. 5). On the other hand, yellow colonies had a low density of polyps ( = 56, +/- 2.5) and the thickest branches ( = 0.202, +/- 2.459, Fig. 5). Magenta colonies had a mid-range of both branch thickness and polyp density (Fig. 5) Finally, branch thickness was most variable, with a very low p-value of 0.0019 (F = 7.948, Table 2). Tukey post-hoc test calculated that the difference was most significant between yellow ( = 0.202 , +/- 0.010) and orange ( = 0.178, +/- 0.005, Fig. 5) as well (SE = 0.00432). Principal Component Analysis

Figure 6. PCA biplot (PCA score plot with loadings as vectors) of PC1 and PC3 for yellow, magenta, and orange variants of L. virgulata.

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Differences in morphological characteristics between colors were also examined in the principal component analysis. The first principal component (PC1) explained 37% of the variance among measurements and was characterized by positive loading of yellow and negative loading of orange (Fig. 6, Supplementary Fig 1 & 2). PC2 explained 21.2 %, and PC3 explained 20.1% of the variance. Due to their similar influence level on the orthogonal axis, an ANOVA was run to analyze the distribution of the means by color for each of the PCs. PC3 had a greater significance in means and higher distinctive loadings of yellow and orange than PC2 (Supplementary Fig 3). Therefore, PC3 was chosen to be graphed with PC1 for clearer visualization (although both display similar groupings). PC3 was characterized by negative loadings of yellow and positive loadings of orange (Supplementary Fig 2). Magenta’s loading values were around zero for all PCs and did not create a clear distinction. The three loading vectors that displayed the greatest influence between colors were PD, BT, SW. Sclerite width had an influence on orange colonies in PC3. Polyp density and branch thickness had a strong influence on yellow colonies in PC1. Furthermore, the angle between the PD vector and BT vector was quite narrow, suggesting a possible correlation between the two variables (Fig. 6). Finally, an ANOVA of the collection of principle components computed that the principal component weights have a strong relationship to color (p = 0.0119). Therefore, the color variants of L. virgulata can likely be explained by their significant morphological differences.

Table 2. Multiple one-way ANOVA tests depicting variation of morphologies between yellow, orange, and magenta colored colonies (N = 10).

Discussion The yellow, orange, and magenta colors of L. virgulata exhibited differences that were statistically significant for three measured morphological trait variables. Bayer mainly characterizes species based upon sclerite shape, branch morphology, and calyx structure including lip projections (Bayer, 1961). Regarding sclerite shape, although the lengths were rather similar, the widths of sclerites measured in this analysis were noticeably dissimilar between colors with a statistically significant difference. Therefore, according to Bayer’s classification method, because of unique differences in sclerite dimensions, it demonstrates that the three-color morphs display a statistically significant difference in sclerite shape. Additionally, although Bayer (1961) did not specifically utilize branch diameter as a major criterion when determining octocoral taxonomic classification, experts in recent years have determined that branch thickness is indeed a meaningful characteristic as it has been utilized in more recent articles when delineating new species of octocoral (Hernandez et al., 2021). When analyzing branch thickness between colors, the values displayed statistically significant

12 differences as well. Results in this study also revealed distinct differences in polyp density in addition to the aforementioned sclerite dimension and branch diameter differences (Fig. 2). Therefore, according to Bayer’s (1961) methodology and more recently accepted methods, the acquired data suggests that the three colors of L. virgulata may be different species, especially yellow and orange. When considering the items measured which did not display significant differences, it may not meaningfully reduce the likelihood of the three colors representing different species. In a transplant study by Prada and colleagues (2008), using Eunicea flexuosa, it was found that when colonies were reciprocally transplanted from one environment to another, they developed similar phenotypic characteristics to the native colonies in the transplanted environment. While it was initially believed to be due to other factors, subsequent genomic analysis determined that they were actually different species (Prada et al., 2008). Thus, the similarities found amongst three of the characteristics measured in this common garden experiment may not detract from the likelihood of these colors representing different species to any meaningful degree at this time. Moreover, while it was noticed incidentally that magenta sclerite pigmentation appears across all colors, studies have suggested that magenta sclerites are found in the presence of disease across a variety of gorgonian species (Leverette et al., 2008, Smith et al., 2008, Kim et al., 2006). Gorgonians are also known for expressing magenta pigmentation as a response to stresses such as an injury (Ivanenko et al., 2017). Therefore, this may explain the reason for magenta sclerites existing in each of the three colors at varying degrees because they were extracted from the same environment. Thus, the presence of morphological characteristics that are statistically significantly different amongst the three colors, along with alternative explanations for similarities, would suggest that the three colors may represent different species. Bayer’s approach for speciation has generally been used since 1961. Since then, a variety of additional traits have been considered for measurement, including branch diameter, sclerite colorization, inter-calyx distance, sclerite proportions, difference in capstan and spindle measurements, and polyp density (C.P., 1978; Hernandez et al., 2021; Lau et al., 2019; Breedy & Guzman, 2007). In recent years, there has been a call for reevaluation of criteria to determine speciation by a number of researchers (Zawada et al., 2019; McFadden et al., 2010; Prada et al., 2008; Silvestri et al., 2019). When considering these notable and striking differences, combined with an ever-growing number of known Leptogorgia species (Hernandez et al., 2021), the results of this study give further insight into the limited knowledge of octocorals compared to scleractinian (Van de Water et al., 2018; Prada et al., 2010; Ward -Paige, 2005). Octocorals are a notable taxon within the marine benthos and have a large ecological role in benthic communities. For example, they provide a habitat for a vast number of organisms, provide structure for symbiotic relationships, and serve as a refuge for diverse species (Ruzicka et al., 2013; Tsounis and Edmunds, 2017). The results of this study further support the suggestion that there could be more significant within Leptogorgia genus than what was previously believed (Zawada et al., 2019; McFadden et al., 2010; Prada et al, 2008; Silvestri et al., 2019). Specifically, this comparative phenotypic analysis of morphological characteristics revealed that instead of color varying independently, color is associated with other morphological differences, suggesting the possibly the colors represent different species, especially yellow and orange.

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Conclusion Eight commonly utilized morphological characteristics were evaluated. The yellow, orange, and magenta colors of L. virgulata exhibited statistically significant morphological differences for three of six variables (SW, BT, PD), more strongly between yellow and orange. These differences appear to be meaningful enough to suggest possible speciation between colors, that may warrant further investigation.

Acknowledgements The author would like to thank Dr. Don Levitan from Florida State University for his guidance, advisership and analytical expertise. Also, the author would like to show gratitude to Rachael Best for her inspiring gorgonian knowledge, personal mentorship, and assistance in fieldwork. Thank you to Dr. Janie Wulff for her leadership and oversight of the HITM program. Finally, the author graciously thanks both Dr. Markus Huettel and Dr. Sophie McCoy for their recommendations, support, and serving on the HITM committee. Also, appreciative regards to the FSU Coastal & Marine Lab for providing supplies and resources.

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Supplemental Figures

Supplementary Figure 1. Bar plot depicting the percentage of variation for each Eigenvector. PC1 explains 37%, PC2 explains 21.2%, PC3 explains 20.1%, PC4 explains 15.5%, and PC5 explains 6.2%.

Supplementary Figure 2. ANOVA box and whiskers plot displaying color variance for each principal component. PC1 and PC3 show a greater distinction between colors than PC2, PC4, or PC5. (Orange PC1 p-value = 0.0166. Yellow PC3 p-value = 0.0057; Orange PC3 p-value = 0.032).

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Supplementary Fig 3. PCA plot of PC1 and PC2. Polyp density and branch thickness had a strong influence on yellow colonies in PC1. Sclerite width has a strong influence on orange colonies in PC2.

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