)ORULGD6WDWH8QLYHUVLW\/LEUDULHV 2021 Coded in Colors: Morphological Differentiation Associated with Color Suggests Further Speciation of Leptogorgia virgulata Chandler Michaele Wright Follow this and additional works at DigiNole: FSU's Digital Repository. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS & SCIENCES CODED IN COLORS: MORPHOLOGICAL DIFFERENTIATION ASSOCIATED WITH COLOR SUGGESTS FURTHER SPECIATION OF LEPTOGORGIA VIRGULATA 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 corals. 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 species. 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), polyp 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 genus 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 taxonomy 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 3 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. 4 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 animals, 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)