Phenotypic and Genetic Diversity in the Sea Urchin Lytechinus Variegatus by Maria Letizia Wise University Program in Ecology Duke University Date:_______________________ Approved: ___________________________ Daniel Rittschof, Supervisor ___________________________ David McClay ___________________________ William Kirby-Smith ___________________________ Mark Fonseca Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Ecology in the Graduate School of Duke University 2011 ABSTRACT Phenotypic and Genetic Diversity in the Sea Urchin Lytechinus Variegatus by Maria Letizia Wise University Program in Ecology Duke University Date:_______________________ Approved: ___________________________ Daniel Rittschof, Supervisor ___________________________ David McClay ___________________________ William Kirby-Smith ___________________________ Mark Fonseca An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Ecology in the Graduate School of Duke University 2011 Copyright by Maria Letizia Wise 2011 Abstract Diversity in coloration is a common phenomenon in marine invertebrates, although the ecological significance of the diversity is often unknown. Patterns of geographic variability, particularly with respect to color phenotypes, are evident in many organisms and may provide visual evidence of natural selection. This dissertation examined the geographic and genetic variability of color patterns and morphology of the sea urchin Lytechinus variegatus. This study had 3 objectives: 1) to describe and quantify phenotypic diversity—color and morphology— throughout the geographic range; 2) to determine the heritability of color in genetic crosses between individuals with similar and differing phenotype; 3) to assess the degree of genetic divergence between and within the regions and congruence between the phylogenetic mitochondrial COI data and color phenotypes seen in the field. The distribution of color phenotypes in the field is highly variable across the geographic range which stretches from Beaufort NC to southern Brazil and throughout the Gulf of Mexico and Caribbean. Urchins in each of the 5 regions sampled (Beaufort, Gulf, Keys, Panama and Brazil) have a distinct phenotypic composition despite the presence of similar color morphs. The two regions at the extremes—Beaufort and Brazil—demonstrate the most homogeneous phenotypes, each with a single dominant color morph. The Keys has the most heterogeneous composition with all 14 color morphs present. Morphological diversity mirrors color diversity in being highly variable iv across the range. Urchins in Beaufort are significantly different morphologically from urchins in the central portion of the range, with thicker, flatter tests and longer more robust spines. The heritability of color phenotypes and morphology suggests that genes rather than environment have a major role in color phenotype and patterning as well as morphology in test, spines and lanterns. F1 and F2 offspring of Beaufort and Tavernier Key crosses resemble the parental phenotypes in both morphology and color phenotype. Hybrids from crosses between regions have a mixed color palette and intermediate morphological characteristics. The crosses establish that in L. variegatus the white phenotype is a dominant autosomal trait and green and purple are recessive and co- expressed. Patterning of the test and spines is dominant to non-patterning. Analysis of the mitochondrial COI gene in urchins from Beaufort, Gulf and Keys regions revealed two clades. Clade 1 composed exclusively of Keys urchins differed significantly (FST = 0.89, P < 0.001) from Clade 2 composed of urchins from Beaufort, Gulf and Keys. Genetic differentiation within Clade 2 was zero, indicating that urchins in these regions are genetically identical. The genetic split between phenotypically indistinguishable Keys urchins suggests cryptic species. Genetic differentiation does not concord with phenotypic and morphological differentiation. No structure was detected with regards to color phenotype in either clade or region of origin in Clade 2. v Contents Abstract ......................................................................................................................................... iv List of Tables ................................................................................................................................. ix List of Figures ............................................................................................................................... xi Acknowledgements ...................................................................................................................xiv 1. Introduction ............................................................................................................................... 1 2. Color variability in Lytechinus variegatus ............................................................................... 9 2.1 Introduction ....................................................................................................................... 9 2.2 Materials and Methods .................................................................................................. 15 2.2.1 Sample sites and urchin habitat .............................................................................. 15 2.2.2 Field surveys of in situ color morphs ...................................................................... 17 2.2.3 In situ color scoring ................................................................................................... 18 2.2.4 Color variability ......................................................................................................... 20 2.2.5 Test color after removal of spines and epidermis ................................................. 21 2.2.6 Data analysis .............................................................................................................. 22 2.3 Results .............................................................................................................................. 23 2.3.1 Field surveys of in situ color morphs ...................................................................... 23 2.3.2 Color variability in live urchins ............................................................................... 30 2.3.3 Color variability after removal of spines and epidermis ..................................... 42 2.4 Discussion ........................................................................................................................ 53 3. Morphological variability in L. variegatus ............................................................................ 63 vi 3.1 Introduction ..................................................................................................................... 63 3.2 Materials and Methods .................................................................................................. 68 3.2.1 Sample Collection ...................................................................................................... 68 3.2.2 Morphological measurements ................................................................................. 69 3.2.3 After removal of spines and epidermis .................................................................. 72 3.2.4 Data analysis .............................................................................................................. 73 3.3 Results .............................................................................................................................. 75 3.3.1 Urchin morphology ................................................................................................... 75 3.3.2 Morphology after removal of spines and epidermis ............................................ 95 3.4 Discussion ...................................................................................................................... 102 4. Genetic crosses....................................................................................................................... 114 4.1 Introduction ................................................................................................................... 114 4.2 Materials and Methods ................................................................................................ 118 4.2.1 Urchin collection ...................................................................................................... 118 4.2.2 Larval culture ........................................................................................................... 119 4.2.3 Juvenile cultures ...................................................................................................... 122 4.2.4 Crosses ...................................................................................................................... 123 4.3.5 F2 and F3 generations ............................................................................................... 125 4.3.6 Morphological and color variability ..................................................................... 126 4.3 Results ............................................................................................................................ 128 4.3.1 Cross color phenotypes .......................................................................................... 128 4.3.2 Cross F1