Sequencing a Gene under Strong Selection Aspartate Aminotransferase in North Atlantic Littorina Felix Mittermayer Degree project for Master of Science (Two Years) in Biology Degree course in Marine Ecology 45 hec Spring and Autumn 2013 Department of Biological and Environmental Sciences University of Gothenburg Examiner: Kerstin Johannesson Department of Biological and Environmental Sciences University of Gothenburg Supervisor: Marina Panova and Mårten Duvetorp Department of Biological and Environmental Sciences University of Gothenburg ABSTRACT 3 INTRODUCTION 4 NATURAL SELECTION, LOCAL ADAPTION AND ALLOZYME VARIATION 4 SPECIES OF LITTORINA IN NORTH ATLANTIC 4 ASPARTATE AMINOTRANSFERASE ALLOZYME VARIATIONS IN L. SAXATILIS 6 AIM 7 MATERIAL AND METHODS 8 SAMPLING, SAMPLE PREPARATION AND PHENOTYPING 8 DNA AMPLIFICATION AND SEQUENCING 8 DATA ANALYSIS 10 RESULTS 10 DISCUSSION 11 FUTURE WORK 13 REFERENCES 14 FIGURES AND TABLES 17 2 Abstract Natural selection is one of the driving forces of evolution, to understand it we must gain inside into the molecular mechanics that create genetics variation. Allozymes are variants of an enzyme coded for by different alleles, they are generally considered to be under neutral or weak selection. Aspartate aminotransferase (Aat, EC 2.6.1.1) in the rough periwinkle, Littorina saxatilis (Olivi, 1792), has however shown to be under a very stringed selection regime, Aat is an essential part of the anaerobe energy production is molluscs. The two variants of Aat are found over a vertical gradient, Aat100 is predominant (0.7-0.8) in the surf zone while Aat120 is mainly found (0.8-0.9) in the splash zone. After an extinction event of L. saxatilis in the surf zone the opening was recolonized by individuals from the splash zone that were mainly Aat120, but within period of a few generations Aat100 was again the dominant allele in the surf zone. A gene under that kind of selection can further provide inside into speciation and local adaption once the coding sequence is available. Sampling of Littorina species, genotyping using acrylamid gel electrophoresis and RNA extraction was prepared prior to the start of the project. All initial primers were designed using the coding sequence from Crossastrea gigas, the closest related species available and later complemented using Aplysia californica genome data as well as assembly data from the IMAGO genome project and transcriptome data from A. Sa Pinto, CIBIO, Portugal. Following primers were based on partial sequences acquired from the partially sequenced samples. Final primers allowed us to characterized the coding sequence for 348 of the 409 amino acids that comprise enzyme based on 118 RNA sequences from 19 individuals (L. saxatilis, Littorina arcana, Littorina compressa) A total of 4 synonymous and 2 non- synonymous mutations were identified to distinguish Aat100from Aat120. Several more synonymous and non-synonymous mutations are found to the coding sequences of Aat in Littorina fabalis and Littorina littorea. Further we calculate the isoelectric point for this part of the enzyme, 8.74 for Aat100 and 8.44 for Aat120, which does explain the different migration speed through the acrylamind gels. We additionally retrieved the available protein and nucleotide sequences for Aat in other molluscs and incorporate them into a phylogenetic tree, revealing that Aat in molluscs is indeed a gene under strong selection 3 Introduction Natural selection, local adaption and allozyme variation Local adaption is a process in which a population or a group of individuals have changed genetically in one or more characters in a way that improves fitness in a local environment in relation to the population living in other environments. This change or adaption to the local conditions, may the selection means be biotic or abiotic, improves the survival and reproductive success of those individuals that are being favoured by the reigning natural selection regime under local conditions (Futuyma 2009). When this local adaption results in reproductive isolation such as assortative mating or intrinsic incompatibilities a speciation event might occur (Butlin et al 2008). Allozymes are variants of an enzyme coded for by different alleles of a gene and distinguished on gel electrophoresis. Variation in allozyme frequencies is common among different populations of one species (Johannesson & Johannesson 1989, Armbruster 2001). These frequency variation is often considered to be the result of random genetic drift (Thorpe & Solé-Cava 1994) but some allozyme variation has shown to be maintained by strong diverging selection (Theisen 1978, Koehn et al. 1983), that is in some cases connected to the habitat (Johannesson & Johannesson 1989, Johannesson et al 1995, Armbruster 2001). Examples of allozymes under selection are cytosolic malate dehydrogenase in limpets (Lottia spp) (Dong & Somero 2009), phosphoglucose isomerase in Colias butterflies (Wheat et al. 2006) and lactate dehydrogenase in fish Fundulus heteroclitus (Powers & Schulte 1998). Another example of adaptive variation in proteins is the haemoglobin of Cod (Gadus morhua) (Andersen et al. 2009) which is selected by temperature differences over many hundreds of kilometres. One of the most striking examples of allozyme variation is the case of aspartate aminotransferase in the intertidal snail Littorina saxatilis the selection occurs at strength over a gradient of few meters (see below). Species of Littorina in North Atlantic In the North Atlantic, there are six species of Littorina: the rough periwinkle L. saxatilis (Olivi, 1792), its sister-species L. compressa (Jeffereys, 1865) and L. arcana (Hannaford Ellis, 1978), two species of flat periwinkles L. obtusata (L) and L. fabalis (Turton, 1825) and the more distantly related species L. littorea (L.) The main focus of this study is on L. saxatilis. 4 The ovoviviparous marine snail L. saxatilis inhabits the littoral and sublittoral shorelines of the Northern Atlantic, it can be found in the Arctic, and the North American east coast as well as on most Northern and Western European shores (Reid 1996), where it can occur in very high densities, with several hundred individuals per m2. Littorina saxatilis is the only species within Littorina genus that has direct development, meaning that the females release fully developed juveniles after carrying them in a brood pouch (Reid 1996). The highly variable intertidal habitat can cause very different stress levels in the various microenvironments with respect to abiotic factors such as temperature and desiccation (Sokolova et al. 2000) or wave- exposure and biotic factors such as predation (Johannesson 2003) . As a consequence of the mode of reproduction and the low mobility of adult snails, migration and gene flow among distant populations is weak. Weak gene flow in combination with strong differential selection have in this species resulted in the formation of wave-exposed, moderate, sheltered and barnacle ecotypes of snails with different morphologies (Reid 1996), in addition to genetic differences as a consequence of genetic drift and selection (Johannesson & Johannesson 1990). The most common ecotypes are the large, thick-shelled one with high spire known as crab type and the small, thin-shelled one with a big aperture, the wave type. Depending on the geographical position the location of the different ecotypes varies; in the UK for instance the crab type is generally found on shores with boulder that allow for the presence of crabs while the wave type is found on cliffs, the gradient is crab type above wave type. In Sweden however the different ecotypes are found on a horizontal distribution patter, as they are found in different habitats along the coastline: crab type in sheltered bays often among boulders and pebbles and the wave type on wave exposed cliffs. Thus, the wave ecotype in Sweden is exposed to further microenvironmental variation between low and high shore. Littorina compressa and L. arcana are considered to be sister species with L. saxatilis (Reid et al 1996). Their distributions are limited to the British Isles, Brittany and northern Norway. Where all three species occur in sympatry, they have different vertical distribution with L. compressa in the lower, L. saxatilis in the middle and L. arcana in the upper part of the littoral zone, however the segregation of the vertical distribution is not total as there is a considerable overlap between vertically neighbouring species. Unlike L. saxatilis these two species are oviparous, i.e. they lay their eggs in gelatinous masses in damp crevices from which metamorphosed juveniles hatch. Further, L. compressa and L. arcana have the same ecotypes as L. saxatilis except a brackish water type since they are not found in that kind of an environment (Reid 1996). 5 The flat periwinkle, L. obtusata, is found all around the north Atlantic, from Russia (White Sea) to Portugal in the eastern and Greenland to New England in the western Atlantic. This species inhabits the eulittoral zone and is closely associated with macrophytes, mostly fucoids. Its sister species L. fabalis has a similar distribution range but is not found in Atlantic Canada or New England. Similar to L. saxatilis and its sister species L. obtusata and L. fabalis lack a planktonic development but lay gelatinous egg masses on thallie of their host fucoids from with fully developed juveniles hatch. Both species produce ecotypes dependent on the exposure of their habitats to wave action, exposed, sheltered and a moderate ecotype