The molecular basis of development of the sword, a sexual selected trait in the genus Xiphophorus Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) an der Universität Konstanz, Mathematisch-naturwissenschaftliche Sektion Fachbereich Biologie vorgelegt von Dipl. Biol. Nils Offen Tag der mündlichen Prüfung: 19.12.2008 Referenten: Prof. Dr. Axel Meyer Prof. Dr. Michael K. Richardson Acknowledgements First, I would like to thank my Ph. D. advisor Prof. Axel Meyer who gave me the opportunity to work on this fascinating organism in his lab. I’m grateful to my supervisor PD. Dr. Gerrit Begemann for his advice, various discussion on the the projects and other stuff, inspiration and for the chance to develop and try out own ideas and to improve my scientific and personal skills. I’m thankfull for a quite fascinating time as his PhD student. I want to thank my current and past collegues of the Meyerlab for various help in the lab and interesting discussions. I’m specially gratefull to (in no particular order) Silke Pittlik, Sebastién Wielgoss, Kai Stölting, Simone Högg, Dominique Leo, Dave Gerrad, Dirk Steincke, Elke Hespeler, Katharina Mebus, Nicola Blum, Matthias Sanetra, Cornelius Eibner, Ylenia Chiari, Maria Buske and Milena Quentin for joining me for a while on my road to Ph. D. and for a memorable time. I also want to thank Helen Gunter and Kathryn Elmer for proof-reading parts of the thesis. And last but not least I’m grateful to Janine Sieling for excellent animal care that made my work so much easier. Some of those people become more than just colleagues to me and I’m very thankful for several activities and happy hours in and outside the lab. In this context I’d like to mention the Badminton group, which I joined for more than two years. Especially, I want to thank my parents, my grandparents, my brother and sister and friends outside the university, in particular Martin Eggert, who gave me great support and fresh motivation to continue this work to the end. I’m also grateful to my better half, Alexandra Schuh, who brightened up my life and supported and motivated me continuously in the last ~two years of my thesis. Last, I like to thank her mother and her stepfather for a place to relax and for delicious food. 2 Table of contents General introduction 4 Swords, sexy males and choosy females 5 Sword evolution and the Sensory Exploitation-Preexisting Bias Hypothesis 9 Sword development 11 The male gonopodium 14 Chapter I: Fgfr1 signalling in the development of a sexually selected trait 19 in vertebrates, the sword of swordtail fish 1.1 Abstract 19 1.2 Background 20 1.3 Results 23 1.4 Discussion 35 1.5 Conclusions 41 1.6 Material and Methods 42 Chapter II: A Subtractive Hybridisation approach to identify novel genes 47 involved in the development of the sword in green swordtail (X. helleri) 2.1 Abstract 47 2.2 Introduction 47 2.3 Results 49 2.4 Discussion 61 2.5 Experimental Procedures 67 Chapter III: Retinoic acid is involved in gonopodium formation in the green 72 swordtail, Xiphophorus helleri 3.1 Abstract 72 3.2 Introduction 72 3.3 Material and Methods 76 3.4 Results 82 3.5 Discussion 94 Summary 100 Zusammenfassung 103 Eigenabgrenzung 106 Literature cited 107 Appendix 120 3 General Introduction Xiphophorus fishes (Fam. Poeciliidae) are small live bearing toothcarps (Cyprinodontiformes) that are endemic to parts of Mexico and Central America [1-3]. The genus itself can be subdivided into four different groups, the northern and southern swordtails and the northern and southern platyfish, depending on their distribution relative to the Trans-Mexican Volcanic Belt in central Veracruz [3]. This classification into northern and southern swordtails, but not that of southern and northern platyfish, is further supported by several molecular phylogenies [4, 5]. However, the platyfish were originally combined in an independent genus ( Platypoecilus ), before Myron Gordon classified the platyfish as a subgroup within the genus Xiphophorus in 1951 [6]. To this day 26 Xiphophorus species have been described [3]. Some species such as X. andersi show a very restricted distribution pattern, whereas others such as the swordtail X. helleri and the two platyfish X. maculatus and X. variatus are the most widely distributed species (Figure 1). The relationship among these 26 species is rather incompletely resolved, since solid phylogenies using both mitochondrial and nuclear markers comprise only 22 of the 26 described species [4, 5]. Interestingly, several Xiphophorus species can be interbred within captivity [7] and hybridization occurs also under natural conditions [5, 8]. Multiple natural hybrid zones were found between X. birchmanni and X. malinche, due to a disruption in chemical communication [8, 9] . In addition, a recent study showed that the swordtail species X. clemenciae resulted from an ancient hybridization event between a swordtail and a platy species [5]. Different populations of Xiphophorus species such as X. helleri or X. maculatus can be amazingly variable in color pattern [10]. Several of these pigment pattern variants showed independent mendelian segregation [11, 12]. The ability to make interspecies crosses and the availability of genetic markers turned a popular aquarium fish into a model for early genetic studies in fish in the first half of the 20th century [11]. Since these days Xiphophorus fishes have become an established model organism for early genetic, as well as for behavioural studies and melanoma formation (reviewed in [13, 14]). 4 Figure 1: Distribution of Xiphophorus species in Mexico and Central America A: Distribution of: (A) X. meyeri , (B) X. gordoni , (C) X. couchianus , (D) X. xiphidium , (E) X. variatus , (F) X. birchmanni , X. continens, X. cortezi , X. malinche , X. montezumae , X. multilineatus , X. nezahualcoyotl , X. nigrensis and X. pygmaeus , (G) X. evelynae , (H) X. andersi , (I) X. milleri and X. kallmani , (J) three species of X. clemenciae , (K) X. helleri and X. maculatus , (K) X. alvarezi , (M) X. signum , (N) X. mayae , (O) “PMH” type of Xiphophorus [15] The satellite map was obtained from google maps. The distribution of Xiphophorus species was adapted from Kallman and Kazianis [3]. Swords, sexy males and choosy females In the second half of the 20 th century Xiphophorus become a valuable organism for behavioural biologists (reviewed in [14]). Male swordtails perform a complex courtship behaviour that consists of several behavioural sequences such as lateral or sexual display [16, 17]. Interestingly, in several Xiphophorus species not all males perform a complex courtship behaviour, but show an alternative mating strategy (reviewed in [14]). In X. nigrensis , only large males court in front of the female and perform sexual display, 5 whereas small males perform a sneak and chase behaviour instead [18, 19]. The difference in body length of mature males that determine the mating strategy of the respective individual seems to result from an allelic variation in the pituitary locus ( P). X. nigrensis males with the PL (large) allele show a higher growth rate and mature later than males with the Ps (small) allele [19]. The term P locus was originally termed by Kallman and Schreibman, who proposed that the P locus controls when the pituitary-gonadal axis is activated and sexual maturation is induced [20, 21]. They hypothesised that the P locus acts on the maturation of gonadotropic hormone producing cells. The gonadotropic hormones that are released by the pituitary gland stimulate Leydig-like cells in the testis to secrete testosterone [22, 23]. Both gonadotropic and sex hormones promote spermatogenesis and sexual maturation (reviewed [24]). Interestingly, the PS allele is maintained in populations of X. nigrensis , even though females prefer large, courting males [25]. This might be due to the fact that both PL and Ps males can have equal fitness [26]. Firstly, Ps males mature and start reproducing earlier that PL males [19]. Secondly, the large PL males need a longer time to reach sexual maturity and are more attractive to predators and therefore likely have a higher mortality rate [19, 27]. However, the genetic nature of the P locus, even though the term was coined by Kallman and Schreibman in 1973 [20] is still unknown, as well as the mechanism how the locus actually times sexual maturation. Besides the attempts to uncover the genetics of behaviour, Xiphophorus is more widely known as a model for sexual selection (reviewed in [14]). Many studies contribute to the understanding of female mate choice and the traits that are involved. The contribution of several traits such as chemical cues [28], colouration [29, 30], vertical bar pattern [31], body size [32] or courtship behaviour itself [33] have been studied by several groups. A first attempt to understand the genetic basis of mate choice was made by M. Cummings and co-workers in a recent study [34]. They identified a couple of genes that are differentially expressed in the brains of X. nigrensis females, when they interact with attractive males. Another sexually selected trait, a colourful extension of the caudal fin called the sword, has been first described by Darwin himself [35]. According to a definition given by Basolo in 1991, the sword is a coloured ventral extension with 0.7-6.0 times the length of 6 the caudal fin that also exhibits a black ventral margin [36]. However, Meyer and co- workers provided an alternative definition that did not include colouration and a black margin [4, 37]. As a consequence, the colourless ventral extension of X. andersi is considered a sword by this definition [4, 37], whereas after Basolo’s definition it is considered to be a protrusion [38]. In fact, Basolo’s sword definition is supported by several studies that showed the biological relevance of both length and a distinct colour pattern [39-42].