PHYLOGENETIC RELATIONSHIPS AND EVOLUTIONARY ECOLOGY OF NICARAGUAN MIDAS-CICHLIDS (AMPHILOPHUS SPP.) DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER FAKULTÄT FÜR BIOLOGIE DER LUDWIG- MAXIMILIANS- UNIVERSITÄT MÜNCHEN VORGELEGT VON MATTHIAS GEIGER AM 08. JUNI 2011 ERSTGUTACHTER: PROF. DR. GERHARD HASZPRUNAR ZWEITGUTACHTER: PROF. DR. GÜNTHER HEUBL TAG DER MÜNDLICHEN PRÜFUNG: 19. DEZEMBER 2011 WORK PRESENTED IN THIS DISSERTATION WAS PERFORMED IN THE LABORATORY OF PROF. DR. G. HASZPRUNAR, DIRECTOR OF BAVARIAN STATE COLLECTION OF ZOOLOGY, MUNICH, GERMANY. WORK WAS PERFORMED UNDER THE SUPERVISION OF PROF. DR. G. HASZPRUNAR AND DR. U. SCHLIEWEN THE THEORY OF EVOLUTION BY CUMULATIVE NATURAL SELECTION IS THE ONLY THEORY WE KNOW OF THAT IS IN PRINCIPLE CAPABLE OF EXPLAINING THE EXISTENCE OF ORGANIZED COMPLEXITY. RICHARD DAWKINS BRITISH ETHOLOGIST, GENETICIST, & POPULARIZER OF GENETICS (1941 ‐ ) EHRENWÖRTLICHE VERSICHERUNG ICH VERSICHERE HIERMIT EHRENWÖRTLICH, DASS DIE VORGELEGTE DISSERTATION VON MIR SELBSTÄNDIG UND OHNE UNERLAUBTE BEIHILFE ANGEFERTIGT IST. ERKLÄRUNG HIERMIT ERKLÄRE ICH, DASS ICH MICH ANDERWEITIG EINER DOKTORPRÜFUNG OHNE ERFOLG NICHT UNTERZOGEN HABE UND DASS DIE DISSERTATION NICHT GANZ ODER IN WESENTLICHEN TEILEN EINER ANDEREN PRÜFUNGSKOMMISSION VORGELEGT WORDEN IST. Publications from the work presented in this dissertation Geiger MF, McCrary JK, Schliewen UK (2010) Not a simple case ‐ A first comprehensive phylogenetic hypothesis for the Midas cichlid complex in Nicaragua (Teleostei: Cichlidae: Amphilophus). Molecular Phylogenetics and Evolution, 56, 1011–1124. Geiger MF, McCrary JK, Stauffer, JR (2010) Description of two new species of the Midas cichlid complex (Teleostei: Cichlidae) from Lake Apoyo, Nicaragua. Proceedings of the Biological Society of Washington, 123, 159–173. Geiger MF, Mayr C, McCrary JK, Schliewen UK Molecular Ecology of the Lake Apoyo Midas Cichlid Species Flock. (submitted to Molecular Ecology) Table of Contents GENERAL INTRODUCTION I ‐ Study System 2 II ‐ Speciation Theory 4 SUMMARY ‐ AIMS OF THIS THESIS 8 MATERIAL & METHODS Materials Examined 9 Taxon Sampling 10 Morphometry 10 DNA Extraction, PCR, mtDNA & AFLP Genotyping 10 Phylogeny Reconstruction & Tests 11 Outlier Locus Detection 12 Inference of Genetic Structure 12 Stable Isotope Analysis 13 Correlating Genetic, Morphometric & Ecologic Differentiation 13 RESULTS & DISCUSSION I ‐ Molecular phylogeny of Nicaraguan Midas Cichlids 15 II ‐ Taxonomic evaluation of Lake Apoyo Amphilophus 18 III ‐ Molecular Ecology of the Lake Apoyo species flock 20 SUMMARY (GERMAN) 23 ACKNOWLEDGEMENTS 24 REFERENCES 25 CURRICULUM VITAE 31 APPENDIX Article I Article II Article III SUPPLEMENTARY MATERIAL CD including online supporting information of Articles I & III + full specimen list + pdf ‐ files of publications General Introduction Part I – Study System GENERAL INTRODUCTION PART I – STUDY SYSTEM Cichlid fish species flocks (Teleostei: Perciformes: Cichlidae) of the African lakes have served for decades as prime model systems for evolutionary biology in general and speciation research in particular (Kocher 2004; Seehausen 2006). Alone the East African cichlid radiations in Lake Victoria, Malawi and Tanganyika harbour estimated 2000 species (Seehausen 2006), which all belong to a single major lineage of predominantly Eastern and Southern African cichlids. The sheer size of these mega‐diverse cichlid species flocks as well as the physical size of the three lakes not only holds promises but also constraints as a model system, because it is difficult to sample these systems in toto. Flocks with fewer species that are endemic to small lakes, e.g. crater lakes, thus represent an interesting alternative model system to study adaptive radiations, if the analysis of complete species richness is also within the focus. Quite well known are the monophyletic cichlid flocks of Cameroonian crater lakes with five to eleven species per lake (Schliewen et al. 2001; Schliewen & Klee 2004; Stiassny et al. 1992; Trewavas 1972). More recently, crater lake species flocks of the Nicaraguan Midas cichlid complex (genus Amphilophus) attracted the attention of evolutionary biologists (Barlow 1976; Barluenga & Meyer 2004; Barluenga et al. 2006a; Elmer et al. 2009 & 2010a; McKaye et al. 2002; Stauffer & McKaye 2002; Stauffer et al. 2008; Wilson et al. 2000). Both, Nicaraguan Amphilophus cichlids as well as Cameroonian crater lake cichlids belong to phylogenetic lineages only distantly related to the great East African cichlid radiations and therefore may serve as comparative systems for the analysis of parallel patterns of speciation. In contrast to the Cameroonian crater lake assemblages, there are no detailed estimates for cichlid species numbers in the Nicaraguan crater lakes, and consequently, there has been no established phylogenetic species‐ level framework for the Midas cichlid species complex prior to this work. Nicaraguan Midas cichlids (Amphilophus spp.) are named after the famous King Midas of the Greek mythology who was given the fatal ability to turn everything he touched into gold – until he starved as wealthy man. The name Midas cichlid derives from the fact that, in some Nicaraguan lakes, brightly colored Amphilophus individuals can be found that have lost the melanophores that usually build patterns of black bars, yielding individuals ranging in color from white to orange or red, and collectively termed ‘gold’. They are found in varying abundances up to 10% in more turbid lakes (Barlow 1976) and are known from at least four of the to date described eleven species Fig. 1 The Pacific Versant of Nicaragua, including the Great Lakes and crater lakes of this study (modified from Barluenga et al. in this species complex. 2006). Midas cichlids occur in all major freshwater‐bodies within the Rio San Juan drainage in Nicaragua: they are very abundant in the two large and old rift lakes Nicaragua and Managua but also inhabit rivers and the majority of the crater lakes west of the two great lakes (Table 1 & Fig. 1). 2 General Introduction Part I – Study System Almost each lake contains several locally endemic Midas cichlid phenotypes or species, which are highly diverse in morphology, color, behavior and ecology (e.g. Barlow 1973, 1976, 1978; Elmer et al. 2010b; McKaye 1977, 1980; McKaye et al. 2002; Wilson et al. 2000; Stauffer et al. 2008). Only until recently, one group of researchers considered only three polymorphic species which are distributed over the whole Lake Nicaragua system including multiple crater lakes, i.e. A. citrinellus, A. zaliosus and A. labiatus (Barlow & Munsey 1976; Barluenga & Meyer 2004; Barluenga et al. 2006a&b; Bunje et al. 2007; Wilson et al. 2000). The opposite view claims that the phenotypic diversity of Midas cichlid species complex falls into multiple species per lake including the crater lakes (McKaye et al. 2002; Stauffer & McKaye 2002; Stauffer et al. 2008). It was also this discrepancy between observed and described diversity that has contributed to the initiation of this PhD study. The multi‐species concept has now gained wider acceptance as judged from most recent publications (Elmer et al. 2010a&b, Lehtonen et al. 2010). Depending on the author, the Midas cichlid complex incorporates three and eleven species at the moment, but may contain 30 or more species (McKaye et al. 2002; pers. obs.). taxon author type locality estimated min. age A. citrinellus Günther 1864 L. Nicaragua > 500.000 y+ A. labiatus Günther 1864 L. Managua > 500.000 y+ A. amarillo Stauffer & McKaye 2002 L. Xiloá < 2.000 – 6.000 y* A. sagittae Stauffer & McKaye 2002 L. Xiloá A. xiloaensis Stauffer & McKaye 2002 L. Xiloá A. astorquii Stauffer, McCrary & Black 2008 L. Apoyo < 21.000 – 25.000 y*° A. chancho Stauffer, McCrary & Black 2008 L. Apoyo A. flaveolus Stauffer, McCrary & Black 2008 L. Apoyo A. globosus Geiger, McCrary & Stauffer 2010 L. Apoyo A. supercilius Geiger, McCrary & Stauffer 2010 L. Apoyo A. zaliosus Barlow & Munsey 1976 L. Apoyo Table 1 Current taxonomy of valid Midas cichlid species. +Bussing 1976; *Freundt et al 2006; ° Sussmann 1982. 3 General Introduction Part II – Speciation Theory GENERAL INTRODUCTION PART II – SPECIATION THEORY Although more than 150 years have now passed since Darwin’s famous book “On the origin of species” has been published (Darwin 1859), one of the central and fundamental issues in evolutionary biology remains the question of how species evolve (e.g. Coyne & Orr 2004). The origin of new species has puzzled evolutionary biologists for decades, and while early studies focused on how species evolved under Darwin’s introduced natural selection within a single habitat (e.g. Woltereck 1931; Brooks 1950), subsequent work sought for alternatives to natural selection (e.g. Mayr 1942; Futuyama & Mayer 1980). Later on, with the advent of molecular biology, underlying genetic aspects of speciation have gained widespread interest (e.g. Coyne & Orr 1998; Via 2002). Today, in the era of genomics where unprecedented amounts of genetic information are readily available (given sufficient resources) and are combined with ecological and morphological data, we will likely see greater advance in our understanding of evolution and speciation than ever before (Butlin 2010). Given the complexity of interactions that individuals and populations experience between conspecifics, with heterospecifics and with the environment, different modes of speciation have been classified to facilitate the study of speciation. The most widely accepted framework is a classification