A Phylogenetic and Transcriptomic Perspective
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Institut für Biochemie und Biologie Evolutionsbiologie/Spezielle Zoologie Adaptive radiation and speciation in African weakly electric fish: a phylogenetic and transcriptomic perspective. (Genus Campylomormyrus, Mormyridae, Teleostei) Dissertation zur Erlangung des akademischen Grades "doctor rerum naturalium" (Dr. rer. nat.) in der Wissenschaftsdisziplin "Evolutionsbiologie" eingereicht an der Mathematisch- Naturwissenschaftlichen Fakultät der Universität Potsdam von Francesco Lamanna Potsdam, März 2015 Gutachter: • 1. Gutachter: Prof. Dr. Ralph Tiedemann (Universität Potsdam) • 2. Gutachter: Prof. Dr. Walter Salzburger (Universität Basel) • 3. Gutachter: PD Dr. Frieder Mayer (Leibniz-Institut für Evolutions- und Biodiversitätsforschung am Museum für Naturkunde, Berlin) Published online at the Institutional Repository of the University of Potsdam: URN urn:nbn:de:kobv:517-opus4-80097 http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-80097 Erklärung der Urheberschaft Ich erKläre hiermit an Eides statt, dass ich die vorliegende Arbeit ohne Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die Arbeit wurde bisher in gleicher oder ähnlicher Form in Keiner anderen Prüfungsbehörde vorgelegt. Potsdam, den Francesco Lamanna Table of contents Table of contents 1 Abstract 1 2 Zusammenfassung 2 3 Introduction 3 Systematic placement of the African weakly-electric fish genus Campylomormyrus 3 3.1 3.2 On Electrogenesis and electroreception 5 3.3 History of an adaptive radiation 6 3.4 Aims of this study 7 4 Summary of articles 10 4.1 Summary of article I 10 4.2 Summary of article II 11 4.3 Summary of article III 12 5 Article I 13 6 Article II 30 7 Article III 68 8 Discussion 95 8.1 Transcriptomic analysis of electric organ and skeletal muscle 95 8.1.1 Sequencing and characterization of reference transcriptomes 95 8.1.2 Patterns of gene-expression between skeletal muscle and electric organ 96 8.1.3 Future perspectives 98 8.2 Campylomormyrus species tree estimation and delimitation 100 8.2.1 Future perspectives 101 9 References 102 10 Acknowledgements 106 iv v 1 Abstract 1 Abstract The rise of evolutionary novelties is one of the major drivers of evolutionary diversification. African weakly-electric fishes (Teleostei, Mormyridae) have undergone an outstanding adaptive radiation, putatively owing to their ability to communicate through species-specific Electric Organ Discharges (EODs) produced by a novel, muscle-derived electric organ. Indeed, such EODs might have acted as effective pre-zygotic isolation mechanisms, hence favoring ecological speciation in this group of fishes. Despite the evolutionary importance of this organ, genetic investigations regarding its origin and function have remained limited. The ultimate aim of this study is to better understand the genetic basis of EOD production by exploring the transcriptomic profiles of the electric organ and of its ancestral counterpart, the skeletal muscle, in the genus Campylomormyrus. After having established a set of reference transcriptomes using “Next-Generation Sequencing” (NGS) technologies, I performed in silico analyses of differential expression, in order to identify sets of genes that might be responsible for the functional differences observed between these two kinds of tissues. The results of such analyses indicate that: i) the loss of contractile activity and the decoupling of the excitation-contraction processes are reflected by the down-regulation of the corresponding genes in the electric organ; ii) the metabolic activity of the electric organ might be specialized towards the production and turn- over of membrane structures; iii) several ion channels are highly expressed in the electric organ in order to increase excitability, and iv) several myogenic factors might be down-regulated by transcription repressors in the EO. A secondary task of this study is to improve the genus level phylogeny of Campylomormyrus by applying new methods of inference based on the multispecies coalescent model, in order to reduce the conflict among gene trees and to reconstruct a phylogenetic tree as closest as possible to the actual species-tree. By using 1 mitochondrial and 4 nuclear markers, I was able to resolve the phylogenetic relationships among most of the currently described Campylomormyrus species. Additionally, I applied several coalescent-based species delimitation methods, in order to test the hypothesis that putatively cryptic species, which are distinguishable only from their EOD, belong to independently evolving lineages. The results of this analysis were additionally validated by investigating patterns of diversification at 16 microsatellite loci. The results suggest the presence of a new, yet undescribed species of Campylomormyrus. 1 2 Zusammenfassung 2 Zusammenfassung Das übergreifende Ziel dieser Arbeit ist das bessere Verständnis der Bedeutung der schwachen Elektrizität für die adaptive radiation der Mormyriden Afrikas. Das gewählte Modell-Taxon, die Mormyriden-Gattung Campylomormyrus, zeigt eine große Vielfalt an elektrischen Entladungsformen. Diese Entladungsformen sind artspezifisch. Die genetische Basis dieses Merkmales ist allerdings noch unbekannt. In dieser Arbeit habe ich transkriptomische Untersuchungen vom elektrischen Organ und Skelettmuskel durchgeführt. Die Ergebnisse dieser Analysen zeigen, dass die phenotypischen Unterschiede zwischen dem elektrischen Organ und dem Skelettmusckel in den jeweiligen transkriptomen gespiegelt sind. Ich habe auch einen phylogenetischen Stammbaum für die Gattung Campylomormyrus hergestellt, durch die Anwendung von „Multispecies Coalescent Models“-basierten Methoden. Außerdem, durch die Anwendung von Mikrosatellitdaten, die als unabhängiger Beweis dienten, konnte ich zeigen, dass die identifizierten phylogenetischen Gruppen reproduktiv isolierte biologische Arten sind. Auf diese Weise konnte ich ein neuen, noch unbeschriebenen Art nachweisen. 2 3 Introduction 3 Introduction. 3.1 Systematic placement of the African weakly-electric fish genus Campylomormyrus. Mormyrids are a family of freshwater weakly-electric fishes endemic to African riverine and, partially, lacustrine systems. They belong to the superorder Osteoglossomorpha, considered one of the phylogenetically basal groups of Teleostei (Near et al. 2012). With more than 180 described species in 20 genera, mormyrid fishes account for almost 90% of the overall diversity within osteoglossomorphs (Lavoué & Sullivan 2004). Together with the monospecific family Gymnarchidae, they constitute the superfamily Mormyroidea (Fig. 1); the most remarkable synapomorphic (derived) characters supporting the monophyly of this taxon are definitely the presence of electric organs, matched electroreceptors and a substantially enlarged cerebellum (Taverne 1972). The monophyletic status of Mormyroidea has been further confirmed by several molecular phylogenetic studies (Alves-Gomes & Hopkins 1997; Sullivan et al. 2000). Within the family Mormyridae two subfamilies are recognized (Fig. 1): Petrocephalinae, formed by a single genus (Petrocephalus) composed of 42 species and Mormyrinae constituted by all the remaining genera and species. Campylomormyrus 1 tamandua Campylomormyrus 2 tamandua Campylomormyrus numenius 14 Campylomormyrus sp. 1 Gnathonemus petersii Genyomyrus donnyi Marcusenius sp. 1 Marcusenius moorii 13 Marcusenius greshoffi Hippotamyrus spp. Marcusenius senegalensis Hyperopisus bebe Hippopotamyrus pictus 12 Brienomyrus niger Brienomyrus DPNP hopkinsi 11 Paramormyrops gabonensis Brienomyrus longicaudatus Brienomyrus sp.2 Marcusenius DPp conicephalus 10 Ivindomyrus opdenboschi Boulengeromyrus knoepffleri 9 Pollimyrus spp. Pa Stomatorhinus spp. 8 Mormyrus spp. 7 Brienomyrus brachyistius 6 Isichthys NPp henryi 5 Mormyrops nigricans 3 4 Mormyrops zanclirostris 2 Myomyrus macrops Ant. Post. Petrocephalus spp. S 1 Gymnarchus niloticus 10 Pp Figure 1 Phylogenetic tree of Mormyroidea. On the left side the different electrocyte morphologies and Electric Organ Discharge (EOD) types are represented, as well as their position on the phylogeny. The abbreviations below each electrocyte indicate its morphology and innervation pattern: S=stalKless; NPp=non penetrating stalKs posteriorly innervated; Pa =penetrating stalKs anteriorly innervated; DPp=doubly penetrating stalKs posteriorly innervated; DPNP=doubly penetrating and non-penetrating stalKs; Pp=penetrating stalKs posteriorly innervated. Ant.=anterior side of the electrocyte; Post.=posterior side (Modified from Sullivan et al. 2000). 3 3 Introduction One of the most peculiar genera of the subfamily Mormyrinae is Campylomormyrus. This genus RULJLQDWHG LQ UHODWLYHO\ UHFHQW WLPHV § 0ya; Lavoué et al. 2012) and is formed by 15 described species (Poll et al. 1982; Feulner et al. 2007). All members of the genus Campylomormyrus are characterized by the presence of prominent, tubular, elongated snouts (Fig. 2). All species are endemic to the Congo River basin, with the highest peaks of diversity observed in the rapids occurring along the last 300 km upstream of the river mouth. Over the past years, few molecular phylogenies of the genus were produced, which allowed to identify several monophyletic groups corresponding to well defined body morphologies and electric features (Feulner et al. 2006, 2007). These studies, however, did not allow to resolve the relationships between few terminal taxa (Fig. 2), associated to short branches, that may have being prone to extensive levels of Incomplete Lineage Sorting (ILS;