Allma Mater Studiiorum – Uniiversiità dii Bollogna DOTTORATO DI RICERCA IN BIODIVERSITÀ ED EVOLUZIONE Ciclo XXIII Settore/i scientifico-disciplinare/i di afferenza: BIO - 05 A MOLECULAR PHYLOGENY OF BIVALVE MOLLUSKS: ANCIENT RADIATIONS AND DIVERGENCES AS REVEALED BY MITOCHONDRIAL GENES Presentata da: Dr Federico Plazzi Coordinatore Dottorato Relatore Prof. Barbara Mantovani Dr Marco Passamonti Esame finale anno 2011 of all marine animals, the bivalve molluscs are the most perfectly adapted for life within soft substrata of sand and mud. Sir Charles Maurice Yonge INDEX p. 1..... FOREWORD p. 2..... Plan of the Thesis p. 3..... CHAPTER 1 – INTRODUCTION p. 3..... 1.1. BIVALVE MOLLUSKS: ZOOLOGY, PHYLOGENY, AND BEYOND p. 3..... The phylum Mollusca p. 4..... A survey of class Bivalvia p. 7..... The Opponobranchia: true ctenidia for a truly vexed issue p. 9..... The Autobranchia: between tenets and question marks p. 13..... Doubly Uniparental Inheritance p. 13..... The choice of the “right” molecular marker in bivalve phylogenetics p. 17..... 1.2. MOLECULAR EVOLUTION MODELS, MULTIGENE BAYESIAN ANALYSIS, AND PARTITION CHOICE p. 23..... CHAPTER 2 – TOWARDS A MOLECULAR PHYLOGENY OF MOLLUSKS: BIVALVES’ EARLY EVOLUTION AS REVEALED BY MITOCHONDRIAL GENES. p. 23..... 2.1. INTRODUCTION p. 28..... 2.2. MATERIALS AND METHODS p. 28..... Specimens’ collection and DNA extraction p. 30..... PCR amplification, cloning, and sequencing p. 30..... Sequence alignment p. 32..... Phylogenetic analyses p. 37..... Taxon sampling p. 39..... Dating p. 43..... 2.3. RESULTS p. 43..... Obtained sequences i p. 44..... Sequence analyses p. 45..... Taxon sampling p. 45..... Maximum Likelihood p. 47..... Bayesian Analyses p. 50..... Dating the tree p. 52..... 2.4. DISCUSSION p. 52..... The methodological pipeline p. 53..... The phylogeny of Bivalvia p. 59..... CHAPTER 3 – PHYLOGENETIC REPRESENTATIVENESS: A NEW METHOD FOR EVALUATING TAXON SAMPLING IN EVOLUTIONARY STUDIES p. 59..... 3.1. BACKGROUND p. 62..... 3.2. RESULTS p. 62..... Algorithm p. 64..... Testing p. 69..... Implementation p. 72..... 3.3. DISCUSSION p. 78..... 3.4. CONCLUSIONS p. 80..... 3.5. METHODS p. 80..... Average Taxonomic Distinctness (AvTD) p. 82..... Test of significance p. 82..... Variation in Taxonomic Distinctness (VarTD) p. 83..... Von Euler’s index of imbalance p. 85..... Shuffling analysis p. 86..... Shuffling phase p. 87..... Analysis phase ii p. 88..... CHAPTER 4 – A MOLECULAR PHYLOGENY OF BIVALVE MOLLUSKS: ANCIENT RADIATIONS AND DIVERGENCES AS REVEALED BY MITOCHONDRIAL GENES p. 88..... 4.1. INTRODUCTION p. 91..... 4.2. MATERIALS AND METHODS p. 91..... Taxon sampling, PCR amplification, and sequencing p. 93..... Assembling the dataset p. 94..... Evaluating phylogenetic signal p. 96..... Model decision tests and tree inference p. 100..... 4.3. RESULTS p. 100..... Sequence data p. 101..... Evaluating phylogenetic signal p. 108..... Phylogenetic reconstructions p. 118..... 4.4. DISCUSSION p. 118..... Phylogenetic signal p. 119..... Bivalve phylogeny p. 124..... Tracing and optimizing major morphological characters on the evolutionary tree p. 127..... 4.5. CONCLUSIONS AND FINAL REMARKS p. 129..... CHAPTER 5 – A TWO-STEPS BAYESIAN PHYLOGENETIC APPROACH TO THE MONOPHYLY OF CLASS BIVALVIA (MOLLUSCA) p. 129..... 5.1. INTRODUCTION p. 133..... 5.2. MATERIALS AND METHODS p. 133..... Assembling the dataset p. 133..... Alignments p. 134..... Preliminary analyses p. 135..... Model decision tests and tree inference iii p. 138..... 5.3. RESULTS p. 138..... Preliminary analyses and phylogenetic signal p. 143..... Phylogenetic trees p. 147..... 5.4. DISCUSSION p. 151..... CHAPTER 6 – CITED REFERENCES p. 188..... CHAPTER 7 – APPENDICES p. 188..... Appendix 2.1 p. 190..... Appendix 2.2 p. 191..... Appendix 2.3 p. 192..... Appendix 2.4 p. 193..... Appendix 2.5 p. 194..... Appendix 2.6 p. 196..... Appendix 2.7 p. 197..... Appendix 2.8 p. 197..... Appendix 2.9 p. 198..... Appendix 2.10 p. 199..... Appendix 2.11 p. 200..... Appendix 3.1 p. 202..... Appendix 4.1 p. 205..... Appendix 4.2 p. 206..... Appendix 5.1 p. 208..... Appendix 5.2 p. 209..... CHAPTER 8 – PUBLISHED PAPERS p. 267..... ACKNOWLEDGEMENTS iv FOREWORD The main scope of my PhD is the reconstruction of the large-scale bivalve phylogeny on the basis of four mitochondrial genes, with samples taken from all major groups of the class. To my knowledge, it is the first attempt of such a breadth in Bivalvia. I decided to focus on both ribosomal and protein coding DNA sequences (two ribosomal encoding genes, 12s and 16s, and two protein coding ones, cytochrome c oxidase I and cytochrome b), since either bibliography and my preliminary results confirmed the importance of combined gene signals in improving evolutionary pathways of the group. Moreover, I wanted to propose a methodological pipeline that proved to be useful to obtain robust results in bivalves phylogenesis. Actually, best-performing taxon sampling and alignment strategies were tested, and several data partitioning and molecular evolution models were analyzed, thus demonstrating the importance of molding and implementing non-trivial evolutionary models. In the line of a more rigorous approach to data analysis, I also proposed a new method to assess taxon sampling, by developing Clarke and Warwick statistics: taxon sampling is a major concern in phylogenetic studies, and incomplete, biased, or improper taxon assemblies can lead to misleading results in reconstructing evolutionary trees. Theoretical methods are already available to optimize taxon choice in phylogenetic analyses, but most involve some knowledge about genetic relationships of the group of interest, or even a well-established phylogeny itself; these data are not always available in general phylogenetic applications. The method I proposed measures the "phylogenetic representativeness" of a given sample or set of samples and it is based entirely on the pre- existing available taxonomy of the ingroup, which is commonly known to investigators. Moreover, it also accounts for instability and discordance in taxonomies. A Python-based script suite, called PhyRe, has been developed to implement all analyses. 1 Plan of the Thesis This Thesis, after a general introduction (Chapter 1), is divided into four parts, each representing the main arguments of my research during PhD. Chapter 2 is the first attempt, with a partial dataset, to draw a phylogeny of Bivalvia, especially of deeper nodes, and to establish a methodological pipeline for further studies. This part has been already published in Molecular Phylogenetics and Evolution (Plazzi and Passamonti, 2010). Chapter 3 is dedicated to the abovementioned "phylogenetic representativeness" and the software PhyRe. This part has also already been published in BMC Bioinformatics (Plazzi et al., 2010). Chapter 4 re-analyzes Bivalvia phylogeny through a larger dataset, and better specifies phylogenetic relationships among the lower level groups of Bivalvia, whenever the dataset was suitable. Chapter 5 will address the ongoing question of the monophyly or polyphyly of Bivavia. Papers from chapters 4 and 5 will be submitted shortly for publication. Finally, Chapter 6 lists cited references in the whole Thesis, Chapter 7 is composed by Appendices, and Chapter 8 includes copies of the papers I published during my PhD, also the ones that are not directly related to the main topic of the present Thesis. 2 CHAPTER 1 INTRODUCTION 1.1. BIVALVE MOLLUSKS: ZOOLOGY, PHYLOGENY, AND BEYOND The phylum Mollusca The outstanding scientific interest for the second richest phylum in the animal kingdom – slightly less than 100,000 species known (Brusca and Brusca, 2003) – and the over time passionate work of collectors and amateur malacologists, led to a stunning abundant literature in the field of mollusk taxonomy and systematics. Georges Cuvier (1769-1832) was the first to establish the group “Mollusca” (in 1795) as something similar to the assemblage we refer to with this name. Since then, barnacles, tunicates, and brachiopods were purged from the phylum: mollusks are now bilaterally symmetrical animals, unsegmented lophotrochozoan protostomes, typically featuring a dorsal visceral mass, a mantle secreting calcareous epidermal spicules, shell plates, or a true shell, a bold muscular foot, and a radula. Despite the lack of a complete agreement in the general classification of mollusks, the phylum can be arranged in seven or eight classes. Some of them are very poorly known, such as the unconventional grouping of Aplacophora, including Chaetodermomorpha (=Caudofoveata) and Neomeniomorpha (=Solenogastres), and the class of Monoplacophora, thought to be extinct until Lemche‟s (1957) discovery of a living species, Neopilina galatheae. Also Chitons (class Polyplacophora) and tusk shells (class Scaphopoda) are better known to museum visitors and zoology students, rather than to non specialists. On the contrary, humans were always very familiar with the remaining three classes of mollusks, which were commonly used as popular tools, musical devices, 3 money, decorations, and – hence the huge economical worth – food: cowries, limpets, snails, slugs (class Gastropoda); cuttlefishes, squids, octopuses, nautiluses (class Cephalopoda); clams, cockles, oysters, quahogs, scallops, mussels (class Bivalvia). Notwithstanding the importance they have for mankind, our knowledge of mollusks‟ evolutionary history is still limited. The sister group of mollusks was variably found in Sipunculida (peanut worms; Scheltema, 1993) or Ectoprocta