UNIVERSITY OF SOUTHAMPTON Systematics and Phylogeny of the Holothurian Family Synallactidae Francisco Alonso Solís Marín Doctor of Philosophy SCHOOL OF OCEAN AND EARTH SCIENCE July 2003 Graduate School of the Southampton Oceanography Centre This PhD dissertation by: Francisco Alonso Solís Marín Has been produced under the supervision of the following persons: Supervisors: Prof. Paul A. Tyler Dr. David Billett Dr. Alex D. Rogers Chair of Advisory Panel: Dr. Martin Sheader “I think the Almighty put synallactids on this earth as some sort of punishment.” Dave Pawson DECLARATION This thesis is the result of work completed wholly while registered as a postgraduate in the School of Ocean and Earth Science, University of Southampton. UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF SCIENCE SCHOOL OF OCEAN AND EARTH SCIENCE Doctor of Philosophy Systematics and Phylogeny of the Holothurian Family Synallactidae By Francisco Alonso Solís-Marín The sea cucumbers of the family Synallactidae (Echinodermata: Holothuroidea) are mostly restricted to the deep sea. They comprise of approximately 131 species, about one-third of all known deep-sea holothurian species. Many species are morphologically similar, making their identification and classification difficult. The aim of this study is to present the phylogeny of the family Synallactidae based on DNA sequences of the mitochondrial large subunit rRNA (16S), cytochrome oxidase I (COI) genes and morphological taxonomy characters. In order to examine type specimens, corroborate distributional data and collect muscles tissues for the DNA analyses, 7 institutions that hold holothurian specimens were visited. For each synallactid species, selected synonymy, primary diagnosis, location of type material, type locality, distributional data (geographical and bathymetrical) and extra biological information were extracted from the primary references. 17 species were screened for mitochondrial DNA in this thesis; 2 Bathyplotes, 5 Mesothuria, 3 Pseudostichopus, 1 Pelopatides, 2 Paroriza, 1 Benthothuria, 1 Zygothuria and the species Isostichopus fuscus (Ludwig), which was used as an outgroup for the phylogenetic analysis. The elasipodid species Deima validum was screened in order to evaluate the phylogenetic relation of the Elasipoda with the Synallactidae. A new species of the genus Pseudostichopus is described based on molecular and morphology evidence. A new species of the genus Mesothuria is proposed based on geographical, morphological and molecular evidence, and a new species of the genus Synallactes is described based on morphological data. The results of this study constitute the first phylogenetic test of the classification of Synallactidae. The family Synallactidae is a polyphyletic group. The family seems to be formed by 2 groups that can have different ancestors, a) a Paroriza-Pseudostichopus- Pelopatides-Mesothuria-Zygothuria group, b) a non-synallactid group with Benthothuria funebris and Paroriza prouhoi. There is morphological consistency in the Synallactidae, but it is not enough to validate the monophyly of the family. When combining elasipodid and synallactid mitochondrial DNA sequences in one analysis the “elasipodid clade”, is separated into two branches. One branch is formed by Deima validum, Paroriza prouhoi and Benthothuria funebris. These three species have no morphological relation, but their mitochondrial DNA reveals their cryptic relationship. This study contributes to the understanding of the deep-sea synallactid holothurian taxonomy. Significant progress has been made in combining morphological and molecular approaches to taxonomy, demonstrating the need for more studies of this nature in the future. CONTENTS CHAPTER ONE – GENERAL INTRODUCTION AND AIMS 1.1. The phylum Holothuroidea (Echinodermata) …………………………………………….………. 1 Figure 1.1. Representative holothurian body types …………………………………………….………. 2 Figure 1.2. Some holothurian ossicles ………………………………………………………………….……….. 3 Figure 1.3. Internal anatomy of a dendrochirotid holothurian, dissected from the left side ………….………….. 5 1.1. The family Synallactidae ……………………………………………………………….………….. 7 1.2.1. General remarks …………………………………………………………………………….…………….. 7 Figure 1.4. Synallactid ossicles …………………………………………………………………….……... 7 1.3. Ecology ………………………………………………………………………………………………… 9 1.3.1. Feeding ………………………………………………………………………………………………… 9 Table 1.1. Comparison of tentacles of synallactids ……………………………………………………… 9 1.3.2. Reproduction …………….………………………………………………………………………………… 11 1.3.3. Swimming …………………………………………………………………………………………... 12 1.3.4. Bathymetric distribution ……………………………………………………………………... 13 1.3.5. Geographical distribution ……………………………………………………………………… 14 1.4. History of exploration and study of the deep-sea holothurians, with a particular reference on the family Synallactidae ………………………………………………………………………………… 15 1.5. Basic problems with holothurian taxonomy ………………………………………………… 24 1.6. Molecular taxonomy ………………………………………………………………………………… 27 1.6.1. Advantages of molecular data ……………………………………………………………………… 28 1.6.2. Advantage of morphological data …………………………………………………………… 29 1.6.3. Integrating molecular and morphological data ………………………………………………… 30 1.6.4. The advantages in using mitochondrial DNA as a phylogenetic tool …………………………… 30 1.7. Aims of the present work ……………………………………………………………………… 32 CHAPTER TWO – GENERAL MATERIALS AND METHODS 2.1. Holothurian collection and fixation ………………………………………………………..…… 33 Figure 2.1. Bathymetric chart of the study area in the North Atlantic with 1000-4000m isobaths ……….. 34 2.2. Dissection of the specimens ……………………………………………………………………….. 35 Figure 2.2. “Fresh” specimen of Benthothuria funebris Perrier dissected on board RRS Discovery ……….. 35 2.3. Photographic records ………………………………………………………………………………….. 36 Table 2.1. Museums and collections visited or referenced during the present study ………………….. 36 2.4. Systematics …………………………………………………………………………………………….. 37 2.4.1. Taxonomic identification ……………………………………………………………………….. 37 2.4.2. Re-examined material from museums collections ………………………………………………….. 37 2.4.3. Ossicle preparation …………………………………………………………………………………… 37 2.5. Reproduction ……………………………………………………………………………………………… 38 2.5.1. Histology ……………………………………………………………………………………………… 38 2.5.2. Staining ……………………………………………………………………………………………… 39 2.5.3. Image analysis …………………………………………………………………………………… 39 2.6. DNA analyses …………………………………………………………………………………… 40 2.6.1. DNA extraction …………………………………………………………………………………… 40 Table 2.2. Oligonucleotide primers sequences used in the present study ………………………………… 41 2.6.2. Thermal cycle amplification ………………………………………………………………………… 42 2.6.3. Product purification …………………………………………………………………………………… 42 2.6.4. Sequencing ……………………………………………………………………………………………… 42 2.7. Phylogenetic analysis inferred from molecular characters ………………………………………… 43 2.7.1. Sequence alignments …………………………………………………………………………………… 43 2.7.2. Phylogenetic analysis …………………………………………………………………………………… 43 2.8. Phylogenetic analysis inferred from morphology …………………………………………………… 44 2.8.1. The ingroup ……………………………………………………………………………………………… 44 2.8.2. The outgroup ……………………………………………………………………………………………… 45 2.8.3. Phylogenetic analysis ……………………………………………………………………………………. 46 CHAPTER THREE – MOLECULAR TAXONOMY, DISTRIBUTION AND REPRODUCTION IN THE GENERA MESOTHURIA AND ZYGOTHURIA (HOLOTHUROIDEA: SYNALLACTIDAE) FROM THE NORTH ATLANTIC OCEAN 3.1. Introduction ……………………………………………………………………………………………… 48 3.2. Materials and methods …………………………………………………………………………………… 49 Table 3.1. List of stations where Zygothuria lactea was collected by RRS Discovery and RRS Challenger …………………………………………………………………………………………………………………… 50 Table 3.2. List of stations where Mesothuria sp. 1. was collected by RRS Discovery and RR Challenger ………………………………………………………………………………………………………………….... 51 3.2.1. Identification ……………………………………………………………………………………………… 51 3.2.2. Molecular analysis …………………………………………………………………………………… 51 Table 3.3. 16S Oligonucleotid primers sequences used in the present study ……………………………… 52 Table 3.4. DNA sequences used in the molecular analysis …………………………………………………… 52 Table 3.5. Nucleotide substitution rate matrix ……………………………………………………………… 53 3.3. Results ………………………………………………………………………………………………… 54 3.3.1. Systematics ……………………………………………………………………………………………… 54 Genus Mesothuria Ludwig, 1894 ………………………………………………………………………… 54 Mesothuria verrilli (Théel, 1886a) ………………………………………………………………………… 55 Table 3.6.. Age variation of ossicles in Mesothuria verrilli …………………………………………………… 56 Figure 3.1. Mesothuria verrilli (Théel, 1886) ………………………………………………………………. 58 Figure 3.2. Distribution of Mesothuria verrilli and Mesothuria sp . 1 ………………………………………… 60 Mesothuria sp. 1 ………….…………………………………………………………………………………… 61 Table 3.7. Age variation of ossicles in Mesothuria sp. 1 …………………………………………………… 62 Figure 3.3. Mesothuria sp. 1 ……………………………………………………………………………………. 63 Mesothuria bifurcata Hérouard, 1901 ………………………………………………………………………… 66 Genus Zygothuria Perrier, 1898 ………………………………………………………………………… 67 Zygothuria lactea (Théel, 1886a) ………………………………………………………………………… 68 Figure 3.4. Zygothuria lactea (Théel, 1886a) ……………………………………………………………… 71 Figure 3.5.. Zygothuria lactea (Théel, 1886a) ……………………………………………………………… 72 Figure 3.6. Distribution of Zygothuria lactea and Z. oxysclera ………………………………………… 73 Zygothuria oxysclera (Perrier, 1902) ………………………………………………………………………… 74 Figure 3.7. Zygothuria oxysclera (Perrier, 1902) ……………………………………………………………… 75 Figure 3.8. Distribution of a group of Mesothuria species …………………………………………………… 79 Figure 3.9. Distribution of a group