Phylogeny of the Family Eunicidae (Polychaete, Annelida) Based on Molecular and Morphological Data

Home , Eunice aphroditois, Eunice norvegica, Eunicida, Eunicidae, Lysidice (annelid), Nereis

PHYLOGENY OF THE FAMILY EUNICIDAE (POLYCHAETE, ANNELIDA) BASED ON MOLECULAR AND MORPHOLOGICAL DATA

by Joana Zanol Pinheiro da Silva

Teaching License in Biology, 1999, Universidade Federal do Rio de Janeiro, Brazil

Master in Science-Zoology, 2002, Universidade Federal do Rio de Janeiro, Brazil

A Dissertation submitted to

The Faculty of Columbian College of Arts and Sciences of The George Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

January 31, 2009

Dissertation directed by

Diana Leigh Lipscomb Professor of Biology

Kristian Fauchald Research Zoologist, NMNH

The Columbian College of Arts and Sciences of The George Washington University certifies that Joana Zanol Pinheiro da Silva has passed the Final Examination for the degree of Doctor of Philosophy as of November 18, 2008. This is the final and approved form of the dissertation.

PHYLOGENY OF THE FAMILY EUNICIDAE (POLYCHAETE, ANNELIDA) BASED ON MOLECULAR AND MORPHOLOGICAL DATA

Joana Zanol Pinheiro da Silva

Dissertation Research Committee:

Diana Leigh Lipscomb, Professor in Biology, Co-Director

Kristian Fauchald, Research Zoologist, NMNH, SI, Co-Director

Sheri A. Church, Assistant Professor of Biological Sciences, Committee

Member

James M. Clark, Ronald B. Weintraub Professor of Biology, Committee

Member

iii Dedication

To my parents and sister for the unconditional support always.

”Nothing in life is to be feared. It is only to be understood” Marie Curie

“An expert is a man who has made all the mistakes which can be made in

a very narrow field” Niels Bohr iv Acknowledgments

This work would not have been possible without the help, support and guidance of many people to whom I am greatly thankful. Foremost, I would like to thank both of my advisors Dr. Diana L. Lipscomb and Dr. Kristian Fauchald for the support, guidance and freedom for independent thinking and for allowing me to pursue my own ideas throughout

this research. I am also thankful to Dr. Kristian Fauchald for believing in me from my

undergraduate years, which pushed me forward and made this graduate experience

possible.

I would like to thank the members of my dissertation committee, Dr. Sheri A.

Church, Dr. James M. Clark, Dr. Gustavo Hormiga and Dr. Allen G. Collins, for the

discussions during the course of the research, the critical review of this work and helpful

suggestions that made it better, and for coping with tight schedules.

I am grateful to Dr. Kenneth M. Halanych for his essential contribution throughout

this work and technical support with the molecular bench work; and Dr. Torsten H. Struck

for sharing unpublished data and information and for the help in my first steps in the

molecular laboratory.

All professors, students and staff of the Department of Biological Sciences at GWU

were of great importance during this work. I am greatly thankful to them, especially the

systematics course professors, Dr. Marc W. Allard, Dr. James M. Clark, Dr. Patrick S.

Herendeen and Dr. Gustavo Hormiga, whose courses provided me with important

knowledge to carry out this research; Dr. Fernando Alvarez, M. Rosario Castañeda, Vinita

Gowda, Lara Lopardo and Dr. Dimitar Dimitrov, for the very fruitful informal discussion

v on systematics; M.Rosario Castañeda and Vinita Gowda, also for the very valuable

comments on the preview of my dissertation seminar; Dr. Patricia Hernandez and Nathan

Bird, for the comments on the confocal laser scanning microscopy results; Dr. Hartmut G.

Doebel and the late Paul Spiegler for the very valuable guidance through the Teaching

Assistant experience; Dr. John R. Burns and Dr. James M. Clark for being engaged graduate advisors; Rashelle Shanon and Hoda Fatah for going out of their way to help; and

Dr. Matjaz Kuntner, Dr. Ingi Agnarsson and Dr. Svetlana Maslakova, for the tips on the graduate life at GWU and in the United States.

I am also greatly thankful to all the staff in Invertebrate Zoology Department at

Natural History Museum, Smithsonian Institution, especially Dr. Jon Norenburg, for

providing important information for field work; Linda Ward, W. Geoff Keel, William

Moser, Paul R. Greenhall, Tim Coffer and Cheryl Bright, for the help with my research and for dealing with all the loans necessary for this project.

I am extremely grateful to all the staff and post-doctorate fellows at the Smithsonian

Marine Station at Fort Pierce, which made it possible to complete the work for Chapter 2 in a short period of time and for making my stay in Fort Pierce very pleasant. I am especially thankful to Dr. Mary Rice, Dr. Valerie Paul, Sherry Reed, Michael Scott Jones, Julie

Piraino, William (Woody) Lee, Hugh Reichardt and Joan Kaminski for all their help.

I am pleased to thank Dr. George von Dassow for the introduction and invaluable help with confocal laser scanning microscopy; Dr. Anja Schulze for important help with confocal laser scanning microscopy techniques; Scott Whittaker, for his unconditional help in all steps of Scanning Electron Microscopy (SEM); Lara Lopardo, for the introduction to

SEM; Nataliya Budaeva for the identification of onuphids; Dr. Christoph Bleidorn, Dr.

vi Tiago B. Quental, Dr. Omar Torres-Carvajal for discussions on phylogenetic analyses; Dr.

Kevin de Queiroz for discussion of various systematics topics and for giving me access to

his computers; Dr. Paulo C. Paiva, for constant encouragement, for dealing with Brazilian collecting permits, and for giving me access to his laboratory; Dr. Alexander Tzetlin, for the comments on the confocal laser scanning microscopy results; Diana Marques for the help with graphic softwares; Dr. Leonard Hirsch for constant encouragement, information on collecting permits and general discussions; and all members in Dr. Halanych’s lab in the

Spring 2005, especially Heather Blasczyk, Dr. Lee Weight, Jeff Hunt, Dr. Jeff Bates, and all other members of the LAB, NMNH, Smithsonian Institution. Institution, without whom the molecular laboratory work would not have been possible.

This work would not have been possible without the extensive donation and loan of specimens by several people from diverse institutions, for this reason I am indebted to Dr.

M. Teresa Aguado (Universidad Autónoma de Madrid, Spain), Dr. Christos Arvanitidis

(Hellenic Centre for Marine Research, Greece), Dr. Nicola Bettoso (Osservatorio Alto

Adriatico ARPA-FVG, Italy), Dr. Luis F. Carrera-Parra (El Colegio de la Frontera Sur,

Mexico), Dr. Danny Eibye-Jacobsen (Zoological Museum, Copenhagen, Denmark), Dr.

Maria Cristina Gambi (Stazione Zoologica Anton Dohrn, Italy), Dr Chris Glasby (Northern

Territory Museum, Australia), Dr. Bert Hoeksema (National Natural History Museum

Naturalis Leiden, Netherlands), Dr. Stéphane Hourdez (Station Biologique de Roscoff,

France), Dr. Ardis Johnston (Harvard Univeristy, USA), Dr. Stephen Keable (Australian

Museum, Australia), Dr. Ceri Lewis (University of the Western Cape, South Africa), Dr.

Eduardo López (Universidad Autónoma de Madrid, Spain), Linda Meurisse (Institut Royal

Des Sciences Naturelles de Belgique, Belgium), Dr. Tarik Meziane (Musée National

vii d'Histoire Naturelle, France), Dr. Birger Neuhaus (Museum für Naturkunde der Humboldt-

Universität zu Berlin, Germany), Anderson Oliveira (Universidade do Estado do Rio de

Janeiro, Brazil), Dr. Julio Parapar (Universidade da Coruña, Spain), Dr. Fredrik Pleijel

(Tjärnö Marine Biological Laboratory, Göteborg University, Sweden), Dr. Alexandra

Rizzo (Museu de Zoologia da Universidade de São Paulo, Brazil), Dr. Anja Schulze,

(Texas A&M University, Galveston Campus, Texas), Dr. Emma Sherlock (The Natural

History Museum, United Kingdom), Dr. Elin Sigvaldadottir (The Swedish Museum of

Natural History, Sweden), Roger Springthorpe (Australian Museum, Australia), Leonne

Vermond (National Natural History Museum Naturalis Leiden, Netherlands), Dr. Miguel

Villena (Museo Nacional de Ciencias Naturales, Spain), Sara C. Watson (America

Museum of Natural History, USA) and Gisela Weigel (Zoologisches Museum, Universität

Hamburg, Germany).

I am also indebted with all those who helped in the sampling of the specimens used here, Leslie H. Harris, Dr. Bruno Pernet, Shelly Walther, David E. Montagne, John, all the staff in the July 2004 monitoring cruise of the County Sanitation District of Los Angeles

County, Dr. Rosebel C. Nalesso, Wilson Franklin Junior, Rossana Sousa, Tatiane Martins

Garcia, Dr. Flavia Mochel, Jane, Luiz, other UFMA undergraduate students, Dr Martin

Christoffersen, Andre Souza, Sherry Reed, Michael Scott Jones, William (Woody) Lee,

Natalyia Budaeva, Geoff Keel, Dr. Fredrik Pleijel and Dr.Greg Rouse. These collections would not have been possible without the kind hospitality of Leslie H. Harris, David Ocker,

Liliane Zanol, Bernardo and Júlia Zanol Xavier.

viii I extend my thanks to all friends and extended family, who were very important providing emotional support and encouragement, especially Vinita Gowda, Diana Marques,

Simone S. O. Pszczol, Tiago B. Quental, Christine Ruta and Paulo C. Paiva.

Finally, I would like to thank my parents for the constant invaluable support and for setting the grounds of whom I am today, always encouraging me to follow my dreams and, to work for the things I want; my sister for always being there, believing in me and encouraging me to move forward; and my late beloved grandma, who was very puzzled with what I was doing but nevertheless very proud of it.

This research was funded by a NSF doctoral dissertation improvement grant, NSF-

WormNet grant (EAR-0120646), Link Foundation/Smithsonian Institution Graduate

Fellowship, Cosmos Club Grants in aid of research and SICB-Libbie H. Hyman Memorial

Scholarship.

Abstract of Dissertation

PHYLOGENY OF THE FAMILY EUNICIDAE (POLYCHAETE, ANNELIDA) BASED ON MOLECULAR AND MORPHOLOGICAL DATA

The bristle worm family Eunicidae (~326 spp. and 9 genera) inhabits soft and hard

marine bottoms. Many burrow into hard corals and calcareous algae or live in crevices in

the reef and play important roles in coral reef communities (Hutchings, 1986). The family

is distributed worldwide, but it is most common in shallow tropical waters (Fauchald,

1992a). The monophyly of Eunicidae is controversial in both morphological and molecular

phylogenies. The spectrum of characters traditionally used is not enough to define and

support a monophyletic Eunicidae or some of its best known genera. As traditionally

recognized Eunicidae has no apomorphic morphological features, nevertheless its anterior

end is characteristic and distinct from the other families in the order Eunicida. This inability to define clear characters is due to inconsistent hypotheses of homology between the different parts of the anterior end in the order Eunicida and poor understanding of morphological variability in the family Eunicidae.

In this study we used α-tubulin immunostaining and confocal Laser Scanning

Microscope to examine the anterior nervous system of five families of Eunicida, thus improving information about morphological and innervation similarities and hypotheses of homology among features such as buccal lips, pharyngeal fold, dorsolateral fold and anterior extension of the dorsolateral fold of these families. Light microscopy and SEM x examination of characters along the body further improved our knowledge of traditional

characters and of their variation. These new approaches of morphological analysis

improved character definitions, increased the number of morphological characters and

improved the morphological phylogenetic signal. Furthermore, sequences of nucleotides of

the genes 16S, COI and 18S added additional phylogenetically informative data at all levels

of the tree and were essential in recovering more stable and better supported relationships.

Eunicidae is monophyletic, supported by at least two unique synapomorphies (dorsal

buccal lip fused to the dorsal side of the prostomium and the anterior extensions of the dorsolateral fold medially connected) with Onuphidae as its sister group. Eunice sensu lato is polyphyletic. However, Eunice sensu stricto cannot be defined because of the ambiguous placement of the type species, Eunice aphroditois. The genera Leodice and Nicidion are resurrected to include species of Eunice sensu lato and Marphysa sensu lato placed in the same clades as their respective type species and diagnosed by synapomorphies supporting the clades. Nematonereis is included in Lysidice, making the latter monophyletic and more inclusive with the lack of palps as one of its unique synapomorphies. Marphysa is emended to include only the species in the Marphysa sensu stricto clade, including Nauphanta mossambica. Nauphanta is valid but it has no diagnostic features at this point.

Aciculomarphysa, Euniphysa, Fauchaldius, and Palola are valid genera and no changes are made to their diagnostic traditional characters.

The absence/presence pattern of peristomial cirri, palps and lateral antennae are due to loss during the evolution of the family and not stepwise addition. The pattern of loss follows the inverted pattern observed for the addition of these appendages in the ontogenetic sequences of species having all appendages. This suggests that the pattern

xi observed in the adults lacking some appendages may be a paedomorphic characteristic in

Eunicidae.

The integrative approach including different method of analyses of the morphology and molecular data, used here was essential to produce well resolved and supported relationships and to identify morphological apomorphies that could be used to redefine monophyletic taxa. Thus it may offer a way to deal with groups for which traditional morphological characters and molecular data alone are insufficient and inconsistent hypotheses of homology have been applied.

xii Table of Contents

Dedication...... iv

Acknowledgments...... v

Abstract of Dissertation ...... x

Table of Contents ...... xiii

List of Figures ...... xiv

List of Tables...... xvii

List of Symbols / Nomenclature...... xviii

Chapter 1: Introduction...... 1

Chapter 2: Cephalic and pharyngeal innervation in the order Eunicida

(polychaete, Annelida) using confocal laser scanning microscopy (cLSM) and its implications for homology...... 11

Chapter 3: Molecular phylogeny of the family Eunicidae (polychaete,

Annelida) based on 16S, COI and 18S nucleotide sequences...... 78

Chapter 4: Total evidence phylogeny and generic reclassification of the family Eunicidae (polychaete, Annelida) ...... 140

Chapter 5: Conclusion...... 263

References...... 266

Appendices ...... 288

xiii List of Figures

Figure 2.1………………………………………………………………………………61

Figure 2.2………………………………………………………………………………63

Figure 2.3………………………………………………………………………………66

Figure 2.4………………………………………………………………………………68

Figure 2.5………………………………………………………………………………70

Figure 2.6………………………………………………………………………………72

Figure 2.7………………………………………………………………………………74

Figure 2.8………………………………………………………………………………76

Figure 3.1………………………………………………………………………………128

Figure 3.2………………………………………………………………………………130

Figure 3.3………………………………………………………………………………132

Figure 3.4………………………………………………………………………………134

Figure 3.5………………………………………………………………………………136

Figure 3.6………………………………………………………………………………138

Figure 4.1………………………………………………………………………………201

Figure 4.2………………………………………………………………………………203

Figure 4.3………………………………………………………………………………205

Figure 4.4………………………………………………………………………………207

Figure 4.5………………………………………………………………………………209

Figure 4.6………………………………………………………………………………211

Figure 4.7………………………………………………………………………………213

xiv Figure 4.8………………………………………………………………………………215

Figure 4.9………………………………………………………………………………217

Figure 4.10..……………………………………………………………………………219

Figure 4.11..……………………………………………………………………………221

Figure 4.12..……………………………………………………………………………223

Figure 4.13..……………………………………………………………………………225

Figure 4.14..……………………………………………………………………………227

Figure 4.15..……………………………………………………………………………229

Figure 4.16..……………………………………………………………………………231

Figure 4.17..……………………………………………………………………………233

Figure 4.18..……………………………………………………………………………235

Figure 4.19..……………………………………………………………………………237

Figure 4.20..……………………………………………………………………………239

Figure 4.21..……………………………………………………………………………241

Figure 4.22..……………………………………………………………………………243

Figure 4.23..……………………………………………………………………………245

Figure 4.24..……………………………………………………………………………247

Figure 4.25..……………………………………………………………………………249

Figure 4.26..……………………………………………………………………………251

Figure 4.27..……………………………………………………………………………253

Figure 4.28..……………………………………………………………………………255

Figure 4.29..……………………………………………………………………………257

Figure 4.30..……………………………………………………………………………259

xv Figure 4.31..……………………………………………………………………………261

xvi List of Tables

Table 1.1………………………………………………………………………………….8

Table 1.2………………………………………………………………………………….9

Table 1.3………………………………………………………………………………….10

Table 2.1………………………………………………………………………………….60

Table 3.1…………………………………………………………………………………117

Table 3.2…………………………………………………………………………………119

Table 3.3…………………………………………………………………………………120

Table 3.4…………………………………………………………………………………121

Table 3.5…………………………………………………………………………………122

Table 3.6…………………………………………………………………………………123

Table 3.7…………………………………………………………………………………125

Table 3.8…………………………………………………………………………………126

Table 4.1…………………………………………………………………………………199

Table 4.2…………………………………………………………………………………200

xvii List of Symbols / Nomenclature

1. AIC- Akaike Information Criterion

2. alp- anterolateral nerve projection

3. alp-dbvr- ventralmost side of the alp composed by the ventralmost nerves of the

dbvr

4. alp-dfm- dorsalmost side of alp composed by nerves of the dfm

5. AM- Australian Museum, Sidney, Australia

6. BF- Bayes Factor

7. BI- Bayesian inference

8. bl- buccal lip

9. BP- nonparametric bootstrap branch support value

10. cas- commissure of the association system

11. cc- circumoesophageal connective

12. CI- consistence index

13. cLSM- confocal laser scanning microscopy

14. COI- cytochrome oxidase I

15. dbl- dorsal buccal lip

16. dbvr- dorsal branch of the ventral root of the circumoesophageal connective

17. dcdr- dorsal commissure of the dorsal root of the circumoesophageal connective

18. dcdrn- nerves branching from the dcdr

19. dcvr- dorsal commissure of the ventral root of the circumoesophageal connective

20. dcvrn- nerves branching from the dcvr

xviii 21. dcvrnL1and2- lateral nerves branching from the dcvr

22. dcvrnM- median nerves branching from the dcvr

23. dfm- dorsal fibril mass

24. dfmn- nerves branching from the dfm

25. dfmnL- lateral nerve branching from the dfm

26. dfmnM1- ventralmost median nerve branching from the dfm

27. dfmnM2- dorsalmost median nerve branching from the dfm

28. dla- dorsolateral antennae

29. dlf- dorsolateral fold

30. dlfae- dorsolateral fold anterior extension

31. dlff- dorsolateral fold fusion

32. dln- dorsolongitudinal nerves branching from nerve cells posterior to the dnrp

33. DM1- pair of muscle bundles extending from the muscularized pharynx into the

posterior extension of the prostomium

34. dnrp- dorsal neuropile

35. drcc- dorsal root of the circumoesophageal connective

36. ESS- effective sample size

37. Γ, gamma shape parameter- rate heterogeneity

38. GTR- General Time Reversible model

39. IBUFRJ- Departamento de Zoologia, Instituto de Biologia, UFRJ, Rio de Janeiro,

Brazil

40. ILD- Incongruence Length Difference test

41. IRSNB- Institut Royal des Sciences Naturelles de Belgique, Brussels, Belgium

xix 42. ivf- inconspicuous ventral furrow on the ventral side of the prostomium

43. K80- Kimura-2-parameter

44. lgvbvr- long nerves branching from the anterior end of the vbvr

45. lnalp- lateral nerve branching from the alp-dbvr

46. lno- lateral no

47. lnon- nerves of lateral no

48. ma- median antenna

49. mdf- mandible fold

50. Mkv- Markov model accounting for variable number of states, conditioned to the

presence of only variable characters in the data set

51. ML- maximum likelihood

52. mlnalp- midlateral nerve branching from the alp-dbvr

53. mnalp- median nerve branching from the alp-dbvr

54. MNCN- Museo Nacional de Ciencias Naturales de Madrid, Madrid, Spain

55. mndla- median nerves of the dorsolateral antennae

56. mno- median no

57. mnon- nerves of the median no

58. mp- median plexus

59. MP- maximum parsimony

60. MR- morphological data

61. MRL- morphological and molecular data

62. MxI- maxillae I

63. MxII- maxillae II

xx 64. MxC- maxillary carrier

65. mxf- maxillary fold

66. MZSP- Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil

67. nbl- nerve of the buccal lip

68. nc- nuchal commissure

69. ndbl- nerve of the dorsal buccal lip

70. ndla- nerve of the dorsolateral antennae

71. nma- nerve of the median antenna

72. nrcl- nerve cells

73. NTM- Northern Territory Museum, Darwin, Australia

74. nvbl- nerve of the ventral buccal lip

75. oc- ocular commissure

76. ocn- ocular nerves

77. oeg- oesophageal ganglion

78. oen- oesophageal nerve

79. p- palp

80. PABA- Partition Addition Bootstrap Alteration

81. PB- posterior probability

82. pcas- posterior commissure of the association system

83. pep- posterior extension of the prostomium

84. phf- pharyngeal fold

85. phn-pharyngeal nerves

xxi 86. phn1- nerve branching from the main phn trunk at the anterior end of the

muscularized pharynx

87. phn2-6- nerves branching from the main phn trunk at the ventrolateral sides of the

muscularized pharynx

88. phn1phn4-uc- u-shaped commissure connecting phn1+phn4 from opposite sides

89. phnmxI- nerve branching from phn1+ phn4 which follows the outline of the

ventroposterior end of MxI

90. phnmxII- nerve branching from phnmxI and extending to the ventroposterior end of

MxII

91. pn5- nerves of the palp branching between vrcc and the vcvr2

92. pn6- nerves of the palp branching from the vcvr1

93. pn8- nerves of the palp branching from the dfm

94. pn12- nerves of the palp branching from the drcc

95. pnalp- posterolateral nerve branching from the alp which extends towards the

posterior end, loops forward and runs on a slant ventro-anteriorly

96. pnr5- nerve roots of pn5

97. pnr6- nerve roots of pn6

98. pnr8- nerve roots of pn8

99. pnr12- nerve roots of pn12

100. pro- prostomium

101. rDNA- ribosomal DNA

102. RI- retention index

103. SH- Shimodaira-Hasegawa test

xxii 104. shvbvr- short nerves branching from the anterior end of the vbvr

105. SPB- Sörensen’s phosphate buffer

106. stdl- stomatogastric dorsolateral nerve

107. stdp- stomatogastric dorsoparamedian nerve

108. stdp1- thick branch of the stdp

109. stdp1c- commissure of the stdp1

110. SEM- scanning electron microscope

111. SMNH- Swedish Museum of Natural History, Stockholm, Sweden

112. stdp1wr- wrapping of the stdp1 around the MxC

113. stdp2- thin nerve fiber branching from stdp

114. stdp2c- commissure of the stdp2 posterior to the muscularized pharynx

115. stn- stomatogastric nerve

116. SYM- Symmetric Model

117. TBR- Tree-Bisection-Reconnection

118. tpe- transverse anterior peristomial extension of the dorsolateral fold

119. TVM- Transversion Model

120. USNM- United States National Museum

121. vacas- ventroanterior commissure of the association system

122. vbl- ventral buccal lip

123. vbvr- ventral branch of the ventral root of the circumoesophageal connective

124. vcdr- ventral commissure of the dorsal root of the circumoesophageal connective

125. vcdrn- nerve branching from vcdr

126. vcvr- ventral commissure of the ventral root of the circumoesophageal connective

xxiii 127. vnrp- ventral neuropile

128. vp- ventral pads on the ventral side of the prostomium

129. vrcc- ventral root of the circumoesophageal connective

130. xc- x shaped connection y

131. Z07- characters unchanged from Zanol et al. (2007)

132. Z07m- characters modifies from Zanol et al. (2007)

133. ZMB- Museum für Naturkunde, Berlin, Germany

134. ZMH- Zoologisches Museum, Hamburg, Germany.

xxiv Chapter 1: Introduction

Polychaetes, bristle worms, (~83 families/~10,000 spp) are among the most diverse groups of marine invertebrates (Hutchings, 1998). They are distributed worldwide and frequently dominate macrobenthic communities in terms of species richness and abundance

(e.g., Grassle and Maciolek, 1992; Diaz-Castañeda and Almeda-Jauregui, 1999; Kröncke et al., 2003). Many species play key roles in communities by changing the environment (e.g., tube dwelling species; Woodin, 1981) and reworking sediment within the sea bed (e.g., burrowing species; Hutchings, 1998; Knox, 1977; François et al., 2002). We now recognize that Polychaeta is a paraphyletic grade at the base of the annelid tree with

Clitellata (Oligochaeta, earth worms, Hirudinea, leeches) as well as the phyla Echiura and

Sipuncula nested within it (McHugh, 2000; Struck et al., 2007). However, the annelid phylogeny is still uncertain, despite the numerous efforts to understand it (Colgan et al.,

2006; Rousset et al., 2007; Struck et al., 2007, Dunn et al., 2008).

The traditional Polychaeta contained at least seven orders (Rouse and Pleijel, 2001).

Eunicidae is the nominal family for the order Eunicida, which is composed of seven extant and thirteen extinct families dating back to the Ordovician (Kielan-Jaworowska, 1966)

(Table 1.1). This order has long been recognized by the presence of a jaw apparatus composed of a ventral mandible and dorsolateral maxillae in a ventral muscularized pharynx (Dales, 1962) but there is dispute as to which taxonomic rank it represents (e.g.,

Hartman, 1944; Day, 1967; Fauchald, 1977). There is general agreement that Eunicida is monophyletic (Rouse and Fauchald, 1997; Struck et al., 2002, 2006).

1 The jaw apparatus, other pharyngeal structures and the shape of the prostomium are the most common features used to identify the different eunicidan1 families, since these

features are conservative within the families and differ considerably among them. The body

is otherwise generally wormlike, slender, long, with relatively inconspicuous appendages,

including notopodial and ventral cirri, parapodial lobes and, in many species, branchiae.

The eunicidan fossil families are known just by the jaw apparatus. Five types of maxillae have been recognized in the order, two of these are only known from extinct taxa, the remaining three types are present both in extinct and extant families (Kielan-Jaworowska,

1966; Mierzejwski and Mierzejwska, 1975). Features used to identify the eunicidan families include the dorsolateral maxillae, prostomial and pharyngeal features but hypotheses of their primary homology are highly controversial (e.g., Pruvot, 1885; Binard

and Jeener, 1929; Hartman, 1944; Kielan-Jaworowska, 1966; Fauchald, 1970; Jumars,

1974; Wolf 1980; Orensanz, 1990; Orrhage, 1995; Paxton, 2004). These controversies are

impediments to understand the evolution of these features as well as for the construction of

analytical morphological phylogenetic hypotheses of the order or its families.

Among the eunicidan families, Eunicidae is one of the most species-rich with

around 326 species grouped in nine currently accepted genera (Table 1.2). Eunicids inhabit

soft and hard marine bottoms; many burrow into hard corals and calcareous algae or live in

their crevices, and play important roles in coral reef communities (Hutchings, 1986). The

family is distributed worldwide, but it is most common in shallow tropical waters

(Fauchald, 1992a). The length of adult eunicids varies from few millimeters up to 6 meters

1 Herein the adjective eunicid denotes members of the family Eunicidae whereas eunicidan denotes members of the order Eunicida. 2 (Fauchald, 1992a). Many eunicid species are economically important as bait for leisure and commercial fishing in diverse regions such as the Mediterranean Sea, Australia, Japan and

United States (Gambi et al., 1994; Olive, 1994). Additionally, the reproductive swimming phase of members of the genus Palola are a delicacy for natives from several of the South

Pacific Islands (Schulze, 2006).

The generally accepted composition of Eunicidae has been stable since it was

proposed by Hartman (1944, modified from Kinberg, 1865). This classification proposes

that the Onuphidae is the closest lineage to Eunicidae, a hypothesis consistently recovered

in both morphological and molecular phylogenies (Rouse and Fauchald, 1997; Struck et

al., 2002, 2006; Rousset et al., 2007; Zanol et al., 2007; Struck et al., 2008). However, this

classification proposes no unique diagnostic features (apomorphies) for the Eunicidae but

instead characterizes it with plesiomorphies or homoplasies (Orensanz, 1990), such as

asymmetric labidognath jaws, one to five prostomial appendages, peristomium double

ringed and a wide bilobed prostomium. Previous molecular and morphological

phylogenetic analyses focusing, respectively, on the order Eunicida and on the genus

Eunice (type genus for the family) (Struck et al., 2002, 2006; Zanol et al., 2007) did not always recover the current Eunicidae as monophyletic. In the morphological and molecular analyses using just 18S rDNA data Eunicidae is paraphyletic with Onuphidae nested within it, while it is monophyletic in molecular analyses using data from more genes (COI, 16S rDNA, 18S rDNA and 28S rDNA) and only two eunicid terminal species. Therefore, under a phylogenetic framework the current composition for Eunicidae is dubious. No study has focused on the phylogeny of the family per se.

3 Eighteen additional genera have been described for eunicid species (Table 1.3;

Fauchald, 1992a). Most of these have been synonymized with other genera and a few are

indeterminable (Fauchald, 1992a). Of the genera later synonymized to the genus Eunice,

Leodice Lamarck, 1818 and Nicidion Kinberg, 1865 had the most widespread use by

several authors. Leodice named for Leodice antennata Lamarck, 1818, was not clearly

differentiated from Eunice, and both names were used interchangeably through the first

third of the 20th century (Hartman, 1944). Nicidion was described for abranchiate species, a characteric currently not considered valid at the generic level (Fauchald, 1992a). The original description of Nicidion includes three species, none of which was identified as the type-species. Two of these species, N. gualapaguensis and N. longicirrata, were placed in the genus Palola and are indeterminable beyond the genus level because of the incomplete original description and poorly preserved type specimens (Fauchald, 1992b). The third species, N. cincta, is the only determinable species and has been referred to Eunice

(Hartman, 1948; Fauchald, 1992a).

The diagnostic characters of most eunicid genera are plesiomorphic (e.g., five prostomial appendages, a pair of peristomial cirri and labidognath jaws in Eunice) (Zanol et al., 2007) or ontogenetically variable (number of prostomial appendages in Lysidice and

Nematonereis, and absence of peristomial cirri in Marphysa) (Åkesson, 1967; Giangrande,

1989). Despite these inconsistencies, this generic classification has remained unmodified for many years (Orensanz, 1990). Just three of the nine currently accepted genera have been revised and had their monophyletic status phylogenetically tested. These more recent studies suggest that the genus Eunice is polyphyletic (Fauchald, 1992a; Zanol et al., 2007),

and Palola (Fauchald, 1992b; Schulze, 2006) and Euniphysa (Lu and Fauchald, 2000) are

4 monophyletic. Eunice can be divided in at least two monophyletic groups Eunice sensu

stricto and probably Leodice, however the composition and diagnostic features of such

groups are still unclear (Zanol et al., 2007).

The taxonomy of Eunicidae at the generic level has traditionally relied on anterior presegmental characters, such as the absence/presence of the dorsal buccal lip (sensu

Orrhage, 1995; e.g., Kinberg, 1865; Ehlers, 1868), the shape of the mandible (curved versus flat; e.g., Hartman, 1944; Fauchald, 1970) and the absence/presence of the head appendages, including palps, antennae and peristomial cirri. Indeed, most authors have considered the presence and absence of the anterior appendages as the main important informative and unambiguous feature at the generic level (e.g., Kinberg, 1865; Ehlers,

1868; Gravier, 1900; Hartman, 1944; Gathof, 1984). The two opposing hypotheses of evolution within Eunicidae are based mainly on these features. These hypotheses advocate that the eunicid evolution proceeded in a stepwise pattern with the addition of anterior appendages, from one median prostomial appendage (Nematonereis-like) to five prostomial appendages and one pair of persitomial cirri (Eunice-like) (Gravier, 1900; Hartman, 1944), or in the opposite direction with the gradual loss of anterior appendages (Orensanz,1990).

In earlier studies, branchial features (absence/presence, distribution and shape) were

the main or only segmental characters considered at the generic level (Kinberg, 1865;

Ehlers, 1868). However, the absence/presence of branchiae is not informative at this level

(Fauchald, 1992a), and in some cases it varies even within the same species (Nogueira et

al., 2001). Currently the shape and distribution of branchiae are features used at the

intrageneric level and in the diagnoses of species (Fauchald, 1992a; Orensanz, 1990). The

absence/presence of different kinds of chaetae are the main segmental characters currently

5 used at the generic level. Palola lacks pectinate chaetae and subacicular hooks (Fauchald,

1992b). Nauphanta lacks compound chaetae and has fan shaped pectinate chaetae

(Fauchald, 1987). Except for this use of the shape of the pectinate chaetae, chaetal

structures are mostly used at the intrageneric level and in the diagnoses of species.

Segmental characters vary along the eunicid body in a structured way (Fauchald,

1992a) and this variation may contain phylogenetic information (Lu and Fauchald, 2000;

Zanol et al., 2007). However, the common practice, probably rooted in the view that

segmental features are homogeneous along the body so that all segments of the body would

convey the same information (e.g., Ehlers, 1868), does not take into account such variation.

Furthermore the homology between segments of individuals with different segment counts

is not straight forward, the number of segments varies within eunicid species and no clear

body regions are recognized. Thus, comparative studies including variation along the body

have primarily to deal with the serial homology issue, which has not been done so far.

Purpose of this study

The purpose of this study was to conduct a detailed analysis of anatomical and along the body variation, develop better understanding of homology in Eunicida and

Eunicidae and test the monophyly of the family and its genera as well as the relationship among them based on molecular and morphological data. The detailed anatomical study of the external morphology and of the nervous system of the anterior end of five families of

Eunicida improved our general understanding of the structures in this region and also of the correspondence and topological similarities among them in the different eunicidan families,

6 These similarities allowed us to refine traditional hypotheses of primary homology and build new ones among features varying across Eunicida, thus important in the rooting of the eunicid tree and in the test of the monophyly of the family. Molecular, morphological and total evidence analyses were conducted to understand the phylogenetic information content of the different kinds of data and their interaction. The molecular phylogenetic topologies were further examined to explore the contribution of the different genes to the different branches of the trees. Finally, the combination of, molecular and morphological data with the novel attempts to account for serial homology in including morphological variation along the body and also to refine primary hypotheses of homology among the families of the order Eunicida with the anatomical studies provided a robust test to the monophyly of the family, its genera and for the relationships among them. The resulting trees also allowed us to evaluate diagnostic and traditional taxonomic characters in a phylogenetic perspective and to make relevant nomenclatural changes in order to define monophyletic genera.

Table 1.1: Families of the order Eunicida and their types of maxillae.

Maxillae type Family Ctenognath Dorvilleidae* Tetrapionidae† Labidognath asymmetric Eunicidae* Onuphidae* Kaloprionidae† Paulitinidae† Polychaetaspidae† Polychaeturidae† Rhamphoprionidae† Labidognath symmetric Lumbrineridae* Symmetroprionidae† Placognath Mochtyiellidae† Rhytiprionidae† Xanioprionidae† Prionognath Oenonidae* Atraktoprionidae† Skalenoprionidae† Prionognath? Histriobdellidae Xenognath Archaeoprionidae† UNCERTAIN Hartmaniellidae *Families examined in the present study. † Extinct families.

Table 1.2: Genera of the family Eunicidae currently accepted and total number of species.

Genera Species #

Eunice Cuvier, 1817 ~220 Marphysa Quatrefages, 1866 ~50 Palola Gray, 1847 ~20 Lysidice Savigny, 1818 ~15 Euniphysa Wesenberg-Lund, 1949 9 Nematonereis Schmarda, 1861 ~5 Nauphanta Kinberg, 1865 2 Fauchaldius Carrrera-Parra and Salazar-Vallejo, 1998 1 Aciculomarphysa Hartmann-Schröeder and Zibrowius, 1998 1

Table 1.3: Genera described for species of the family Eunicidae but not currently accepted.

Genus Type Species Type Locality Current Status Amphiro Kinberg 1865 Amphiro atlantica La Plata River, Argentina Synonym of Marphysa Aphelothrix Chamberlin, Eunice mossambica Mozambique Synonym of Marphysa 1919a Peters, 1854 Blainvillea Quatrefages, Blainvillea filum France Synonym of Nematonereis 1866 Euriphyle Kinberg, 1865 Eurphyle capensis Cape of Good Hope, South Synonym of Eunice Africa Heteromarphysa Verrill, Heteromarphysa tenuis Bermuda Island Indeterminable 1900 Leodice Lamarck 1818 Leodice antennata Gulf of Suez, Red Sea Synonym of Eunice Litognatha Stewart, 1881 Litognatha worslei Singapore Synonym of Palola Lysibranchia Cantone, 1983 Lysibranchia Sicily, Italy, Mediterranean Synonym of Lysidice paucibranchiata Sea Macduffia McIntosh, 1885 Macduffia bonhardi West Indies Synonym of Marphysa Mayeria Verrill 1900 Staurocephalus Florida Indeterminable gregaricus Mayer, 1900 Nausicaa Kinberg, 1865 Nausicaa striata East Paicific Ocean, Synonym of Marphysa Panama Nereidice Blainville, 1828 Erected for Lysidice Invalid Nereidonta Blainville, 1828 Nereidonta paretti Mediterranea Sea Synonym of Palola Nicidion Kinberg, 1865 Nicidion cincta, N. Society Islands, Galapagos Synonym of Eunice gualapaguensis and N. Islands and Hawaii longicirrata Palpiglossus Wagner, 1885 Palpiglossus labiatus White Sea Indeterminable Paramarphysa Ehlers, 1887 Paramarphysa longula Off Cuba Synonym of Marphysa Pseudopalolo Friendlander Eunice dubia Woodworth, Samoa Indeterminable in Woodworth, 1907 1907 Tibiana Lamarck, 1816 Tibiana fasciculata and T France and Australia Indeterminable ramosa

10 Chapter 2: Cephalic and pharyngeal innervation in the order Eunicida (polychaete, Annelida) using confocal laser scanning microscopy (cLSM) and its implications for homology

INTRODUCTION

The nervous system is generally considered a uniform organ-system in invertebrates

(Bullock, 1965). Hence, it is a useful source of information on the topological similarities of different structures across taxa at different taxonomic levels. Such topological similarity information provides a test for hypotheses of primary homology and allows for a clear delimitation of morphological characters because it goes beyond superficial

morphological similarity (Rieppel and Kearney, 2002). Hypotheses of primary homology

(sensu de Pinna, 1991) are conjectures of common evolutionary origin among structures

recognized by similarities and subject to further testing by congruence under

phylogenetic analyses.

In polychaetes, studies of the nervous system have been an important source of

information on topological similarity, improving explicit hypotheses of primary

homology (e.g., Binard and Jeener, 1929; Orrhage, 1980, 1990, 1991, 1993, 1995;

Orrhage and Müller, 2005) and allowing those to be tested in future studies. Structures

such as the palps in polychaetes, which have a variety of shapes could be hypothesized as

homologous because they are always innervated by the same nerves (Orrhage, 1980,

1990, 1991, 1993, 1995).

The order Eunicida, diagnosed by the presence of a ventral mandible and dorsolateral

maxillae in a ventral muscularized pharynx (Dales, 1962), is composed of thirteen extinct

11 and seven extant families (Table 1.1). The extant families are commonly identified by the shape of the prostomium and the pharyngeal structures, which differ considerably among the families (Fig. 2.1). The body is otherwise generally wormlike, slender, long with relatively inconspicuous appendages, including notopodial and ventral cirri, parapodial lobes and, in many species, branchiae.

In eunicidans1 the current hypotheses of primary homology concerning conspicuously variable elements among the families such as the dorsolateral maxillae, prostomial and pharyngeal features are highly controversial (e.g., Pruvot, 1885; Binard and Jeener, 1929;

Hartman, 1944; Kielan-Jaworowska, 1966; Fauchald, 1970; Jumars, 1974; Wolf 1980;

Orensanz, 1990; Orrhage, 1995; Paxton, 2004). These controversies are impediments to understand the evolution of these features as well as to the construction of analytical morphological phylogenetic hypotheses of the order or its families. Therefore it is necessary to focus on the improvement of more explicit hypotheses of primary homology among the different features as, for example, in detailed studies of the nervous system (e.g., palps in Orrhage, 1995)

The eunicidan nervous system conforms to the typical polychaete architecture

(Müller, 2006). It is composed of an anterior supraoesophageal ganglion or brain, dorsal and ventral roots of the circumoesophageal connectives fused at their posterior end, and a ventral nerve cord from which the peripheral nerves branch. In adult eunicidans the ventral nerve cord is trineural (Pruvot, 1885), probably due to the fusion of pairs of lateral nerves of the original pentaneural larval nerve cord (Müller and Westheide, 2002).

1 Herein the adjective eunicidan denotes members of the order Eunicida whereas eunicid denotes members of the family Eunicidae.

12 At least in the eunicidan families Eunicidae, Onuphidae and Dorvilleidae, the

neuropiles of the brain are composed of commissures connecting the circumoesophageal

connective roots from both sides, and include cells from the association system in the

first two families (Orrhage, 1995; Müller and Hennig, 2004). Eunicidae and Onuphidae

bear at least a dorsal commissure and a dorsal fibril mass connecting the dorsal roots and

four commissures (one dorsal and three ventral) connecting the ventral roots of the

circumoesophageal connective (Orrhage, 1995). All eunicidan families have two main

pairs of stomatogastric nerves branching from the ventralmost part of the

supraoesophageal ganglion (Spengel, 1882; Purschke, 1987; Orrhage, 1995) and

innervating the oesophagous and muscularized pharynx. The median stomatogastric

nerve pair (oesophageal nerve sensu Orrhage, 1995) is connected in an oesophageal

ganglion at the anterior end of the pharynx (Fig. 2.2B,C), posterior to which they run

independently on each side of the stomatogastric system in the dorsolateral fold (Pruvot,

1885; Binard and Jeener, 1929; Purschke, 1987).

In the present study we formulate hypotheses of primary homology among features

of the anterior end of the five most studied and species-rich families of Eunicida

(Eunicidae, Onuphidae, Dorvilleidae, Oenonidae and Lumbrineridae).

Immunohistochemical staining and confocal laser scanning microscopy (cLSM) were used to examine the innervation pattern and light microscopy was used to examine internal and external morphological features of the anterior end. The observed similarities were considered evidence supporting the primary hypotheses of homology discussed here, such as buccal lips, dorsolateral and pharyngeal folds, maxillae I and maxillary carriers.

Controversial hypotheses of homology at the anterior end of Eunicida

Buccal lips. The frontal and ventral structures of the onuphid prostomium (Fig.

2.1C) were historically named as frontal and ventral (or labial) palps (e.g., Pruvot, 1885;

Paxton, 1998; Orensanz, 1990). However these structures are not innervated by the same nerves as the palps in other polychaete families and may not be homologous to them

(Binard and Jeener, 1929; Orrhage, 1995). These structures have a unique innervation which is similar to that of the “ventral pads” of the eunicid prostomium. The structures in both families (Fig. 2.1A) are currently considered homologous and referred to as buccal lips (Orrhage, 1995) supporting the hypotheses in previous studies (e.g., Pruvot, 1885;

Pruvot and Racovitza, 1895; Binard and Jeener, 1929). The pair of ventral pads on the prostomium of lumbrinerids and dorvilleids (vp, Fig. 2.1G,I) also have been considered homologous to the onuphid buccal lips (Pruvot, 1885; Orensanz, 1973), but these hypotheses have not been further studied or tested.

Dorsolateral fold. The dorsolateral fold is a ciliated eversible fold present on the

dorsal side of the pharynx in various polychaete families (Purscke and Tzetlin, 1996).

Purscke and Tzetlin (1996) described it in dorvilleids and cited its presence in other

eunicidan families, however no further studies have been done to examine the similarities

and differences of this fold among the families. In all eunicidan families that have been

studied to date, the continuation of the oesophageal nerve posterior to the oesophageal

ganglion (sensu Orrhage, 1995) runs through the dorsolateral folds (Ehlers, 1868;

Spengel, 1882; Heider, 1925; Haffner, 1959; Purschke, 1987).

14 Maxillary elements. The dorsolateral maxillae in eunicidan are currently classified into five types (Table 1.1), each family has just one type of maxilla. However, maxillae classified as the same type may show variation in detailed structure among and within families; and some features and elements of the maxillae (carriers, maxillary plates, denticles and their arrangement) appear to vary independently of each other. The comparison among and within the different maxillary types is hampered by the controversial hypotheses of homology among the different elements. Therefore the simplistic classification of the maxillae in five types probably conceals phylogenetic information, and focusing on identifying the homology among elements of different maxillae may be more informative (Struck et al., 2006). The number, shape and placement of elements of ctenognath maxillae (Dorvilleidae) vary within this type and are distinctly different from labidognath (Eunicidae, Onuphidae and Lumbrineridae) and prionognath (Oenonidae) types. Therefore the hypotheses of primary homology of the elements of ctenognath maxillae are the most controversial among the three maxillae types of the families examined here. The presence and structure of maxillary carriers and maxilla I in Dorvilleidae have been disputed subjects in the literature. Maxillary carriers have been hypothesized as absent (e.g., Kielan-Jaworowska, 1966; Wolf, 1980) or present as the posteriormost dorsal structures of the maxillae (e.g., Fauchald 1970;

Jumars, 1974; Oug, 1978; Orensanz, 1990; Eibye-Jacobsen and Kristensen, 1994). For

Wolf (1980) these posteriormost dorsal structures should be named carrier-like and not carrier. Since they differ in function and surrounding muscles from the maxillary carriers of other eunicidans and just the topological similarity is not sufficient evidence to support this hypothesis homology (Wolf, 1980). The presence of maxilla I in the dorvilleids and

15 the possible homology to the structures called maxilla I in the other eunicidan families are also controversial issues (e.g., Kielan-Jaworowska, 1966; Fauchald, 1970; Jumars,

1974, Paxton, 2004). The posteriormost dentate and elongated plate in ctenognath jaws was considered homologous to maxilla I in Kielan-Jaworowska (1966), but other studies consider this plate as the base plate of maxilla I and the dorsal row of free denticles anterior to it as maxilla I (Fauchald, 1970; Jumars, 1974). However, many studies avoid the issue of homologies by referring to the posteriormost dentate and elongated plate as superior basal plate (e.g., Westheide and Nordheim, 1985; Wolf, 1986).

MATERIAL AND METHODS

Specimens examined

External morphology and pharyngeal dissection. We observed and photographed under a dissecting microscope prostomial structures, ventral dissections of the muscularized pharynx and median parasagital cuts of the anterior end of specimens of

Eunice valens (Chamberlin, 1919b) (Eunicidae, USNM xxxxxx), Nothria iridescens

(Johnson, 1901) (Onuphidae, USNM 22407), Arabella iricolor (Montagu, 1804)

(Oenonidae, USNM 61732), Dorvillea sociabilis (Webster, 1879) (Dorvilleidae, USNM

33830), Lumbrineris fragilis (Müller, 1776) (USNM 30097) and Lumbrineris latreilli

Audouin and Milne Edwards, 1834 (USNM 53018). Pictures and dissections were analyzed for morphological and topological similarities. We also examined the relevant literature to confirm that the examined species represent the general morphology of the prostomial and muscularized pharynx features present in each family.

16 Innervation. We examined five species of the five target eunicidan families collected near Fort Pierce, Florida, USA (Table 2.1). Adults of Dorvilleidae,

Lumbrineridae and Oenonidae are small enough to be examined under cLSM. However, due to the large size of adults of most species of Eunicidae and Onuphidae, juveniles of both families were examined. The examined specimens of Eunicidae (unidentified to genus and species) had only the median antenna present, adult maxillae, juvenile mandibles, lacked peristomial cirri and the prostomium was entire distally and dorsally with a ventromedian longitudinal groove. The onuphid juveniles, belonging to

Kinbergonuphis simoni (Santos, Day and Rice, 1981) had all adult anterior features except for the mandibles.

The Eunicidae gen. sp. juveniles were collected in gelatinous drop-shaped egg masses attached to the substrate. Similar egg masses have been described only for species of Marphysa (e.g., Aiyar, 1931) so we, tentatively, identify these juveniles as belonging to a species of Marphysa.

Immunohistochemical staining and cLSM

Immunohistochemical staining allows the fluorescent staining of specific systems, which in the cLSM are excited by a laser beam and emit fluorochromes. The cLSM captures only the in-focus light emitted by the fluorochromes and takes stacks of two- dimensional pictures, which can later be mounted in three-dimensional images. Therefore the combination of such staining technique and microscopy allows a detailed, three- dimensional view of systems in a less time-consuming fashion than traditional methods

(Müller, 2006), making it an ideal tool for comparative studies of anatomical features.

17 The nervous system has been a frequent target of such studies (e.g., Müller and

Westheide, 2002; Purschke and Hessling, 2002). In invertebrates the antibody acetylated

α-tubulin is enough to reconstruct the complete nervous system, but 5-HT serotonin and

FMRFamide are also used (Müller, 2006).

Fixation and staining. Specimens were relaxed in MgCl2 (7 %) isotonic to

seawater and fixed overnight (8-12 hours) at 4o C in 4% paraformaldehyde in 0.1 M

Sörensen’s phosphate buffer (SPB) containing 8% sucrose. Specimens were rinsed three times (20 minutes total) in 0.1M SPB with 0.1% sodium azide and conserved in the same solution at 4oC.

Nerves were stained by immunohistochemical methods for acetylated α-tubulin and serotonin; and muscles with Phalloidin Alexa Fluor 488 or BODIPY FL phallacidin

(which is more resistant to Murray Clear method using isopropanol, G. von Dassow, personal communication) (Molecular Probes-Invitrogen). Specimens were made more permeable by incubating them in 0.1M PBT (0.1M SPB and 1% Triton-X) for 2-5 hours

at room temperature or overnight at 37oC with collagenase 1000U/ml (Sigma-Aldrich ) in

1mM CaCl2, 0.1% Triton-X and 0.1M Tris-HCl, PH 7.5 (Yoshida-Noro et al., 2000). All further steps were carried out in a rotary table. Unspecific sites were blocked by incubating specimens in 5% normal goat serum in PTA (blocking solution) for 8-12 hours at room temperature. Specimens were rinsed once with 0.1M PTA and incubated in primary antibodies, respectively, mouse monoclonal anti-acetylated α-tubulin (Sigma-

Aldrich; dilution 1:100) and rabbit anti-serotonin 5HT (Immunostar; dilution 1:100); diluted in 0.1M PTA for 12 hours at room temperature. The specimens were then rinsed in 0.1M PTA twice (for a total of one hour) and four times (for a total of nine hours) in

18 blocking solution; and incubated for 12 hours at room temperature in secondary antibody

solutions diluted 1:100 in blocking solution. Secondary antibodies were directed against mouse in the case of acetylated α-tubulin and against rabbit for serotonin. Secondary antibodies were conjugated with Alexa Fluor (Invitrogen). Afterwards specimens which

had dual nervous system immuno-staining were rinsed in 0.1M PTA twice (for a total of

one hour) and four times (for a total of nine hours) in blocking solution.

The protocol was repeated starting with the addition of the second primary

antibody. Specimens which had a dual label (immuno plus phalloidin staining) were

rinsed twice in 0.1M PTA (for a total of one hour), incubated in phalloidin for an hour at

room temperature and rinsed three times in 0.1M SPB for no longer than 15 minutes each

rinse. All specimens were cleared and mounted in Murray Clear (2:1 benzyl benzoate and

benzyl alcohol) and observed under cLSM BioRad Radiance 2100. We collected confocal

Z-series images for the stained structure as well as Z-series transmission images of all

observed specimens.

We observed three specimens of the eunicid juvenile, K. simoni (Onuphidae) and

Lumbrineris verilli Perkins, 1979 (Lumbrineridae) stained with antibody against

acetylated α-tubulin and/or serotonin; two specimens of Schistomeringos cf. pectinata

Perkins, 1979 and one specimen of Arabella cf. mutans (Chamberlin, 1919a) stained with

antibody against acetylated α-tubulin. To test specificity of secondary antibodies control

specimens of each species were processed through the protocol but primary antibodies

were not added.

Images resulted from cLSM Z-series were analyzed using Zeiss LSM Image

Browser (version 4.0.0157). Even though we also stained the muscles of some specimens

19 with phalloidin, in the present study we only examined the nervous system and

transmission images. We based our primary hypothesis of homology on correspondence

and topological similarities of the internal and external morphological features of the

anterior end and the innervation pattern of the prostomium and muscularized pharynx

structures.

RESULTS

In this description of the anatomy, we use the terminology of Orrhage (1995) and

Orrhage and Müller (2005). In descriptions of placement with two qualifiers, the first

qualifier is the main one, as for example, ventroanterior is the anterior end of the ventral

side while anteroventral is the ventral end of the anterior side. Features referred to as

unidentifiable were not observed due to poor staining signal or background noise.

Prostomium and selected pharyngeal structures

E. valens (Eunicidae) has a round, frontally bilobed prostomium with a small dorsomedian furrow and a conspicuous ventromedian longitudinal groove (Fig. 2.1A).

On the ventral side, an inconspicuous furrow follows the shape of the prostomium and outlines the buccal lip (sensu Orrhage, 1995), which is narrower and shorter than the

prostomium (Fig. 2.1A). A pair of muscle bundles (DM1 sensu Wolf, 1980) extends from

the muscularized pharynx into the posterior extension of the prostomium (pep) and

connects medially (Fig. 2.1A, B). The DM1 is slanted in a ventral to dorsal direction becoming increasingly dorsal towards the posterior end.

20 The prostomium of N. iridescens (Onuphidae) is round and has a pair of conspicuous drop-shaped, dorsal buccal lips and a pair of rectangular ventral buccal lips

(Fig. 2.1C). A pair of posterior extensions of the prostomium (pep) is present on the ventral side posterior to the ventral buccal lip (Fig. 2.1C). A DM1 bundle runs in each pep. A ventroposterior median groove is present in between the pair of ventral buccal lips and it extends posteriorly in between the pair of posterior extensions of the prostomium

(pep, Fig. 2.1C).

E. valens and N. iridescens have five prostomial appendages, three median antenna and two lateral palps. Both species also have the dorsolateral fold (dlf) terminating at the anterior end of the muscularized pharynx (Fig. 2.1B,D) where it connects to the posterior extension of the prostomium (pep).

The prostomium of A. iricolor (Oenonidae) and of both L. fragilis and L. latreilli

(these two species are similar in the features described and therefore they will be referred together hereafter as Lumbrineris sp.) has no appendages, is pointed, and has an inconspicuous ventral furrow (ivf) that outlines its ventromedian region (Fig. 2.1I). A. iricolor has a pair of longitudinal inconspicuous ventral furrows which terminate posterior to the anterior end of the prostomium and do not connect at their anterior end

(Fig. 1E). In Lumbrineris sp., the inconspicuous ventral furrow is continuous and follows the outline of the prostomium but it terminates before the anterior tip of the prostomium.

In both A. iricolor and Lumbrineris sp., the dorsolateral fold (dlf) has an anterior extension which extends forward further than the anterior end of the muscularized pharynx (“Mundwülste” sensu Spengel, 1882; “Rüssel” sensu Wolf, 1980). However, in

A. iricolor the dorsolateral fold anterior extension (dlfae) is connected anteriorly forming

21 a narrow bridge to the base of the prostomium, while in Lumbrineris sp. the dlfae anterior

end has paired transverse anterior peristomial extensions (tpe, “Mundpolstern” sensu

Ehlers, 1868; buccal lip sensu Orensanz, 1973) (Fig. 1E, I). DM1 was not identifiable in

either of these species.

D. sociabilis has a round prostomium with a pair of lateral antennae and a pair of

ventral palps. The ventral side of the prostomium bears a pair of pads bounding the

mouth (sensu Pruvot, 1885) which are anterior extensions of the pharyngeal fold (phf)

(sensu Purschke, 1987) (Fig. 2.1G). The dorsolateral fold (dlf) terminates at the anterior

end of the muscularized pharynx (Fig. 2.1H). A short inconspicuous pair of folds is

present anterior to dlf and dorsally to phf.

All species have four folds in the pharynx (Fig. 2.1). The dorsalmost fold is the

dorsolateral fold (dlf). Its anterior end is always ventral in relation to its posterior end.

The dlf usually ends posteriorly close to the posterior end of maxillae I (MxI). At this end,

both dlf fuse (dlff) and separate the lumen of the pharynx from the oesophagus. Ventral to

the dlf are the paired maxillary folds (mxf), which bear the maxillary plates. The

pharyngeal fold (phf) is ventral to the mxf and dorsal to the mandible fold (mdf), which supports the mandibles. The mdf is connected to the outer edge of pharyngeal fold (phf) along its whole length and appears to be a ventral extension from phf. In A. iricolor, the mandible fold is small and it appears that the mandibles are connected directly to the phf.

Cephalic and selected anterior nerves

The descriptions of the cephalic and stomatogastric nervous system are based on α-

tubulin and serotonin stained structures. Here we provide a description of the

22 commissures in the supraoesophageal ganglion, circumoesophageal connectives (cc),

selected stomatogastric nerves and the structures they innervate. Since just one species

was examined for each family we refer to them by their family name.

Circumoesophageal connectives. Circumoesophageal connectives (cc) are the

nerves connecting the neuropiles of the supraoesophageal ganglion with the ventral nerve

cord. Before reaching the supraoesophageal ganglion these connectives split into a dorsal

(drcc) and a ventral (vrcc) root. The drcc and vrcc from opposite sides of the body

connect with their respective counterparts through commissures present in the neuropiles

of supraoesophageal ganglion. In all families, the vrcc bifurcates into a dorsal (dbvr) and

a ventral branch (vbvr). In Eunicidae this happens just anterior to the separation from

drcc while in the other families it occurs almost at, or at, the site of the commissures

(Figs. 2.2-2.4). In Dorvilleidae, we were not able to distinguish dbvr and vbvr. In this

family a thin and short nerve diverges from vrcc posterior to the connection of vrcc to the

supraoesophageal ganglion and connects to the third ventral commissure of the vrcc

(vcvr3) (anterior connection of the stomatogastric system with circumoesophageal

connective sensu Müller and Henning, 2004).

In all families, the dorsal roots of the circumoesophageal connectives (drcc) connect through a dorsal commissure (dcdr) and a dorsal fibril mass (dfm), which may contain ventral commissures (e.g., Oenonidae, Dorvilleidae and Lumbrineridae). The dorsal branch of the vrcc (dbvr) connects through a dorsal commissure (dcvr), first and second ventral commissures (vcvr1 and vcvr2). In all families the dorsalmost nerves of dbvr connect forming the dcvr. The ventralmost nerves of dbvr form the ventral side of the anterolateral

23 nerve projection (alp-dbvr), vcvr1 and vcvr2. The ventral branch of the vrcc (vbvr) connects through the third and fourth ventral commissures (vcvr3 and vcvr4).

The relative placement of the connections of the roots of the circumoesophageal connectives with the respective commissures varied among families. In Eunicidae and

Lumbrineridae, the ventral root of the circumoesophageal connectives (vrcc) connects to the commissures at a position anterior and almost as far dorsal as the connection of the dorsal root of the cc (drcc). In both of these families as well as in Oenonidae, the connection of the dorsal branch of the vrcc (dbvr) is dorso-posterior to the one of the ventral branch of the same root (vbvr) (Figs. 2.2B,J,M, 2.3C, 2.4C,D). In Onuphidae, vrcc connection to the commissures is ventro-posterior to drcc connection and dbvr connection is dorso-anterior to the vbvr one (Figs. 2.2F, 2.3B). Finally, in Dorvilleidae and Oenonidae the vrcc connection to the commissures is ventro-anterior to drcc connection (Figs. 2.2D,J, 2.4A-C). In Dorvilleidae, vrcc connects at the ventral side of the dorsal commissure of the vrcc (dcvr) and on the posterior dorsal side of ventral commissures of the vrcc (vcvr).

Supraoesophageal ganglion. Two main neuropiles (dorsal and ventral) surrounded by nerve cells (nrcl) compose the supraoesophageal ganglion. The content of such neuropiles does not correspond directly to the dorsal and ventral roots of the circumoesophageal connectives (drcc and vrcc) or nerve functions (e.g., motor versus sensory nerves). The nerves branching from the dorsal neuropile innervate only sensory structures however the ones from the ventral neuropile innervate both sensory and motor structures.

24 In all families, the dorsal neuropile (dnrp) contains the dorsal commissure of the drcc (dcdr), nuchal commissure (nc), the dorsal fibril mass (dfm) and the dorsal

commissure of the vrcc (dcvr). In Dorvilleidae, it also contains the ocular commissure

(oc) (Fig. 2.4A). We could not clearly distinguish oc in Eunicidae, Onuphidae and

Oenonidae, however Eunicidae and Oenonidae had two pairs of eyes and Onuphidae one

pair. The Lumbrineridae examined here had no eyes. The ventral neuropile (vnrp) bears

the first, second, third and fourth ventral commissures of the vrcc (vcvr1, vcvr2, vcvr3 and

vcvr4) (Figs. 2.3, 2.4). In Oenonidae, vcvr2 was not found.

In all families, the dorsal and ventral neuropiles (dnrp and vnrp) are connected

laterally by the dorsal branch of the vrcc (dbvr) (vrcc in the case of Dorvilleidae) and,

except in Onuphidae, are distinctly separated medially between the dorsal and first

ventral commissures of vrcc (dcvr and vcvr1). This separation forms a median dc-vcvr

intrasupraoesophageal ganglion cavity. The dnrp is the longest and most massive of the

neuropiles. In Eunicidae, Oenonidae and Lumbrineridae dnrp is dorso-posterior to vnrp

(Figs. 2.2B,J,L, 2.3B, 2.4C,D). In Onuphidae and Dorvilleidae, the dnrp is dorsal to vnrp

(Figs. 2.2D-G, 2.3A,B, 2.4A,B), in the latter dnrp is longer and extends forward further

than vnrp.

Onuphidae lacks a median dc-vcvr intrasupraoesophageal ganglion cavity in that the

first and second ventral commissures of the vrcc (vcvr1 and vcvr2) are closely associated with dorsal commissure of the same root (dcvr). The family bears a unique plexus and X shaped connection (xc) between vcvr1-vcvr2 and dcvr. In this family a cavity is present in

the center of the supraoesophageal ganglion, however, it is located between vcvr1-vcvr2 and the third and fourth ventral commissures of the vrcc (vcvr3 and vcvr4) (vcvr

25 intrasupraoesophageal ganglion cavity). We refer to the dorsal and ventral parts of the

onuphid supraoesophageal ganglion respectively as the dorsoventral neuropile

(dnrp+dcvr+vcvr1+vcvr2) and the third-fourth ventral commissures of the vrcc

(vcvr3+vcvr4).

In Eunicidae, Dorvilleidae, Oenonidae and Lumbrineridae, a pair of anterolateral nerve projections (alp) also connect the ventral and dorsal neuropiles. In Eunicidae,

Oenonidae and Lumbrineridae the alp is composed at the dorsalmost side by extensions of the dorsal fibril mass nerves (alp-dfm) and at the ventral side by nerves extending from the dorsal branch of the vrcc (alp-dbvr) (Figs. 2.2A,I,M, 2.3C, 2.4C,D). Dorvilleidae bears only the ventralmost side of the alp (alp-dbvr), composed of nerves from dbvr

(Figs. 2.2D, 2.4B).

In Lumbrineridae one pair of dorsolongitudinal nerves (dln) branching off from nerve cells posterior to dorsal neuropiles (dnrp) run laterally and innervate the dorsoposterior end of the prostomium (Fig. 2.2L). Another pair of dorsolongitudinal nerves branch slightly lateral to the first ones and run ventrally ending at the ventroposterior end of the prostomium. In Lumbrineridae most of the dnrp and the posterior end of third ventral commissure of the vrcc (vcvr3) are located in the peristomium. The main innervations of the anterior two thirds of the prostomium are long nerve fibers branching out from the anterolateral nerve projections (alp) on either side.

All families have nerve cells located dorsal, lateral and anterior to the dorsal neuropile (dnrp), dorsal and lateral to the anterolateral nerve projections (alp) (except in

Onuphidae which lacks alp) and lateral to the ventral neuropile (vnrp) (except in

Dorvilleidae). In Lumbrineridae nerve cells are also present anterior and ventral to vnrp

26 and in Oenonidae these cells are numerous, massive and take up most of the prostomium.

In Onuphidae nerve cells are also numerous and massive at the anterior end of the

dorsoventral neuropile, having about the same volume as the fibril mass at the median

section, and at the anterolateral side of the third ventral commissure of the vrcc (vcvr3).

Orrhage (1995) named these nerve cells in Onuphidae “association system cells”. In

Onuphidae two commissures of the association system are present (cas, sensu Orrhage,

1995), one is ventroanterior and the other posterior to the dorsoventral neuropile (vacas

and pcas) (Figs. 2.2F, 2.3A). The ventral roots of the nerves of the median antenna (nma)

and lateral nerves of the dorsolateral antennae (ndla) branch out from the posterior

commissures of the association system (pcas, Fig. 2.3A). In the other families, the

commissures of the association system appear to be absent.

Dorsal neuropile- The dorsal commissure of the drcc (dcdr) is the dorsalmost

structure of the dorsal neuropile (dnrp). In all families, except Lumbrineridae, the dcdr is located at the posterior end of the prostomium (Figs. 2.2-2.4). In Lumbrineridae, the dcdr

is located posterior to the nuchal organs in the peristomium and connects to the drcc at its

anterior end (Figs. 2.2L, 2.4D). In Dorvilleidae, a pair of nerves branch from each side of

the dcdr (dcdrn) and innervate the dorsoanterior side of the prostomium (Figs. 2.2C,

2.4A); nerves branching from dcdr and innervating the dorsolateral antennae were not

observed. In Eunicidae and Onuphidae, the nerves of the median antenna (nma) (Figs.

2.2A,E, 2.3A,C) branch from dcdr, in the latter median nerves of the dorsolateral

antennae (mndla) also branch from dcdr (Figs. 2.2E, 2.3A). The eunicids examined here

were juveniles bearing only the median antenna and we could not distinguish the nerves

27 of the lateral antennae and palps. Nerves branching from dcdr appear to be absent in

Oenonidae and Lumbrineridae.

The nuchal commissures (nc) are placed ventrally to ventro-posteriorly to the dorsal

commissures of the drcc (dcdr, Figs. 2.2A,C,E,H,L, 2.3A,C, 2.4A,C,D). In all families

the nerves branching from it innervate the nuchal organs directly. In Oenonidae the

nuchal nerves bifurcate, the ventralmost branch innervates a ventral ganglion and the

dorsalmost the nuchal organs, which are long and extend posteriorly beyond the anterior end of the dorsolateral fold anterior extension (dlfae) (Fig. 2.2H). In Eunicidae and

Onuphidae nerves branching from the nc also innervate the posterior end of the prostomium (Fig. 2.2A,E). In Dorvilleidae, the median and lateral nerves branching from the nc innervate, respectively, the median and lateral nuchal organs (Figs.2.2C, 2.4A).

The nc nerves in Lumbrineridae loop dorso-anteriorly to innervate the nuchal organs,

which are located anterior to the nc (Figs. 2.2L, 2.4D).

In Dorvilleidae, the ocular commissure (oc) is as deep as the nuchal commissures

(nc) but is placed at the anterior end of the dorsal neuropile (dnrp) (Fig. 2.4A). The

ocular nerves (ocn) branching from oc innervate the dorsal and lateral pairs of eyes. In

Onuphidae, a commissure from which branching nerves run to the sides of the

prostomium is located posterior to the nc. This commissure may be the ocular

commissure but we cannot be certain since eyes are not visible in the stained specimens

and it is posterior to the position described in Orrhage (1995) and that observed in

Dorvilleidae. In Oenonidae, the median eyes are directly dorsal to the dorsal nerve cells

of the supraoesophageal ganglion and the lateral ones are dorsal to the region where the

28 dorsal root of the circumoesophageal connective (drcc) connects to the dorsal neuropile

(dnrp).

In all families, the dorsal fibril mass (dfm) is ventro-anterior to the dorsal

commissure of the drcc (dcdr) and nuchal commissures (nc, Figs. 2.2-2.4).Oenonidae,

Dorvilleidae and Lumbrineridae have conspicuous ventral commissures of the drcc (vcdr)

in the dfm. Oenonidae has two vcdr (Fig. 2.2I), Dorvilleidae and Lumbrineridae have at

least one (Figs. 2.2C,L, 2.4A,D). Eunicidae has fibrils in the dfm positioned in the same

direction as the vcdr but these are not grouped in a denser commissure as observed in

Oenonidae, Dorvilleidae and Lumbrineridae. In Onuphidae, vcdr was not identified

either. In Eunicidae, Oenonidae and Lumbrineridae the dfm extends to the anterior region

of the prostomium forming the dorsalmost part of the anterolateral nerve projections (alp-

dfm) (Figs. 2.2A,H,L, 2.3C, 2.4,C,D). In Oenonidae and Lumbrineridae the vcdr in the

dfm are the ones extending forward to form the alp-dfm (Figs. 2.2I,L, 2.4C,D). In

Dorvilleidae the vcdr extends forward innervating the anterior end of the prostomium

(Figs. 2.2C, 2.4A), however it does not connect to ventral part of the anterolateral nerves

projections (alp).

The anterolateral nerves projections (alp) extend dorso-ventrally connecting the dorsal fibril mass (dfm), dorsal commissure (dcvr) and the first ventral commissure of the vrcc (vcvr1). The alp is composed dorsally of nerves branching from the dfm (alp-dfm;

absent in Dorvilleidae) and ventrally of nerves branching from the dorsal branch of the

ventral root of the vrcc (alp-dbvr) (or vrcc in the case of Dorvilleidae). Onuphidae lacks

alp, it has nerves branching from the dfm and dbvr but they do not merge in a nerve mass.

The nerves branching from the alp innervate dorsal, lateral, anterior and ventroanterior

29 sides of the prostomium. In Oenonidae the innervation of the ventroanterior side of the

prostomium by nerves branching from the alp were unidentifiable. The alp-dfm curve towards the center of the body but do not meet in Eunicidae (Figs. 2.2A, 2.3C), it is wide,

palmate with numerous long nerve fibers extending from it in Lumbrineridae (Figs. 2.2L,

2.4D), and aliform and long in Oenonidae (Figs. 2.2H, 2.4C), extending from the

posterior end of the prostomium to its anterior most quarter (Figs. 2.2H-J, 2.4C).

Furthermore, in Oenonidae a nerve branches from the posterolateral end of the alp

(pnalp), extends towards the posterior end, loops forward and runs on a slant ventro-

anteriorly (Figs. 2.2I, 2.4C).

In Onuphidae three pairs of dorsoanterior nerves (dfmn) (Figs. 2.2E, 2.3A) branch

from the dorsal fibril mass (dfm) to innervate the dorsal and anterior sides of the

prostomium. These dfmn connect at different depths to the dfm, the ventralmost and

dorsalmost branches are median (dfmnM1 and dfmnM2, respectively) while the middle branch is lateral (dfmnL) (Fig. 2.3A).

In Dorvilleidae the two nerve roots of the dorsolateral antennae (ndla) and the three palp dorsal nerve roots (pnr8 sensu Orrhage and Müller, 2005) branch from the dorsal fibril mass (dfm)(Figs. 2.2D, 2.4B), ndla is dorsal and pnr8 ventral to the ventral commissure of the drcc (vcdr). In Onuphidae the palp dorsal nerve root branches from the drcc (pnr12 sensu Orrhage and Müller, 2005). Both in Onuphidae and Dorvilleidae the ventral nerves of the palp branch from the same place, the first ventral commissure of the vrcc (vcvr1) (pnr6 sensu Orrhage and Müller, 2005) and between the vrcc and the

second ventral commissure of the vrcc (vcvr2) (pnr5 sensu Orrhage and Müller, 2005).

30 In all families the dorsal commissure of the vrcc (dcvr) is the most ventral structure

in the dorsal neuropile (dnrp), but how far dorsal they reach varies among families. In

Onuphidae, Oenonidae and Dorvilleidae the dcvr are about as dorsal as the ventralmost nerves of the dorsal fibril mass (dfm) (Fig. 2.2A,H). In Eunicidae the dcvr dorsalmost nerves are about as dorsal as the dfm dorsalmost nerves. In Lumbrineridae the dcvr extends from the dorsalmost to the ventralmost side on the anterior end of the dnrp (Fig.

2.2L). In all families the dfm and dcvr are closely connected laterally and by the median plexus (mp) (sensu Müller and Henning, 2004), making it difficult to recognize their boundaries. Two lateral gaps (fiber free regions) are present on the sides of the mp. The median gap described in Müller and Henning (2004) for Dorvilleidae was not observed.

In Onuphidae three pairs of nerves branch from the dorsal commissure of the ventral vrcc (dcvr) and innervate the anterior end of the prostomium, the most dorsal is median (dcvrnM) and the two ventral are lateral (dcvrnL1 and dcvrnL2) and sit at the same depth (2.2F, 2.3B).

Ventral neuropile- In all families (except Oenonidae, in which vcvr2 was not observed) the first ventral commissure of the vrcc (vcvr1) is anterior, more extensive and thicker than the second ventral commissure for the same root (vcvr2) (Figs. 2.3, 2.4). In

Eunicidae and Lumbrineridae both commissures are at the same depth, while in

Onuphidae and Dorvilleidae dorsalmost nerves of the vcvr1 are more ventral than the vcvr2 ones.

In Eunicidae, Dorvilleidae, Oenonidae and Lumbrineridae the nerves emerging

from the first ventral commissure of the vrcc (vcvr1), do not branch from the commissure

fibers. Instead they branch from the ventro-anterolateral nerves projection (alp-dbvr)

31 (Figs. 2.2B,D,J,M, 2.3C, 2.4B-D), which extends from the dorsal branch of the vrcc

(dbvr) (or vrcc in the case of Dorvilleidae). In Eunicidae and Lumbrineridae the nerves of

the dbvr extend antero-ventrally forming the alp-dbvr and vcvr1. In Oenonidae the ventral

nerves of the dbvr also extend antero-ventrally forming the ventral side of the alp-dbvr

but loop to the ventro-posterior side, where they connect, forming vcvr1. The nerves

branching from vrcc to form the alp-dbvr in Dorvilleidae extend antero-dorsally

becoming as dorsal as the ventral side of the dorsal fibril mass (dfm). The alp-dbvr in

Dorvilleidae connects on the dorsal side with the dorsal commissure of the vrcc (dcvr)

and on the ventral side with the dorsoanterior extensions of the fourth ventral commissure

of the ventral branch of the vrcc (vcvr4). The alp-dbvr and the dorsoanterior extensions of

the vcvr4 are closely connected, making their boundaries difficult to discern.

In Eunicidae at least two pairs of median (mnalp), one pair of midlateral (mlnalp) and one pair of lateral nerves (lnalp) branch from the ventro-anterolateral nerves projection (alp-dbvr) (Figs. 2.2B, 2.3C). All these nerves innervate the ventral side of the prostomium, with exception of the dorsal mnalp which innervates the anterior end. In

Lumbrineridae around 12 pairs of nerves branch out from alp-dbvr and innervate the anterior and ventral sides of the prostomium (Figs. 2.2M, 2.4D). In Dorvilleidae several nerves branching from the alp-dbvr innervate the anteroventral and ventroanterior sides of the prostomium. Finally, in Oenonidae, several nerves branching from alp-dbvr innervate at least the anterior and lateral sides of the prostomium but possibly also the ventral side, the immuno-staining signal of the innervations of the ventroanterior side of the prostomium was not clear.

32 In Eunicidae, Onuphidae and Lumbrineridae the third ventral commissure of the

vrcc (vcvr3) is ventral to both the first and second ventral commissures of the same root

(vcvr1and vcvr2) (Figs. 2.2B,G,M, 2.3B,C, 2.4D). In Dorvilleidae and Oenonidae vcvr3 is located, respectively, at the same depth as vcvr2 and vcvr1 (Figs. 2.2D,J, 2.4B,C). In

Oenonidae vcvr1 and vcvr3 are closely connected by a plexus, while in the other families

there is no nerve tissue connecting these commissures. In all families the ventral branch

of the vrcc (vbvr) connect to vcvr3. Vcvr3 is horseshoe shaped in all families with the arch end towards the anterior end. Both main stomatogastric nerves, oesophageal nerves (oen) and pharyngeal (phn), branch from vcvr3 at the median and lateroposterior end,

respectively (Figs. 2.2B,D,F,J,M, 2.3, 2.4). No nerves branch form the anterior end of

vcvr3.

In Eunicidae, Onuphidae, Dorvilleidae and Lumbrineridae, we observed a fourth

ventral commissure of the vrcc (vcvr4), it is the ventroanteriormost commissure and bears several pairs of nerves innervating the prostomium. Vcvr4 is always anterior to the third ventral commissure of the vrcc (vcvr3). The two commissures are closely connected in all

families. In Eunicidae and Onuphidae vcvr4 is completely ventro-anterior to vcvr3. In

Dorvilleidae the dorsoanterior extensions of vcvr4 and in Lumbrineridae the dorsal end of

vcvr4 are as dorsal as vcvr3, however vcvr4 is deeper than vcvr3, terminating more ventrally. Vcvr4 connects posteriorly to vcvr3 and dorsally to the first ventral commissure of the vrcc (vcvr1) (except in Onuphidae, which has s a cavity between vcvr1- vcvr2 and vcvr3-vcvr4). It is difficult to distinguish the boundary between vcvr4 and vcvr1 in all families in which they are connected (Fig. 2.2B,D,M).

33 In Eunicidae approximately six pairs of nerves branch from the fourth ventral

commissure of vrcc (vcvr4) and innervate the buccal lips (nbl), which is medio-posterior to the region innervated by the nerves branching from the first ventral commissure of the vrcc (vcvr1) (Figs. 2.2B, 2.3C). In Onuphidae nerves branching from vcvr4 also innervate

the buccal lips, roughly four pairs innervate the ventral buccal lip (nvbl) and at least three pairs innervate the dorsal buccal lip (ndbl) (Figs. 2.2G, 2.3B). A few nerves innervating the dorsal buccal lip in Onuphidae appear to branch from the anterior association system.

In Lumbrineridae around 16 nerve pairs branch out from vcvr4 and innervate the ventro-

midposterior side of the prostomium. Similarly, in Dorvilleidae several pairs of nerves

branching out from vcvr4 innervate the ventro-midposterior side of the prostomium.

We did not observe the fourth ventral commissure of the vrcc (vcvr4) in Oenonidae,

however, two thin pairs of nerves (a long and a short pair) branching out from the ventral branch of the vrcc (vbvr) are roughly in the appropriate position and innervate the ventral side of the prostomium (Fig. 2.5). The long nerves (lgvbvr) (Fig. 2.2J) connect to each

other through a commissure in the anterior half of the prostomium and innervate the

ventromedian side of the prostomium (Fig. 2.5). The short nerves innervate (shvbvr) the

ventro-midposterior half of the prostomium (Fig. 2.5). The latero-ventroanterior sides of the prostomium are innervated by nerves branching from a commissure formed by the connection of nerves branching from the lateral side of vbvr, first and third ventral commissures of the vrcc (vcvr1 and vcvr3).

Stomatogastric nerves - oesophageal and pharyngeal nerves

34 Stomatogastric nerves such as the oesophageal nerves (oen) and ramifying nerves

(stdl and stdp) as well as the pharyngeal nerves (phn) generally show similar innervation

patterns in the different families. These nerves branch from the third ventral commissure

of the vrcc (vcvr3) and innervate parts of the feeding apparatus and digestive system such as the muscularized pharynx or the dorsal walls of the gut.

In all families, after branching from the third ventral commissure of the vrcc

(vcvr3), the oesophageal nerve pair (oen) runs parallel to each other towards the postero-

dorsal end and connects in a ganglion (oeg) (soesg sensu Orrhage, 1995) at the anterior

end of the muscularized pharynx (oeg is unidentifiable in Oenonidae) (Figs. 2.2A,C,E,

2.3A-C, 2.4A,D). Immediately posterior to the oeg the oen enters the dorsolateral fold

(dlf) and bifurcates into a thin stomatograstric dorsolateral nerve (stdl sensu Müller and

Henning, 2004), which innervates the anterior half of ventral side of the dlf where it ends,

and a thicker stomatograstric dorsoparamedian nerve (stdp sensu Müller and Henning,

2004), which runs in and innervates the dlf toward its posterior end. The poor signal yield

by the immuno-staining on the muscularized pharynx of Oenonidae and Lumbrineridae

made it impossible to distinguish structures such as stdl in these two families and the oeg

and stdp in Oenonidae.

The stomatograstric dorsoparamedian nerve (stdp) pair extends posteriorly parallel

to each other in the dorsolateral fold (dlf) until the posterior end of maxillae I (MxI)

where the two dlf fuse (this last step is unidentifiable in Oenonidae and Dorvilleidae). At

this site a thin nerve fiber branches out from stdp (stdp2) (dsn1 sensu Purschke, 1987),

and continues dorsally in the dorsolateral walls of the gut towards the posterior end,

while the remaining thick branches of stdp (stdp1) (dsn2 sensu Purschke, 1987) run

35 towards the mid-ventral side, becoming as ventral as the anterior end of the oesophageal

nerves (oen) (Fig. 2.6) (unidentifiable in Oenonidae and Dorvilleidae). At this point the branches of the stdp1 converge in the median region and continue posteriorly parallel to

each other until the anterior quarter of the maxillary carrier (MxC), where they diverge,

run towards the lateral side, wrap around the MxC (stdp1wr) and finally connect with the pharyngeal nerves (phn) ventrally (Fig. 2.6) (unidentifiable in Oenonidae and

Dorvilleidae). From the site of the divergence of the stdp1 to the site at the ventral side where it connects to phn, nerves branch off from the stdp1 and innervate the muscles

associated with MxC.

In Eunicidae and Onuphidae, stdp2 bifurcates posterior to the muscularized pharynx; one branch continues posteriorly in dorsolateral walls of the gut and the other forms a commissure with the stdp2 branch from the opposite side (stdp2c) (Fig. 2.7D,E).

In Eunicidae, stdp2c is immediately posterior to the muscularized pharynx (in the same

chaetiger as the posterior end of the muscularized pharynx) and as dorsal as the convergence of the stdp1. While in Onuphidae, stdp2c is approximately one chaetiger posterior to the posterior end of the muscularized pharynx and is ventral to the

convergence of the stdp1. This combination of bifurcation and commissure of the stdp2 were unidentifiable in the other families.

In Dorvilleidae the α-tubulin stain signal of the cilia placed on the dorsolateral fold

(dlf) masked the stomatograstric dorso-paramedian nerve (stdp) signal, for this reason it was not clear how the thick branches of stdp (stdp1) run to the ventral side at the posterior

end. However it was possible to determine that part of the stdp continues dorsally in dorsolateral walls of the gut and a faint signal similar to the ventroposterior divergence of

36 the stdp1 and wrapping around the maxillary carrier (MxC) is present about midway through the second half of the maxillae (Fig. 2.6E,F).

The oesophageal nerves (oen) and ramifying nerves may differ among the different families in the distance between the two nerves of the oen pair, the site they go through anterior to the oesophageal ganglion (oeg) and in the way the thick branches of the stomatograstric dorsoparamedian nerve (stdp1) connect and/or fuse.

Anterior to the oesophageal ganglion (oeg) the oesophageal nerves (oen) are placed differently in the five families. In Eunicidae and Oenonidae the pair of oen come close together immediately after branching from the third ventral commissure of the vrcc

(vcvr3) and extend posteriorly into the prostomium side by side, almost merging (Figs.

2.2B,K, 2.3C, 2.4C). In Eunicidae the oen run close together until the dorsolateral fold

(dlf) while in Oenonidae they are close together in the narrow anterior end of the dorsolateral fold anterior extensions (dlfae) and separate once the dlfae separate. In the other three families, the oen are conspicuously separated along their entire course. After branching from vcvr3 and before reaching the dlf, the oen run through the posterior extension of the prostomium (pep) in Eunicidae and Onuphidae (Fig. 2.2F) and through the dlfae in Oenonidae and Lumbrineridae. In Dorvilleidae the oen do not enter directly from the vcvr3 in the dlf, but first run through a structure dorsal to the pharyngeal fold, which we could not clearly identify. In Eunicidae and Onuphidae, the two thick branches of the stomatograstric dorsoparamedian nerve (stdp1) branches connect to form a commissure (stdp1c) (second ganglion sensu Quatrefages, 1844; infraoesophageal ganglion of visceral nervous system sensu Heider, 1921) when they converge towards the

37 mid-ventral side at the posterior end of maxillae I (MxI), in Lumbrineridae both stdp1 approach each other but continue posteriorly as distinct nerves (Figs. 2.6A-D,G,H).

In all families, the pharyngeal nerve (phn) extends ventro-posteriorly in the lateral edge of pharyngeal fold (phf). Posterior to phf, this nerve follows the terminal connection of the posteriormost ventral transverse muscles of the muscularized pharynx, which are posterior to the posterior end of the mandibular carriers and terminate posterior to the posterior end of the maxillary carrier (MxC). At the posterior end of the muscles the phn from the opposite sides connect in a commissure (Fig. 2.7A,E,H). Along its way the phn gives off small dead-end nerves which innervate the phf and laterodorsal muscles of the muscularized pharynx (Fig. 2.7B,F,H). Phn also branches off six larger nerves which innervate the muscles close to MxC and the maxillary fold (mxf) (Fig. 2.7B,F,H). The most anterior of these nerves (phn1) diverges from the phn at the anterior end of the muscularized pharynx, all other nerves (phn2-6) branch off from phn at the ventrolateral sides of the muscularized pharynx. Such features of the phn were unidentifiable in

Oenonidae and Lumbrineridae. In Lumbrineridae a nerve splits from phn immediately posterior to its origin at the third ventral commissure of the vrcc (vcvr3) and innervates the transverse anterior peristomial extension of the dorsolateral fold (tpe). In Oenonidae we could only observe the left phn, the lack of the right one is probably a staining artifact

(Fig. 2.2J,K).

In Eunicidae and Onuphidae the anteriormost large nerve to branch off from the

pharyngeal nerve (phn1) is median and continues towards the posterior end in the

maxillary fold (mxf) until the mid-posterior end of mxf, and the posterior end of maxillae

I (MxI), where it fuses with the fourth anteriormost large nerve branching off from phn

38 (phn4) and connects to the fifth anteriormost large nerve branching off from phn (phn5).

Anterior to the fusion of phn1 and phn4, nerves branching off from phn1 and from the third anteriormost large nerve branching off from phn (phn3) innervate the ventralmost side of the mxf. From the site at which phn1 and phn4 fuse they extend dorso-posteriorly until the anterior end of maxillary carrier (MxC), where the phn1+ phn4 from both sides

of the body connect in a U-shaped commissure (phn1phn4-uc). Phn1+ phn4 connect along its way with the posteriormost large nerve branching off from phn (phn6) and the posterior ventral wrap of the thick branches of the stomatograstric dorsoparamedian nerve (stdp1wr) respectively. At the U-shaped commissure at the posterior end of phn1+

phn4, two midposterior nerves split off and diffusing nerves innervate the median muscles at the ventral side of MxC. Dorso-posterior to the connection of phn6 with phn1+ phn4, a thin nerve comes off from phn1+ phn4 extending to the anterior end (phnmxI) (Fig.

2.7C,G). At its anterior end phnmxI follows the outline of the ventroposterior end of MxI and gives rise to several small blind-end nerves and to a nerve that runs anteriorly

(phnmxII) until the ventroposterior end of maxillae II (MxII), where it innervate the muscles.

Phn3 runs obliquely in a mid-posterior direction and connects to phn4 at its posterior end. Phn4 also extends in the mxf obliquely at a mid-posterior direction. However it is at a more dorsal position and slanted at a greater angle than phn3. Phn5 and phn6 branch off close together from phn at a site posterior to the U-shaped commissure at the posterior

end of phn1+ phn4.They run in a dorso-midanterior direction and become conspicuously

separated before connecting to phn1+ phn4. The second anteriormost large nerve

39 branching off from phn (phn2) extends forward and ventrally, innervating the ventroanterior end of the pharyngeal fold.

In Dorvilleidae, we were only able to recognize phn1, phn4 and phn5phn6. Phn1 is thicker than in Eunicidae and Onuphidae and runs obliquely towards the posterior end. At its posterior end phn1 loops dorso-posteriorly to connect with what appears to be the

posterior ventral wrap of the thick branches of the stomatograstric dorsoparamedian

nerve (stdp1wr). We could not distinguish a posterior U-shaped commissure nor the separation between phn5 and phn6.

DISCUSSION

The comparative observations of the morphology and innervation described above allowed for a broad understanding of the variation of the anterior nerves within Eunicida and for hypotheses, that can be tested under a phylogenetic framework, of the evolution of this system within the order and in relation to what is considered to be the polychaete ground pattern and typical architecture. The similarities of the innervation of anterior features among the different families combined with correspondence and topological similarities of the morphology made it possible to formulate and support the explicit

hypotheses of primary homology discussed below that can also be further tested in a

phylogenetic framework and possibly become synapomorphies to family level and more

inclusive clades, improving the resolution within the ordinal phylogeny.

Cephalic and Stomatogastric nerves

40 The cephalic nervous system of the families examined here is similar to that of the

typical polychaete in that the dorsal and ventral roots of the circumoesophageal

connectives (drcc and vrcc) are partially fused (Müller, 2006).

All families examined bear five to seven commissures of the circumoesophageal

connectives, two of the dorsal and three to five of the ventral root (of which two or three

belong to the dorsal branch and one or two to the ventral branch). In Onuphidae and

Eunicidae, this pattern has been considered derived from the typical polychaete supraoesophageal ganglion (Orrhage, 1995). The typical polychaete supraoesophageal ganglion and ground pattern is characterized by the presence of four commissures of the

circumoesophageal connective, two of the dorsal and two of the ventral root (Müller,

2006). This pattern of four commissures was not observed in the eunicidan families nor

in some other families of polychaete, putting the ground pattern hypothesis to the test

(Müller, 2006). The ground pattern hypothesis assumes that taxa having fewer

commissures in the adult have lost them or that they became fused, as observed in

regenerating specimens (Müller and Hennig, 2004), and that the presence of more

commissures in some taxa is due to ramifications of the original four (e.g., Orrhage and

Müller, 2005). The topological similarities observed in the present study support the

primary hypothesis of homology between the commissures of the roots and branches of

the circumoesophageal connective in Eunicida and the commissures of the respective

roots and branches in other families. However the hypotheses of evolution and homology

of the commissures as well as the ground pattern hypothesis have to be tested in the light

of the Annelida phylogeny, especially since Polychaeta is paraphyletic (McHugh, 2000),

before any conclusion can be drawn.

41 The general morphology of the cephalic system and relative position of the

connection of the roots of the circumoesophageal connective with the different

commissures varied among families of Eunicida. However it appears to be conservative

within families when the current results are compared to previous studies (e.g., Spengel,

1882; Pruvot, 1885; Binard and Jeener, 1929; Orrhage, 1995; Müller and Hennig, 2004).

Similar external morphology of the prostomium did not result in similar morphology of

the central nervous system. Lumbrinerids and oenonids are the families with the most

similar external morphology of the prostomium. However they have conspicuously

different supraoesophageal ganglion morphology (see also Spengel, 1882) including

differences in the position of the supraoesophageal ganglion (partially located under the

peristomium fold in Lumbrineridae), the shape of the anterolateral nerve projections

(alp), the relative positions of the commissures and oesophageal nerves (oen) and the

number of the ventral commissures (the second and fourth ventral commissures of the

ventral root of the circumoesophageal connective, vcvr2 and vcvr4, are not observed in

Oenonidae). In oenonids usually the prostomium is more flattened and tapering than in lumbrinerids which could account for some of these differences.

The supraoesophageal ganglion is widest, shortest and most anterior in Onuphidae, where the commissures of the dorsal root of the circumoesophageal connective sits roughly on top of the ventral root commissures, and anterolateral nerves projections (alp) are missing. Only in onuphids, the dorsal root and the dorsal branch of the ventral root of the circumoesophageal connectives (drcc and dbvr) are anterior to their ventral

complements, the ventral root (vrcc) and ventral branch of the ventral root (vbvr), respectively (Fig. 2.3A,B). The onuphid and eunicid supraoesophageal ganglia have been

42 considered very similar in previous studies (e.g., Pruvot, 1885; Orrhage, 1995). They are certainly the most similar among the five families studied here, however we observed additional differences between the two families not yet described in the literature. Such differences, which also distinguish Onuphidae from the other families studied here, include the relative position of the commissures of the roots of the circumoesophageal connectives, the lack of a dc-vcvr intrasupraoesophageal ganglion cavity between the dorsal and ventral commissures of the vrcc (dcvr and vcvr) and of alp in Onuphidae.

These differences observed in Onuphidae in relation to the remaining Eunicida families could be due to a relative decrease in the size of the prostomial cavity of onuphids, perhaps linked to the conspicuous evaginations of the dorsal and ventral buccal lip. A deeper comparative and evolutionary study is necessary to test such hypothesis.

All eunicidan families have a cavity between the commissures of the ventral root of the circumoesophageal connective (vrcc) (Spengel, 1882; Pruvot, 1885; Hanström, 1927;

Binard and Jeener, 1929). In Onuphidae the cavity (vcvr intrasupraoesophageal ganglion cavity) is between the first-second ventral commissures of the vrcc (vcvr1 and vcvr2) and the third-fourth ventral commissures of the same root (vcvr3 and vcvr4), while in all other examined families the cavity (dc-vcvr intrasupraoesophageal ganglion cavity) is placed between the dorsal and ventral commissures of the vrcc (dcvr and vcvr) and conspicuously separates the dorsal and ventral neuropiles of the supraoesophageal ganglion. Onuphidae has a nerve plexus between dcvr and vcvr1-vcvr2 which is absent in the other families and it lacks the plexus connecting vcvr1and vcvr4 which is present in the other eunicidan families examined. The onuphid vcvr intrasupraoesophageal ganglion cavity is probably a derived condition in this family, since Onuphidae is nested within the

43 eunicidan clade (Struck et al., 2006) and its sister family (Eunicidae) and all more basal families (Dorvilleidae, Oenonidae and Lumbrineridae) bear a dc-vcvr intrasupraoesophageal ganglion cavity. Most studies do not describe an intrasupraoesophageal ganglion cavity for other annelid families or indeed for the families used in this study (e.g., Orrhage, 1995). Therefore it is not clear how widespread and varied in position these cavities may be in Annelida. An intrasupraoesophageal ganglion cavity appears to be present also in Eunice(Eunicidae, polychaete), Euphrosyne

(Euphrosinidae, polychaete) (Raw , 1949) and Harmothoe imbricata (Polynoidae, polychaete) (Binard and Jeener, 1929). Raw (1949) described what seems to be the intrasupraoesophageal ganglion cavity as a muscle gap in Eunice and Euphrosyne prostomia. Further studies on the presence and placement of the intrasupraoesophageal ganglion cavity in other annelids could provide interesting information for a better understanding of the supraoesophageal ganglion morphology and evolution.

The current division of the supraoesophageal ganglion in two main neuropiles

(dorsal and ventral) clearly separated by the dc-vcvr intrasupraoesophageal ganglion cavity is utilitarian rather than topologically or functionally important. It does not coincide with the connections of the dorsal and ventral roots of the circumoesophageal connective (drcc and vrcc) in the supraoesophageal ganglion, supporting Binard and

Jeener (1929) observations and contradicting Pruvot (1885), nor with nerve functions

(e.g., motor versus sensory nerves). The nerves branching from the dorsal neuropile innervate sensory structures, such as the nuchal organs, eyes, and sensory structures associated with antennae and part of the palps. However, the nerves from the ventral neuropile have sensory as well as motor function, innervating the prostomium, buccal

44 lips, stomatogastric system and part of the palps, confirming prior results in Spengel

(1882), Pruvot (1885), Binard and Jeener (1929) and Orrhage (1995). This division of the

neuropiles also does not support any hypothesis of segmentation of the supraoesophageal ganglion even though our dorsal neuropile is equivalent to the median and hind supraoesophageal ganglia sensu Heider (1925) (“centre antennaire” sensu Pruvot, 1885) and the ventral neuropile is equivalent to Heider’s anterior supraoesophageal ganglion

(“centre stomato-gastrique” sensu Pruvot, 1885).

The classification of the first and second ventral commissures of the vrcc (vcvr1 and vcvr2) as ventral is also not topologically accurate. Even though both vcvr1 and vcvr2 are

ventral to the dorsal commissure of the ventral root of the circumoesophageal connective

(dcvr), the three of them (vcvr1, vcvr2 and dcvr) are commissures of the dorsal branch of the vrcc (dbvr). The only commissures of the ventral branch of the vrcc (vbvr) are the third and fourth ventral commissures (vcvr3 and vcvr4).

The fourth ventral commissure of the vrcc (vcvr4) bearing the nerves innervating the buccal lips (nbl) is described for the first time in the present study. Previous studies considered that the closely placed nbl, oesophageal and pharyngeal nerves (oen and phn) emanated from the same commissure, the third ventral commissure of the vrcc (vcvr3)

(Heider, 1925; Binard and Jeener, 1929; Orrhage, 1995). In the examined species these nerves are certainly closely placed, however they branch from different commissures of the ventral branch of the vrcc (vbvr), oen and phn branch from vcvr3, while nbl branches

from vcvr4. In Oenonidae we did not observe a vcvr4 as in the other families. However we observed thin nerves also branching from vbvr (lgvbvr and shvbvr, Fig. 2.5) and innervating the ventral side of the prostomium; we consider these nerves homologous to

45 vcvr4. The present observation of the origin of oen and nbl from different commissures cast doubt on the hypothesis that the buccal lips in Eunicidae and Onuphidae are part of the alimentary canal (Orrhage, 1995). Since only the oen or nbl can have a corresponding

placement to the alimentary canal or stomatogastric nerves of other families (e.g.,

Acoetidae, Polynoidae, Spionidae and Trochochaetidae).

The main nerves and the general innervation of the muscularized pharynx and

anterior end of the digestive system in Eunicida are conservative characteristics present in

most or all of the observed eunicidan families in the present and previous studies. (e.g.,

Quatrefages, 1844; Heider, 1925; Orrhage, 1995; Müller and Hennig, 2004). Such

conservative characteristics are the presence of oesophageal nerves (oen); the placement

of the oesophageal ganglion (oeg) on the anterior side of the muscularized pharynx;

stomatogastric dorsolateral and dorsoparamedian nerves (stdl and stdp) innervating the

dorsolateral fold, the dorsal side of the maxillary carrier region of the muscularized

pharynx (unidentifiable in Oenonidae and Dorvilleidae) and the dorsal walls of the

oesophagus and gut (unidentifiable in Oenonidae and Dorvilleidae); and pharyngeal

nerves (phn) branching from the third ventral commissure of the vrcc (vcvr3), extending posteriorly in the pharyngeal fold (phf) and outlining the ventral side of the muscularized pharynx (unidentifiable in Oenonidae and Lumbrineridae). Most polychaete families have some form of stomatogastric nerves branching from the same relative position as in

Eunicida (Orrhage and Müller, 2005). However the hypotheses of homology between these nerves in Eunicida and in the other families are not clear. The commissure of the stomatogastric dorsoparamedian nerve posterior to the muscularized pharynx (stdp2c) in

Eunicidae and Onuphidae may indicate the edge between two different gut regions, such

46 as for example, the transition from the oesophagus to intestine in some Eunicida (Ehlers,

1868) and the differing gut structures along the gut in other polychaete (Tzetlin and

Purschke, 2004).

Within Eunicida some of the stomatogastric nerves (stn), dorsoparamedian and pharyngeal nerves, vary respectively in relative position and branching nerves among families with different jaw types, which require different muscle systems. These differences in the stn may be due to the muscle differences and to evolutionary relatedness. Eunicidae and Onuphidae, families with the most similar stn and closest in evolution, have asymmetric labidognath maxillae in a folded maxillary fold, the commissure of the thick branch of the stomatograstric dorsoparamedian nerve (stdp1c)

and six identically large nerves emanating from phn. Dorvilleidae bears ctenognath

maxillae with multiple denticles in a straight maxillary fold and only four thicker large

nerves emanating from phn (stdp1c is unidentifiable), while Lumbrineridae have

symmetric labidognath maxillae in a straight maxillary fold and lack stdp1c (nerves

emanating from phn are unidentifiable). We could only observe the stn in full detail in

Eunicidae and Onuphidae, the poor staining signal at this region in Oenonidae and

Lumbrineridae restrained us from observing parts of the stn, and in Dorvilleidae,

background noise mainly due to ciliary staining also did not allow a detailed observation

of the posterior end of stn, parts of phn and of the large branching nerves. Therefore before more detailed information is available on the stn of the eunicidan families and the phylogeny of the order is considered we cannot further consider the source of similarity of these nerves. In the family Eunicidae the relative position of the oesophageal nerves

(oen) in relation to each other, the oesophageal ganglion (oeg), stomatogastric

47 dorsolateral nerve (stdl), the anterior end of the stomatogastric dorsoparamedian nerve

(stdp) and stomatograstric dorsoparamedian nerve commissure (stdp1c) are conservative

features (e.g., Marphysa sanguinea in Quatrefages, 1844; Eunice punctata in Heider,

1925). The existing differences described below between our observations and those of

Quatrefages (1844) and Heider (1925) are due to misinterpretation of the nerves by

previous authors. Their descriptions contradict each other and differ from what we

observed in Eunicidae and other eunicidan families.

The anterolateral nerve projection (alp) described here for Eunicidae, Dorvilleidae,

Oenonidae and Lumbrineridae bear the roots for nerves innervating the anterior end,

dorsal, lateral and part of the ventral side of the prostomium of these families (e.g.,

Pruvot, 1885; Heider, 1925; Haffner, 1962). Heider (1925) and Haffner (1962)

considered the alp equivalent to the nerves innervating the dorsal and ventral buccal lips

(ndbl and nvbl) in Onuphidae. Orrhage (1995) may also have considered some of these nerves as innervating buccal lips, since one of the differences he described between the

Eunicidae and Onuphidae supraoesophageal ganglia was the larger number of nerves innervating the buccal lips of Eunicidae than in Onuphidae. We did not observe this difference in the number of nerves in the two families when only the nerves branching from the fourth ventral commissure of the vrcc (vcvr4),as the ones innervating the buccal lips of the Onuphidae, are considered. The alp observed in Dorvilleidae contrasts to that of the other eunicidan families by including only the ventral side (alp-dbvr) and for projecting nerves to the anterodorsal side, not anterior or anteroventral sides as in the other families. This pattern of nerve projection of the alp-dbvr of Dorvilleidae is probably due to the more ventral connection of the ventral root of the circumoesophageal

48 connective (vrcc) to its dorsal and ventral commissures (dcvr and vcvr) than in the other

families. Pruvot (1885, plate XIII figure 12) illustrates a pair of ovoid projections at the

anterior end of the supraoesophageal ganglion that may represent the alp in Dorvilleidae,

alp has otherwise not been described in other previous studies of the cephalic nervous

system of Dorvilleidae (e.g., Müller, 2004; Müller and Henning, 2004).

Dorvilleidae and Onuphidae, which lack at least the dorsal part of the anterolateral

nerve projection (alp), have nerves branching from the same nerves (dorsal fibril mass and dorsal commissure of the ventral root of the circumoesophageal connective, the latter only in Onuphidae) and innervating similar regions of the prostomium as the alp. These similarities suggest that the nerves innervating the prostomium in both families are homologous to the ones in the alp. Therefore considering the current hypothesis of phylogeny of the order Eunicida (Struck et al., 2006), the nerve fibers forming the dorsal or complete alp have been lost independently in these two families. The loss of the alp nerves may be related to the relatively smaller prostomia in these two families than in the other three families examined here.

The dorvilleid observed here is unique in having a pair of nerves emanating towards

the anterior end from the dorsal commissure of the dorsal root of the circumoesophageal

connective (dcdrn) and innervating the dorsoanterior end of the prostomium. In eunicid

and onuphid a similar pair of nerves branch from dcdr but innervate the median antenna

(nma). The similarity in topology and the direction they extend suggest that dcdrn and

nma are homologous. However because of the uncertain position of Dorvilleidae in the

eunicidan phylogeny (Struck et al., 2006) it is not possible to resolve the evolution of

these nerves, if, for example, the nerves were originally associated with a median antenna

49 or with other portions of the dorsoanterior prostomium. To the best of our knowledge dcdrn have not been reported in previous studies of dorvilleid nervous system (e.g.,

Pruvot, 1885; Müller, 2004; Müller and Henning, 2004)

Additional observations of the present study contrasting with previous studies.

1. The intrasupraoesophageal ganglion cavity in the Lumbrineridae examined here is

the smallest among all families and not the largest as described in Pruvot (1885).

This difference could be due to variation within Lumbrineridae as well as

procedural artifacts.

2. We could not distinguish the three splits of the nuchal commissures described for

Eunicidae and Onuphidae as well as the additional nuchal nerves extending from

the dorsal commissure of the drcc (dcdr) (Orrhage and Müller, 2005).

3. Orrhage (1995) described five palp nerve roots in the Onuphidae

supraoesophageal ganglion, two branching from the dorsal and three from the

vrcc. In the present study we observed only one palp nerve emanating from each

root of the circumoesophageal connective in both Onuphidae and Dorvilleidae.

This difference could be due to a higher resolution of the sections examined by

Orrhage than in the images yielded by cLSM in the present study or to

interspecific variation. Even though we did not observe as many nerves

innervating the palps as Orrhage (1995) did, our observation support the status of

these structures as palps in Onuphidae and Dorvilleidae. Their innervation are in

accord with the palp innervation definition, in that at least one palp nerve root

branch from each of the roots of the circumoesophageal connective (Orrhage and

Müller, 2005).

50 4. We did not observe in Onuphidae a nerve branching from the first ventral

commissure of the ventral root of the circumoesophageal connective (vcvr1) and

innervating the dorsal buccal lips as described in Orrhage (1995). Our results

show that most nerves innervating the dorsal buccal lip branch from the fourth

ventral commissure of the vrcc (vcvr4) plus a few from the association system.

5. The oesophageal ganglion (oeg) has been described and characterized as being

surrounded by ganglia cells (e.g., Heider, 1925; Binard and Jeener, 1929; Haffner,

1959; Orrhage, 1995). In the present study we did not observe any ganglia cells

surrounding it. In Dorvilleidae the lack of ganglia cells in oeg makes it resemble a

chiasm (Pruvot, 1885). Müller and Henning (2004) described the oeg in

Dorvilleidae as stomatogastric connective and ring, suggesting that they also did

not observe ganglion cells in this location.

6. In the species observed here the pharyngeal nerve (phn) branches from the lateral

sides of the third ventral commissure of the vrcc (vcvr3) close to the ventral branch

of the same root (vbvr) as described in Binard and Jeener (1929) and not from the

circumoesophageal connective (cc) (Heider, 1925; Orrhage, 1995).

7. In Eunicidae the commissure of the thick branches of the stomatogastric

dorsoparamedian nerve (stdp1c) (infraoesophageal ganglion of visceral system

sensu Heider, 1925) is directly connected to the wrap of the same nerves (stdp1wr)

(second ganglion sensu Quatrefages, 1844; x-shaped ganglion sensu Heider,

1925), However Heider (1925) describe these two structures as if they were

independent while Quatrefages (1844) has no mention of the stdp1c. The stdp1wr

ventral connection with two large nerves branching from the pharyngeal nerves

51 (phn1 and phn4) was also observed and described in Quatrefages (1844) and

Heider (1925). We could not identify the posteriormost oesophageal ganglion

described in Heider (1925), which is supposed to be posterior to stdp1wr. The thin

branches of the stomatogastric dorsoparamedian nerve (stdp2) which extend

posteriorly in the dorsal walls of the gut (lateral oesophageal nerves sensu Heider,

1925) were not mentioned in Quatrefages (1844). Quatrefages’ (1844) inferior

system of the proboscis has the same nerve shapes as our system of pharyngeal

nerves, however the author illustrated the system posterior to and as dorsal as the

oesophageal nerve system. Such position of the system of pharyngeal nerves

would be possible only if he had folded the pharyngeal system backward before

illustrating it, however no mention of this is presented in his text.

8. Our observations on the posterior end of the cephalic nerves in Oenonidae are

similar to the description in Spengel (1882). However he described the pair of

oesophageal nerves (oen) extending apart from each other before entering the

anterior extension of the dorsolateral fold (dlfae). This difference may be due to

interspecific variation or to a misinterpretation of the observed nerve. The nerve

Spengel (1882) labeled as oen in his figure of the prostomium nerves in ventral

view (sn figure 52 in Spengel, 1882) is similar to the nuchal nerves of the species

we studied (Fig. 2.2H), which reaches the posterior end of the nuchal organ

beyond the anterior end of the dlfae.

Hypotheses of primary homology at the anterior end of Eunicida

52 Even though the external morphology of the anterior end and cephalic nerves vary

considerably among the Eunicida families examined here, we observed similarities in the

innervation of the prostomium and pharyngeal structures that give support to hypotheses

of primary homology of buccal lips, dorsolateral and pharyngeal folds and the

Dorvilleidae maxillae basal plates and carrier like structure.

Buccal lip, antennae and palps. Our observations corroborate Orrhage’s (1995) in

supporting the homology of the buccal lips between Eunicidae and Onuphidae. In both

families they are minimally innervated by nerves branching from the fourth ventral

commissure of the vrcc (vcvr4). However we did not observe any nerves branching from

the first ventral commissure of the vrcc (vcvr1) and innervating the dorsal buccal lip as

described in Orrhage (1995). In Onuphidae a few dorsal buccal lips nerves also branch

from the ventral side of the anterior association system.

Some Eunice (Eunicidae) species have a dorsal partition in the prostomium forming two dorsal lobes, leading some authors to describe such prostomia as four lobed (e.g.,

Kinberg, 1865). These dorsal lobes have been considered homologous to dorsal buccal lips in the Onuphidae (e.g., Pruvot and Racovitza, 1895; Haffner, 1959). The Eunicidae

juveniles observed in the present study lack such dorsal lobes; thus we cannot evaluate

this hypothesis. Future studies on species bearing such dorsal lobes will provide

important new insights in the buccal lip homology.

In Lumbrineridae the area of the prostomium innervated by nerves branching from

the fourth ventral commissure of the vrcc (vcvr4) (ventro-midposterior side of the

prostomium) (Fig. 2.2M) corresponds to the region of the prostomium of the dissected L.

latreilli bounded by the inconspicuous ventral furrow (Fig. 2.1I). In Oenonidae the region

53 equivalent to the one bounded by the inconspicuous ventral furrow (Fig. 2.1E) observed

in the dissected A. iricolor and in other species of Oenonidae (Pettibone, 1957) is

innervated by the long and short nerves also branching out from the anterior most side of

ventral branch of the vrcc (vbvr) which we consider homologous to vcvr4. Finally, in

Dorvilleidae the nerves branching from vcvr4 innervate the ventral region of the prostomium directly anterior to the pharyngeal folds (Fig. 2.1G). These similarities in the innervation and location in the prostomium suggest that in both Lumbrineridae and

Oenonidae the region of the prostomium bounded by the inconspicuous ventral furrow and the region directly anterior to the pharyngeal fold in Dorvilleidae are homologous to the buccal lip in Eunicidae and Onuphidae and should be referred to as the buccal lips

(Fig. 2.8).

The transverse anterior peristomial extension of the dorsolateral fold (tpe) in

Lumbrineridae and the pair of ventral pads on the prostomium of Dorvilleidae are not

innervated by the fourth ventral commissure of the vrcc (vcvr4) nerves and have similar

innervations to the anterior extension of the dorsolateral and pharyngeal folds,

respectively, see discussion below. This contradicts Pruvot’s (1885) hypothesis of homology with the ventral buccal lip in the Onuphidae.

Dorsolateral fold anterior extension and pharyngeal folds. In all families examined here the stomatogastric dorsoparamedian nerve (stdp) runs through the dorsolateral folds as previously described (Ehlers, 1868; Spengel, 1882; Heider, 1925;

Haffner, 1959; Purschke, 1987), supporting the previous description and hypothesis of homology of this feature among the eunicidan families (Tzetlin and Purschke, 2005).

54 In all families examined here, except Eunicidae, the oesophageal nerves (oen) extend

postero-dorsally through two folds (pep in Onuphidae) before reaching the oesophageal

ganglion and the dorsolateral folds (Fig. 2.1 D, F,H, J). In Eunicidae the oesophageal

nerves also run postero-dorsally through the pep, however in this family the pep is a

transverse band because the DM1 muscles that run in it are medially fused (Wolf, 1980).

In Oenonidae and Lumbrineridae, these folds are the anterior extension of the

dorsolateral fold (dlfae) (“Mundwülste” sensu Spengel, 1882). In Dorvilleidae the folds

through which oen run are inconspicuous and dorsal to the pharyngeal fold.

Spengel (1882) gave the same name to the anterior extension of the dorsolateral fold

(dlfae) of both Oenonidae and Lumbrineridae because of their innervation and

topological similarities. Because of the topological and innervation similarities of this

structure among all families we here expand Spengel’s (1882) homology hypothesis to

include Eunicidae and Onuphidae pep as well. We refer to it as the anterior extension of

the dorsolateral fold (dlfae) because of its close connection with the dorsolateral fold.

Further and more detailed observation of the dorvilleid folds the oen run through are

necessary before any hypothesis can be drawn about its homology.

Both species of Dorvilleidae examined here have a pair of ventral pads on the

prostomium around the mouth opening. Pruvot (1885) claimed homology for this

structure with buccal lips; in contrast, our analyses of the cephalic and stomatogastric

nervous system and dissections of the anterior end show, respectively, that the dorvilleid

ventral pads are not innervated by the fourth ventral commissure of the vrcc (vcvr4) as are the buccal lips of the other families and that they are directly connected to the pharyngeal fold (Fig. 2.1G,H). For this reason. it is here considered to be an anterior prolongation of

55 the pharyngeal fold (Fig. 2.8). However, this is probably not the case in the whole family, because in a species of Ophryotrocha, the dorsolateral fold, rather than the pharyngeal fold is present in the mouth opening (Purschke, 1987).

This variability within Dorvilleidae makes the schematic representation of the dorvilleid foregut presented by Purschke and Tzetlin (1996) showing the dorsolateral fold on the dorsal side of the mouth, inapplicable to dorvilleid taxa in which the anterior prolongation of the pharyngeal fold is present. Tzetlin and Purschke (2005) expanded the generalization of the same schematic drawing to the whole order Eunicida. This generalization is supported here for Eunicidae, Onuphidae, Oenonidae and

Lumbrineridae. In all these families we observed the dorsolateral fold anterior extension

(dlfae) present around the mouth opening (Fig. 2.8).

Dorvilleidae superior basal plates and maxillary carrier-like structure. In the families with labidognath and prionognath jaws examined here, with distinct maxillae I and maxillary carrier, the posterior fusion of the dorsolateral folds separating the pharyngeal lumen and the oesophagus is always at the posterior end of maxillae I (Fig.

2.1A,C,E,I). This is also the region where in the labidognaths the stomatogastric dorsoparamedian nerve (stdp) runs ventrally and wrap around the maxillary carriers

(unidentifiable in oenonid prionognath jaws).

In Dorvilleidae the fusion of the dorsolateral folds (Fig. 2.1G) and a faint signal of the stomatogastric dorsoparamedian nerve (stdp) wrapping around the maxillae were also observed. However, in Dorvilleidae the stdp wraps around the maxillae further posteriorly, in the region where the superior basal plates and carrier like structures are placed. Purschke (1987) also described the oesophageal nerve wrapping at the region of

56 the carrier-like structure - “The nerve dsn2 (oen) innervates the maxillary fold at the carrier-like structure…”.

These observations appear to increase the source of topological similarity between the ctenognath superior basal plates and carrier-like structures of the dorvilleids and maxillae I and maxillary carriers of the labidognaths. However the staining background noise at this region of the dorvilleid jaw does not allow enough detailed information to support any conclusion at this point. Further studies which can recover the details of the oesophageal nerves at this region are necessary.

CONCLUSIONS

The present study is the first to compare cephalic and stomatogastric innervations among the five eunicidan families examined here using immunohistochemical staining and cLSM and to use this information to draw hypotheses of homology. All families have

distinctive structures on the ventral side of the prostomium or in the anterior end of the pharynges, some of them have superficially similar external morphology but are innervated by different nerves and connected to different structures. The structures supported and proposed here as hypotheses of primary homology have correspondence and topological, external and innervation, similarities. However the topological similarities given by the innervation similarities were decisive in the hypotheses of primary homology of the buccal lip of the Dorvilleidae and of the dorsolateral fold anterior extension in all families.

The dorsolateral fold is present in all eunicidan families examined here. In all families, except Dorvilleidae, the dorsolateral fold has an anterior extension (dlfae), a

57 region anterior to the oesophageal ganglion. In Oenonidae and Lumbrineridae its posterior end is at or posterior to the peristomial posterior end, in Eunicidae and

Onuphidae it is short, terminating at the anterior half of the peristomium or anterior to it

(Fig. 2.8). In Eunicidae this anterior extension is a transverse band while in the other families we observed it is a pair of folds (Fig. 2.8).

The ventral pads on the Dorvilleidae prostomium are anterior prolongations of the pharyngeal fold (phf) and not buccal lips as previously suggested (Fig. 2.8).

Buccal lips are present in all families (Fig. 2.8). They are almost inconspicuous and entire in Dorvilleidae, Oenonidae and Lumbrineridae; have a median furrow in Eunicidae and Onuphidae; and in the latter they are conspicuous and divided in dorsal and ventral buccal lips.

We compared adults of some species with juveniles of others and examined the nervous system of just one species of each family under the assumptions that ontogenetic variation would be minimal since the structures of interest were of the adult shape or close to it; and that within family variation is expected to be smaller than among families.

These assumptions are supported by the fact that our observations were similar to the ones described in previous studies on other species of all families and adults of Eunicidae and Onuphidae (e.g., Pruvot, 1885; Binard and Jeener, 1929; Orrhage, 1995).

Future studies on more species representing the diversity of prostomium morphology within the families, examining different ontogenetic stages and using specimens at the same ontogenetic stage are necessary to test our results. This is especially important in Eunicidae and Dorvilleidae in which the shape of prostomia vary considerably and in the Dorvilleidae the variation in the maxillary plates creates further

58 problems. The prostomia and maxillae of Ophryotrocha like dorvilleid genera are different from those of the Schistomeringos like genera. Therefore some of the hypotheses of homology proposed here based on a Schistomeringos species may not be applicable to all dorvilleid genera. Detailed studies on the anterior end innervations of

Ophryotrocha will probably give important additional information for hypotheses of homology in this body region.

Table 2.1: Species examined under cLSM and their collection localities.

Family Species Locality Depth Life stage examine d Dorvilleidae Schistomeringos cf. pectinata Pepper Park, Fort 3 m adult Perkins, 1979 Pierce 27o 29′ 31″ N 80 o 17′ 49″ W Eunicidae Eunicidae gen. sp. Port Canaveral, intertidal juvenile 28 o 24′ 35″ N 80 o 37′ 59″ W Lumbrineridae Lumbrineris verilli Perkins, 1979 Indian River Lagoon, 1.5 m adult 27 o 23′ 55″ N 80 o 16′ 57″W Oenonidae Arabella cf. mutans (Chamberlin, Pepper Park, Fort 3 m adult 1919a) Pierce 27o 29′ 31″ N 80 o 17′ 49″ W Onuphidae Kinbergonuphis simoni (Santos, East side of the Indian intertidal-0.3 juvenile Day and Rice, 1981) River Lagoon, m Sebastian

Fig. 2.1. Ventral dissection and sagital median section of the prostomium and pharynx.

A,B: Eunice valens. C,D: Nothria iridescens. E,F: Arabella iricolor. G,H: Dorvillea sociabilis. I: Lumbrineris latreilli. J: Lumbrineris fragilis. A,C,E,G,I: Ventral dissection.

B,D,F,H,J: Sagittal section. bl, buccal lip; dbl, dorsal bl; dlf, dorsolateral fold; dlfae, dlf anterior extension; dlff, dlf fusion; DM1, pair of muscle bundles extending from the muscularized pharynx into the pep; ivf, inconspicuous ventral furrow; ma, median antenna; mdf, mandible fold; mxf, maxillary fold; p, palp; pep, posterior extension of the prostomium; phf, pharyngeal fold; pro, prostomium; tpe, transverse anterior peristomial extension of the dlf; vbl, ventral bl; vp, ventral pad. *- features discussed as such for the first time in the present study.

62 Fig. 2.2. Dorsal view of the cephalic and anterior stomatogastric nerves in the prostomium and anterior end of the pharynx, α-tubulin immunoreactivity, Z-series sections color coded to depth, red deepest, blue shallowest. A,B: Eunicidae gen. sp. A: sections 35.2 to 63.2 µm of 188 µm. B: sections 35.2 to 86.4 µm of 188 µm. C,D: Schistomeringos cf. pectinata C: sections 49.6 to 90.4 µm of 160.8 µm. D: sections 90.4 to 127.2 µm of 160.8 µm. E-G:

Kimbergonuphis simoni. E: sections 22.4 to 51.2 µm of 160 µm. F: sections 50.4 to 81.6

µm of 160 µm. G: sections 63.2 to 128.8 µm of 160 µm. H-K: Arabella cf. mutans H: sections 47.9 to 129.8 µm of 172.6 µm. I: sections 62.4 to 91.2 µm of 170.4 µm. J: All sections, 170.4 µm. K: sections 69.3to 143.1 µm of 175.5µm. L,M: Lumbrineris verilli. L: sections 25.6 to 49.6 µm of 105.6 µm. M: sections 48.8 to 75.2 µm of 105.6 µm. alp, anterolateral nerve projections; alp-dbvr, ventralmost side of the alp composed by the ventralmost nerves of the dbvr; cc, circumoesophageal connective; dbvr, dorsal branch of the vrcc; dcdr, dorsal commissure of the drcc; dcdrn, nerves branching from dcdr; dcvr, dorsal commissure of the vrcc; dcvrn, nerve branching from dcvr; dfm, dorsal fibril mass; dfmn, nerves branching from dfm; dln, dorsolongitudinal nerves branching from nerve cells posterior to the dorsal neuropile; DM1, pair of muscle bundles extending from the muscularized pharynx into the posterior extension of the prostomium; drcc, dorsal root of the cc; lgvbvr, long nerves branching from the anterior side of vbvr; lnalp, lateral nerves branching from the alp-dbvr; lno, lateral no; mlnalp, midlateral nerves branching from the alp-dbvr; mnalp, median nerve branching from the alp-dbvr; mno, median no; nbl, nerves of the buccal lip; nc, nuchal commissure; ndbl, nerve of the dorsal buccal lip; ndla, nerves of dorsolateral antennae; nma, nerves of the median antenna; no, nuchal organs; non, nerve of the nuchal organs; nvbl, nerve of the ventral buccal lip; oeg, oesophageal ganglion; oen,

63 oesophageal nerves; pcas, posterior commissure of the association system; phn, pharyngeal

nerves; pn5, nerves of the palp branching between vrcc and the vcvr2; pn6, nerves of the palp branching from the vcvr1; pn8, nerves of the palp branching from the dfm; pn12, nerves of the palp branching from the drcc; pnalp, posterolateral nerve branching from the alp; stdl, stomatogastric dorsolateral nerve ; stdp, stomatogastric dorsoparamedian nerve; vbvr, ventral branch of the vrcc; vcdr, ventral commissures of the drcc; vcdrn, nerve branching from vcdr; vcvr, ventral commissure of the vrcc; vcvr1, first ventral commissure

of the vrcc; vcvr2, second ventral commissure of the vrcc;vcvr3, third ventral commissure of the vrcc; vcvr4, fourth ventral commissure of the vrcc; vrcc, ventral root of the cc; xc, x shaped connection.

Fig. 2.3. Schematic drawing of selected cephalic and anterior stomatogatric nerves, not all

branching nerves are represented. A: Onuphidae dorsal view, black most dorsal, lightest gray most ventral. B: Onuphidae ventral view, black most ventral, lightest gray most dorsal. C: Eunicidae dorsal view, black most dorsal, lightest gray most ventral. alp, anterolateral nerve projections; alp-dbvr, ventralmost side of the alp composed by the ventralmost nerves of the dbvr; alp-dfm, dorsalmost side of alp composed by nerves of the dfm; cc, circumoesophageal connective; dbvr, dorsal branch of the vrcc; dcdr, dorsal commissure of the drcc; dcvr, dorsal commissure of the vrcc; dcvrnL, lateral nerve branching from dcvr; dcvrnM, median nerve branching from dcvr; dfm, dorsal fibril mass; dfmnL, lateral nerves branching from dfm; dfmnM, median nerves branching from dfm; drcc, dorsal root of the cc; nbl, nerves of the buccal lip; nc, nuchal commissure; ndbl, nerve of the dorsal buccal lip; ndla, nerves of dorsolateral antennae; nma, nerves of the median

antenna; no, nuchal organs; non, nerve of the nuchal organs; nvbl, nerve of the ventral

buccal lip; oeg, oesophageal ganglion; oen, oesophageal nerves; pcas, posterior

commissure of the association system; phn, pharyngeal nerves; pn5, nerves of the palp

branching between vrcc and the vcvr2; pn6, nerves of the palp branching from the vcvr1; pn12, nerves of the palp branching from the drcc; stdp, stomatogastric dorsoparamedian nerve; vbvr, ventral branch of the vrcc; vcvr, ventral commissure of the vrcc; vcvr1, first

ventral commissure of the vrcc; vcvr2, second ventral commissure of the vrcc;vcvr3, third ventral commissure of the vrcc; vcvr4, fourth ventral commissure of the vrcc; vrcc, ventral root of the cc; xc, x shaped connection.

67 Fig. 2.4. Schematic drawing of selected cephalic and anterior stomatogatric nerves, not

all branching nerves are represented. A: Dorvilleidae dorsal view, black most dorsal,

lightest gray most ventral. B: Dorvilleidae ventral view, black most ventral, lightest gray

most dorsal. C: Oenonidae dorsal view, black most dorsal, lightest gray most ventral; one of the vcdr omitted for clarity. D: Lumbrineridae dorsal view, black most dorsal, lightest gray most ventral. alp, anterolateral nerve projections; alp-dbvr, ventralmost side of the alp composed by the ventralmost nerves of the dbvr; alp-dfm, dorsalmost side of alp composed by nerves of the dfm; cc, circumoesophageal connective; dbvr, dorsal branch of the vrcc; dcdr, dorsal commissure of the drcc; dcdrn, nerves branching from dcdr; dcvr, dorsal commissure of the vrcc; dfm, dorsal fibril mass; drcc, dorsal root of the cc; lno, lateral no; lnon, nerves of lateral no; mno, median no; mnon, nerves of the median no; mp, median plexus; nbl, nerves of the buccal lip; nc, nuchal commissure; ndla, nerves of dorsolateral antennae; no, nuchal organs; non, nerve of the nuchal organs; oc, ocular commissure; ocn, ocular nerves; oeg, oesophageal ganglion; oen, oesophageal nerves; phn, pharyngeal nerves; pn5, nerves of the palp branching between vrcc and the vcvr2; pn6, nerves of the palp branching from the vcvr1; pn8, nerves of the palp branching from the dfm; pnalp, posterolateral nerve branching from the alp; stdp, stomatogastric dorsoparamedian nerve; vbvr, ventral branch of the vrcc; vcdr, ventral commissures of the drcc; vcdrn, nerve branching from vcdr; vcvr, ventral commissure of the vrcc; vcvr1, first ventral commissure of the vrcc; vcvr2, second ventral commissure of the vrcc;vcvr3, third ventral commissure of the vrcc; vcvr4, fourth ventral commissure of the vrcc; vrcc, ventral root of the cc.

Fig. 2.5. Arabella cf. mutans, ventral nerves of the prostomium, α-tubulin immunoreactivity, projection of Z-series sections 153.6 to 160.8 µm. lgvbvr, long nerves branching from the anterior side vbvr; oen, oesophageal nerves; shvbvr, short nerves branching from the anterior side of vbvr; vbvr, ventral branch of the ventral root of the circumoesophageal connective.

Fig. 2.6. Dorsal view of the dorsal stomatogastric nerves of the muscularized pharynx.

A,C,E,G: α-tubulin immunoreactivity, Z-series sections and transmission image of the jaws superimposed. B,D,F,H: α-tubulin immunoreactivity Z-series sections color coded to depth, red deepest, blue shallowest. A,B: Eunicidae gen. sp. C,D: Kimbergonuphis simoni. E,F: Schistomeringos cf. pectinata. G,H: Lumbrineris verilli. MxI, maxillae I;

MxC, maxillary carrier; oeg, oesophageal ganglion; oen, oesophageal nerves; stdl, stomatogastric dorsolateral nerve ; stdp, stomatogastric dorsoparamedian nerve; stdp1, thick branch of stdp; stdp1c, commissure of the stdp1; stdp1wr, wrapping of the stdp1 around the MxC; stdp2, thin nerve fiber branching from stdp.

73 Fig. 2.7. Dorsal view of the pharyngeal nerves of the muscularized pharynx and of the

commissure of the dorsal stomatogastric nerves posterior to the muscularized pharynx, α- tubulin immunoreactivity, Z-series sections. A-D: Eunicidae gen. sp. A: anterior end, color coded to depth, red deepest, blue shallowest, sections from 71.2 to 116 µm of 199

µm. B: muscularized pharynx, color coded color coded to depth, red deepest, blue shallowest, sections from 48.8 to 96 µm of 196 µm. C: muscularized pharynx, sections from 55.2 to 66.4 µm of 196 µm. D: anterior end, color coded to depth, red deepest, blue shallowest, sections from 55.2 to 66.4 µm of 199 µm. E-G: Kimbergonuphis simoni. E: anterior end, color coded to depth, red deepest, blue shallowest, sections from 105.3 to

162.4 µm of 219 µm. F: muscularized pharynx, color coded color coded to depth, red deepest, blue shallowest, sections from 43.2 to 102.4 µm of 194 µm. G: muscularized pharynx, sections from 60 to 72.8 µm of 196 µm. H: Schistomeringos cf. pectinata. H: muscularized pharynx, color coded color coded to depth, red deepest, blue shallowest, sections from 37.6 to 64.8 µm of 117 µm. cc, circumoesophageal connective; MxI, maxillae I; MxC, maxillary carrier; oeg, oesophageal ganglion; oen, oesophageal nerves; phn, pharyngeal nerves; phn1, nerve branching from the main phn trunk at the anterior end of the muscularized pharynx; phn2-6, nerves branching from the main phn trunk at the ventrolateral sides of the muscularized pharynx; phn1phn4-uc, u-shaped commissure connecting phn1+phn4 from opposite sides; phnmxI, nerve branching from phn1+ phn4

which follows the outline of the ventroposterior end of MxI; stdp, stomatograstric

dorsoparamedian nerve; stdp2, thin nerve fiber branching from stdp; stdp2c, commissure of the stdp2 posterior to the muscularized pharynx.

Fig. 2.8. Ventral view of the prostomium and dissected muscularized pharynx. Hypotheses

of primary homology supported or proposed in this study surrounded my the different lines.

A: Eunice valens. B: Nothria iridescens. C: Arabella iricolor. D: Dorvillea sociabilis. E:

Lumbrineris latreilli.

Chapter 3: Molecular phylogeny of the family Eunicidae (polychaete, Annelida) based on 16S, COI and 18S nucleotide sequences

INTRODUCTION

Eunicidae are benthic marine annelids inhabiting soft and hard marine substrates; many burrow into hard corals and calcareous algae or live in their crevices and play important roles in coral reef communities (Hutchings, 1986). The family is distributed worldwide, but it is most common in shallow tropical waters (Fauchald, 1992a). Many eunicid1 species are economically important as bait for leisure and commercial fishing in diverse regions such as along the Mediterranean Coast, in Australia, Japan and the United

States of America (Gambi et al, 1994; Olive, 1994). Additionally, the reproductive swimming phase of members of the genus Palola are a delicacy for natives from several of the South Pacific Islands (Schulze, 2006).

The current generally accepted composition of Eunicidae has been stable since it was proposed by Hartman (1944, modified from Kinberg, 1865). This classification proposes that the Onuphidae is the closest lineage to Eunicidae, a hypothesis consistently recovered in both morphological and molecular phylogenies (Rouse and Fauchald, 1997; Struck et al., 2002, 2006; Rousset et al., 2007; Zanol et al., 2007; Struck et al., 2008). However, this classification proposes no unique diagnostic features (apomorphies) for the Eunicidae but instead characterizes it with plesiomorphies or homoplasies (Orensanz, 1990), such as asymmetric labidognath jaws, one to five prostomial appendages, peristomium double

1 Herein the adjective eunicid denotes members of the family Eunicidae whereas eunicidan denotes members of the order Eunicida. 78 ringed and wide bilobed prostomium. Previous molecular and morphological phylogenetic

analyses focusing, respectively, on the order Eunicida and on the genus Eunice (type genus

for the family) (Struck et al., 2002, 2006; Zanol et al., 2007) did not always recover the current Eunicidae as monophyletic. In both the morphological analysis of Eunice using 59

characters and molecular analysis of Eunicida using just 18S rDNA data Eunicidae is

paraphyletic with Onuphidae nested within it, while it is monophyletic in molecular

analyses of Eunicida using data from more genes (COI, 16S rDNA, 18S rDNA and 28S

rDNA) and only two eunicid terminal species. Therefore, under a phylogenetic framework

the current composition for Eunicidae is dubious. No study has focused on the phylogeny of the family per se.

Species of Eunicidae (~326 spp.) are grouped in nine currently accepted genera

(Table 1.2). Proposed diagnostic characters of most genera appear to also be plesiomorphic

(e.g., five prostomial appendages, a pair of peristomial cirri and labidognath jaws in

Eunice) (Zanol et al., 2007) or ontogenetically variable (number of prostomial appendages in Lysidice and Nematonereis, and absence of peristomial cirri in Marphysa) (Åkesson,

1967; Giangrande, 1989). Despite these inconsistencies, this generic classification has remained unmodified for many years (Orensanz, 1990). Just three of the nine currently accepted genera have been revised and had their monophyletic status phylogenetically tested, with these results: Eunice is polyphyletic (Fauchald, 1992a; Zanol et al., 2007), and

Palola (Fauchald, 1992b; Schulze, 2006) and Euniphysa (Lu and Fauchald, 2000) are

monophyletic. Eunice can be divided in at least two monophyletic groups Eunice sensu

stricto and Leodice, however the composition and diagnostic features of such groups are still unclear (Zanol et al., 2007).

79 Eighteen additional genera have been described for eunicid species (Fauchald,

1992a). Most of these have been synonymized with other genera and a few are

indeterminable (Fauchald, 1992a). Of the genera later synonymized to the genus Eunice,

Leodice Lamarck, 1818 and Nicidion Kinberg, 1865 had the most widespread use by

several authors. Leodice named for Leodice antennata Lamarck, 1818, was not clearly differentiated from Eunice, and both names were used interchangeably through the first third of the 20th century (Hartman, 1944). Nicidion was described for abranchiate species, a characteric currently not considered valid at the generic level (Fauchald, 1992a). The original description includes three species, none of which was identified as the type- species. Two of these species, N. gualapaguensis and N. longicirrata, were placed in the

genus Palola and are indeterminable beyond the genus level because of the incomplete

original description and poorly preserved type specimens (Fauchald, 1992b). The third

species, N. cincta, is the only determinable species and has been referred to Eunice

(Hartman, 1948; Fauchald, 1992a).

According to Hartman (1944), eunicid phylogeny has a basal split separating Palola

from a clade containing the remaining genera recognized by her ((Eunice, Nicidion)

(Nematonereis (Lysidice (Marphysa, Paramarphysa)))). In an 18S rDNA phylogeny of

Eunicida, Eunicidae is paraphyletic and grouped in three clades (Lysidice, Nematonereis),

(Eunice species with yellow subacicular hook) and (Marphysa, Eunice torquata) (Struck et

al., 2006). The first scenario (Hartman, 1944) is completely hypothetical, lacking any

analytical foundation. The second (Struck et al., 2006) is based on a small fraction of the

eunicid diversity and on just one molecular marker, which can be biased by biological

processes (e.g., natural selection, horizontal gene transfer) (Rokas et al., 2003).

80 In the present study we tested the monophyly of Eunicidae and of some of its genera

(Eunice, Marphysa, Palola, Lysidice and Nematonereis) using comprehensive taxon

sampling designed to sample the diversity within the family. We employed nucleotide

sequences of mitochondrial 16S ribosomal DNA (rDNA), mitochondrial cytochrome

oxidase I (COI) and nuclear 18S rDNA, because of their informative nature in a previous

study (Struck et al., 2006) and differing evolutionary rates which can be expected to

provide phylogenetic information at different levels of the tree (Hillis and Dixon, 1991;

Halanych and Janosik, 2006). We also briefly explore and discuss the phylogenetic utility

of these genes in the context of the combined data sets trees resulting from this study.

MATERIAL AND METHODS

Taxon sampling

We sampled 50 ingroup species representing six (Eunice, Marphysa, Palola,

Lysidice, Euniphysa and Nematonereis) of the nine currently valid genera of the family

Eunicidae. No sample was available for the remaining three genera (Aciculomarphysa,

Fauchaldius, Nauphanta). The six genera sampled are also the most species-rich and well- studied genera in the family (Table 1.2). The choice of species took into account the diversity of characters within the different genera and attempted to include the type species of each genus or the species deemed most similar to it when specimens of the type species were not available. A single specimen was sampled for all species with the exception of

Nematonereis unicornis Schmarda, 1861, for which two individuals were used. Table 3.1 lists the terminal taxa employed in this study and their collection locality information.

81 Results of previous studies (e.g., Struck et al., 2006; Zanol et al, 2007) are

inconsistent on the monophyly of Eunicidae and on the placement of Onuphidae within

Eunicidae. Therefore we included species representing the main onuphid clades among the

outgroup species to test the placement of Onuphidae in relation to Eunicidae. Besides the

species of Onuphidae, the outgroup taxa for the analyses were species of the other three

best-known families of the order Eunicida, Dorvilleidae, Lumbrineridae and Oenonidae; and species of the other aciculate clades Amphinomida and Phyllodocida (Rouse and

Pleijel, 2001, Struck et al., 2007) (Table 3.1).

Species identification

Species identification within Eunicidae is difficult due to the poor understanding of

intraspecific and ontogenetic variations. Therefore, we initially identified some species

herein as cf. and others as sp. The specimen identified as Euniphysa sp. is a short anterior

end, missing the information about chaetal distribution necessary to identify species of this

genus.

Here we create a species complex for three species of Eunice originally described

from Caribbean specimens: filamentosa Grube, 1856, denticulata Webster, 1884 and conglomerans Ehlers, 1887. The holotypes of these species are small (width at chaetiger 10

1.5mm), median (width at chaetiger 10 3mm) and large (width at chaetiger 10 3.5mm), respectively (Fauchald, 1992a). One of our samples of this species complex from Belize includes several specimens collected in the same sponge varying in size from small to large and presenting similar character variation to the one present among the three species. Our

82 current knowledge is insufficient to distinguish the three species or lump them into a single

one; consequently a species complex, hereafter referred to as the Eunice filamentosa

complex, is the most appropriate way to identify them until further studies provide more

information.

DNA extraction, PCR amplification and DNA sequencing

Genomic DNA was extracted from specimens fixed in ethanol 95-100% or frozen at -

80oC using DNeasy Tissue Kit (Qiagen) following the manufacturer’s instructions. Parts of

two mitochondrial genes, 16S rDNA (~ 1,100 bp) and cytochrome c oxidase subunit I

(COI, ~1,200 bp), and one complete nuclear gene, 18S rDNA (~1,800 bp), were amplified and sequenced using the primers listed on Table 3.2.

PCR reactions took place in 25 µl solution containing, ddH2O, 10x buffer (Promega),

25mM MgCl2, 2mM dNTP, 10 µM of each primer, 0.15-0.25 µl Taq DNA Polymerase (5

U/µl) (Promega) and 2 µl DNA template. In some reactions, we replaced 10x buffer and

Taq DNA Polymerase (5 U/µl) (Promega) for NH4 10x Buffer and Taq DNA Polymerase

(Biolase), respectively. All amplification cycles used HotStart PCR-protocol by adding the

Taq polymerase after an initial denaturation at 94oC for 3-5 minutes. In addition the

amplification cycle for 16S rDNA used a touchdown PCR protocol under the following temperature profile: initial denaturation 3 min 94oC; 1 cycle 1 min 94oC; 40 cycles 30 s

94oC, 30 s start temp 51 oC (lowering 0.2-0.3 oC/cycle), 2 min 72oC; 1 cycle 7 min 70oC.

The temperature profile for the COI amplification cycle was: initial denaturation 3-5 min

94oC; 1 cycle 1-2 min 94oC; 35 cycles 1 min 94oC, 1 min 45-48oC, 2 min 72oC; 1 cycle 7

min 72oC. The temperature profile for the 18S rDNA amplification cycle was: initial

83 denaturation 3-5 min 94oC; 1 cycle 3 min 94oC; 40 cycles 1 min 94oC, 1 min 30 s 35-42oC,

1 min 30 s 70oC; 1 cycle 7 min 70oC.

PCR products were purified using ExoSAP-IT (USB). PCR products with more than

one band were run through a 1.2% Sea Plaque Low Melting Agarose TAE gel, target bands

were excised and purified using Gelase (Epicentre). Sequencing reactions for both strands

of amplified genes used BigDye Terminator Cycle Sequencing Kit v3.1 (Applied

Biosystems). Products were cleaned up using Sephadex columns, dried and sequenced

using an Applied Biosystems automated sequencer. Some products were sequenced using

CEQ dye terminator chemistry and a CEQ 8000 Genetic Analysis System (Beckman

Coulter). We assembled sequence contigs using Sequencher, version 4.5 (Gene Codes, Ann

Arbor, Michigan) and SeqMan (DNA*).

The target fragment of 16S was sequenced for 56 taxa and nine shorter sequences of

16S were obtained from GenBank (Table 3.1). We sequenced the target fragment of COI

for 26 terminals, partial target fragment for 17 terminals and obtained 13 shorter sequences

from GenBank (Table 3.1). The complete 18S was sequenced for 48 terminals, partially

sequenced for three terminals, and 11 sequences were obtained from GenBank (Table 3.1).

Twenty of the 69-terminals were missing data for at least one complete gene partition

(Table 3.1), four of these were missing 16S, 13 lacked COI and 7 lacked 18S sequences.

All missing data was scored as question marks.

We were only able to sequence 16S data for Euniphysa tridontesa (Shen and Wu,

1990) and 18S data for Euniphysa sp. We assumed monophyly of the genus (see

morphological evidence of Lu and Fauchald, 2000) and concatenated the sequences of both

species in a single terminal in the combined analyses.

Sequence alignment

We aligned the nucleotide sequences of 16S and 18S rDNA using two different

approaches to check the sensitivity of our data to different alignment parameters. The first

alignment approach was a ClustalX (Thompson et al., 1997) alignment using default

settings (0.50 DNA transition weight, 30% delay divergent sequences, 10 gap opening

penalty and 0.20 gap extension penalty) followed by a manual adjustment in BioEdit (Hall,

1999) (hereafter referred to as the Clustal alignment). The second one used MAFFT (online

version; Katoh et al., 2002; Katoh et al., 2005) under default settings to align the nucleotide

sequences (Moderately accurate FFT-NS-2, FFT-NS-i or L-INS-i; 1.53 gap opening penalty; and 0 offset value, gap extension penalty) (hereafter referred to as the MAFFT alignment). MAFFT implements progressive and iterative alignment and adds extra steps to improve the alignment guide tree (Katoh et al., 2005). It has been shown to improve nucleotide alignment accuracy compared to other alignment methods (Morrison, 2006;

Ahrens and Vogler, 2008).

COI nucleotide sequences were translated into amino acid sequences and hand aligned in MacClade 4.07 (Maddison and Maddison, 2000). The COI nucleotide sequences of seven species included in the present study have multiple-base insertions, which are not part of the gene open reading frames, are placed between codons (phase 0) and would not cause frame shift in the translation of the proten if spliced from the mRNA. These insertions were excluded from the data set before the analyses. In the COI alignment used in the present study these nucleotide insertions are placed between positions 660-661 in

Eunice thomasiana Augener, 1922 (~703 bp), Eunice torquata Quatrefages, 1866 (703 bp),

85 Onuphis elegans (Johnson, 1901) (313bp), Onuphis cf. iridescens. (Johnson, 1901) (313bp)

and Mooreonuphis pallidula (Hartman, 1965) (~350bp), 687-688 in Eunice norvegica

(Linnaeus, 1767) (454 bp) and 1092-1093 in Eunice cf. antillensis Ehlers, 1887 (365 bp).

The nucleotide sequences at the 5’and 3’ ends of these insertions are similar to those of the group II self-splicing intron recently described for the mtDNA genome of an annelid

(Vallès et al., 2008), which is one of the four diagnostic features of group II self-splicing introns (Vallès et al., 2008). It was not possible to identify the other three diagnostic features in these insertions.

Incongruence among genes

We tested the incongruence between the different data sets using the Incongruence

Length Difference test (ILD) (Mickevich and Farris, 1981; Farris et al., 1995) and

Shimodaira-Hasegawa test (SH) (Shimodaira and Hasegawa, 1999) as implemented in

PAUP* 4.0b10 (Swofford, 2001).

The ILD tests ran under the default settings for the homogeneity test in PAUP*

4.0b10 for 100 replicates holding 10 trees at each step. We ran ILD tests for all three possible gene pairs for each alignment method and between the 16S and 18S alignments produced by the different alignment methods. Uninformative characters were excluded before running the tests (Cunningham, 1997) as well as terminals missing at least 50% of the data for at least one of the genes of the tested pair (Table 3.1). The null hypothesis of congruence between the pair of data set being tested was rejected at a 5% significance level.

86 We ran from 5-6 SH tests for each alignment for 1,000,000 bootstrap replicates using

RELL approximation. In each test, we examined the congruence of a single gene or a

combined (16S+18S+COI) data set with the trees resulting from all maximum likelihood

(ML) analyses (single genes and combined data sets from both alignment methods). Such

tests allowed us to test the congruence between genes but also between the two different

alignment methods. All matrices and trees used in the SH test were generated with the most

restricted data sets (excluding the 20 terminals missing all data for at least one of the genes,

Table 3.1). ML analyses performed to generate the single gene trees tested in the SH test

used the 49-terminal data sets and followed the procedures described below. The data sets

were considered incongruent when the null hypothesis that all trees are equally good

explanations of the data was rejected at a 5% significance level (Nygren and Sundberg,

2003; Struck et al., 2006).

Phylogenetic analyses

We analyzed the gene data sets separately as well as combined. For each alignment

method, we analyzed two different combined data sets, a complete data set including all

taxa regardless of how complete they were (from here on referred to as 69-terminal) and

one including only the 49 terminals which had data for all genes (from here on referred to as 49-terminal). This allowed us to explore the phylogenetic information content of each of the markers, the stability and the congruence of the trees generated by them, and to check for artifacts in the tree that could be due to missing data.

In total nine data sets (Table 3.3) were analyzed under the three criteria described below. We used WinClada (Nixon, 2002) to edit matrices and concatenate the different

87 nucleotide sequences to form the combined matrix and MEGA3.1 (Kumar et al., 2004) to

determine the characteristics of the sequences.

The saturation of each gene and of the different codon positions of the COI were

examined in saturation curves built in DAMBE v4.5.50 (Xia, 2000) using Kimura-2-

parameter (K80) corrected distances. Taxa missing 50% or more of the data for one of the

partitions were removed prior to the saturation curve analyses. Three sets of saturation

curves were built, for the whole data set, just for Eunicidae and just for Onuphidae.

Maximum Parsimony (MP). Characters were unordered and equally weighted and

gaps were treated as unknowns (“?”). Tree search was heuristic, using the option Tree-

Bisection-Reconnection (TBR)+TBR in NONA (Goloboff, 1999). Analyses started with a

stepwise addition tree with taxa randomly added in 5000 replicates and 50 trees held on

each step of the TBR. During searches branches were collapsed when minimum branch

length was zero.

The trees of the MP analyses resulting in more than one most parsimonious tree were

summarized in strict consensus. Branch support for the combined data sets was estimated

with nonparametric bootstrap values (BP) (Felsenstein, 1985) (5000 replicates) and Bremer

support (Bremer, 1988) calculated in TNT (Goloboff et al., 2008). Bremer support was

calculated using the suboptimal search option in the menu created with the macro in the file

“BREMERtnt.run” distributed with the software. The suboptimal search followed the

default parameters and looked for trees longer than the most parsimonious in increments of

one step per search up to 20 steps.

Maximum Likelihood (ML). We used the Akaike Information Criterion (AIC) as implemented in ModelTest (Posada and Crandall, 1998) to choose substitution models for

88 all data sets. The General Time Reversible model, with rate heterogeneity and estimated proportion of invariable sites (GTR+I+ Γ model) was the chosen model for all data sets

(genes and combined as well as 69 and 49-terminals), except for the 16S rDNA data sets for which the Transversion Model (TVM), with rate heterogeneity and estimated proportion of invariable sites (TVM+I+Γ) was chosen.

ML analyses for all data sets, except 16S rDNA were run in GARLI v0.951 (Zwickl,

2006) using default settings. We ran 20 different searches for each data set to ensure convergence on the tree with the highest maximum likelihood. The initial tree for ML analyses was randomly generated and initial parameters values were estimated from this tree. Nonparametric bootstrap branch support values (BP) were calculated for all data sets

(including 16S data sets under the GTR+I+Γ model ) in GARLI v0.951 running 500 replicates.

ML analyses of 16S rDNA were run in PAUP*4.0b10 (Swofford, 2001) because the model TVM+I+Γ can only be implemented in GARLI v0.951 if the rates of transition are fixed a priori. Tree search was heuristic, using TBR and 200 replicates. Analyses started with a neighbor joining tree and initial parameter values for the model TVM+I+Γ were estimated from this tree (Clustal alignment, Pinvar= 0.189389, Gamma distribution shape=

0.622749; MAFFT alignment, Pinvar= 0.183606, Gamma distribution shape= 0.595314).

Bayesian Inference approach. For Bayesian analyses, the substitution models for the 16S rDNA data sets and different codon positions of the COI were determined using the

AIC in MrModelTest (Nylander, 2004). GTR+I+Γ was the preferred model for the 16S rDNA data sets and the second codon position of the COI. The best substitution model for the first codon position of the COI under AIC was the Symmetrical Model with rate

89 heterogeneity and estimated proportion of invariable sites (SYM+I+Γ model) and the

GTR+Γ for the third position. For all other data sets we used the models determined in

ModelTest (Posada and Crandall, 1998).

Bayesian analyses were run in MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003).

We ran two Bayesian analyses for each of the four combined data sets. One treated each

gene as a different partition (from now on referred to as gene partition). The other also included the different codon positions of the COI as different partitions (from now on referred to as gene+codon partition). We chose not to partition the ribosomal genes into stems and loops due to the ambiguity of secondary structure models for 18S and 16S ribosomal genes of annelids (e.g., Nereis limbata GenBank accession number U36270 in http://www.psb.ugent.be/rRNA/ssu/query/index.html and http://rdp8.cme.msu.edu/html/).

We also ran two Bayesian analyses (partitioned and non-partitioned in codon position) for the COI data set.

In all analyses, model parameter values were estimated during the runs. In partitioned

analyses such parameter values were estimated independently for each partition, but tree

topology and branch length were the same for all partitions. All analyses had two

independent and simultaneous runs with flat prior probabilities, four chains and other default settings. Every 100th tree was sampled, the first 25% trees were discarded as “burn in”. The remaining trees were summarized in a majority rule consensus with the Bayesian

Posterior Probability support for each clade.

An average standard deviation of split frequencies <0.02 calculated in MrBayes

v3.1.2 (Ronquist and Huelsenbeck, 2003) as well as the graph of the log likelihood of the

sampled trees plotted in Tracer v.1.4 (Rambaut and Drummond, 2007) were used to

90 estimate convergence between the runs (stationarity of parameters). We verified the mixing

quality of all parameters by examining the plot of the log likelihood versus sampled trees as

well as the effective sample size (ESS) for all parameters calculated in Tracer v.1.4. An

ESS higher than 200 for the log likelihood and higher than 100 for all other parameters

when the two runs were combined was considered a good mixing and the result of the

analyses were accepted.

The different data sets reach convergence at different generations: COI no partition at

45x105, codon partition at 10x106. ClustalX alignment: 16S at 15x105 generations, 18S for

60x105, 16SCOI18S 69-terminals gene partition at 67x105, 16SCOI18S 69-terminals

gene+codon partition at 80x105, 16SCOI18S 49-terminals gene partition at 67x105 and

16SCOI18S 49-terminals gene+codon partition at 40x105. MAFFT alignment: 16S r DNA at 25x105 generations, 18S at 70x105, 16SCOI18S 69-terminals gene partition at 90x105,

16SCOI18S 69-terminals gene+codon partition at 60x105, 16SCOI18S 49-terminals gene partition at 40x105 and 16SCOI18S 49-terminals gene+codon partition at 80x105.

We used Bayes Factor (BF) formula 2lnBF= 2(HMlnLH1-HMlnLH0) to calculate the evidence (2lnBF) supporting the results of the gene+codon partition analyses (H1) as opposed to the results of gene partition analyses (H0) for each of the combined data sets

(different alignments, 69 and 49-terminals) (e.g., Kass and Raftery, 1995; Nylander et al.,

2004; Brown et al., 2007). The harmonic means of lnL for the two combined runs were calculated after the removal of the “burn in” value using MrBayes v3.1.2 (Ronquist and

Huelsenbeck, 2003). The table in Kass and Raftery (1995) was used as the guide to the strength of the evidence supporting H1, 2lnBF <0 no evidence (evidence supports H0),

0<>2 insignificant, 2<>6 positive, 6<>10 strong, >10 very strong.

Hypothesis testing

In order to test traditional and ambiguous hypotheses of evolutionary relationships, we conducted ML analyses in GARLI v0.951 (Zwickl, 2006) following the above descriptions, the combined 69-terminals data sets generated by both alignments and constraining the trees to fit each of the tested hypotheses (Table 3.7). SH-test was used to determine if the constrained trees were significantly different from the best tree found for the combined 69-terminals data set of the two different alignments. SH test was run as described above. The hypotheses tested were: Lysidice monophyletic, Marphysa monophyletic, Palola and Lysidice as sister groups and Eunicidae paraphyletic.

Effect of combining data sets on the support of the clades

We classified the variation in the ML bootstrap support from single gene trees to the combined ones in three categories. No changes (=), bootstrap support for that branch in the combined analyses is in the same interval 0-49%, 50-84% or 85-100% as the bootstrap support values in at least one of the single gene partition trees. Improved support (+), bootstrap support for that branch in the combined analyses is on an upper interval when compared to the maximal bootstrap support values for that clade in single gene partition trees. Inferior support (-), bootstrap support for that branch in the combined analyses is on a lower interval when compared to the minimal bootstrap support values for that clade in single gene partition trees. After the classification, we tallied the amount of each category for both alignments. Clades with improved support are more strongly supported by the combined data set than by any of the single gene data sets. Therefore improved support was

92 considered evidence favoring combined analyses over single gene partitions. The 49- terminal data sets were used to guarantee that all terminals would be present in single partition and combined analyses.

Contribution of gene data sets to the different clades

Partition Addition Bootstrap Alteration (PABA) (Struck et al., 2006) identifies the possible source of incongruence among partitions and the contribution of the different markers to branches of trees resulting from combined analyses. It examines the alteration

(δ) of nonparametric bootstrap branch support values (BP) in different nodes of the tree under all possible combinations of partition addition. Here we were only concerned with the contribution of each gene to the different branches of the combined data set

(16SCOI18S). Therefore we calculated the alteration in BP (δ) only between the combined tree and the trees resulting from the data sets produced from the three possible combinations of pair of genes (gene pair data sets). Positive δ when a partition is added means that the added partition contributes to the support of that node and negative means no contribution of the added partition to the support of that node.

We calculated PABA from trees resulting from the 49-terminal data set ML analyses.

We ran additional ML bootstrap analyses for the three different possible combinations of gene data sets (e.g., 16S+18S, 16S+COI and 18S+COI) for each alignment including only the 49-terminals and following the procedure described above. The substitution models as determined by the Akaike Information Criterion (AIC) in ModelTest (Posada and Crandall,

1998) were GTR+I+Γ for 16S+18S (both alignments), 18S+COI (both alignments) and

16S+COI (Clustal alignment) data sets and TVM+I+Γ for the 16S+COI (MAFFT

93 alignment) data set. Because the model TVM+I+Γ can only be implemented in GARLI

v0.951 (Zwickl, 2006) if the rates of transition are fixed a priori, we also used the GTR+I+

Γ model in the ML bootstrap analysis of the 16S+COI (MAFFT alignment) data set.

We calculated δ for each of the genes as described in Struck et al. (2006), subtracting

the ML BP value for each branch resulted from the combined data set (16SCOI18S) from

the ML BP value for the same node in each of the trees of the gene pair data sets. The

resulting number is the δ of the gene not present in the gene pair data set. The average δ for

each of the genes for all clades was calculated to examine the general trend of the δ for

each gene. Average δ for each of the genes was also calculated for Eunicida branches

outside the (Eunicidae, Onuphidae) clade, Onuphidae, Eunicidae and all clades nested

within clades 11 and 30. δ values of zero when the BP of the combined data set

(16SCOI18S) was 100 and δ values for clades with BP in the combined data set <5 were

removed from average calculations.

RESULTS

Sequence characteristics and Incongruence

After the removal of the COI nucleotide insertions, the combined data set had 4966 aligned positions in the MAFFT alignment and 4911 in the Clustal alignment. Of these

2165 and 2145 sites, respectively, were variable and 1680 and 1685 sites were parsimony informative (Table 3.3). We did not remove any region of the alignments before running the analyses. The alignments are available online at TreeBASE.

94 The 16S alignments from MAFFT and Clustal were respectively 1206 and 1165 long, had 863 and 855 variable sites, 744 and 751 parsimony informative sites and 630 and 442 sites with gaps (Table 3.3). 16S sequences were A biased and G poor. Mean nucleotide composition was A 0.357, C 0.228, G 0.154 and T 0.261. The overall p-distance among

16S sequences was 0.3 for both alignments (standard error 0.008-0.009). The 16S sequences saturation curves showed tranversion saturation for all sites as well as for the eunicid ones (Appendix 1, 2). 16S sequences are not saturated in Onuphidae.

The COI alignment after the removal of the nucleotide insertions was 1284 sites long, had 677 variable sites, 607 parsimony informative sites and three sites with gaps (Table

3.3). First, second and third codon positions had, respectively, 139, 55 and 413 parsimony informative sites. The nucleotide composition varied among the three codon positions.

None of the nucleotides predominated in the first codon position (A 26.2 C 23.9 T 20.9 G

28.9), while the second position was T biased, A and G poor (A 15.8 C 25.4 T 42.4 G 16.4) and the third position was A and C biased and G poor (A 38.6 C 30.1 T 27.1 G 4.2). The overall p-distance among COI sequences was 0.23 (standard error 0.007); however it varied among codon positions. The overall p-distance for the first, second and third codon positions were respectively 0.124 (standard error 0.01), 0.046 (standard error 0.006) and

0.526 (standard error 0.008). The saturation curve for all sites of the different codon positions of the COI showed transition saturation for all positions and tranversion for the third one (Appendix 1, 2). In eunicid sequences, the first and third codon positions are saturated for tranversion and the latter is also saturated for transitions. Onuphid sequences are saturated for transitions at the second codon position.

95 The 18S alignments from MAFFT and Clustal were respectively 2476 and 2465 long,

had 625 and 613 variable sites, 329 and 327 parsimony informative sites and 806 and 738

sites with gaps (Table 3.3). None of the nucleotides predominated in the 18S sequences (A

24.8 C 22.7 T 24.7 G 27.9). The overall p-distance among 18S sequences was 0.04 for both alignments (standard error 0.002). 18S sequences are not saturated in any of the sequences analyzed (Appendix 1, 2).

In ILD tests, both 18S alignments are congruent with COI, but 18S is congruent with

16S only in the MAFFT alignment (Table 3.4). COI and 16S are incongruent. In the SH test the combined data sets resulting from the different alignments are congruent with each

other as well as with the 16S and 18S data sets from the same alignment. The COI

alignment is congruent with the MAFFT combined data set but it is incongruent with the

Clustal combined data set. All single gene data sets were significantly incongruent with

each other independent of the alignment (Table 3.5).

Phylogenetic analyses

MP statistics for all analyses are presented in Table 3.3. The clade numbers cited

herein refer to the number on the branches of Figures 3.1 and 3.2. Clade numbers are

equivalent in the 69-terminal trees and in the ones with more exclusive taxon sampling

(i.e., do not include all 69-terminal taxa). Same number clades in different analyses may

have different taxa composition solely due to the absence/presence of taxa not included in

all analyses. Branch support values for all combined analyses are in Table 3.6 and for the

single gene ML trees in Table 3.8.

96 Combined data set. Eunicidae is monophyletic (MP, ML BP<85% and PB <95%)

(Table 3.6) with Onuphidae as its sister group in all combined trees (69 and 49-terminals,

Figs. 3.1, 3.2, 3.4, 3.5) with the exception of the trees resulting from the 69-terminal (both

alignments, Fig. 3.3) and 49-terminal (both alignments, results not shown) Bayesian

analyses with gene partition only and the 49-terminal Bayesian analyses with gene+codon

partition (MAFFT alignment, Fig. 3.4C). However there is very strong evidence supporting the results of the Bayesian gene+codon partition analyses (H1) over the results from the gene partition for both the 69 (MAFFT alignment 2lnBF= 2252.92; Clustal alignment 2ln

BF= 2235.74) and 49 (MAFFT alignment 2lnBF= 2238.3; Clustal alignment

2lnBF=2240.64) terminal analyses (Kass and Raftery, 1995). For this reason, we do not consider the results of Bayesian gene partition (Fig. 3.3) any further.

The two most basal eunicid clades (Figs. 3.1, 3.2, 3.5 clades 11 and 30) are the same in almost all recovered trees. Clade 11 contains Eunice species mostly distributed in two

major clades present in all recovered trees (Figs. 3.1A, 3.2A clades 21 and 23) and Lysidice

sp.1 (absent in the 49-terminal analyses), for which we only had 18S sequence. All

remaining eunicid species are placed in clade 30, except Eunice fucata Ehlers, 1887 (absent

in the 49-terminal analyses) which is sister to clade 30 in the trees of the MAFFT

alignment and sister to clade 11 in the trees of the Clustal alignment (Figs. 3.1, 3.2). The

basal placement of Lysidice sp.1 makes Lysidice polyphyletic in all 69-terminal trees. The

remaining Lysidice species form a clade nesting Nematonereis species (absent in the 49-

terminal analyses) in almost all 69-terminal combined trees (Figs. 3.1, 3.2, 3.5 clade 33).

The strict consensus tree of MP analyses of the 69-terminal Clustal matrix (Fig. 3.2B) is the

97 only tree lacking clade 11 (Figs. 3.1, 3.2, 3.4) and the Lysidice+Nematonereis clade (Figs.

3.1, 3.2, 3.4 clade 33).

Eunice and Marphysa are polyphyletic and Palola monophyletic (ML BP>85%, MP

BP< 85% and PB>95%) in all combined trees (Figs. 3.1-3.5). Two Marphysa species are

nested with Eunice species within clade 38 (Figs. 3.1, 3.2), which is also present in all

combined trees (ML BP>85%, MP BP< 85% and PB>95%). All the remaining Marphysa

species are grouped with Marphysa sanguinea (Montagu, 1815), type species for the genus,

as the sister clade to clade 38 (Figs. 3.1, 3.2 clade 39). This Marphysa clade (clade 39) has

a clade of M. sanguinea-like species in all combined trees (Figs. 3.1-3.4 clade 46), which is

sister to a clade of Marphysa bellii (Audouin and Milne Edwards, 1833)-like species in all

69-terminal combined trees (Figs. 3.1, 3.2, 3.4 clade 47). Only one species of the M. bellii

like-clade (Figs. 3.1, 3.2, 3.4 clade 47) is present in the 49-terminal matrix. It is sister to the

clade of M. sanguinea like species in the 49-terminal trees. The hypothesis of a

monophyletic Marphysa was the only hypothesis rejected in SH-test with a 5% confidence

interval (P<0.001) (Table 3.7).

In the 69-terminal phylogenetic analyses, most methods used here (MP, ML and

Bayesian gene+codon partition) resulted in similar trees when the same alignment was

analyzed (Figs. 3.1, 3.2). These trees contrasted mainly in localized sister group

relationships of terminal branches (mostly with two to four species) which were not

necessarily poorly supported clades (BP <85%, Table 3.6). The only basal ambiguity is the

sister group to the (Eunicidae, Onuphidae) clade in the MAFFT trees (Fig. 3.1A,C), clade

70 in the Clustal alignment trees (Fig. 3.2A,C) and the strict consensus tree of MP analyses

of the 69-terminal Clustal alignment. The MP strict consensus tree of the 69-terminal

98 Clustal alignment has a basal polytomy in the Eunicidae clade (10, Fig. 3.2B) because of

the ambiguous placement of Lysidice sp.1 and (Eunice violaceomaculata Ehlers, 1887,

Eunice roussaei Quatrefages, 1866) clade. Lysidice sp.1 has three most parsimonious

placements, within the Lysidice+Nematonereis clade (33, Figs. 3.1, 3.2), at the base of

clade 11 (Figs. 3.1, 3.2) and at the base of clade 19 (Figs. 3.1, 3.2). The clade (E.

violaceomaculata, E. roussaei) (57, Figs. 3.1, 3.2) is basal to clade 30 (Figs. 3.1, 3.2) or

grouped with the other Eunice species placed in clade 52 of the MAFFT tree (Fig. 3.1).

The 69-terminal trees of the different alignments analyzed with the same method

contrasted mainly in the placement of the E. fucata (Figs. 3.1 clade 12, 3.2 clade 67),

Euniphysa sp. (Figs. 3.1 clade 58, 3.2 clade 73), Lysidice+Nematonereis clade (Figs. 3.1 clade 31, 3.2 clade 73) and the monophyly of clade 52 (Fig. 1). Such contrasting placements led to the different basal Eunicidae relationship observed in the trees yielded by the different alignments (Figs. 3.1, clades 12, 31, 32, 52; 3.2, clades 67, 68, 69, 75).

The differences described above for the results of the 69-terminal analyses were also

observed among the 49-terminal analyses when the same sets of trees were compared.

However, the 49-terminal trees of the different analyses of the same alignment had

additional divergences only observed in 69-terminal trees of different alignments (Fig. 3.4).

Even though the 69-terminal data sets included 20 taxa missing data for a complete

gene partition it recovered more consistent trees across the different analyses and among

the different alignments (Figs. 3.1, 3.2) than the trees of the 49-terminal data set (Fig. 3.4).

Decrease in resolution due to missing data is not inevitable, it depends on the phylogenetic

information content and the number of characters present (Wiens, 2003). The characters

present in the taxa missing data in the 69-terminal data set, with the exception of Lysidice

99 sp.1, E. fucata and Euniphysa sp., were informative enough to consistently place them in the different combined data set trees. Furthermore the improved representation of the diversity within Eunicidae in the 69-terminal data sets was important in improving the congruence among the different trees of the same alignment.

As expected in all 49-terminal trees most clades are more strongly supported than in the 69-terminal trees (Table 3.6). This is especially verified in the two main clades containing eunicid species (Figs. 3.1A, 3.2A clades 11 and 30, Table 3.6) which have poor

ML BP support in the 69-terminal trees and are strongly supported on the 49-terminal trees

(Table 3.6).

Single genes data sets. 16S mt rDNA- All Clustal trees (MP, ML and Bayesian) and the ML and Bayesian MAFFT trees resulted in a monophyletic Eunicidae. However only the Clustal MP tree and the MAFFT ML tree support a monophyletic Onuphidae, the family is polyphyletic in all other trees of both alignments. Eunicidae is paraphyletic with

Onuphidae nested within it in the MAFFT MP strict consensus tree. The outgroup relationships of both the 16S Clustal and MAFFT trees contrasts with the trees in the combined analyses. In the 16S trees the Lumbrineridae clade (Figs. 3.1, 3.2 clade 5) is sister to the (Eunicidae, Onuphidae) (Figs. 3.1, 3.2 clade 8) clade, to Oenone fulgida

Savigny, 1818 or to the Oenonidae clade (Figs. 3.1, 3.2 clade 7). Dorvillea erucaeformis

Malmgren, 1865 is either the most basal Eunicida or else groups with the Amphinomidae clade (Figs. 3.1, 3.2 clade 2). The within-eunicid relationships in the 16S trees (except the

MAFFT MP strict consensus tree) are similar to the ones of the combined analyses of the same alignments (Figs. 3.1, 3.2, 3.5; Appendix 3). In the 16S Clustal trees, the only exceptions are the relationships within clades 11 and 71 (the latter only in the 16S MP and

100 16S Bayesian trees) and the collapse of clades 71(16S ML tree) and 67 (16S MP tree). The

Clustal alignment produced more robust trees, similar across the three methods of analyses,

than the MAFFT alignment (Appendix 3).

COI mtDNA- The (Eunicidae, Onuphidae) clade as well as monophyletic Palola

(clade 53) and Onuphidae are not present in any of the COI trees resulting from the four

analyses (MP, ML, Bayesian no partition and Bayesian codon partition) (Appendix 4).

Eunicidae is monophyletic in the ML and Bayesian no partition trees, which also support

clade 30 present in all combined trees (Fig. 3.5) and clades 31, 52 of the MAFFT combined

trees. ML and both Bayesian trees recovered most current genera and clades present in all

combined trees with the exception of the monophyly of Palola (clade 53), the clade of M.

sanguinea-like species (46) and clade 34. Clades 11, 21 and 23 (Figs. 3.1A, 3.2A) are the

only deep clades present in all COI trees as well as in the combined ones. All the other

clades shared among all these trees (43, 48, 55 and 62) are shallow branches with four

terminals at most. This low number of shared basal clades in all COI trees is due to the

divergent COI MP trees. The COI MP strict consensus is well resolved, however most

clades are unique to this tree, they are not present in any of the other trees from this study

or previous ones (e.g., Struck et al., 2006).

18S rDNA- In all 18S trees, Palola (clade 53) and Onuphidae are monophyletic

(clade 9), Eunicidae is paraphyletic with Onuphidae nested within it and sister to the clade

(D. erucaeformis, Oenonidae) (clade 66) and Lumbrineridae (clade 5) is the most basal clade in Eunicida (Fig. 3.5; Appendix 5). Most of these relationships are present in all trees of the combined data sets, the exceptions are clade 66, present only in the trees resulting from the Clustal alignment, and the paraphyletic Eunicidae, present only in the Bayesian

101 analyses with gene partition only. Besides these relationships, the only clades shared

among all 18S trees and the combined analyses trees are shallow with four taxa at the most

(clades 21, 25, 28, 37, 41, 42, 43, 44 and 53). Most clades within the (Eunicidae,

Onuphidae) clade in the 18S trees are unique to those and are not present in any of the other trees resulting from this study or previous ones (e.g., Struck et al., 2006).

Eight branches (out of 45 examined) were more strongly supported in the ML trees of the combined data set than in the ML tree of single gene partitions in both alignments

(Table 3.8). One clade was more strongly supported in the ML trees of single gene partition for both alignments than in the combined ones (Table 3.8). When branch support was considered for a single alignment at a time, 13 and 10 branches, respectively in the MAFFT and Clustal alignment, are more strongly supported in the combined analyses than in the single gene partition trees. Only three and five, respectively, are more strongly supported in the single gene partition trees versus the combined ones (Table 3.8).

Contribution of gene data sets to the different clades

Here we consider that a gene has a strong signal for a clade when its contribution for the ML BP of that clade in the combined analyses is positive or indeterminable (na, Table

3.8) and the ML BP for the clade in that gene tree is higher than 85%.

All genes were phylogenetically informative and their phylogenetic signal was generally distributed in different areas of the tree (Table 3.8). The three genes combined enhanced their phylogenetic utility in different depths of the tree, their phylogenetic signal added to each other and hidden phylogenetic signal became apparent. Twenty-six out of the

45 clades for which we calculated PABA had their support enhanced or not altered by all

102 three genes (Table 3.8). Clades 45 in the MAFFT alignment, 49 in both alignments and 75

in the Clustal alignment were the only clades strongly supported in single gene trees (16S

trees) but poorly supported in the combined trees (Table 3.8). While no single gene tree

strongly supports clades 1 in the Clustal alignment, 4, 23, 25, 30 and 59 in both alignments,

they are strongly supported in the combined trees with positive BP contributions from all three genes, with exception of clade 23 for which 18S contributions is zero.

The monophyly of Eunicidae is supported by 16S and COI genes, which were

essential in supporting it with BP> 50% in combined trees (Table 3.8). Both of these genes

are also the ones supporting the eunicid monophyletic genera and clades 11, 31, 38, 39 and

53 (Figs. 3.1, 3.2; Table 3.8). Onuphid monophyly as well as most relationships within this family are mainly supported by the 18S gene, which yielded strong phylogenetic signal for all of these relationships (Table 3.8, clades 9, 14, 15, 17). The (Eunicidae, Onuphidae) clade is also strongly supported by the 18S gene. The contribution of the other genes to this relationship is indeterminable. However in the 16S gene trees, the (Eunicidae, Onuphidae) clade has BP of 56% and 86%, respectively, in the MAFFT and Clustal alignments (Table

3.8).

16S was strongly informative from basal (e.g., Lumbrineridae and Oenonidae, Table

3.8 clades 5, 7) to terminal branches. The strongest phylogenetic signal of 16S is concentrated on branches within clade 30 (Table 3.8). However, it also strongly supports

Lumbrineridae, Oenonidae, terminal clades within Onuphidae, and clades 21, 28 and 29.

All the clades strongly supported by 16S placed outside clade 30 have three terminals at the most and are usually composed of closely related species. 16S had the highest positive

contribution to the BP of most clades within Eunicidae in the combined trees (Table 3.8)

103 exceptions are nested within clade 11. It also positively contributed to the most basal eunicidan clades (Table 3.8, clades 1,4)

COI phylogenetic signal only strongly supported clades within Eunicidae (Table 3.8).

Most of them are shallow containing two to four species, with the exception of Marphysa sensu stricto (clade 39) and clade 11 (most basal eunicid clade). In the combined analyses

COI was essential for the strong support (BP>85%) of Onuphidae and clades within it.

18S was the main gene contributing to the support of the relationships basal to

(Eunicidae, Onuphidae) and it strongly supported (positive or indeterminable BP alteration and ML BP>85% in the 18S tree) Lumbrineridae and Oenonidae (Table 3.8, clades 5, 7). It also provided strong support to few eunicid terminal clades containing three terminals at the most and nested in clades 11 and 34 (Table 3.8). 18S was essential in supporting clades within clade 11 with BP> 50% and clade 46 with BP> 85% (Table 3.8).

DISCUSSION

Congruence and the phylogenetic utility of the different genes

The contribution of each gene to clades in different specific areas of the trees may explain the improved combined analyses, which resulted in better resolved and supported clades due to additive phylogenetic signal, even though most single gene data sets are incongruent in the ILD and SH tests. These results further support the idea that congruence is not a requirement for combining different partitions (Cunningham, 1997). Data set congruence does not guarantee accuracy of combined analyses. Incongruent data sets may

104 result in accurate trees, while congruent data sets may result in strongly supported

inaccurate trees (Hipp et al., 2004).

Furthermore, the results of incongruence tests may be influenced by different

characteristics of the data sets, not only incongruence (see Ramírez, 2006 for a review). In

this study the effect of reduced taxon sampling may have affected some of the results of the

incongruence tests (ILD and SH test). In both tests we used the most restricted data sets

(i.e., all taxa containing data for all the genes combined in each of the individual test).

Therefore, the data sets and trees tested for congruence may be incongruent due to the

artifacts caused by the reduced taxonomic sample (Hillis, 1998) and not by incongruence

among the data sets.

16S was the most informative gene, being essential for nonparametric bootstrap

branch support (BP)>50% and 85% at all levels of the combined tree, from basal eunicidan

to terminal eunicid clades (Table 3.8). Here we used longer 16S sequences than previous

studies (e.g., Schulze, 2006; Struck et al., 2006) and did not remove ambiguously aligned regions. These regions usually evolve at a faster rate than the clearly aligned, more conservative ones, therefore the inclusion of all regions of the 16S in phylogenetic analyses may make this gene informative for both more recent and older divergences (Simmon et al., 1994) and could account for its wide range of phylogenetic information observed here.

Furthermore, in recent studies the removal of ambiguously aligned or saturated regions of rDNA decreased tree resolution (for 12S and 16S, Hertwig et al., 2004; for 18S and 28S,

Struck et al., 2008). In the present study 16S was informative at intrageneric levels as previously observed in other polychaetes (Halanych and Janosik, 2006) as well as at the

105 intergeneric, family and order levels. Onuphidae was the only eunicidan family for which16S dercreased BP in combined analyses.

The different branch levels at which COI contributes positively to the branch support values (Table 3.8) are probably supported by different codon positions, which have different rates of evolution (Halanych and Janosik, 2006). However, substantial positive contributions of the COI are concentrated on more recent divergences. It is responsible for a BP>50% only in terminal clades within Eunicidae and for a BP>85% for Onuphidae, a clade within it and clades within Eunicidae. This could be a result of the saturation of the

COI sequences observed here. Saturation increases with increase in age of divergence, therefore increasing homoplasy and decreasing the phylogenetic signal towards the base of the tree. Homoplasies are especially problematic in the faster evolving third codon position

(Halanych and Janosik, 2006), which here is completely saturated (Appendix 1, 2) and makes up most of the informative characters (68%) of the COI sequences.

18S is among the genes most used to resolve the phylogeny of annelid groups

(Halanych and Janosik, 2006). It is usually useful to resolve ancient relationships (Hillis and Dixon, 1991), such as the Ordovician diversification of the order Eunicida (Kielan-

Jaworowska, 1966), as observed here (Table 3.8). Furthermore 18S has regions evolving at different rates, which allow for informative phylogenetic signal at different levels of the tree (Halanych and Janosik, 2006). Although comparison of taxonomic levels may be meaningless in a phylogenetic context, in previous studies on annelid groups as well as in our results, 18S was informative at the family level, supporting families and its internal branches (Bleidorn et al., 2005; Burnette et al., 2005). In our results and in Struck et al.

106 (2006), Eunicidae is the only eunicidan family not supported by 18S even though its sister

group, Onuphidae, and most of onuphid internal branches are strongly supported by 18S.

The difference observed in the 18S signal between the two sister families Eunicidae and Onuphidae could be due to different factors: 1) Faster radiation of Eunicidae. However, in the combined trees both Eunicidae and Onuphidae are supported by short branches with about the same length (Figs. 3.1, 3.2), suggesting that both family diversifications happened at about the same time. 2) Saturation of nucleotide positions in Eunicidae.

However 18S is not saturated in Eunicidae (Appendix 2). 3) Incorrect topology. However, in the topologies with Onuphidae nested within Eunicidae, Onuphidae is sister group to clades that are not supported by 18S either. 4) Different rates of evolution of this gene in both families. The 18S transversion saturation curve for Eunicidae has a lower slope than those of Onuphidae, suggesting a slower rate for this gene in Eunicidae. The difference in rates seems to be the most likely explanation for this difference of phylogenetic signal between Eunicidae and Onuphidae, however further analyses are necessary before definite conclusions can be drawn. In Annelida substitution rates across lineages in 18S are not well known (Halanych and Janosik, 2006). In mollusks the variation of 18S rates across lineages is widespread (Passamaneck et al., 2004).

Generalizations of the phylogenetic information content of genes at certain depths of the trees should be read with caution because they generally assume a constant substitution rate across lineages. Our study suggests that at least for two sister families of Annelida such assumption may not hold.

Phylogeny of Eunicidae

107 The eunicid relationships discussed here are based on the 69-terminal trees, which are

the preferred ones. These trees include a larger taxon sample (i.e., better representation of eunicid diversity), which usually improves topology even when taxa are missing data

(Gauthier et al., 1988; Zwickl and Hillis, 2002; Poe, 2003; Wiens, 2005); and combined phylogenetic information from three independent molecular markers that improved the resolution and clade support when combined. We only recognize the relationships congruent in most of the 69-terminal combined trees resulting from both alignments. The only relationships of 69-terminal combined trees not considered here are those resulting from Bayesian analyses with gene partition only, which were strongly overruled, according to BF results, by the Bayesian trees of gene+codon partition.

Eunicidae is monophyletic and sister group to Onuphidae corroborating the four gene analyses in Struck et al. (2006). Here the family Onuphidae, as in this previous study, diverged basally into the subfamilies Hyalinoeciinae and Onuphinae (Paxton, 1986).

However, in contrast to Paxton’s (1986) phylogeny, Mooreonuphis is basal in Onuphinae,

and the relationships among Onuphis, Diopatra and Paradiopatra are inconsistent (Figs.

3.1A,B, 3.2C). The sister group to the (Eunicidae, Onuphidae) clade is ambiguous, it varied with the alignment. It is Dorvilleidae or a (Dorvilleidae, Oenonidae) clade, both possibilities were also the ones recovered, respectively, in the least species-rich four gene tree or the more species-rich 18S tree in Struck et al. (2006).

The monophyly of Eunicidae is poorly supported (Figs. 3.1, 3.2; Table 3.5) and the hypothesis of paraphyly was not rejected (Table 3.6). The monophyletic status of Eunicidae is sensitive to partition in Bayesian analyses (Figs. 3.1-3.3), taxon (Figs. 3.1, 3.4C) and gene sampling (Appendices 3.3-3.6; Struck et al., 2006). The latter appears to be the most

108 influential in affecting the recovery of a monophyletic Eunicidae, since the phylogenetic

status of Eunicidae recovered here for each of the genes corroborates the ones in Struck et al. (2006) even though only two or four eunicid species were included in those analyses.

The poor support for Eunicidae and the instability between Eunicidae and Onuphidae could be due to the short length of the branches separating both families and supporting the most basal Eunicidae clades, since these have few synapomorphies, which can be more easily overwhelmed by homoplasy in the longer external branches. These short internal branch lengths may have different causes: insufficient sample of informative data, sequence saturation, inappropriate model of evolution, character conflict and rapid radiation (Whitfield and Lockhart, 2007). As for most phylogenetic ambiguities, in most cases of short branches increasing the data used in the analyses, using models of evolution better fitted to different partitions (Baurain et al., 2007), adding other sort of data less prone to homoplasy (e.g., genomic features) (Rokas and Carroll, 2006; Whitfield and Lockhart,

2007) as well as the adition of taxa in order to break long external branches (Hillis, 1996;

Graybeal, 1998) are likely to make the resolution more robust in future studies but may be less effective in case of rapid radiation (see Whitfield and Lockhart, 2007 for a review).

No biogeographic pattern can be characterized in basal eunicid clades, most clades have widespread distributions (Fig. 3.1; Table 3.1). Of the five eunicid genera for which we tested the monophyly, only Palola is monophyletic. Eunice and Marphysa are polyphyletic

and Lysidice is paraphyletic with Nematonereis nested within it. These results correspond

well to the robustness of the morphological diagnostic features of these genera. Palola is

the only one with clear unique synapomorphies (e.g., strongly curved ventral mandibles

and lack of subacicular hooks) (Zanol et al., 2007). The other four genera are mainly

109 diagnosed by plesiomorphies (e.g., five prostomial appendages, peristomial cirri and double rings in Eunice) or ontogenetically variable features (e.g., lack of peristomial cirri in

Marphysa, Lysidice and Nematonereis; lack of palps in Lysidice and Nematonereis and the lack of lateral antennae in Nematonereis) (Orensanz, 1990). We do not consider the placement of Lysidice sp. 1 as evidence of a polyphyletic Lysidice because we only had

18S sequence data for this species, which was not enough to consistently place or group it with Lysidice species (Appendix 5). For this reason, the placement of this species in the phylogeny is doubtful and we do not discuss it further.

The polyphyletic status of Eunice recovered in the present study as well as the placement of the most similar species to the type species of Leodice (Figs. 3.1, 3.2 clade s28) and Eunice (Fig. 3.1, 3.2 clade 57), respectively, in conspicuously different clades support the morphological phylogeny of the genus (Zanol et al., 2007). This phylogeny suggested the breakup of Eunice in a few monophyletic genera and the resurrection of

Leodice.

In the present trees Leodice could encompass at least the species grouped in clade 23

(Figs. 3.1, 3.2) since it includes the clade of the species most similar to Eunice antennata

(Larmarck, 1818) which is the type species of Leodice (clade 28). This clade is consistent in all 69-terminal trees and strongly supported in most analyses (Figs. 3.1, 3.2). The placement of the clade of E. torquata-like species (clade 21) in clade 11 as sister to clade

20 (Fig. 3.1) or 23 (Fig. 3.2) is different from any previous results. It contradicts the poorly supported hypothesis of a unique derived origin to the dark color of the subacicular hooks

(Zanol et al., 2007), since all Eunice species outside clade 11 have dark subacicular hooks.

Clade 11(Figs. 3.1, 3.2) includes only Eunice species with articulated prostomial

110 appendages, grouping some of the species with cylindrical articulation and all of the ones with moniliform articulations included in the analyses. Clade 20 (Fig. 3.1) contains all the

Eunice species with yellow subacicular hooks included in the analyses, such as the major

Eunice clade in the18S tree in Struck et al. (2006). The low support for this clade is due to the inconsistent placement of E. cf. antillensis (Figs. 3.1, 3.2 basal to clade 11 or 20). The presence of yellow subacicular hook in other Eunicidae (e.g., N. cf. unicornis and

Marphysa viridis Treadwell, 1917) make this feature unlikely to yield a unique synapomorphy for this clade.

Eunice sensu stricto includes at least clade 57, which bears the most similar species to

Eunice aphroditois (Pallas, 1788), type species of the genus. It is not clear if any of the other species included in the analyses would also be members of this genus. Clade 52 which nests clade 57 in the MAFFT alignment trees (Fig. 3.1) is poorly supported in most preferred trees with the exception of the Bayesian tree. The other Eunice species in clade

52 are clustered together in all combined analyses but the relationship among them is not consistent as well as the placement of Euniphysa in this Eunice clade. Each of the terminal clades in clade 52 and E. norvegica may represent different genera including additional species not present in our analyses. The inconsistent placement of Euniphysa within clade

52 or sister to the Lysidice+Nematonereis clade (Figs. 3.1, 3.2) is probably due to missing data since we had only 50% of the data for this taxon. However, regardless of this inconsistency in placement Euniphysa is always placed in the sister group to Palola.

Eunice fucata has the longest branch among Eunice species in the current trees. None of the species considered similar to it based on traditional morphological features were included in the analyses (e.g., Eunice sebastiani Nonato, 1965 and Eunice sonorae

111 Fauchald, 1970). Therefore in addition to the different hypotheses of homology between the alignments and the missing data for COI sequences, the inconsistent placement of this species between the 69-terminal alignments may also be due to an incomplete sample of appropriate eunicid species in the present study (see also Gauthier et al., 1988). The inclusion of closely related species could potentially break this long-branch and provide improved phylogenetic information on the placement of the group (Poe, 2003).

The species of Marphysa nested among Eunice species in clade 38 (Figs. 3.1, 3.2) support the idea that the lack of peristomial cirri, which are also absent in Lysidice and

Nematonereis, is not enough to define a monophyletic group. This feature is probably more variable (e.g., lack of peristomial cirri in younger specimens) than traditionally accepted

(e.g., Eunice insularis, Nogueira et al., 2001). Few species in clade 38 have been associated with the currently defunct genus Nicidion Kinberg, 1965. E. cariboea was often referred to as Nicidion (e.g., Hartman, 1944) and species of Nicidion have been synonymized with it

(e.g., Monro, 1930). Additionally the subacicular hooks of Eunice cincta (Kinberg, 1865), one of the species in the original description of Nicidion, resemble those of Eunice mutilata

Webster, 1884 (Fauchald, 1992a), a species also nested within clade 38. For these reasons clade 38 may represent Nicidion, if E. cincta is considered the type species of this genus and the diagnostic features are broadened beyond the original one, lack of branchiae, to include the branchiated species (e.g., Marphysa hentscheli Augener, 1931 and E. mutilata) in clade 38.

The second clade containing Marphysa species (Figs. 3.1, 3.2 clade 39) is Marphysa sensu stricto, including M. sanguinea, the type species of the genus. The two basal clades in Marphysa sensu stricto represent, respectively, the groups “sanguinea”-like (Figs. 3.1,

112 3.2 clade 46) and “bellii”-like (Figs. 3.1, 3.2 clade 47) taxa as described in Orensanz

(1990).

The consistent placement of Nematonereis, nested within the Lysidice clade, makes a monophyletic Lysidice improbable unless Nematonereis is synonymized with it. A better representation of Nematonereis diversity could help resolve this problem, since the current

analyses included only one Nematonereis species and the hypotheses of a monophyletic

Lysidice was not rejected (Table 3.7).

The phylogenetic relationships recovered for the genus Palola are almost identical to those recovered by Schulze (2006). The main difference between the two results is due to the addition of P. cf. siciliensis, which grouped at the base of Palola with a specimen from

Panama which was sister to clade 61 in Schulze (2006). P. cf. siciliensis is the only

Mediterranean specimen included in the analyses, all others are from the Caribbean Sea

and Pacific Ocean.

Palola and Lysidice+Nematonereis are derived within Eunicidae in all trees, supporting the apomorphic condition of the curved mandibles (Orensanz, 1990), which are probably convergent in these clades as recovered in Zanol et al. (2007). The close relationship between Palola and Lysidice+Nematonereis is recovered only in the 69- terminal Clustal trees (Fig. 3.2 clade 75). Even then the nonhomoplasious synapomorphy status of the curved mandible remains ambiguous since Euniphysa, which lacks curved mandibles, is sister group to (Lysidice, Nematonereis) in these trees. However the

Euniphysa sister group relationship is inconsistent among 69-terminal alignments and the hypothesis of sister group relationship between Palola and the clade (Lysidice,

Nematonereis) was not rejected (Table 3.7).

113 The basal variation in sister group relationships within clade 30 (Figs. 3.1, 3.2) is

mainly due to the uncertain placement of the Lysidice+Nematonereis clade and the Eunice

clade 57. Consequently the only consistent basal relationships within clade 30 in all 69-

terminal trees is the close relationship between clades 38 and 39 (Figs. 3.1, 3.2 clade 34)

and the closer relationship between Palola, Euniphysa, E. filamentosa complex, E. impexa

and E. norvegica than between any of them and clade 34.

The evolution of Eunicidae did not happen by the stepwise addition of features after the early separation of Palola as proposed in Hartman (1944). She proposed starting from

Nematonereis (one median prostomial appendage), through Lysidice (three median prostomial appendages), and Marphysa (five prostomial appendages) to Eunice (five

prostomial appendages and one pair of peristomial cirri). However, the present results show

the evolution of Eunicidae happening in the opposite direction and not exclusively in a stepwise mode. First, Palola is not at the base of the eunicid clade. The most basal forms

had both five prostomial appendages and a pair of peristomial cirri (Fig. 3.6) supporting the

framework proposed in Orensanz (1990). The peristomial cirri were lost independently at

least twice (Fig. 3.6) while the loss of the pair of palps (the most lateral and usually anterior of the prostomial appendages) and lateral antennae (medio-lateral paired prostomial

appendages) appears to be unique to the Lysidice+Nematonereis clade; this reduction

appears to have happened in a stepwise fashion (Fig. 3.5). During ontogenesis species of

Eunice sensu lato always go through a sequence of states, adding prostomial appendages starting with the median antenna, (“Nematonereis” stage) followed by the addition of the

lateral antennae, (“Lysidice” stage), then palps (“Marphysa” stage) and finally the

peristomial cirri (“Eunice” stage) (Åkesson, 1967; Giangrande, 1989; Orensanz, 1990).

114 Because the development of the head appendages is conservative and the lack of these

structures in adults always follows the same pattern (i.e., adult specimens lacking structures

that develop early, always lack the ones that develop later also), the lack of the head

appendages features may be a paedomorphic characteristic in Eunicidae; such as suggested

for the simplifications of a variety of morphological traits in other polychaete groups

(Westheide, 1987; Struck et al., 2002; Bleidorn, 2007).

CONCLUSION

All three genes used here (16S, COI and 18S) were informative at different levels of

the trees. 16S and COI were important in recovering a monophyletic Eunicidae and the

relationships within the family, while 18S was important in the resolution of basal

eunicidan relationships (i.e., in the rooting of the tree), the monophyly of Onuphidae and

the basal relationships within this family.

Eunicidae is monophyletic, however, with low support and the monophyly is not

present in all recovered trees. Eunice sensu lato is polyphyletic, divided in at least three

independent clades, probably representing at least the defunct genera Leodice, Nicidion and a monophyletic Eunice sensu stricto. Marphysa is also polyphyletic. Two species of

Marphysa sensu lato are nested within one of the Eunicidae clades as sister clade to the

Marphysa sensu stricto clade. The two basal groups in Marphysa s.s. represent a

“Marphysa sanguinea-like” and a “Marphysa bellii-like” group. Lysidice is paraphyletic with Nematonereis nested within it. Palola is the only monophyletic genus among the ones

115 we tested for monophyly. The sister group relationship of Euniphysa is uncertain, however, it was always placed within the sister clade to Palola.

116 Table 3.1: List of terminal taxa used in this study, sampling locality and length of the sequences determined in this study or GenBank accession numbers for downloaded sequences. COI5’, 5’ end of the COI fragment sequenced. COI3’, 3’ end of the COI fragment sequenced. Type species or most similar species to type underlined.

Length of the Sequences/GenBank Accession Numbers

Family Species Locality Voucher 16S COI 18S

5’ 3’ Eunicidae Eunice

Eunicidae E. americana Hartman, 1944 California, USA USNMXXXXXXX 889 612 616 1717 Carrie Bow Cay, Eunicidae E. amoureuxi Rullier, 1974 USNMXXXXXXX 960 684 1666 Belize 64°41.3'S, Eunicidae E. antarctica Baird, 1869 USNMXXXXXXX 947 1128 1733 65°55.6'W, Antarctic Carrie Bow Cay, Eunicidae E. cf. antillensis Ehlers, 1887 USNMXXXXXXX 924 1602 1772 Belize SEM Carrie Bow Cay, Eunicidae USNMXXXXXXX 902 501 1717 E. cariboea Grube, 1856 Belize E. filamentosa Grube, 1856 Carrie Bow Cay, Eunicidae SEM USNMXXXXXXX 933 529 480 1715 complex CBC Belize E. filamentosa Grube, 1856 Eunicidae E, EILD Ceará, Brazil MZSP890 971 625 complex BR E,SEM Carrie Bow Cay, Eunicidae USNMXXXXXXX 938 1706 E. fucata Ehlers, 1887 Belize E. harassii Audouin and Milne Eunicidae Roscoff, France USNMXXXXXXX 978 1200 1772 Edwards, 1933 Port Lincoln, Eunicidae E. impexa Grube, 1878 USNMXXXXXXX 949 377 651 1765 Australia E. cf. insularis Nogueira et al., Eunicidae Ceará, Brazil MZSP889 931 1253 1671 2001 Eunicidae E. cf. limosa Ehlers, 1868 Catalonia, Spain not vouchered 950 606 1668

Eunicidae E. lucei Grube, 1856 California, USA USNMXXXXXXX 908 1194 1629

E. marcusi Zanol et al., 2000 Eunicidae E,EILD Espírito Santo, BrazilMZSP893 952 1239

E. mikeli Carrera-Parra and Carrie Bow Cay, Eunicidae SEM USNMXXXXXXX 1035 574 233 1716 Salazar-Vallejo, 1998 Belize E. miurai Carrera-Parra and Carrie Bow Cay, Eunicidae USNMXXXXXXX 956 1106 1759 Slazar-Vallejo, 1998 Belize Carrie Bow Cay, Eunicidae E. mutilata Webster, 1884 USNMXXXXXXX 1008 1253 1704 Belize Eunicidae E. norvegica (Linnaeus, 1767) Trondheim, Norway USNMXXXXXXX 952 1639 1693 Carrie Bow Cay, Eunicidae E. notata (Treadwell, 1921) USNMXXXXXXX 1001 630 1778 Belize Eunicidae E. roussaei Quatrefages, 1866 Funtana, Croatia not vouchered 938 676 1758 Eunicidae E. rubra Grube, 1856 Ceará, Brazil MZSP888 971 1220 1712 Carrie Bow Cay, Eunicidae E. thomasiana Augener, 1922 USNMXXXXXXX 956 1058 585 1771 Belize

E. torquata Quatrefages, 1866 Eunicidae SEM Catalonia, Spain USNMXXXXXXX 978 1878 1717

E. valens (Chamberlin, 1919b) Eunicidae SEM Washington, USA USNMXXXXXXX 929 1233 1761

E. cf. violaceomaculata Ehlers, Carrie Bow Cay, Eunicidae SEM USNMXXXXXXX 1015 665 1803 1887 Belize Eunicidae Marphysa M. angeli Carrera-Parra and Carrie Bow Cay, Eunicidae USNMXXXXXXX 935 1251 1771 Salazar-Vallejo, 1998 Belize M. cf. bellii (Audouin and Milne E,EILD, Eunicidae Edwards, 1833), juvenile Greece not vouchered 1761 MOL M. bellii (Audouin and Milne Eunicidae Roscoff, France USNMXXXXXXX AY8388351 AF4127892 Edwards, 1833)E M. brevitentaculata Treadwell, Quintana Roo, Eunicidae USNMXXXXXXX 983 1115 1772 1921 Mexico Eunicidae M. californica Moore, 1909 California, USA USNMXXXXXXX 967 1274 1761 Eunicidae M. disjuncta Hartman, 1961 California, USA USNMXXXXXXX 976 1269 1756

Eunicidae M. fallax Marion and Bobretzky, Tuscany, Italy not vouchered 917 1758 117 E,EILD,SEM 1875

M. cf. hentscheli Augener, 1931 Eunicidae SEM Ceará, Brazil MZSP892 971 1266 1761

Eunicidae M. regalis Verrill, 1900 Ceará, Brazil MZSP896 971 662 493 1754

M. sanguinea (Montagu, 1815) Eunicidae SEM Roscoff, France USNMXXXXXXX 944 1287 1770

Eunicidae M. viridis Treadwell, 1917 Ceará, Brazil MZSP894 978 1266 1760 Eunicidae Palola

Eunicidae P. cf. siciliensis (Grube, 1840) Catalonia, Spain not vouchered 958 1189 1723 9 SEM Eunicidae P.viridis Gray in Stair, 1847 Kosrae, Micronesia USNM1084405-9C 989 1262 1761 9 Eunicidae Palola sp. A7Pohnpei 142 Pohnpei, Micronesia USNM1084377-7A 1012 1260 1760 9,E,EILD 3 3 Eunicidae Palola sp. A1Perlas57 Isla Perlas, Panama DQ317900 DQ317842

Palola sp. 3 3 Eunicidae 9E,EILD,MOL Kosrae, Micronesia DQ317884 DQ317826 A9Kosrae161 9,E,EILD 3 3 Eunicidae Palola sp. A3Guam102 Guam DQ317879 DQ317821 9,E,EILD 3 3 Eunicidae Palola sp. B7Bocas78 Panama DQ317872 DQ317817 * 9,E,EILD 3 3 Eunicidae Palola sp. B1Bocas70 Panama DQ317870 DQ317815 9,E,EILD 3 3 Eunicidae Palola sp. B5Belize14 Belize DQ317863 DQ317809 Eunicidae Lysidice

Eunicidae L. collaris Grube, 1870 Catalonia, Spain not vouchered 952 669 1771 L. ninetta Audouin and Milne Eunicidae SEM Catalonia, Spain not vouchered 908 607 533 1772 Edwards, 1833 E, EILD Carrie Bow Cay, Eunicidae USNMXXXXXXX 1774 Lysidice sp.1 Belize E, EILD Eunicidae Lysidice sp.2 Ceará, Brazil MZSP884 980 1056 Eunicidae Nematonereis N. unicornis Schmarda, 1861 Eunicidae E,SEM Catalonia, Spain not vouchered 953 1773 A , N. unicornis Schmarda, 1861 Eunicidae E,EILD,MOL Catalonia, Spain not vouchered 1001 1750 B Eunicidae Euniphysa E. tridontesa (Shen and Wu, off Sumatra, Eunicidae E,EILD WO19209 943 1990) Indonesia E,EILD Eunicidae Euniphysa sp. Timika, West Papua WO19206 650 Onuphidae Diopatra cf. ornata Moore, 1911 California, USA USNMXXXXXXX 974 624 635 1775 Adelaide, SA, Onuphidae Diopatra dentata Kinberg, 1865 USNMXXXXXXX 911 1148 1767 Australia Onuphidae Hyalinoecia sp. Massachusetts, USAUSNMXXXXXXX 918 1278 1775 Mooreonuphis pallidula Onuphidae Massachusetts, USAUSNMXXXXXXX 965 908 1759 (Hartman, 1965)

Nothria conchylega (Sars, 1835) 4 5 Onuphidae E, EILD,MOL AF321417 AY176295 Onuphis elegans (Johnson, Onuphidae Washington USA USNMXXXXXXX 950 1400 1652 1901) Onuphis cf. iridescens (Johnson, Onuphidae California, USA USNMXXXXXXX 949 1489 1656 1901) Paradiopatra quadricuspis (Sars, Onuphidae Trondheim, Norway USNMXXXXXXX 960 1180 1791 1872) Arabella semimaculata (Moore, Oenonidae not vouchered 1033 AY8388662 AY8388442 1911)

Oenone fulgida Savigny, 1818 2 2 Oenonidae SEM not vouchered 941 AY838872 AY838853

Dorvillea erucaeformis Dorvilleidae USNMXXXXXXX 991 AY8388682 AY8388462 Malmgren, 1865 Lumbrineridae Lumbrineris inflata Moore, 1911 AY8388322 AY3665206 AY5256227

Lumbrineris latreilli Audouin and 2 6 7 Lumbrineridae SEM AY838833 AY364855 AY525623 Milne Edwards, 1834

Eurythoe complanata (Pallas, 6 Amphinomidae E,EILD,MOL AY364851 1766) Hipponoe gaudichaudi Audouin Amphinomidae and Milne Edwards, 1833 AY5778818 E,EILD,MOL Paramphinome jeffreysii Amphinomidae USNMXXXXXXX 889 AY8388752 AY8388562 (McIntosh, 1868) Glycera dibranchiata Ehlers, Glyceridae USNMXXXXXXX 960 AY9952102 AY9952082 1868 1Sequences from Struck et al. 2002. 2Sequences from Struck et al, 2006. 3Sequences from Schulze, 2006. 4Sequences from Dahlgreen et al. 2001. 5Sequences from Worsaae et al. 2005. 6Sequences from Joerdens et al. (2004). 7Sequences from Struck and Purschke, 2005. 8Sequences from Rouse et al., 2004. 9Specimens from Schulze, 2006. ESpecimens excluded from SH test and PABA analyses. EILDSpecimens excluded from ILD test in Chapter 4. MOL, coded only for molecular data in Chapter 4. SEM, species examined under the SEM in Chapter 4. 118

Table 3.2: Amplification and sequence primers. Positions refer to the ones in an alignment of mollusk and annelid species (16S and COI) and Homo sapiens (18S).

Gene Sequence (5’-3’) Position Direction Reference 16S 16SAN-F TAC CTT TTR CAT CAT GG 307 forward This study 16SEU-R ACC TTT GCA CGG TCA GGR TAC CGC 967 reverse This study 16SarL CGC CTG TTT ATC AAA AAC AT 883 forward Palumbi et al. 1991 16SAN-R GCT TAC GCC GGT CTG AAC TCA G 1471 reverse This study COI ACOIAF CWA ATC AYA AAG ATA TTG GAAC 23 forward Colgan et al., 2001 ACOIAR AAT ATA WAC TTC WGG GTG ACC 709 reverse Colgan et al., 2001 COIEU-R TCD GGR TGD CCA AAR AAT CA 704 reverse This study Myz-m-F CTT TGA TCC AGC TGG AGG AGG TGACC 651 forward Dahlgren, unpublished COIAN-F GAC CCW GCH GGR GGA GGM GAC C 659 forward This study COIAN-R GCA TCN GGR TAR TCH GAR TAN GG 1309 reverse This study 18S 18e CTG GTT GAT CCT GCC AGT 3 forward Hillis and Dixon, 1991 18L GAA TTA CCG CGG CTG CTG GCA CC 609 reverse Hillis and Dixon,1991 18F509 CCC CGT AAT TGG AAT GAG TAC A 548 forward Struck et al., 2002 18R GTC CCC TTC CGT CAA TTY CTT TAA G 1191 reverse Hillis and Dixon, 1991 18F997 TTC GAA GAC GAT CAG ATA CCG 1044 forward Struck et al., 2002 GGA TCC AAG CTT GAT CCT TCT GCA GGT 18R1843 TCA CCT AC 1843 reverse Elwood et al., 1985

119

Table 3.3: Sequences characteristics and maximum parsimony analyses statistics. CI, consistency index. RI, retention index.

Data sets Number of aligned positionsTree length Number of most CI RI (parsimony informative) parsimonious trees COI 1283 (607) 6337 150 0.20 0.33 MAFFT 16S 1206 (744) 8706 3 0.20 0.42 18S 2476 (329) 1270 3848 0.67 0.74 16SCOI18S 4966 (1680) 16490 10 0.24 0.40 69-terminal 16SCOI18S 4966 (3336) 14550 1 0.26 0.38 49-terminal Clustal 16S 1165 (751) 8664 1 0.21 0.42 18S 2465 (289) 1242 1280 0.67 0.74 16SCOI18S 4911 (1685) 16435 42 0.23 0.40 69-terminal 16SCOI18S 4911(3273) 14519 2 0.26 0.38 49-terminal

120

Table 3.4: Incongruence length difference test results. The three different gene partitions for all alignments were tested against it other. We also test the 16S and 18S rDNA of the different alignment against each other, these results are denoted by *, ◊, ‡. Numbers in bold face refer to significant incongruent data sets on 5% significance level.

Clustal+hand adjusted MAFFT 16S 18S 16S 18S 16S 0.77◊ 0.05* - 0.01** 18S 0.01 1.00‡ 0.10 - COI 0.01 0.31 0.02 0.37 *16S MAFFT alignment and 18S Clustal alignment. **16S Clustal alignment and 18S MAFFT alignment. ◊16S MAFFT alignment and Clustal alignments. ‡18S MAFFT alignment and Clustal alignments.

121

Table 3.5: Shimodaira-Hasegawa test results comparing the different data sets with the maximum likelihood trees resulted from the respective alignments. Numbers in bold face refer to significant incongruent data sets on 5% significance level.

Clustal+hand adjusted MAFFT Hand Trees/data set 16S 18S 16SCOI18S 16S 18S 16SCOI18S COI 16S Cl - 0.000012 0.023827 0.395105 0.000016 - 0.012253 18S Cl 0.000000 - 0.000000 0.000000 0.764763 - 0.000000 16S Mf 0.420035 0.000020 - - 0.000023 0.020210 0.002603 18S Mf 0.000000 0.744862 - 0.000000 - 0.000000 0.000000 COI 0.000001 0.000000 0.000000 0.000121 0.000000 0.000000 - 16SCOI18S Cl 0.270497 0.070768 - - - 0.724927 0.009797 16SCOI18S Mf - - 0.738417 0.482281 0.081421 - 0.106510 Cl- tree resulted from the hand adjusted ClustalX alignment for the 16S and 18S genes; Mf- tree resulted from the MAFFT alignment for the 16S and 18S genes.

122 Table 3.6: Branch support values for the clades present in the topologies resulted from the analyses of the 69 and 49-terminals data sets. Clade numbers refer to the numbers on figures 3.1 and 3.2. Clade number and support values shaded in grey are well supported

(one of the bootstrap values >85%) on the 49-terminal topologies and poorly supported on the 69 one. Clade number and support values underlined are present in all topologies resulted from the 49 and 69-terminal topologies. BI PB, Bayesian inference posterior probability. ML BP, maximum likelihood nonparametric bootstrap value. MP BP, maximum parsimony nonparametric bootstrap value. na, non applicable, clade not present in the topology from such analyses.

69-terminals 49-terminals

MAFFT alignment Clustal alignment MAFFT alignment Clustal alignment clades ML BP MP BP Bremer BI PB ML BP MP BP Bremer BI PB ML BP MP BP Bremer BI PB ML BP MP BP Bremer BI PB support support support support

1 81 <50 9 1 93 na na 1 78 <50 4 1 90 na na 1 2 98 94 15 1 99 99 13 1 na na na na na na na na 3 52 <50 0 0.56 62 na na 0.59 na na na na na na na na 4 90 58 9 1 97 na na 1 92 67 12 1 97 na na 1 5 100 100 42 1 100 100 40 1 100 100 45 1 100 100 42 1 6 <50 na na na na na na na <50 na na na na na na na 7 100 81 9 1 99 91 23 1 99 79 10 1 99 92 27 1 8 98 95 48 1 99 99 49 1 100 100 65 1 100 100 61 1 9 84 <50 3 1 76 <50 14 1 86 <50 2 1 78 65 8 1 10 60 <50 4 0.83 65 54 4 0.87 57 <50 7 na 71 53 4 0.77 11 67 <50 0 0.67 65 <50 0 0.91 90 81 16 0.99 94 95 19 0.99 12 <50 <50 1 0.61 na na na na na na na na na na na na 13 100 93 10 1 100 94 11 1 na na na na na na na na 14 91 <50 8 1 87 59 10 1 88 <50 4 1 84 60 12 1 15 94 82 20 1 93 75 13 1 95 79 25 1 92 72 15 1 16 <50 na na 0.64 60 na na na <50 na na na <50 <50 3 na 17 100 100 49 1 100 100 39 1 100 100 48 1 100 100 40 1 18 83 na na 1 89 <50 1 1 80 <50 1 1 84 <50 3 1 19 66 <50 1 0.64 64 na na 0.94 na na na na na na na na 20 <50 na na <50 na na na na 52 na na na <50 na na na 21 100 99 4 1 99 99 4 1 100 100 46 1 100 100 42 1 22 100 99 4 1 99 99 4 1 na na na na na na na na 23 98 50 5 1 97 <50 2 1 99 58 15 1 99 60 7 1 24 52 50 2 0.99 <50 na na 0.97 56 <50 1 0.98 <50 na na 0.98 25 99 52 9 1 95 <50 1 1 97 <50 5 1 94 <50 3 1 26 55 <50 2 1 <50 na na 1 59 <50 1 1 <50 na na 1 27 56 na na 0.97 50 <50 <50 1 58 na na 0.94 56 na na 1 28 100 99 19 11 100 99 19 1 100 100 44 1 100 100 39 1 29 100 99 38 1 100 99 33 1 100 99 49 1 100 99 35 1 30 <50 <50 1 0.64 <50 na na 0.95 97 <50 3 1 95 72 18 1 (31,53) na na na na na na na na <50 na na 0.72 na na na na 31 <50 <50 1 0.64 na na na na 82 na na 1 na na na na 32 <50 <50 1 0.95 na na na na na na na na na na na na 33 89 51 1 0.81 96 na na 1 100 99 31 1 100 100 60 1 34 <50 na na 0.73 82 <50 1 1 <50 <50 4 0.50 88 <50 5 1 35 <50 na na na na na na na na na na na na na na na 36 71 <50 2 1 62 na na 0.95 na na na na na na na na 37 100 99 118 1 100 99 101 1 na na na na na na na na 38 96 <50 3 1 94 62 4 1 100 <50 2 1 100 <50 7 1 39 92 50 1 1 92 57 1 1 100 85 14 1 100 95 21 1 40 85 <50 2 1 94 <50 2 1 91 na na 1 92 <50 1 1 41 100 100 53 1 100 100 57 1 100 100 92 1 100 100 57 1 42 100 99 128 1 100 99 117 1 100 100 43 1 100 100 118 1 43 97 85 15 1 97 92 3 1 100 84 4 1 100 90 11 1 44 100 100 61 1 100 100 62 1 100 100 57 1 100 100 61 1 45 76 <50 1 1 <50 <50 3 na 83 <50 1 1 60 <50 1 0.69 46 97 73 2 1 97 62 2 1 95 <50 2 1 92 55 5 1 123 47 90 na na 0.88 91 na na 0.9 na na na na na na na na 48 99 95 4 1 99 95 4 1 100 97 24 1 100 98 26 1 49 73 <50 2 1 79 <50 <50 1 71 na na 1 67 na na 1 50 <50 na na na <50 na na na <50 na na na 59 na na 0.60 51 62 na na 0.74 76 na na 0.66 na na na na na na na na 52 70 <50 1 0.97 na na na na 76 <50 4 0.74 <50 na na na 53 97 69 4 1 97 66 7 1 100 81 11 1 97 90 10 1 54 53 na na na na na na na na na na na <50 <50 2 na 55 100 96 95 1 100 94 80 1 na na na na na na na na 56 <50 na na na na na na na na na na na na na na na 57 99 67 1 1 100 67 1 1 100 99 27 1 100 99 30 1 58 86 <50 1 1 na na na na na na na na na na na na 59 71 <50 4 1 75 <50 4 1 94 63 11 1 99 85 20 1 60 82 76 8 1 80 75 8 1 na na na na na na na na 61 76 <50 4 1 75 <50 5 1 na na na na na na na na 62 99 73 5 1 99 73 5 1 na na na na na na na na 63 62 <50 4 0.97 58 53 3 0.92 na na na na na na na na 64 98 56 4 1 97 57 4 1 na na na na na na na na 65 100 98 43 1 100 99 44 1 na na na na na na na na 66 na na na na <50 na na 0.85 na <50 4 0.93 <50 na na 0.81 67 na na na na <50 <50 0 0.83 na na na na na na na na 68 na na na na <50 <50 0 0.84 na <50 3 0.81 na na na 0.94 69 na na na na <50 na na 0.95 na na na na na na na 1 70 na na na na <50 na na na na na na na <50 na na na 71 na na na na <50 na na 0.80 na na na 0.64 na na na 0.97 72 na na na na <50 na na na na na na na na na na na 73 na na na na 92 na na 0.96 na na na na na na na na 74 na na na na 80 na na 1 na na na na na na na na 75 na na na na 86 na na 0.95 na na na na 79 na na 1 76 na <50 2 na na <50 2 0.51 na <50 1 0.61 na na na 0.76 77 na <50 4 na na na na na na <50 8 na na <50 2 na 78 na <50 2 0.89 na <50 3 0.50 na na na 0.74 na <50 3 na 79 na na na na na 51 12 na na na na na na 53 12 na 80 na na na na na <50 1 na na na na na na <50 2 na 81 na na na na na <50 3 0.97 na na na 0.95 na na na 0.98

124

Table 3.7: Shimodaira-Hasegawa test results comparing maximum likelihood trees from unconstrained and constrained analyses to test different hypotheses of relationship.

Numbers in bold face refer to significant different results between the constrained and unconstrained results on 5% significance level.

Hypothesis MAFFT Clustal 16SCOI18S 16SCOI18S Lysidice sp. 1 in (Lysidice, Nematonereis) 0.702031 0.633220 Eunicidae paraphyletic 0.803426 0.865337 Lysidice monophyletic 0.259125 0.175637 (Lysidice, Nematonereis) sister group to Palola 0.505743 0.200435 Marphysa monophyletic 0.000000 0.000000

125

Table 3.8: Alteration of bootstrap support (δ) values for 16S, 18S and COI genes in the different clades of the topologies resulted from the 49-terminal combined data set maximum likelihood analyses and nonparametric bootstrap branch support of these clades on the topologies resulted from single gene partition. Clade numbers are equivalent to the ones in figure 3.1 and 3.2 but are missing taxa excluded from the 49-terminal data set due to missing data of a complete gene partition. Positive δ, added partition contributes in supporting that node. Negative δ, added partition does not contribute in supporting that node. Numbers underlined and/or in grey boxes, added partition is essential in order to obtain a bootstrap higher that 50% and/or 85%, respectively, for that node in the trees of combined (16S18SCOI) analyses. Clade numbers in bold face, well supported

(bootstrap>85%) in all analyses. Grey colored nonparametric bootstrap values, below 50%

. BP, nonparametric bootstrap value. na, non applicable because alteration of bootstrap is from 100 to 100 or from <50 to <50. =, bootstrap support for that branch in the combined analyses is in the same interval 0-49%, 50-84% or 85-100% as the bootstrap support values in at least one of the single gene partition trees. +, bootstrap support for that branch in the combined analyses is on an upper interval when compared to the maximal bootstrap support values for that clade in single gene partition trees. -, bootstrap support for that branch in the combined analyses is on a lower interval when compared to the minimal bootstrap support values for that clade in single gene partition trees.

126

MAFFT alignment Clustal alignment BP δ BP δ BP δ BP BP δ BP δ BP δ BP Clades 16S/ add 16S 16S add COICOI add 18S 18S 16S/ add 16S 16S add COICOI add 18S18S 18S/ 18S/COI 16S/18S 16S/COI 18S/ 18S/COI 16S/18S 16S/COI COI COI

Outgroup 1=/+ 78= 21 <50 6 52 29 57 90+ 13 53 1 52 31 73 4+ 92+ 44 <50 5 79 77 62 97+ 35 <50 na 79 93 73 Lumbrineridae 5= 100= na 96 na <50 1 100 100= na 92 na <50 2 100 Oenonidae 7= 99= 3 86 1 <50 3 86 99= 2 <50 3 <50 42 91 66-/= 27- 1 <50 -19 <50 26 53 41= 7 <50 -19 <50 41 <50 Average over 79.20 17.25 -1.75 27.20 85.4 14.25 -3.75 41.8 outgroup clades

Eunicidae + Onuphidae 8= 100= na 56 na <50 14 100 100= na 86 na <50 1 100 Onuphidae 9=/- 86= -1 <50 3 <50 42 92 78- -1 <50 -1 <50 52 91 14=/- 88= -3 60 11 <50 28 95 84- -5 <50 13 <50 46 96 15= 95= -3 <50 2 <50 87 100 92= -7 <50 4 <50 84 100 16= 33= 16 <50 -4 <50 27 <50 46.4= 30 <50 2 <50 30 <50 17= 100= 14 100 na <50 1 98 100= 15 100 na <50 na 99 18= 80= 46 63 -1 <50 14 <50 84= 50 67 4 <50 9 <50 Average over 83.14 11.50 2.20 35.33 83.49 13.67 4.40 44.20 Onuphidae clades

Eunicidae 10+ 57+ 22 <50 16 <50 1 <50 71+ 23 <50 16 <50 -14 <50 Eunice sensu lato basal 11= 90= 9 59 28 93 -6 <50 94= 13 <50 23 93 -5 <50 20=/- 52= 11 <50 12 57 5 <50 47.8- 8 <50 34 57 2 <50 21= 100= 1 99 na 99 na 97 100= 1 100 na 99 na 96 23+ 99+ 4 60 15 84 0 <50 99+ 5 61 18 84 na <50 24+/= 56+ 5 <50 28 <50 35 <50 39.8= 4 <50 25 <50 14 <50 25+ 97+ 0 <50 23 82 8 <50 94+ -4 <50 46 82 27 61 26+/= 59+ 7 <50 9 <50 37 <50 34= -1 <50 17 <50 31 <50 27+ 58+ -4 <50 na <50 53 <50 56+ -2 <50 7 <50 53 <50 28= 100= na 97 na 98 na 100 100= na 98 na 98 na 100 29= 100= 6 98 na 78 na 87 100= 6 99 na 78 na 88 Average over clades within clade 81.1 4.33 16.43 18.86 76.46 3.33 24.29 17.43 11 30+ 97+ 49 <50 9 <50 6 <50 95+ 42 <50 4 <50 -1 <50 31+/= 82+ 42 <50 45 <50 -3 <50 11.2= -33 <50 10 <50 -11 <50 Lysidice 33= 100= 18 97 1 56 2 <50 100= 19 100 na 56 na <50 34=/+ 42= -0.4 <50 -13 <50 16 <50 88+ 47 65 -1 <50 13 <50 Eunice+Marphysa <50 sensu lato 38= 100= 55 99 na <50 1 <50 100= 54 98 na <50 1 Marphysa sensu <50 stricto 39= 100= 5 100 na 86 na <50 100= 7 100 na 86 na 40= 91= 21 96 6 69 -1 <50 92= 21 98 6 69 -4 <50 41= 100= na 98 na 60 na 100 100= na 100 na 60 na 100 42= 100= na 100 na 100 na 98 100= na 100 na 100 na 98 43= 100= 1 85 8 99 na <50 100= na 79 11 99 na <50 44= 100= na 100 na 99 na 100 100= na 100 na 99 na 99 45-/= 83- 65 95 -14 <50 1 <50 60= 42 82 -34 <50 0 <50 M. sanguinea like <50 species 46= 95= 54 98 -2 <50 24 <50 92= 56 98 -4 <50 5 48= 100= 1 96 5 85 na <50 100= 1 96 5 85 na <50 49- 71- 40 93 -13 76 -17 <50 67- 38 92 -14 76 -20 <50 50+ 50+ 14.4 <50 1.2 <50 24.6 <50 59+ 25 <50 13 <50 31 <50 Eunice sensu lato 52+/= 76+ 26.2 <50 56 <50 4 <50 37.2= -13 <50 35 <50 1 <50 Palola 53= 100= 48 95 na <50 1 73 97= 42 98 2 <50 na 73 57= 100= 83 100 na 62 na <50 100= 82 99 na 62 na <50 59+ 94+ 31 59 25 <50 3 <50 99+ 35 65 4 <50 1 <50 75=/- 1= na <50 na <50 na <50 79- 78 94 1 <50 7 <50 Average over clades within clade 30 83.45 33.18 9.23 7.3 82.73 31.94 2.6 2.77 Average over Eunicidae clades 81.94 23.51 11.83 11.03 80.47 22.43 9.80 6.86 Average over ALL clades 81.82 20.95 8.52 18.34 81.49 20.18 7.25 18.52

127

Fig. 3.1: Trees from the analyses of the combined data set of the MAFFT alignment. A.

Maximum likelihood phylogram. Terminals underlined have the insertion in the COI sequence. Numbers on branches are (clade) maximum likelihood bootstrap support/ maximum parsimony bootstrap support/ maximum parsimony Bremer support/ Bayesian posterior probability. Only high branch support shown on clades (bootstrap>85% and posterior probability>95%), for all other support values see Table 3.6. na- clade not present in the strict consensus of the maximum parsimony tree, in the Bayesian tree or in neither.

Bootstrap and posterior probability lower than 85% and 95%, respectively, represented by a – if other high supports are shown for the clade. Clades with numbers in bold face and italic are present in the trees resulted from all analyses of the MAFFT alignment. Clades with numbers underlined are present only in the maximum likelihood tree of the MAFFT alignment. Vertical black bars indicate possible genus level clades. B. Differences between the strict consensus of the most parsimonious trees and the maximum likelihood tree.

Number on branches are maximum parsimony bootstrap support/ maximum parsimony

Bremer support. C. Differences between the strict consensus of the most parsimonious trees and the Bayesian gene+codon tree and the maximum likelihood tree. Numbers on branches are maximum parsimony bootstrap support/ maximum parsimony Bremer support/ Bayesian posterior probability. D. Differences between the Bayesian gene+codon tree and the maximum likelihood tree. Number on branches is Bayesian posterior probability. B-C. Bootstrap lower than 50% represented by a – if other high supports are shown for the clade.

128

129

Fig. 3.2: Trees from the analyses of the combined data set of the Clustal alignment A.

Maximum likelihood phylogram. Terminals underlined have the insertion in the COI sequence. Numbers on branches are (clade) maximum likelihood boostrap support/ maximum parsimony bootstrap support/ maximum parsimony Bremer support/ Bayesian posterior probability. Only high branch support shown on clades (bootstrap>85% and posterior probability>95%), for all other support values see Table 3.6. na- clade not present in the strict consensus of the maximum parsimony tree, in the Bayesian tree or in neither.

with numbers underlined are present only in the maximum likelihood tree of the MAFFT

alignment. Vertical black bars indicate possible genus level clades. B. Differences between the strict consensus of the most parsimonious trees and the maximum likelihood tree.

Numbers on branches are maximum parsimony bootstrap support/ maximum parsimony

Bremer support. C. Differences between the strict consensus of the most parsimonious

trees and the Bayesian gene+codon tree and the maximum likelihood tree. Numbers on

branches are maximum parsimony bootstrap support/ maximum parsimony Bremer

support/ Bayesian posterior probability. D. Differences between the Bayesian gene+codon

tree and the maximum likelihood tree. Number on branches is Bayesian posterior

probability. B-C. Bootstrap lower than 50% represented by a – if other high supports are

shown for the clade.

130

131

Fig. 3.3: Bayesian trees from the gene partition analyses. A. Tree yielded in the analysis of

the 69-terminals combined data set of the Clustal alignment. B. Tree yielded in the analysis of the 69-terminals combined data set of the MAFFT alignment. Posterior probabilities placed on branches.

132

133

Fig. 3.4: Trees from the analyses of the 49-terminal data set for both alignments (MAFFT

and Clustal). A-C. MAFFT alignment. A. Maximum likelihood. B. Most parsimonious

tree. C. Bayesian, gene+codon partition. D-F. Clustal alignment. D. Maximum likelihood.

E. Strict conesnus of the most parsimonious trees. F. Bayesian, gene+codon partition.

Clade numbers on branches. Clade numbers in italic and bold face, bootstrap>85% or posterior probability>95%). Clade not numbered, absent in the 69-terminal trees. Light grey box, Onuphidae. Dark grey box, Eunicidae. See Table 3.6 for branch support measures.

134

135

Fig. 3.5: Presence of clades in the trees yielded by the different analyses and data sets.

Clade numbers refer to those in figures 3.1 and 3.2. Clade numbers underlined may represent genera level clades. Open boxes, absent. Black boxes, present. MP, strict consensus of the most parsimonious trees. BIc, Bayesian inference codon partition. BIg,

Bayesian inference gene partition. BIgc, Bayesian inference gene+codon partition. na, non- applicable because species are not present in the matrix. * clade 33, clade 33 is present but includes only Lysidice species because Nematonereis is not present in the matrix. * clade

75, clade 75 is present but includes only Palola and Lysidice species because Euniphysa sp. is not present in the matrix.

136

137

Fig. 3.6: Most parsimonious reconstruction of the presence and absence of peristomial cirri, palps and lateral antennae in the 69-terminal combined maximum likelihood tree. These traits are also absent in sequential juvenile stages A-C. MAFFT alignment. D-F. Clustal alignment. A,D. Peristomial cirri. B,E. Palps. C,F. Lateral antennae. White branches, absent. Black branches, present.

138

139 Chapter 4: Total evidence phylogeny and generic reclassification of the family Eunicidae (polychaete, Annelida)

INTRODUCTION

Eunicidae is a family of marine annelids distributed worldwide in diverse types of

benthic habitats. The family is well known for the long length some of its species can reach

(around 6 meters, Fauchald, 1992a), for its value as a food delicacy in several South Pacific

Islands (Schulze, 2006) and for its economic use as bait for commercial and leisure fishing

(Gambi et al., 1994; Olive, 1994).

There is general agreement that Eunicids are related to the other families of the order

Eunicida, having the family Onuphidae as its closest relative (Hartman, 1944; Rouse and

Fauchald, 1997; Struck et al., 2002, 2006; Chapter 3). Traditionally, eunicid2 (~326 spp.) species have composed a stable, readily identifiable group, regardless of its taxonomic rank

(Kinberg, 1865; Ehlers, 1868; Hartman, 1944). However, formally it is characterized by no unique diagnostic features (apomorphies) but instead by plesiomorphies or homoplasies

(Orensanz, 1990), including asymmetric labidognath jaws, one to five prostomial appendages, double ringed peristomium and wide, frontally bilobed prostomium. Grouping the eunicid species in a single taxon was recently supported by the recovery of a monophyletic Eunicidae (although not strongly supported) in the first study focusing on the phylogeny of the family per se based on both mitochondrial (16S rDNA and COI) and nuclear genes (18S rDNA) (Chapter 3). Previous molecular and morphological phylogenies

(Struck et al., 2002, 2006; Zanol et al., 2007) had focused, on the phylogeny of the order

2 Herein the adjective eunicid denotes members of the family Eunicidae whereas eunicidan denotes members of the order Eunicida. 140 Eunicida and of the eunicid genus Eunice, respectively, and were conflictive regarding the

monophyly of the family.

Eunicid species are currently grouped in nine genera (Table 1.2). Of the five genera

that have had their phylogenetic status tested only two are monophyletic, Euniphysa and

Palola (Lu and Fauchald, 2000; Schulze, 2006; Zanol et al. 2007; Chapter 3). Eunice and

Marphysa are polyphyletic and Lysidice, paraphyletic (Zanol et al. 2007; Chapter 3).

Euniphysa and Palola are also the only genera clearly supported by unique diagnostic features (apomorphies) (Lu and Fauchald, 2000; Zanol et al., 2007). The other four genera are mainly diagnosed by plesiomorphies (e.g., five prostomial appendages, peristomial cirri and double rings in Eunice) or ontogenetically variable features (e.g., lack of peristomial cirri in Marphysa, Lysidice and Nematonereis; lack of palps in Lysidice and Nematonereis

and the lack of lateral antennae in Nematonereis) (Orensanz, 1990). Stable clades that

could represent monophyletic Lysidice, Marphysa sensu stricto and revive two defunct

genera Leodice Lamarck, 1818 and Nicidion Kinberg, 1865 were identified in the

molecular phylogeny of the family (Chapter 3). However this study did not include Eunice

antennata (Lamarck, 1818), which is the type species of Leodice, or Eunice cincta

(Kinberg, 1865), here considered as the type species of Nicidion since it is the only species

of the three named in the original description that can be reliably identified to species (see

Hartman, 1959 for a different view).

The taxonomy of Eunicidae at the generic level has traditionally relied on anterior

presegmental characters, such as the absence/presence of the dorsal buccal lip (sensu

Orrhage, 1995; e.g., Kinberg, 1865; Ehlers, 1868), the shape of the mandible (curved

versus flat; e.g., Hartman, 1944; Fauchald, 1970) and the absence/presence of the head

141 appendages, including palps, antennae and peristomial cirri. Indeed, most authors have

considered the presence and absence of the anterior appendages as the main informative and unambiguous feature at the generic level (e.g., Kinberg, 1865; Ehlers, 1868; Gravier,

1900; Hartman, 1944; Gathof, 1984). The two opposing hypotheses of evolution within

Eunicidae are based mainly on these features. These hypotheses advocate that the eunicid evolution proceeded in a stepwise pattern with the addition of anterior appendages, from one median prostomial appendage (Nematonereis-like) to five prostomial appendages and one pair of persitomial cirri (Eunice-like) (Gravier, 1900; Hartman, 1944), or in the opposite direction with the gradual loss of anterior appendages (Orensanz,1990).

In earlier studies, branchial features (absence/presence, distribution and shape) were the main or only characters associated with the segments considered at the generic level

(Kinberg, 1865; Ehlers, 1868). However, the absence/presence of branchiae is not informative at this level (Fauchald, 1992a), and in some cases it varies even within the same species (Nogueira et al., 2001). Currently the shape and distribution of branchiae are features used at the intrageneric level and in the diagnoses of species (Fauchald, 1992a;

Orensanz, 1990). The absence/presence of different kinds of chaetae are the main segmental characters currently used at the generic level. Palola lacks pectinate chaetae and subacicular hooks (Fauchald, 1992b). Nauphanta lacks compound chaetae and has fan shaped pectinate chaetae (Fauchald, 1987). Except for this use of the shape of the pectinate chaetae, chaetal structures are mostly used at the intrageneric level and in the diagnoses of species.

Segmental characters vary along the eunicid body in a structured way (Fauchald,

1992a) and this variation may contain phylogenetic information (Lu and Fauchald, 2000;

142 Zanol et al., 2007). However, the common practice, probably rooted in the view that segmental features are homogeneous along the body so that all segments of the body would convey the same information (e.g., Ehlers, 1868), does not take into account such variation.

Furthermore the homology between segments of individuals with different segment counts is not straightforward, the number of segments varies within eunicid species and no clear body regions are recognized. Thus, comparative studies including variation along the body must confront the serial homology issue, which has not been done so far.

In the present study we used a combination of broader taxon sampling than

previously used for the family, molecular and morphological data to further test the

monophyly of the family and to investigate the phylogenetic relationships among the

eunicids. The morphological data included a novel attempt to account for serial homology,

thus to include information about morphological variation along the body. The resulting

phylogeny allowed us to also evaluate diagnostic and traditional taxonomic characters in a

phylogenetic perspective and to make relevant nomenclatural changes in order to define

monophyletic genera.

MATERIAL AND METHODS

Taxon Sampling

We sampled 60 species representing all nine currently valid genera of the family

Eunicidae (see Appendix 6 for the complete list of the specimens and their locality of collection). The choice of species took into account the diversity of morphology and included the type species of the different valid genera when possible as well as of Leodice

143 and Nicidion. Tables 3.1 and 4.1 list the taxa used in this study. Uncertain identification of

some species as “cf.” or “sp.” was due to the poor understanding of intraspecific or

ontogenetic variations or to a lack of complete specimens.

Outgroup taxa are species of the four major non-eunicid families of the order

Eunicida: Onuphidae, Dorvilleidae, Lumbrineridae and Oenonidae; and species of two

other aciculate clades, Amphinomida and Phyllodocida (Rouse and Pleijel, 2001; Struck et al., 2007) (Table 3.1 and 4.1).

Specimen Processing

Specimens were fixed in 4% formaldehyde or 95% ethanol and preserved,

respectively, in 70% and 95% ethanol. Each specimen was examined under stereo and

compound microscopes. Specimens were dissected as described by Day (1967) to code

characters of the buccal apparatus. In order to account for serially homologous characters,

segmental characters were coded independently for four selected chaetigers of each

specimen, one chaetiger from the initial (P1) and one from the final (P6) 2.5% of the body

(determined by number of chaetigers), the median chaetiger from the first quarter (P2) and

one median chaetiger from the posterior three quarters of the body (P3-5). The posterior

three quarters of the body were lumped into a single set for most segmental characters

based on preliminary observations that showed no variation of the selected characters along this region. We quantified branchial distribution as the percentage of continuous chaetigers in which branchiae are present, as used by Fauchald (1992a). Characters varying intraspecifically or within the same specimen (e.g., differently shaped pectinate chaetae in the same parapodia) were coded as polymorphic. Character descriptions are given in the

144 results section because it contains several new observations which otherwise would not be

mentioned in the results or would have to be duplicated in the results section.

One specimen each from 21 species of ingroup and outgroup taxa were examined

under the Scanning Electron Microscopy (SEM) (Table 3.1 and 4.1) in order to improve the

understanding of structural details of morphological characters. This examination allowed

us to refine the hypotheses of primary homology (sensu de Pinna, 1991) of characters that

could be observed and scored using light microscopy for the remaining species.

Specimens examined with SEM were cleaned ultrasonically for 30 seconds in

distilled water with 0.01% Tween 80. They were then rinsed 2x with distilled water for five

minutes each, postfixed with 1% osmium tetroxide (OsO4) for 60 minutes and subsequently

rinsed in distilled water 4x for 10 minutes each. After the last rinse, specimens were

dehydrated in increasing concentrations of ethanol, 50% (10 minutes), 75% (10 minutes),

95% (30 minutes) and 100% (4 times for 30 minutes each), and critical point dried.

Specimens were mounted on SEM stubs and coated with platinum before being examined

with a Leica Stereoscan 440 with LaB6 electron source scanning electron microscope.

Phylogenetic Analyses

We analyzed the morphological data set by itself as well as combined with molecular data (16S, COI and 18S genes, including both alignments of the 69 terminal data sets, see chapter 3 for more details) (total evidence data set). For each of those, two different data sets with alternative taxon composition were analyzed under the different analyses criteria described here. The 79 (73 morphological data only) terminal included all terminals, while the 77 (71 morphological data only) terminal excluded Aciculomarphysa comes Hartmann-

145 Schröder, 1998 and Fauchaldius cyrtauloni Carrera-Parra and Salazar Vallejo, 1998, which had, respectively, 20% and 49% of the morphological characters coded and no molecular data. This large number of uncertain characters results from both species being described from morphologically simplified forms leading to many missing and inapplicable states.

Even though Eunice cincta (Kinberg, 1865) was coded for a similarly small percentage of

morphological characters (24%) it was included in all analyses. In this case missing

characters were mainly caused by unobserved features due to the poor preservation of the

specimen examined; despite the minimal scoring E. cincta was unambiguously placed in all

analyses. Ten species were coded only for morphological data (Table 4.1), while another

six were only coded for molecular data (Table 3.1).

Morphological Data Only Analyses. The morphological data set was analyzed

separately under the maximum parsimony optimization criteria (MP). Characters were

treated as unordered and equally weighted. Tree search was heuristic, using the option

Tree-Bisection-Reconnection (TBR)+TBR in NONA (Goloboff, 1999). Analyses started

with a stepwise addition tree with taxa randomly added in 5000 replicates and 50 trees held

on each step of the TBR. During search branches were collapsed when minimum branch length was zero.

The trees of MP analyses resulting in more than one most parsimonious tree were summarized in a strict consensus tree. Branch support for the 73-terminal (79-terminal in the total evidence) trees total evidence data sets was estimated with nonparametric

bootstrap values (BP) (Felsenstein, 1985) (5000 replicates) and Bremer support (Bremer,

1988) calculated in TNT (Goloboff et al., 2008). Bremer support was calculated using the

suboptimal search option in the menu created with the macro file “BREMERtnt.run”

146 distributed with the software. The suboptimal search followed the default parameters and searched trees longer than the most parsimonious in increments of one step per search up to

20 steps.

Total Evidence Data Analyses. We used the Incongruence Length Difference test

(ILD) (Mickevich and Farris, 1981; Farris et al., 1995) to test the congruence among the morphological and molecular (all three genes combined, Chapter 3) data sets for both alignment approaches. It was run under the default settings for the homogeneity test in

PAUP* 4.0b10 (Swofford, 2001) with 100 replicates holding 10 trees at each step.

Uninformative characters (Cunningham, 1997) and terminals missing at least 50% of the data for at least one of the genes of the tested pair were excluded before running the tests

(see Table 3.1 for a list of excluded terminals). The null hypothesis of congruence between the pairs of data sets being tested was rejected at a 5% significance level.

We used WinClada (Nixon, 2002) to edit matrices and concatenate morphological and molecular data sets. The matrix combining morphological and MAFFT alignment

(Chapter 3) data sets are referred to below as “morphMAFFT” and the one combining morphological and Clustal alignment (Chapter 3) data sets as “morphClustal”. The combined morphological and molecular data sets (total evidence data sets) were analyzed using both MP and Bayesian methods. MP analyses followed the above description for the analyses of the morphological data set.

Characters were optimized in the MP strict consensus tree using the MP criterion and the optimization mode (unambiguous, ACCTRAN or DELTRAN) that increased unique origins and secondary losses as opposing to convergences (de Pinna, 1991) in WinClada

(Nixon, 2002) The best optimization for each character was determined case by case since

147 the optimization increasing unique origins depends on the phylogenetic status of the

character as well as on the inapplicable codings (Agnarsson and Miller, 2008).

We ran two Bayesian analyses in MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003)

for each of the four total evidence data sets (two different alignments and two different

taxon compositions). One treated the morphological data and each gene as a different

partition (from now on referred to as “morphgene partition”). The other also included the

different codon positions of the COI as different partitions because of their distinct

saturation curves (Chapter 3) (from now on referred to as “morphgene+codon partition”).

We chose not to partition the ribosomal genes into stems and loops due to the ambiguity of

secondary structure models for 18S and 16S ribosomal genes of annelids (e.g., Nereis limbata GenBank accession number U36270 in http://www.psb.ugent.be/rRNA/ssu/query/index.html and http://rdp8.cme.msu.edu/html/).

The morphological data partition was analyzed under the Markov model accounting for variable number of states, conditioned to the presence of only variable characters in the

data set (Mkv) with rate heterogeneity (Γ) (Lewis, 2001). The different molecular partitions

were analyzed with the model selected by the Akaike Information Criterion (AIC) as

implemented in ModelTest (Posada and Crandall, 1998) and described in Chapter 3.

The model parameter values were estimated independently for each partition during

the analyses, but tree and branch length were the same for all partitions. In all analyses two

independent and simultaneous runs with flat prior probabilities, four chains and other default settings were performed. An average standard deviation of split frequencies <0.015 calculated in MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003) as well as the graph of the log likelihood of the sampled trees plotted in Tracer v.1.4 (Rambaut and Drummond, 2007)

148 were used to estimate convergence between the runs (stationarity of parameters). We

verified the mixing quality of all parameters (i.e., the efficiency with which the MCMC

algorithm samples a parameter) by examining the plot of the log likelihood versus sampled

trees as well as the effective sample size (ESS) for all parameters calculated in Tracer v.1.

An ESS higher than 200 for the log likelihood and higher than 100 for all other parameters

when the two runs were combined was considered a good mixing and the result of the

analyses were accepted. The 79-terminal analyses were finished at 10x106 generations even if the parameters had not converged (standard deviation of split frequencies morphMAFFT morphgene partition= 0.016890; morphClust morphgene partition= 0.016398).

The different 77-terminal data sets reach convergence at different generations.

MorphMAFFT: total evidence morphgene partition at 5x106, total evidence morphgene+codon partition at 10x106. MorphClustal: total evidence morphgene partition at

8x106, total evidence morphgene+codon partition at 10x106. Every 100th tree was sampled, the first 25% of the trees were discarded as “burn in”. The remaining trees were summarized in a majority rule consensus with the Bayesian Posterior Probability (PB) support for each clade.

RESULTS

Morphological Characters

Morphological characters are partially based on those described in Zanol et al. (2007)

(see original paper for character descriptions, comments and figures). Characters

unchanged from this study are marked Z07 and modified characters are indicated with

149 Z07m. Many of the characters used are formally described and coded for the first time in this study, for this reason are not always available from earlier published descriptions. Soft body part characters are influenced by the state of preservation of the specimen making the coding ambiguous in poorly preserved specimens. Uninformative characters are underlined. Uninformative characters were not removed from the matrix for improved branch length estimation in the Bayesian analyses (Lewis, 2001).

1. Shape of anterior body cross-section: (0) round; (1) dorsoventrally flattened; (2)

dorsally flattened; (3) ventrally flattened. A round body in cross section is the most

common state in the three regions of the body in both ingroup and outgroups

species. Most of the included Onuphidae species have dorsally flattened cross

sections in the anterior region of the body, however median and posterior regions

are round in cross section. All studied Palola species are ventrally flattened

anteriorly, a condition described for almost all types of Palola (Fauchald, 1992b).

Most Marphysa species as well as Nauphanta mossambica (Peters, 1854) observed

here are dorsally flattened in at least one of the body regions, most commonly in the

median and posterior regions, as opposed to being dorsoventrally flattened

(Fauchald, 1970; 1987). N. mossambica is only dorsoventrally flattened in the

anterior region as are Euniphysa spp. (Lu and Fauchald, 2000). The shape of the

body cross-section is here coded in three characters because it varies independently

along the body. The examined species have diverse combinations of the different

shapes of the body cross-section along the body (i.e., having a certain shape of the

150 body cross-section at one of the regions does not determined the shape of the other

regions).

2. Shape of median body cross-section: (0) round; (1) dorsoventrally flattened; (2)

dorsally flattened; (3) ventrally flattened. See character 1 for description.

3. Shape of posterior body cross-section: (0) round; (1) dorsoventrally flattened; (2)

dorsally flattened; (3) ventrally flattened. See character 1 for description.

4. Z07, Body shape: (0) abruptly tapering posteriorly (Fig. 4.1A); (1) evenly tapering

from anterior-median chaetigers (Fig. 4.1B). Most Marphysa species taper abruptly

at the posterior end. This state was also observed in Diopatra ornata Moore, 1911,

some Eunice species, Eunice amoreuxi Rullier, 1974, F. cyrtauloni, N. mossambica

and has been described in some Palola species (Fauchald, 1992b). However this

feature varies intraspecifically as we found for Eunice cariboea Grube, 1856,

Eunice cf. insularis Nogueira et al., 2001 and Marphysa regalis Verrill, 1900.

5. Z07, Relative width and length of chaetigers: (0) at most 10 times wider than long;

(1) more than 10 times wider than long.

6. Z07, Posterior extension of prostomium, caruncle: (0) absent; (1) present. A caruncle

is only present in the outgroup family Amphinomidae among the included taxa.

7. Mediotransversal deep groove of the buccal lips (sensu Orrhage, 1995): (0) absent

(Fig. 4.2C-E); (1) present (Fig. 4.2A,B). The buccal lips in the outgroup families

Amphinomidae and Onuphidae and in the ingroup are medially separated by a deep

groove present at least ventrally; the lips are innervated by the buccal lip nerves,

which branch from a ventral commissure of the ventral root of the

circumoesophageal connective (Orrhage, 1995; Chapter 2). In the remaining

151 eunicidan outgroup families, Dorvilleidae, Lumbrineridae and Oenonidae, the same

nerves innervate at least the median part of the ventral side of the prostomium

which is therefore considered to represent the buccal lips in these families (Chapter

3). However in these families the buccal lips lack the median groove and are at least

in part surrounded by an inconspicuous furrow. Buccal lips are unknown in the

outgroup family Glyceridae.

8. Dorsal buccal lips (sensu Orrhage, 1995): (0) absent; (1) present. The dorsal buccal

lips of Onuphidae are conspicuous free structures on the dorsoanterior end of the

prostomium (Fig. 4.3D). In all eunicids studied, except for Nematonereis unicornis

Schmarda, 1861, the dorsal prostomial region anterior to the prostomial appendages

is puffed up (Fig. 4.3A-C). The same section of the prostomium is more

inconspicuous, even appearing absent, in small species such as the ones in Lysidice

(Fig.4.3A) and very conspicuous, forming a distinct lobe in larger species such as

Eunice aphroditois (Pallas 1788) (Fig. 4.3C). We here consider this puffed up

region of the eunicid prostomia homologous to the dorsal buccal lips in Onuphidae

as had been previously suggested (e.g., Pruvot and Racovitza, 1895; Haffner, 1959).

This hypothesis of homology is based on topological and correspondence

similarities made more evident in SEM observations. In most eunicid species the

dorsal buccal lips are clearly noticeable under the SEM, even if they may be almost

indiscernible in light microscopy. No structures corresponding to the dorsal buccal

lips were observed in any of the outgroup species other than in the onuphids. In

Zanol et al. (2007), the onuphids dorsal buccal lips were called frontal lips (sensu

Paxton, 1998) and were homologized to the anterior end of the eunicid prostomia of

152 the Eunicidae and not to the dorsal buccal lips as we do here. Our coding of the

dorsal buccal lips in eunicid species contrasts with early studies that considered the

absence/presence of buccal lips as important generic level characters (Kinberg,

1865; Ehlers, 1868). These studies considered dorsal buccal lips present only when

these formed a lobe such as in Fig. 4.3C. In eunicid species with less developed

dorsal buccal lips these were considered absent.

9. Shape of dorsal buccal lips: (0) conspicuous, free, attached to the prostomium by

narrow bases (usually digitiform or tapering, Fig. 4.3D); (1) mostly inconspicuous,

fused to the dorsal side of the prostomium (Fig. 4.3A-C).

10. Z07m, Anterior shape of the prostomium in taxa with buccal lips medially separated

and dorsal buccal lip fused to the dorsal side of the prostomium: (0) round (Fig.

4.4A); (1) steep truncate (Fig. 4.4B); (2) round multilobed (Fig. 4.3C); (3) dorsally

entire (Fig. 4.4D); (4) expanded truncate (Fig. 4.4C). The two opposite sides of

round prostomium resemble semicircles, the median region of each half is the

longest. Round multilobed prostomium have a series of ventral lobes. In truncate

prostomia the median end of each side is the longest, the length decreases evenly

from the inner to outer sides (Fig. 4.4B,C). In the steep truncate prostomium the

lateral edge is in addition distinctly ventral to the median edge and may not be

clearly seen from a dorsal view while in the expanded truncate, the lateral edge is

almost or as dorsal as the median edge and is clearly seen in a dorsal view. Dorsally

entire prostomium does not have a dorsal median groove.

11. Z07m, Dorsal shape of the prostomium in taxa with buccal lips medially separated

and dorsal buccal lip fused to the dorsal side of the prostomium: (0) inflated (Fig.

153 4.5C,D); (1) flat (Fig. 4.5E,F); (2) with thickened lateral margins (dimpled) (Fig.

4.3B); (3) flat with higher anterior end (Figs. 4.4C, 4.5A,B). In flat prostomia with

elevated anterior end, the ventral buccal lips rise beyond the rest of the prostomium

on the dorsal side.

12. Relative width of prostomium in taxa with buccal lips medially separated and dorsal

buccal lip fused to the dorsal side in relation to peristomium: (0) as wide as; (1)

narrower; (2) wider. In most Palola species observed here the prostomium is wider

than the peristomium being expanded anterolaterally.

13. Pharyngeal fold, anterior end connects to: (0) anterior extension of dorsolateral fold

(Fig. 4.2A-C,E); (1) buccal lips (Fig. 4.2D). Eunicidan species have four folds in

the pharynx placed at different dorso-ventral positions (Purschke, 1987; Tzetlin and

Purschke, 2005). From dorsal to ventral these folds include the dorsolateral folds

(innervated by the anterior end of the stomatogastric nerves),the maxillary folds

(carrying the maxillary plates), the pharyngeal folds (innervated by the pharyngeal

nerve branching from a ventral commissure of ventral root of the

circumoesophageal connective) and finally the mandible folds (supporting the

ventral mandibles) (Chapter 2).

14. Dorsolateral folds, anterior extension medially: (0) separated (Fig. 4.2B,C,E); (1)

connected (Figs. 4.2A, 4.6). In Eunicidae the muscles that run in the anterior

extension of the dorsolateral fold (DM1 sensu Wolf, 1980) are completely fused

at their anterior end, connecting both sides of the anterior extensions of the

dorsolateral fold. In the outgroup families Lumbrineridae, Oenonidae and

Onuphidae, the DM1 muscles are not medially fused and the anterior extensions

154 of the dorsolateral fold from the two sides are medially separated. The

oesophageal nerves always run through the anterior extension of the dorsolateral

fold before connecting to the oesophageal ganglion (Chapter 2). This anterior

extension is unknown in Dorvilleidae, Amphinomidae and Glyceridae.

15. Anterior notch on the dorsolateral fold anterior extension with both sides

connected: (0) absent (Fig. 4.6A); (1) present (Fig. 4.6B). In Eunicidae the

connection of the anterior extension of the dorsolateral fold may have a notch

anteriorly.

16. Z07, Relative placement of the five prostomial appendages: (0) antennae and palps

evenly spaced; (1) median antenna isolated by a gap from the other antennae and

palps; (2) palps isolated by a gap from the antennae; (3) head appendages on

conspicuously different regions of the prostomium.

17. Arrangement of the five prostomial appendages: (0) all in a straight line; (1)

antennae in straight line, palps located more anteriorly; (2) forming a semicircle

(Fig. 4.4A-C); (3) forming a horseshoe (Fig. 4.3D); (4) lateral antennae and palps in

a straight line, median antenna posterior to them. In the semicircular arrangement,

the palps are anterior and at least partially lateral to the lateral antennae. This is the

most common arrangement among the eunicids we examined. In the horseshoe

arrangement the palps are directly anterior to the lateral antennae. This is the most

common condition among the onuphids examined here.

18. Z07, Median antenna: (0) absent; (1) present.

19. Z07, Lateral antennae: (0) absent; (1) present.

155 20. Z07, Antennal ceratostyles: (0) digitiform, tapering or clavate; (1) medially

inflated (fusiform); (2) button shaped.

21. Z07m, Articulations of median and lateral antennal styles: (0) absent; (1) present.

Here we have broadened our definition of “articulated” from the usage in Zanol et

al (2007). Antennae previously described as wrinkled, but which have at least

some grooves distinct around the whole circumference are here considered

articulated but with irregular wrinkles which is one of the two alternative ways of

having articulate styles (see character 22 for further description).

22. Shape of the articulation of median and lateral antennal styles: (0) irregularly

(wrinkled, inconspicuous) (Figs. 4.3A-C, 4.4A-C, 4.5C,D, 4.8); (1) regular,

conspicuous (no irregular grooves present) (Figs. 4.5E,F, 4.7A,B). The number of

articles in irregularly articulated antennae is difficult to determine because of the

presence of irregular grooves that are not distinct around the whole

circumference. In some species, such as E. cariboea, irregularly articulated

antennae appear to be wrinkled. The articles of regular articulations are distinct so

that the only grooves present are the ones separating the articles. Regular

articulations are only present in some species of Eunice sensu lato.

23. Z07m, Shape of conspicuous regular articulation in median and lateral antennal

styles: (0) short or long cylinders (Fig. 4.7A); (1) moniliform (Fig. 4.7B).

24. Z07, *Antennal ceratophore: (0) absent; (1) present.

25. Z07m, Shape of antennal ceratophore: (0) short, usually ring-shaped (Figs. 4.4A-C,

4.5A,C,E); (1) relatively long, usually articulated (Fig. 4.3D); (2) long, not

articulated (Fig. 4.8).

156 26. Z07, Palpal styles: (0) absent; (1) present.

27. Z07, Palpal styles: (0) digitiform, tapering or clavate; (1) fusiform.

28. Z07m, Articulation of palpal styles: (0) absent; (1) present.

29. Shape of the articulations of palpal styles: (0) multiple articulations, irregular

(wrinkled, inconspicuous); (1) multiple articulations, regular, conspicuous (no

irregular grooves present); (2) a single articulation present. See character 22 for

description. Palps of Dorvillea species never have more than a single articulation

when articulated (Jumars, 1974).

30. Z07, Shape of conspicuous regular articulations in palpal styles: (0) short or long

cylinders (Fig. 4.7A); (1) moniliform (Fig. 4.7B).

31. Z07, Palpophores: (0) absent; (1) present.

32. Z07m, Shape of palpophores: (0) short, usually ring-shaped (Figs. 4.4A-C,

4.5A,C,E); (1) relatively long, usually articulated (Fig. 4.3D); (2) long, not

articulated (Fig. 4.8).

33. Z07m, Peristomial ventro-lateral lips: (0) inconspicuous (Fig. 4.9A); (1) set off by

distinct grooves (Fig. 4.9B); (2) visible only as elevated surfaces (Fig. 4.9C); (3)

medially short, about half as long as the lateral sides of the peristomium (Fig.

4.9D).

34. Z07, Peristomium: (0) a single ring; (1) two rings distinct at least dorsally; (2)

reduced to region around the mouth.

35. Z07, Peristomial cirri: (0) absent; (1) present. Peristomial cirri are present in most

Eunicidae and Onuphidae, however some species in both families lack them.

157 36. Position of peristomial cirri on the posterior peristomial ring: (0) at anterior edge

(Figs. 4.4B, 4.5A, 4.8); (1) clearly posterior to the anterior edge (Figs. 4.5E,

4.7A,B).

37. Z07, Mouth: (0) extending posteriorly to the ventral side of anterior chaetigers; (1)

restricted to peristomium.

38. Dorsolateral maxillae in a ventral muscularized pharynx: (0) absent; (1) present.

39. Z07, Number of rows of maxillary plates: (0) one pair; (1) two or more pairs.

Eunicid species always have just one pair of rows. Dorvilleidae is the only family

examined here to have two or more pairs.

40. Z07, Calcium carbonate mineralizing jaws: (0) absent; (1) present. Dorvilleidae and

Oenonidae have no calcium carbonate in the jaws (Colbath, 1986). However it is

present in the jaws of Eunicidae, Onuphidae and Lumbrineridae. The first two have

calcite and the latter aragonite mineralizing their jaws (Colbath, 1986).

41. Z07, Crystal form of calcium carbonate mineralizing jaws: (0) calcite; (1) aragonite.

42. Z07, Maxillary carrier: (0) ctenognath; (1) prionognath; (2) labidognath. See

Orensanz (1990) for illustration on the different carriers.

43. Z07, Maxillae (Mx) I: (0) dentate (Fig. 4.10D); (1) fang shaped (Fig. 4.10A-C,E).

From here on, the maxillary characters are considered inapplicable to Dorvilleidae.

The homologies of the dorvilleids jaws to those of the other eunicidan families have

not been finally resolved and are debated in the literature (e.g., Kielan-Jaworowska,

1966; Fauchald, 1970; Jumars, 1974; Orensanz, 1990).

44. Z07, Fang shaped MxI: (0) facing each other; (1) facing dorsally.

158 45. Teeth of MxII present on the: (0) anterior end only (at most on the anterior half)

(Fig. 4.10A); (1) anteriormost 2/3 to 3/4 of the plate (Fig. 4.10B-D); (2) whole

length of MxII, from anterior to posterior end (Fig. 4.10E).

46. Teeth of MxII distally: (0) distinctly blunt (Fig. 4.10F); (1) triangular, pointed (Fig.

4.10A-E).

47. Teeth of MxII: (0) curved (Fig. 4.10A-E); (1) straight (Fig. 4.10F).

48. Z07m, Shape of left MxIII: (0) arched (Fig. 4.10A-C; figure 5A in Zanol et al.,

2007); (1) flat with median edged curved towards the dorsal side (Fig. 4.10E; figure

5B in Zanol et al., 2007); (2) boomerang shaped (figure 5D,E in Zanol et al., 2007);

(3) triangular (Fig. 4.2E); (4) completely flat (Fig. 4.2C); (5) fang shaped with a

small proximal tooth (Fig. 4.10D).

49. Z07, Right MxIII: (0) present; (1) absent. The absence of right MxIII is a unique

synapomorphy of the (Eunicidae, Onuphidae) clade.

50. Z07m, Left MxIV: (0) arched dorsoventrally (Fig. 4.10A-C); (1) arched

anteroposteriorly (Fig. 4.10E); (2) attachment base is narrower than the distance

between the proximal and distal ends of the plate (Fig. 4.10F; Fig. 4.5D,E in Zanol

et al., 2007); (3) triangular (Fig. 4.2E); (4) completely flat (Fig. 4.2C); (5) fang

shaped (Fig. 4.10D). The arched plate running dorsoventrally is attached to the

maxillary fold in a dorsal to ventral direction. The ventral end is about as far

anterior as the dorsal one and it is located on the inner side of the left maxillary

fold, facing the right side fold. When arched and running anteroposteriorly the

plates are attached at an anterior to posterior direction. Both ends are at about the

same depth and the inner end is posterior to the outer one.

159 51. Teeth of MxIV arched dorsoventrally on the: (0) dorsal quarter of the plate (Figs.

4.10B, 4.11A); (1) dorsal half only (Figs. 4.10C, 4.11B); (2) dorsal half and part of

the ventral half of the plate (Fig. 4.11C); (3) whole length of the plate (Fig. 4.11D).

52. Z07, MxV: (0) fang shaped (Figs. 4.2C, 4.10D); (1) plate like, a small tooth may

be present (Fig. 4.10A-C,F).

53. Z07, MxVI (Fig. 4.10B,C): (0) absent; (1) present.

54. Z07, Relative size of labidognath maxillary carrier: (0) at least 1/3 longer than half

the length of MxI (figure 5D,G in Zanol et al., 2007); (1) shorter than half the

length of MxI (Fig. 4.10A-D); (2) about half the length of MxI (Fig. 4.10E).

55. Z07, Mandibles: (0) absent; (1) present.

56. Z07, Shape of the mandibles: (0) flat; (1) strongly curved, forming an open scoop,

anterior end strongly calcified, anterior outer edge and carriers from opposite sides

parallel to each other (figure 6B,C in Zanol et al., 2007); (2) curved, poorly

calcified, anterior outer edge and carriers from opposite sides diverging from each

other, X shaped (figure 6D,E in Zanol et al., 2007).

57. Z07, Mandibles: (0) fused medially; (1) in two pieces linked by ligaments.

58. Z07m, Muscle fibers complex F1+F2: (0) absent; (1) present.

59. Z07m, Placement of the muscle fibers complex F1+F2 on the ventral muscularized

pharynx: (0) posterior to mandibular carriers; (1) above mandibular carriers; (2)

between mandibular carriers.

60. Z07m, Branchiae: (0) absent; (1) present.

61. Z07m, Shape of the branchiae: (0) as single filaments (or rarely two filaments); (1)

linearly distributed along shaft; (2) spiraled along shaft; (3) bushy (shaft very

160 short or missing). Single here refers to the shape of the branchiae and not number

of filaments, even though single branchiae usually have just one filament. Some

species may have single branchiae with up to two filaments scattered along the

body without a clear pattern.

62. Linearly distributed branchiae: (0) palmate; (1) pectinate. In palmate branchiae the

branchial shaft is so short that filaments appear that branch from a central origin.

The shaft of the linear (pectinate) branchiae stem may be short or long when

compared to the branchial filaments. However, the filaments always clearly

branch linearly from the shaft.

63. Relative length of the longest branchial filament in relation to the branchial shaft

where branchiae are best developed: (0) shorter; (1) about the same length; (2)

longer.

64. Z07m, Branchiae start: (0) on chaetiger 3; (1) between chaetigers 4 and 9; (2) after

chaetiger 10; (3) on first chaetiger.

65. Z07, Branchial distribution: (0) mostly limited to the first third of the body (present

on less than 55% of the body segments); (1) in most of the body (present on more

than 65% of the body); (2) mostly limited to the second third of the body (present

on less than 55% of the body segments). Percentages refer to number of chaetigers

in which branchiae are continuously present in relation to the total number of

chaetigers in the body.

66. Relative placement of parapodia on first chaetiger in relation to parapodia of the

next following chaetigers: (0) ventral; (1) as dorsal as; (2) dorsal.

67. Z07m, Notopodia: (0) absent; (1) present.

161 68. Z07m, Notopodia: (0) reduced to notopodial cirri; (1) with complete chaetal lobe.

69. Inflated base of the ventral cirri: (0) absent; (1) present.

70. Distribution of the inflated base of the ventral cirri present: (0) in median

chaetigers; (1) along the whole body.

71. Inflated base of the ventral cirri: (0) partially wrinkled (Fig. 4.12A-C); (1)

completely wrinkled (Fig. 4.12D). The appearance we here are naming wrinkled

is not random. These wrinkled regions are present in a consistent pattern even if

the appearance is very simple. The wrinkled area of the inflated base of the

ventral cirri in Eunicidae is clearly visible in SEM, however, in light microscopy

it is only distinguishable as a lighter-whitish region of the inflated base of the

ventral cirri.

72. Position of the wrinkled region in the partially wrinkled inflated base of the ventral

cirri: (0) on the anterior half (Fig. 4.12A,B); (1) restricted to an anterior shield like

region (Fig. 4.12C).

73. Z07, Calcareous chaetae: (0) absent; (1) present.

74. Z07, Color of neuropodial aciculae: (0) dark (brown, dark brown or black); (1) light

(nearly clear or yellow). The darkest color observed was coded.

75. Patterns of variation in darker colors of the neuropodial aciculae along the body: (0)

color of aciculae vary slightly along the body but without a clear pattern; (1)

anteriormost aciculae is the lightest and it has the same color shade as posteriormost

ones; (2) anteriormost aciculae is the lightest and its color shade is considerably

different from more posterior ones; (3) posteriormost aciculae is the lightest and it

has the same color shade as most anterior ones; (4) posterior most aciculae is the

162 lightest and its color shade is considerably different from more anterior ones; (5)

anterior and posteriormost aciculae are the lightest and it has the same color shade

as most median ones; (6) anterior and posteriormost aciculae are the lightest and

their color shade is considerably different to the median ones; (7) anterior most

aciculae are the darkest. Lighter and darker variations of the light and dark hooks

are considered the same color shade. A variation from dark to light is coded as a

considerably different color shade.

76. Maximum number of subacicular hooks per parapodium: (0) one; (1) two; (2)

three; (3) four; (4) five. This is scored as the maximum number of subacicular

hooks observed in a parapodium independent of how often it was observed along

the body.

77. Z07, Color of subacicular hooks (darkest observed): (0) light (nearly clear or

yellow); (1) dark (brown, dark brown or black). The darkest color observed was

coded.

78. Subacicular hook present from: (0) P2; (1) P3; (2) P5; (3) P6; (4) P1. P1 and P6 are

the parapodia from the anterior and posterior, respectively, 2.5% of the body. P2,

P3 and P5 are, respectively, the median parapodia of the first, second and fourth

quarters of the body. Relative body placements were calculated using the number of

chaetigers.

79. Z07, Distribution of subacicular hooks: (0) present in every, or nearly every

segment after first occurrence; (1) may be missing irregularly, sometimes in many

segments.

163 80. Pattern of color variation of the subacicular hooks along the body: (0) color of

hooks vary slightly along the body but without a clear pattern; (1) posteriormost

hook is the lightest and it has the same color shade as more anterior one; (2)

posteriormost hook is the lightest and its color shade is considerably different color

shade than anteriormost one. See comments on character 74.

81. Lateral black dot between posterior parapodia: (0) absent; (1) present. This black

dot is inconspicuous, but appears to be consistently present in the taxa in which it

occurs. It looks like an ink dot (Fig. 4.13).

82. Z07, Ventral pygidial cirri: (0) absent; (1) present.

83. Pygidial ventral shield: (0) absent; (1) present. Pygidium ventral shield is a distinct

ventral expansion of the pygidium to which the cirri attach (Fig. 4.14A).

84. Placement of the pygidial cirri on the pygidium: (0) lateral (Fig. 4.14C); (1) ventral

(Fig. 4.14A,B).

The following characters are directly linked to different body regions (see definition above); note that in some cases the states are not identical in parallel features, but certain states may be missing in a given region

85. P1, Shape of the branchial filaments: (0) medially inflated (fusiform); (1) filiform.

See Fig. 4.15 for equivalent shapes of the notopodial cirri. Fusiform filaments are

medially inflated and narrowed at both ends. Filiform filaments are slender and

threadlike throughout.

164 86. P2, Shape of the branchial filaments: (0) tapering; (1) digitiform; (2) leaf shaped.

See Fig. 4.15 for equivalent shapes of the notopodial cirri. Tapering filaments taper

towards the distal end. Digitiform filaments are finger-like with about the same

diameter throughout their extension with a round distal end, tip may taper. Leaf

shaped filaments are flat, widest at the median region and narrow towards the

proximal and distal ends.

87. P3-5, Shape of the branchial filaments: (0) tapering; (1) digitiform; (2) button

shaped; (3) tapering flat. See Fig. 4.15 for equivalent shapes of the notopodial cirri.

Button shaped filaments are shorter than the chaetal lobe, barely extending from the

body. Tapering flat filaments are flattened and taper towards the distal end. See

comments on character 85.

88. P6, Shape of the branchial filaments: (1) digitiform; (2) button shaped. See Fig.

4.15 for equivalent shapes of the notopodial cirri. See comments on character 86

and 87.

89. P1, Shape of the notopodial cirri: (0) tapering (Fig. 4.15D,J); (1) digitiform (Fig.

4.15 B,I); (2) leaf shaped (Fig. 4.15A); (3) button shaped (Fig. 4.15F); (4) medially

inflated (fusiform) (Fig. 4.15C); (5) thumb shaped (Fig. 4.15E). Thumb shaped cirri

are narrowest on the proximal end, reach the maximum diameter at the proximal

half, maintaining about the same diameter until the distal end, which may be blunt

or taper. See comments on characters 85-87.

90. P2, Shape of the notopodial cirri: (0) tapering; (1) digitiform; (2) leaf shaped; (3)

button shaped; (4) filiform (Fig. 4.15G); (5) medially inflated (fusiform); (6) thumb

shaped. See comments on characters 85-87 and 89.

165 91. P3-5, Shape of the notopodial cirri: (0) tapering; (1) digitiform; (2) leaf shaped; (3)

thumb shaped; (4) button shaped; (5) filiform; (6) medially inflated (fusiform). See

comments on characters 85-87 and 89.

92. P6, Shape of the notopodial cirri: (0) tapering; (1) digitiform; (2) leaf shaped; (3)

button shaped; (4) filiform. See comments on characters 85-87 and 89.

93. P1 and P2. 94. P3-5 Neuropodial pre-chaetal lobes: (0) absent; (1) present.

95. P1. 96. P2. 97. P3-5. 98. P6, Relative length of the neuropodial pre-chaetal lobe in

relation to the chaetal lobe: (0) shorter (Fig. 4.15B-G,J); (1) longer (Fig. 4.15A,H);

(2) about the same length. Shorter pre-chaetal lobes are lower than the base of the

most anterior chaeate, allowing these to be easily observed in an anterior view of

the parapodia. Longer pre-chaetal lobes are higher than at least the base of all

chaetae and the aciculae. Pre-chaetal lobes that are as long as the chaetal lobes are

higher than the base of the most anterior chaetae but lower than the base of the

aciculae.

99. P1, P2 and P3-5, Neuropodial post-chaetal lobe: (0) absent; (1) present.

100. P1. 101. P2. 102. P3-5. 103. P6, Relative length of the neuropodial post-chaetal

lobe in relation to the chaetal lobe: (0) shorter (Fig. 4.15E,G); (1) longer (Fig.

4.15A,C,D,H); (2) about the same length (Fig. 4.15B,F,I,J). Shorter post-chaetal

lobes are lower than the base of the most posterior chaetae. Longer postchaetal

lobes are higher than the aciculae. Post-chaetal lobes that are as long as the chaetal

lobes vary from higher than the base of the most posterior chaetae to as high as the

base of the aciculae.

166 104. P1, Shape of longer neuropodial post-chaetal lobe: (0) tapering (Fig. 4.15A); (1)

round (Fig. 4.15D); (2) digitiform (Fig. 4.15I); (3) fusiform (Fig. 4.15C); (4)

truncate. See comments on characters 85-87 and 89. Truncate long post-chaetal

lobes decrease in length continuously from the dorsal to the ventral side with dorsal

side being the longest.

105. P2, Shape of longer neuropodial post-chaetal lobe: (0) tapering; (1) round; (2)

digitiform; (3) truncate. See comments on characters 85-87, 89 and 104.

106. P3-5, Shape of longer neuropodial post-chaetal lobe: (0) tapering; (1) round; (2)

digitiform. See comments on characters 85-87, 89 and 104.

107. P6, Shape of longer neuropodial post-chaetal lobe: (0) tapering; (1) round; (2)

digitiform

108. Median ridge connecting pre- and post-chaetal lobes separating supra- and

subacicular chaetae (Fig. 4.16): (0) absent (1) present.

109. P1. 110. P2. 111. P3-5 and P6, Acicular lobe in parapodia lacking median ridge

(Fig. 4.18E): (0) absent; (1) present. Acicular lobe contains exclusively the

aciculae, it lacks all other chaetae.

112. P1. 113. P2, Neuropodial chaetal lobe: (0) absent; (1) present.

114. P1. 115. P2. 116. P3-5, Shape of the neuropodial chaetal lobe lacking acicular

lobe and median ridge: (0) rounded (Fig. 4.17A); (1) truncate (Fig. 4.15J); (2)

pointed (Fig. 4.15E); (3) bilobed (Fig. 4.17B).

117. P6, Shape of the neuropodial chaetal lobe lacking acicular lobe and median ridge:

(0) round; (1) truncate; (2) pointed.

167 118. P1. 119. P2, Dorsal fleshy knob in the neuropodial chaetal lobe (Fig. 4.19): (0)

absent; (1) present. The presence of this fleshy knob is one of the diagnostic

features of the genus Euniphysa (Lu and Fauchald, 2000). It is also present in some

Eunice species and Palola cf. siciliensis (Gube, 1840), however, in these species it

is smaller.

120. P1 and P2, Shape of the dorsal fleshy knob in the neuropodial chaetal lobe: (0)

bulging, continuous with the remaining chaetal lobe (Fig. 4.19A); (1) ovoid,

distinctly separated from the chaetal lobe by a groove (Fig. 4.19B).

121. P1 and P2, Placement of the neuropodial chaetal lobe dorsal fleshy knob in

relation to aciculae: (0) anterior; (1) dorsal.

122. P1. 123. P2. 124. P3-5. 125. P6, Placement of aciculae on the neuropodial chaetal

lobe: (0) median; (1) dorsal.

126. P1. 127. P2, Orientation in the chaetal lobe of multiple neuropodial aciculae: (0)

dorsal to ventral; (1) anterior to posterior.

128. P1, Distal end of neuropodial acicula: (0) blunt-tipped or conical (Fig. 4.20A); (1)

hammer-headed or bidentate (Fig. 4.20B,C); (2) mucronate (Fig. 4.20D).

129. P2. 130. P3-5, Distal end of neuropodial acicula: (0) blunt-tipped or conical; (1)

hammer-headed or bidentate; (2) mucronate; (3) arrow shaped (Fig. 4.20E).

131. P6, Distal end of neuropodial acicula: (0) blunt-tipped, conical or irregularly

knobbed; (1) hammer-headed or bidentate; (2) mucronate.

132. P1. 133. P2. 134. P3-5 and P6, Ventral neuropodial limbate chaetae: (0) absent;

(1) present.

168 135. P1. 136. P2. 137. P3-5 and P6, Dorsal neuropodial limbate chaetae: (0) absent;

(1) present.

138. P1. 139. P2. 140. P3-5. 141. P6, Placement of dorsal neuropodial limbate chaetae

on the chaetal lobe in relation to aciculae: (0) posterior-dorsal; (1) posterior; (2)

dorsal. Limbate chaetae placed posterior-dorsally are posterior and dorsal to the

aciculae. They are not behind the aciculae, in an anterior view of the parapodium,

as the limbate placed posterior to the aciculae are.

142. P1. 143. P2. 144. P3-5. 145. P6, Neuropodial pectinate chaetae: (0) absent; (1)

present.

146. P1. 147. P2. 148. P3-5. 149. P6, Shape of the neuropodial pectinate chaetae: (0)

narrow, shaft narrower than limbate chaetae on the same parapodia (Fig. 4.21A-

F,I,J); (1) wide, shaft as wide as or wider than limbate chaetae on the same

parapodia (Fig. 4.21A,G,H). Wide pectinate chaetae correspond to the fan shaped

pectinate chaetae (sensu Fauchald, 1987) and the spatula-shaped pectinate chaetae

(sensu Zanol et al., 2007).

150. P1. 151. P2. 152. P3-5. 153. P6, Relative width of the distal end of the

neuropodial narrow pectinate chaetae: (0) narrow (narrower than or as wide as three

times the shaft width) (Fig. 4.21C,I,J); (1) wide (wider than three times the shaft

width) (Fig. 4.21A,B,D-F).

154. P1. 155. P2 156. P3-5. 157. P6, Outline of the neuropodial narrow pectinate

chaetae: (0) symmetric (Fig. 4.21A-F,I,J); (1) asymmetric (Fig. 4.21L).

169 158. P1. 159. P2. 160. P3-5. 161. P6, Placement of the neuropodial narrow pectinate

chaetae on the chaetal lobe in relation to acicula: (0) anterior; (1) dorsal; (2)

posterior.

162. P1. 163. P2. 164. P3-5, Teeth of neuropodial narrow pectinate chaetae: (0) in a

tranverse row (Fig. 4.21A-E,I,J); (1) in an oblique row (Fig. 4.21F).

165. P1. 166. P2. 167. P3-5. 168. P6, Relative length of the teeth of the neuropodial

narrow pectinate chaetae: (0) both outer teeth longer (Fig. 4.21B); (1) one of the

outer teeth longer (Fig. 4.21C,D,F,I,J); (2) all teeth are the same length (Fig. 4.21E).

169. P1, P2 and P3-5, Length of the inner teeth of the neuropodial narrow pectinate:

(0) equal (Fig. 4.21A-F); (1) unequal, increasing in length from one side to the

other (Fig. 4.21I,J). Pectinate chaetae having inner teeth of unequal length have also

been reported in Marphysa sp.A in Gathof (1984), which was described with the

same prostomial shape and branchial distribution as M. bellii-like species studied

here.

170. P1, 171. P2. 172. P3-5. 173. P6, Curvature of the neuropodial narrow pectinate

chaetae: (0) flat (Fig. 4.21D,F); (1) curved (Fig. 4.21A-C,E).Flat pectinate chaetae

may have the outer edge slightly folded.

174. P2. 175. P3-5. 176. P6, Teeth of the neuropodial wide pectinate chaetae: (0) wide,

usually separated by gaps, tapering from the base (Fig. 4.21G,H); (1) thin side by

side, tapering at the distal end, almost indistinguishable as separate units at the base

(Fig. 4.21H). Neuropodial wide pectinate with wide teeth are the spatula-shaped

pectinate chaetae (sensu Zanol et al., 2007). They had only been previously

reported for some Marphysa species (Orensanz, 1990; Steiner and Amaral, 2000).

170 We observed them in N. mossambica for the first time in the present study.

Neuropodial wide pectinate chaetae with teeth thin side by side, tapering at the

distal end, almost indistinguishable as separate units at the base are the fan shaped

pectinate chaetae (sensu Fauchald, 1987) characteristic of Nauphanta. The presence

of this kind of chaetae together with the lack of compound chaetae were considered

distinct enough to recognize Nauphanta as a valid genus (Fauchald, 1987).

177. P2. 178. P3-5. 179. P6, Placement of the neuropodial wide chaetae on the chaetal

lobe in relation to aciculae: (0) anterior; (1) dorsal.

180. P2. 181. P3-5. 182. P6, Curvature of the neuropodial wide pectinate chaetae: (0)

flat; (1) curved. Flat pectinate chaetae may have the outer edge slightly folded.

183. P1, 184. P2. 185. P3-5. 186. P6, Neuropodial compound chaetae: (0) absent; (1)

present.

187. P1, 188. P2. 189. P3-5. 190. P6, Neuropodial compound chaetae: (0)

pseudocompound (Fig. 4.22A,B); (1) true-compound (Fig. 4.22C-L). In

pseudocompound chaetae the article and shaft are not separate articulated parts, the

fracture in between them extends only halfway through the chaeta (Fig 4.22B). In

true-compound chaetae the article and shaft are clearly separate articulated parts,

even if completely connected, the fracture between them extends all the way

through (Fig. 4.22C,G,J). We observed pseudocompound chaetae only in

Euniphysa aculeata Wesenberg-Lund 1949. True-compound chaetae are present in

most genera of Eunicidae, Dorvilleidae, Lumbrineridae and Onuphidae. The

Onuphidae anterior compound chaetae are traditionally classified as

pseudocompound, however, they are true-compound in that the fracture clearly runs

171 all the way through separating the article and shaft. The different appearance

between the anterior compound chaetae in Onuphidae and those present in

Eunicidae is due to the difference in shaft to articles articulation. In Onuphidae and

Lumbrineridae the article and shaft are completely connected and the proximal end

of the base of article is connected directly to the shaft (Fig. 4.22C-E,H-J). In

Eunicidae and Dorvilleidae the shaft and article are directly connected only at one

edge; the opposite edge, the proximal end of the base of the article, fits into a socket

in the shaft and connects to it through a thin ligament (Fig. 4.22F,G,K). We found

true-compound chaetae with shaft and article connected just at the edges in

parapodia P6 of Mooreonuphis pallidula (Hartman, 1965). They resemble the

onuphid juvenile compound chaetae (Paxton, 1986). These differences among the

compound chaetae are clearly observed in SEM images. However, in light

microscopy, it is also possible to distinguish between pseudocompound and true-

compound as well as between the two kinds of articulations of the latter.

191. P1, 192. P2, Junction of the shaft and article of the neuropodial true-compound

chaetae: (0) completely connected, proximal end of the article directly connected to

the shaft, without the presence of a socket (Fig. 4.22C-E,H-I) (1) connected at both

edges, proximal end of the article fit in a socket (Fig. 4.22F,G,K,L). True-

compound chaetae with shaft and article completely connected are only present in

anterior chaetigers (P1 and P2). All true-compound chaetae present in parapodia

P3-5 and P6 are connected at both edges, with the proximal end of the article fitting

in a socket.

172 193. P1, Distal end of the article of the neuropodial compound chaetae: (0) falcigerous

and bidentate, longest tooth proximal (Fig. 4.22G,E); (1) falcigerous and bidentate,

longest tooth distal (Fig. 4.22D,F); (2) falcigerous and tridentate, all teeth towards

the side, longest tooth distal (Fig. 4.22E); (3) falcigerous and multidentate (Fig.

4.22H); (4) spinigerous (Fig. 4.22L); (5) falcigerous and bidentate, teeth

inconspicuous (appears unidentate if not very carefully examined under high

magnification) (Fig. 4.22K).

194. P2, Distal end of the article of the neuropodial compound chaetae: (0) falcigerous

and bidentate, longest tooth proximal; (1) falcigerous and bidentate, longest tooth

distal ; (2) falcigerous and multidentate; (3) spinigerous; (4) falcigerous and

bidentate, teeth inconspicuous (appears unidentate if not very carefully examined

under high magnification).

195. P3-5. 196. P6, Distal end of the article of the neuropodial compound chaetae: (0)

falcigerous and bidentate, longest tooth proximal; (1) falcigerous and tridentate, at

least one tooth pointing towards the distal end (Fig. 4.22M); (2) spinigerous.

197. P1, Placement of neuropodial compound chaetae on the chaetal lobe in relation to

aciculae: (0) in several rows mostly ventral to acicula, few chaetae of the most

anterior row may be as dorsal as the aciculae; (1) in several rows in a distinct lobe

completely ventral to the acicula; (2) in several rows on both dorsal and ventral

sides of the aciculae; (3) single row parallel to aciculae lobe on the anterior and

posterior face of the parapodia.

198. P2, Placement of neuropodial compound chaetae on the chaetal lobe in relation to

aciculae: (0) in several rows mostly ventral to acicula, few chaetae of the most

173 anterior row may be as dorsal as the aciculae; (1) in several rows in a distinct lobe

completely ventral to the acicula; (2) in several rows on both dorsal and ventral

sides of the aciculae.

199. P3-5, Placement of compound chaetae on the neuropodial chaetal lobe: (0) several

rows mostly ventral; (1) several rows on the dorsal and ventral sides of the aciculae.

200. P1, 201. P2. 202. P3-5. 203. P6, Subacicular hooks: (0) absent; (1) present.

204. P2. 205. P3-5. 206. P6, Distal end of subacicular hooks: (0) falcate (Fig. 4.23A);

(1) bidentate (Fig. 4.23C-F); (2) tridentate (Fig. 4.23B); (3) spinigerous (Fig.

4.23F). The subacicular hooks in Eunice fucata Ehlers, 1887 are not completely

falcate as traditionally described (e.g., Fauchald, 1992a). They have two almost

inconspicuous teeth, thus this species was coded here as bidentate.

207. P1, 208. P2. 209. P3-5. 210. P6, Subacicular hook guards: (0) absent; (1) present.

211. P1, 212. P2. 213. P3-5. 214. P6, Subacicular hook guards covering: (0) proximal

tooth only (Fig. 4.23B,D,E); (1) covering all teeth (Fig. 4.23C,F).

215. Subacicular guards: (0) disassociated fibrils (Fig. 4.23D); (1) solid, sheet-like (Fig.

4.23A-C,E,F). Subacicular guards as loose fiber were only observed in F.

cyrtauloni.

216. P2. 217. P3-5. 218. P6, Placement of the subacicular hook: (0) within the chaetal

lobe; (1) ventral to the chaetal lobe.

219. P3. 220. P4. 221. P6, Pattern of dark color on the subacicular hooks: (0) darkest

color shade near distal end (Fig. 4.24C); (1) darkest color shade present on most of

the length of the hook, but color more concentrated towards proximal end, (Fig.

4.24A,B); (2) darkest color shade present on the distal half.

174 222. P1 and P2. 223. P3-5 and P6, Ventral cirri: (0) absent; (1) present.

224. P1, Shape of the ventral cirri: (0) thumb shaped (Fig. 4.15B,J); (1) tapering (Fig.

4.18C); (2) digitiform (Fig. 4.15I); (3) oval (Fig. 4.18G); (4) bottle shaped (Fig.

4.18D); (5) leaf shaped; (6) medially inflated (fusiform) (Fig. 4.18E); (7)as a ridge

with no distinct tip. See comments on characters 85-87 and 89. Bottle shaped cirri

have a round proximal base and a digitiform tip which is about the same length as

the base, shorter or as long as the chaetal lobe. The base tapers smoothly into the

tip.

225. P2, Shape of the ventral cirri: (0) leaf shaped; (1) cushion shaped without a

distinct tip (Fig. 4.18F); (2) digitiform; (3) round inflated base with a distinct tip

(Fig. 4.12A,D); (4) round inflated base with a continuous tip (Fig. 4.18A); (5)

round inflated base without a distinct tip (Fig. 4.12C); (6) ventral ridge with a

distinct, usually conical, tip (Fig. 4.12C); (7) button shaped (Fig. 4.18B). See

comments on characters 85-87, 89 and 224.

226. P3-5, Shape of the ventral cirri: (0) thumb shaped; (1) tapering; (2) digitiform; (3)

bottle shaped; (4) button shaped; (5) leaf shaped; (6) round inflated base with a

continuous tip; (7) round inflated base with a distinct tip; (8) bowling pin shaped

(Fig. 4.15G). See comments on characters 85-87, 89 and 224. Bowling pin shaped

ventral cirri are similar to bottle shaped; they differ in that they outreach the chaetal

lobe.

227. P6, Shape of the ventral cirri: (0) thumb shaped; (1) tapering; (2) digitiform; (3)

button shaped; (4) bowling pin shaped; (5) bottle shaped. See comments on

characters 85-87, 89, 224 and 225.

175 228. P1, 229. P2. 230. P3-5. 231. P6, Hayashi’s (1994) sensory organ: (0) flat (Fig

4.25A); (1) bulging (Figs. 4.19A, 4.25B); (2) slightly elevated (Fig. 4.25C). Ciliary

sensory organ placed on the ventral side of the base of the notopodial cirri. In

Lumbrineridae, which lack notopodial cirri, it is placed on the dorsal side of the

base of the neuropodia.

232. P2, Notopodial aciculae: (0) absent; (1) present. Notopodial aciculae can be

inconspicuous in small specimens, mainly when they are light in color, leading to

inconsistent scoring. The parapodia in section P2 was the only one for which we

could consistently score it.

Total evidence Data set

The data sets had 5198 (morphMAFFT) and 5143 (morphClustal) characters, of which 232 were morphological (Appendix 7). The number of informative morphological characters was 192, representing about 10% of the total number of informative characters

(1872 in the morphMAFFT and 1877 in the morphClustal). We did not remove any region of the alignments before running the analyses (see Chapter 3 for the characteristics of the nucleotide sequences). Morphological and molecular (both alignments) data sets were found to be incongruent in the ILD test (P=0.02, morphMAFFT, and P=0.01, morphClustal).

Phylogenetic analyses

Statistics for all MP analyses are presented in Table 4.2. Aciculomarphysa comes and

F. cyrtauloni are the only ingroup species with deeply ambiguous placement among and

176 within the different analyses (Figs. 4.27-4.31). In the combined analyses, the differences observed between the 79 (73 in the morphological data set) and 77 (71 in the morphological data set) -terminal trees are restricted to the clades in which these two species are placed. The MP trees yielded by the morphological data analyses of the 73- terminal are more resolved at shallower levels and represent a subset (if only the relationships not including A. comes and F. cyrtauloni are considered) of the MP trees yielded in 71-terminals analyses. For these reasons we do not consider the results of the 79- terminal (73 in the morphological data set) trees any further. All relationships presented below are from the 77-terminal (71 in the morphological data set) trees. Clade numbers are equivalent to those in Chapter 3. An asterisk (*) following the clade number denotes differences among the current clade and the equivalent clade in Chapter 3 due to the new placement of a single taxon or the inclusion of additional species not present in the analyses in Chapter 3.

Total Evidence Trees. Eunicidae is monophyletic with Onuphidae as its sister group in all trees (Figs. 4.28-4.31) and strongly supported in the Bayesian trees (PB>95). Several morphological synapomorphies support the eunicid monophyly (Appendices 3 and 4) and three of these are unique to this clade: dorsal buccal lip fused to the dorsal side of the prostomium (character 9, Figs. 4.3A,B), anterior extensions of the dorsolateral fold medially connected (character 14, Figs. 4.2A) and the presence of Mx VI (character 53).

The latter appears to have been secondarily lost at least three times within Eunicidae.

The phylogenetic status of all genera is congruent among all trees (Figs. 4.28-4.31):

Palola and Euniphysa are monophyletic; Lysidice is paraphyletic with Nematonereis nested

177 within it; Eunice and Marphysa sensu lato are polyphyletic; and Marphysa sensu stricto is

paraphyletic with Nauphanta mossambica (Peters, 1854) nested within it. Furthermore, all

analyses also recovered the most basal divergence within Eunicidae (Figs. 4.28-4.31, clades

11* and 30*) and the clades that could represent monophyletic redefinitions of current and

defunct genera as suggested in Chapter 3 (Figs. 4.28-4.31, clades 11*, 33*, 38*, 39*, 53*; herein referred to as generic level clades).

The generic level clades which may represent the defunct genus Leodice (Figs. 4.28-

4.31, clade 11*) contains almost the same species and relationships as clade 11 in the

molecular analyses (Chapter3, Figs. 4.1, 4.2). The differences include the addition of E.

antennata, the type species for Leodice and not present in the molecular dataset, and the placement of Lysidice sp.1 in another clade (see below). Among the morphological

synapomorphies supporting clade 11* (Appendices 3 and 4), three are unique, regularly

articulated antennae and palps (characters 22 and 29, Fig. 4.7) and the presence of the

lateral black dot in between posterior parapodia (character 81, Fig. 4.13). Both are

secondarily lost at least once within this clade.

Clade 30* includes most eunicid species like its equivalent clade in the molecular

analyses (Chapter 3, clade 30). These two clades differ in species composition mainly

because of the inclusion of additional species to the current analyses (Euniphysa aculeata,

Eunice aphroditois, Eunice cincta, N. mossambica and Palola sp.A9). The only exception

is Lysidice sp.1, which is here grouped in the Lysidice+Nematonereis clade in all trees

(Figs. 4.27-4.31, clade 33*) as opposed to clade 11 in the molecular analyses (Chapter 3).

Lysidice+Nematonereis is supported by several synapomorphies (Appendix 8 and 9), of

which the shape of the curved mandibles (character 56 state 2) is unique to the clade. The

178 most inclusive stable clade containing E. aphroditois (Figs., 4.27-4.31, clade 57*), the type

species of Eunice, is inconsistently placed among the trees of the different alignments and analyses of the morphMAFFT alignment. E. cincta, the type specie of Nicidion, is

consistently grouped with E cariboea and E. cf. insularis within the generic level clade

proposed to represent the defunct genus Nicidion (Chapter 3; Figs. 4.27-4.31, clade 38*).

The trees of the different alignments (morpMAFFT and morphClust) differed mainly

in the placement of Euniphysa, Lysidice+Nematonereis clade and in the monophyly of clade 52* (Fig. 4.28-4.31). The different analyses of the same alignment differed mainly in the position of terminal taxa (Figs. 4.28, 4.29 and 4.30, 4.31). However, the MP strict consensus tree and Bayesian tree of the morphMAFFT differed also in the relationship between clades 33*, 52* and 53* (Figs. 4.28, 4.29). The Bayesian analyses of the same

data set using the different partition schemes, morphgene+codon (Figs. 4.29, 4.31) and

morphgene (results not shown), resulted in trees with the same topology (see Fig. 4.29B for

the only exception) and similar posterior probability values for the branches.

Morphological Data Trees. All generic level clades were also recovered in the MP

strict consensus tree of the morphological data, with exception of the Leodice-like clade

(Figs. 4.28-4.31, clade 11*). The Leodice-like clade collapsed because of the inconsistent

placement of E. fucata within this clade or as its sister group (Fig. 4.27). These trees also

differed from the total evidence and molecular (Chapter 3) trees in the relationships among

and within the generic level clades. Most relationships are poorly supported by both BP

(BP<85%) and Bremer support (Fig. 4.27).

179 DISCUSSION

The total evidence approach used here was useful in consistently placing taxa missing most of the molecular data and behaving as wild card taxa in molecular analyses (e.g.,

Lysidice sp.1, Chapter 3) and in enhancing the support of certain branches, such as the eunicid branch, when compared to both morphological and molecular trees (Figs. 4.27-

4.31). However, this approach was not able to resolve the incongruent relationships observed among the molecular trees of the different alignments (Chapter 3) and between these and the morphological trees (Fig. 4.27; Chapter 3). As in the molecular trees (Chapter

3), the inconsistent placement of the Lysidice+ Nematonereis clade and of the species of

Eunice and Euniphysa grouped in clade 52* led to the incongruence within clade 30* between the trees of the different alignments (Figs. 4.28-4.31), but here also between the

MP and Bayesian trees of the morphMAFFT data set (Figs. 4.28, 4.29).

The phylogenetic relationships discussed here are based on the total evidence trees.

However, the morphological data trees (Fig. 4.27) recovered the same phylogenetic status for the family and most generic level clades. We only recognize those relationships that are congruent in all trees from both morphMAFFT and morphClustal data sets.

The unambiguous recovery of a monophyletic Eunicidae here supported by unique synapomorphies provides further evidence for the identity of the family, a contradictory issue in previous studies (Struck et al., 2006; Zanol et al., 2007; Chapter 3). The consistent polyphyletic status of Eunice (Struck et al., 2006; Zanol et al., 2007; Chapter 3), the type genus of the family, is not surprising since it is diagnosed only by plesiomorphies. Eunice has been traditionally used as the “trash can” taxon for the family containing a disproportional number of species (Table 1.2) artificially grouped by plesiomorphies and

180 the lack of the diagnostic features characterizing other genera. Here we are yet unable to

redefine a monophyletic Eunice. The inconsistent placement of the most inclusive stable

clade containing E. aphroditois (Figs. 4.27-4.31, clade 57*; herein referred to as the E.

aphroditois-clade), the type species of the genus, does not allow for reliable determination

of the diagnostic features of the genus as well as its composition. The other two species in

the E. aphroditois-clade, Eunice roussaei Quatrefages, 1866 and Eunice violaceomaculata

Ehlers, 1887 (also placed in clade 57*), have the same color pattern as described by

Fauchald (1992a) for E. aphroditois, dark, purplish red to black, with white or pale gray dots scattered over the surface and one, sometimes two, anterior chaetigers white. All three species are iridescent with the iridescence arising from a crisscross pattern in the cuticle.

The species Eunice filamentosa complex Grube, 1856 and Eunice impexa Grube

1878, which are sister to the E. aphroditois-clade in the trees of the morphMAFFT

alignment (Figs. 4.28, 4.29; Chapter 3), may actually represent a separate genus which

could also contain Euniphysa (Figs. 4.27-4.31). Their close relationship is supported by three unique and very distinct synapomorphies, MxIV running from dorsal to ventral with

teeth restricted to the dorsal quarter (character 51) and compound falcigers bidentate with

teeth inconspicuous (appears unidentate if not carefully examined under high

magnification) in P1 and P2 (characters 193 and 194). In addition to the species included in

the E. filamentosa complex, the same kind of falcigers has also been recorded for other species such as Eunice tubicola (Treadwell, 1922) and Eunice tubifex Crossland, 1904

(Fauchald, 1992a), which also have a similar shape of the prostomium and branchial pattern. Because of the inconsistent placement of the clade containing E. impexa and E. filamentosa (Figs. 4.27-4.31), for the time being we leave them in Eunice. Further analyses

181 are necessary to consistently place them and provide a stronger test of these potential

relationships.

Most Eunice sensu lato species included in the present analyses are not closely

related to the E. aphroditois clade in any of the trees (Figs. 4.27-4.31). They are

consistently distributed in clades containing the type species of Leodice and Nicidion,

respectively, thus corroborating the generic propositions of Chapter 3 (Figs. 4.27-4.31,

clades 11* and 38*). Here we resurrect both genera to include the species in the clades with

their respective type species and diagnosed by synapomorphies supporting the clades.

Other taxonomic changes undertaken here based on the consistent phylogenetic

relationships (Figs. 4.27-4.31; Chapter 3) are the inclusion of Nematonereis in Lysidice,

making the latter more inclusive and the redefinition of a monophyletic Marphysa

including only the species in the Marphysa sensu stricto clade, inclusive N. mossambica

(Fig. 4.27-4.31, clade 46*). See the taxonomic accounts section for diagnostic features and

further discussion.

Aciculomarphysa and Fauchaldius are monotypic genera described for species with a very simple morphology characterized mainly by the lack of features (Carrera-Parra and

Salazar Vallejo, 1998 ; Hartmann-Schroeder, 1998). These characteristics led to high amounts of missing data and coded characters that are mainly plesiomorphies for the family or autapomorphies, therefore not informative about the position of these genera within the family. The ambiguous placement of A. comes and F. cyrtauloni in the different trees is

probably not just a missing data problem, but also a problem of the quality of coded data

since in some cases accurate relationships can be recovered even if high proportions of

missing data are present (Wiens, 2003). E. cincta, which has similar amounts of missing

182 data and plesiomorphies as A. comes and F. cyrtauloni, has characters coded that are informative enough (i.e., unique synapomorphies of the clade it is nested in) to consistently

place it as the closest relative of E. cariboea and E. cf. insularis in all trees. Such

relationship corroborates the traditional association between E. cariboea and E. cincta (e.g.,

Monro, 1930; Hartman, 1944) and the observed similarities with Eunice mutilata Webster,

1884 (Fauchald, 1992a), another closely related species in the recovered trees. Molecular

data is not subject to the kind of simplification observed in the morphology of A. comes and

F. cyrtauloni, therefore future studies also including molecular data are likely to be more

informative about their position within Eunicidae.

Phylogenetic information content and evolution of selected characters

Herein the names of the genera refer to genera as emended in the present study.

Characters of features of the prostomium and pharynx (characters 6-15) were especially

informative for basal relationships in the tree and in supporting the monophyly of

Eunicidae. Eunicidan families in general have consistently different anterior ends and

robust hypotheses of primary homology were possible by the examination of innervation

similarity (Pruvot and Racovitza, 1895; Haffner, 1959; Orrhage, 1995; Chapter 2). Two of

these characters yielded the only unique synapomorphies to Eunicidae and consistently

present in all species examined here for the family (with exception of N. unicornis which

lacks the dorsal buccal lip), dorsal buccal lip fused to the dorsal side of the prostomium

(character 9, Fig. 4.3A-C) and the anterior extensions dorsolateral fold medially connected

(character 14, Fig. 4.2A).

183 The absence/presence of anterior appendages (characters 19, 26, 35) is not informative in the diagnosis of genera as traditionally considered (e.g., Kinberg, 1865;

Ehlers, 1868; Gravier, 1900; Hartman, 1944; Gathof, 1984). Only the lack of palps is a

unique synapomorphy of a eunicid genus, Lysidice. The lack of lateral antennae is a unique

synapomorphy within Eunicidae for Nematonereis. However it is not enough to support it

as a separate genus, since Nematonereis is nested within Lysidice in all trees, therefore

representing a subset of the latter genus. The lack of the peristomial cirri has at least two

independent origins, at the base of Marphysa or of the (Marphysa, Lysidice) clade and

within Nicidion. Furthermore, intraspecific variation in absence/presence of peristomial

cirri may occur in E. insularis (Nogueira et al., 2001). Therefore having three antennae, a

pair of palps and lacking peristomial cirri is not unique to Marphysa even as a combination.

The lack of palps and lateral antennae on the prostomium and of peristomial cirri are

due to secondary losses within Eunicidae as proposed in Orensanz (1990). The stepwise

pattern of such losses is supported in morphMAFFT MP strict consensus tree, which

includes Marphysa, and Lysidice (Fig. 4.28) as sistergroups, the lack of peristomial cirri is a synapomorphy for this clade, the lack of palps a synapomorphy for Lysidice and the lateral antennae are lost within Lysidice. In the morphClust trees (Figs. 4.30, 4.31) the loss of peristomial cirri has happened three times independently, in Lysidice, Marphysa and within Nicidion (Fig. 4.30, 4.31, clades 33*, 29*, 44) and the stepwise pattern is only present in Lysidice. As we suggested in Chapter 3, paedomorphosis may have played a role in the evolution of such patterns, since the pattern for loss of the anterior appendages during evolution follows the inverse pattern of the growth of these appendages during the ontogeny of eunicid species with the full set of anterior appendages. In these, the

184 appendages develop by stepwise addition starting by the median antenna, lateral antennae,

palps and finally the peristomial cirri.

The absence/presence of the articulations of the prostomial appendages and their

shapes have been suggested to be characters useful to group species in Eunice sensu lato

(Ehlers, 1868). These characters are consistently used in the taxonomy of the family

(Fauchald, 1992a) but had not been particularly informative in supporting phylogenetic

relationships in the family (Zanol et al., 2007). Here the articulations of these appendages

were coded in a different manner (irregular versus regular, characters 22, 29) before coding

for the shape of the articulation, which was coded only for regularly articulated

appendages. This separation in regular and irregular articulation produced more

phylogenetically informative characters, which support the Leodice clade. Regularly articulated antennae and palps are two of the unique synapomorphies for this clade.

Irregularly articulated prostomial appendages are plesiomorphic in Eunicidae and present in all other eunicid species examined here except for species of Euniphysa.

The two independent secondary losses of branchiae (within Nicidion and at the base of Lysidice) observed here support the idea put forward by Fauchald (1992a) that absence/presence of branchiae are not acceptable generic characters. Consequently, genera diagnosed just on this character continue to be considered invalid here (e.g., Paramarphysa

Ehlers, 1887; Lysibranchia Cantone, 1983). Palola is the only genus to have a unique pattern of branchial distribution, when present, branchiae are mostly limited to the second third of the body (present on less than 55% of the body segments). The other patterns have evolved convergently several times within Eunicidae. Pectinate branchiae (three or more filaments present) restricted to the anterior 1/3 of the body (present in less than 55% of the

185 body) is the plesiomorphic state of the branchiae in Eunicidae. This contradicts Miura’s

(1986) hypothesis that the ancestral state of the branchiae in Eunice sensu lato is poorly

developed branchiae with few filaments (most branchiae with 1-2 filaments, 3-4 filaments

occasionally scattered) present along most of the body.

The color and dentition of subacicular hooks (characters 77, 204-206) are the main

characters traditionally used to divide Eunice sensu lato in informal groups combined with

information about the distribution of the branchiae (Hartman, 1944; Fauchald, 1970). The phylogenetic study of Eunice sensu lato (Zanol et al., 2007) did not recover any of these groups with exception of a unique origin for the dark colored hooks, contradicting the idea that these groups are stable enough to perhaps correspond to subgeneric or generic-level taxa (Miura, 1986). Our results support the artificial nature of these groups but contradict the unique origin of dark subacicular hooks. Light colored subacicular hooks are the plesiomorphic state for Eunicidae, dark colored hooks have at least two convergent origins in the two basal eunicid clades (Leodice and its sister clade containing the remaining eunicid, Fig. 4.28-4.31, clades 11*, 30*) and secondarily reverse to light color at least twice in the clade containing all eunicid species with exception of Leodice and E. fucata (Figs.

4.28-4.31, clade 30*). The tridentate dentition of subacicular hooks is the only traditional characteristic (color and dentition) of the hooks informative at the generic level since all species with tridentate subacicular hooks are placed in Leodice.

The pattern of dark color along the subacicular hook (Fig. 4.24), coded for the first

time in the present study, along with the coding for different regions of the body (characters

219-221) was informative at the generic level. It yielded one of the synapomorphies for

Nicidion (darkest color of the subacicular hook near the distal end in P3). Furthermore, the

186 distribution along the body of the pattern of dark color along the subacicular hook is a

unique diagnostic feature for this genus, since it is the only clade in which the darkest color

shade on the subacicular hooks is at the distal half or end of the hook from P3 to the end of

the body. The coding of segmental characters for different regions of the body also yielded

one of the synapomorphies for Marphysa, neuropodial post-chaetal lobes longer than

chaetal lobes in P1 (character 100) and the only unique diagnostic feature for the genus,

narrow pectinate chaetae with both outer teeth longer than inner teeth along the whole body

(characters 165-168).

The coding of segmental characters for the different parts of the body explicitly

account for the variation of these characters along the body. This yielded more

phylogenetic information and morphological data trees more congruent with molecular and total evidence trees than the trees in previous studies (e.g., Zanol et al., 2007). If just a single character is coded for any of the segmental characters that vary along the body, information about topological similarity, which may be informative as observed here, is lost. Coding for different regions of the body accounts for both correspondence and topological similarities, which are likely to yield more robust primary hypotheses of homology and consequently more phylogenetic information.

Taxonomic accounts

We propose changes to the generic classification of eunicid species necessary to account for the evolution of the family and to reflect monophyletic groups. In addition to the genera emended below we consider the following as valid genera in Eunicidae:

Aciculomarphysa Hartmann-Schröeder and Zibrowius, 1998, Eunice Cuvier, 1817 (see

187 Fauchald, 1992a for a revision), Euniphysa Wesenberg-Lund, 1949 (see Lu and Fauchald,

2000 for a revision and phylogeny), Fauchaldius Carrrera-Parra and Salazar-Vallejo, 1998 and Palola Gray, 1847 (see Fauchald, 1992b; Schulze, 2006 for revision and phylogeny).

See Appendix 10 for a taxonomic key for the genera of the family considered valid here.

Eunice sensu lato is polyphyletic, but it is still not clear what taxa would be members of the genus and the diagnostic features of a monophyletic Eunice. Therefore this genus is not emended here and its traditional plesiomorphic diagnostic characteristics are retained.

Eunice species have five prostomial appendages (three antennae and a pair of palps), one pair of peristomial cirri and a complete set of chaetae (aciculae, compound, limbate, pectinate and subacicular hooks), but lack all the combination of characters diagnostic of other genera in the family.

Aciculomarphysa and Fauchaldius are both monotypic and simplified forms. This simplification and the lack of molecular data for both species did not allow for certainty about their placement in the phylogeny and their generic status. They are retained as valid genera here but such status may not be stable in further studies, which should include molecular data for both species. In addition to the lack of most chaetae and neurochaetal lobes in anterior chaetigers, F. cyrtauloni has one unique autapomorphy, the subacicular hook guards consist of disassociated fibrils (Fig. 4.23D, observed here for the first time).

Features underlined are unique in Eunicidae to the species within that genus; while unique to the genus they may not be present in all species. P1, parapodia from the initial

2.5% of the body. P2, parapodia from the median chaetiger of first quarter of the body. P3-

5, parapodia from the median chaetiger of the posterior three quarters of the body. P6,

188 parapodia from the final 2.5% of the body. All relative positions were determined by number of chaetigers.

EUNICIDAE Berthold, 1827

Diagnostic features: Buccal lips (sensu Orrhage, 1995) present. Dorsal buccal lip fused to

the dorsal side of the prostomium (Fig. 4.3A-C). Ventral buccal lip with a mediotransverse deep groove (Fig. 4.2A). Anterior extensions of the dorsolateral fold medially connected

(Figs. 4.2A, 4.6). Median prostomial antenna always present. Two peristomial rings.

Asymmetric labidognath jaws. Birramous parapodia, notopodia reduced to notopodial cirri.

Limbate chaetae present. One or two pairs of pygidial cirri.

Leodice Lamarck, 1818

emended

Type species: Leodice antennata Lamarck, 1818

Diagnostic features: Three antennae (one median and two lateral) and a pair of lateral palps

on the prostomium. Antennal and palpal styles regularly articulated (Fig. 4.7) (few species

with irregular articulation). Prostomium truncate and steep (Fig. 4.4B). A pair of

peristomial cirri present on the posterior peristomial ring. MxII with teeth present on the

anterior 2/3 to 3/4 of the plate, posterior 1/3-1/4 lacking teeth (Fig. 4.10B-D). Lateral black

dot between posterior parapodia present (Fig. 4.13). Inflated base of the ventral cirri round

189 with conspicuous tip. Notopodial aciculae present at least in the anterior end of the body.

Neurochaetae: limbate; pectinate chaetae narrow and symmetric; true compound falciger

bi- or tridentate, aciculae and subacicular hook present. Compound tridentate chaetae

present only on median to posterior parapodia. Acicula light or dark, in the latter the most

anterior is always the lightest but have the same color shade. Subacicular hooks light or dark, bi- or tridentate. Pygidial cirri placed on the pygidial shield (Fig. 4.14A).

Species studied in the present paper belonging to this genus: Leodice americana (Hartman,

1944), Leodice antarctica (Baird, 1869), Leodice antennata Lamarck, 1818, Leodice antillensis (Ehlers, 1887), Leodice harassii (Audouin and Milne Edwards, 1933), Leodice limosa (Ehlers, 1868), Leodice lucei (Grube, 1856), Leodice marcusi (Zanol et al., 2000),

Leodice miurai (Carrera-Parra and Salazar-Vallejo, 1998), Leodice rubra (Grube, 1856),

Leodice thomasiana (Augener, 1922), Leodice torquata (Quatrefages, 1866) and Leodice

valens Chamberlin, 1919.

Remarks: Leodice was originally not clearly differentiated from Eunice, and both names

were used interchangeably through the first third of the 20th century (Hartman, 1944). Here

Leodice encompasses all species previously considered Eunice with regularly articulated

prostomial appendages, tridentate true-compound falcigers, light subacicular hooks (groups

A and C sensu Fauchald,1970), dark aciculae with the most anterior ones being the lightest

but yet having the same shade, and a lateral dot present between posterior parapodia. Not

all Leodice have all these characteristics, but they have at least one. The absence or

presence of the lateral dot varied within a few species. The genus is worldwide distributed.

190 Clade 21 (Figs. 4.28-4.31) may be distinct enough to be classified as a separates genus in

future analyses with more species and better resolution. Clade 21 is consistently separated

from clade 23*. However the ambiguous placement of L. antillensis makes this distinction uncertain.

Lysidice Savigny, 1818

emended

Type species: Lysidice ninetta Audouin and Milne Edwards, 1833

Synonym: Nematonereis Schmarda, 1861

Diagnostic features: One (median) or three antennae (one median and two lateral) on the prostomium. Prostomial lateral palps absent. Peristomial cirri on the posterior peristomial

ring absent. Teeth on MxIII round, distinctly blunt. MxVI absent. Mandibles curved, poorly

calcified, anterior outer edge and carriers from opposite sides diverging from each other, X

shaped. Muscle fiber complex F1+F2 placed between mandibular carriers on the ventral

muscularized pharynx. Inflated base of the ventral cirri round with conspicuous tip.

Notopodial aciculae absent at least in P2. Neurochaetae: limbate, narrow pectinate, true compound bidentate falcigers, aciculae and subacicular hooks. Subacicular hooks light or dark, bidentate, first present on P2. Darkest shade in dark subacicular hooks close to the proximal end. Ventral pygidial cirri present.

191 Species studied in the present paper belonging to this genus: Lysidice collaris Grube, 1870,

Lysidice ninetta Audouin and Milne Edwards, 1833and Lysidice unicornis (Schmarda,

1861).

Remarks: Lysidice is traditionally identified by the lack of palps. Here we make

Nematonereis a subset of Lysidice, which makes the lack of palps within Eunicidae a unique feature of this genus. This feature as well as the lack of lateral antennae in

“Nematonereis-like forms” are also characteristic of some ontogenetic stages of other eunicid genera. Therefore the lack of palp or lateral antennae should not be the only feature used to identify this genus. Other unique features to this genus present in all species are the shape of the curved mandible and the placement of the muscle fiber complex F1+F2 (the latter is unique among eunicid species, but is shared with oenonids). All trees resulting from the present analyses and previous ones with just molecular data (Chapter 3) show

Nematonereis nested within the Lysidice clade. Therefore recognizing Nematonereis makes

Lysidice paraphyletic. Just the lack of palps is not enough to characterize a genus.

Marphysa Quatrefages, 1866 emended

Type species: Marphysa sanguinea (Montagu, 1815)

Diagnostic features: Three antennae (one median and two lateral) and a pair of lateral palps on the prostomium. Peristomial cirri on the posterior peristomial ring absent. MxVI absent.

192 P1 neuropodial post-chaetal lobes longer than chaetal lobes. Neurochaetae: limbate, narrow

or wide pectinate, aciculae and subacicular hooks. Narrow pectinate chaetae have both

outer teeth longer than the other teeth along the whole body. Aciculae dark or light.

Subacicular hooks light or dark, falcate or bidentate. In dark subacicular hooks, darkest shade closest to the proximal end. Ventral pygidial cirri present.

Species studied in the present paper belonging to this genus: Marphysa bellii (Audouin and

Milne Edwards, 1833), Marphysa brevitentaculata Treadwell, 1921, Marphysa californica

Moore, 1909, Marphysa disjuncta Hartman, 1961, Marphysa fallax Marion and Bobretzky,

1875, Marphysa mossambica (Peters, 1854), Marphysa regalis Verrill, 1900, Marphysa

sanguinea (Montagu, 1815), Marphysa viridis Treadwell, 1917.

Remarks: The lack of peristomial cirri is not enough to characterize a monophyletic

Marphysa sensu stricto (Figs. 4.21-4.31). The additional features listed above should also

be used in the identification of the genus. Marphysa sensu stricto has two unique kinds of

pectinate chaetae: narrow pectinate chaetae with unequal inner teeth, increasing from one

side to the other (Fig. 4.21I,J) and wide pectinate chaetae with wide teeth, usually separated

by gaps, tapering from the base (Fig. 4.21G,H,). However, these kinds of pectinate chaetae

are not present in all species of the genus. The first shape was observed only in M. bellii

and M. disjuncta, while the latter is only present in the derived species of the M. sanguinea-

like clade. Most species of Marphysa sensu stricto observed here also have a body abruptly

tapering at the posterior end (except M. fallax), a flat prostomium with higher anterior end

(exceptions are M. fallax and M. regalis) and true-compound spinigerous chaetae on P1 and

193 P2 (except in M. regalis and M. mossambica). M. mossambica is the only species lacking

narrow pectinate chaetae and true-compound chaetae (either bidentate falcigers or

spinigers). The two species identified as Marphysa but placed in another clade (Figs. 4.27-

4.31, clade 38*) contrast with Marphysa sensu stricto in having body evenly tapering, dark

subacicular hook with darkest shade closest to the distal end, true compound chaetae

falcigerous and bidentate only and narrow pectinate with one or both outer teeth longer, a

condition which varies along the body.

Nauphanta Kinberg, 1865

Type species: Nauphanta novahollandiae Kinberg, 1865

Remarks: Nauphanta mossambica is nested within the Marphysa sensu stricto clade in all

trees resulted from the present study. Therefore we consider N. mossambica a species of

Marphysa sensu stricto. This result suggests that Nauphanta is a junior synonym of

Marphysa as previously proposed (e.g., Gravier, 1900; Crossland, 1903; Chamberlin, 1919)

before the resurrection of the genus in Fauchald (1987). Also the presence of wide

pectinate chaetae with narrow teeth side by side, tapering at the distal end, almost

indistinguishable as separate units at the base and the lack of compound chaetae are not

enough to distinguish Nauphanta from Marphysa as previously suggested (Fauchald,

1987). However at this time we are not able to synonymize Nauphanta to Marphysa

because the type species, N. novahollandiae, was not included in our analyses. Most of the

characters placing N. mossambica inside Marphysa sensu stricto are shared with N.

194 novahollandiae, but some of them such as wide pectinate with wide teeth had not been recorded in Nauphanta species before the present study and thus remains unknown for the

type of Nauphanta. Nauphanta now is a monotypic genus including only the type species.

Nematonereis Schmarda, 1861 emended

Type species: Nematonereis unicornis Schmarda, 1861

Remarks: Nematonereis is here included in Lysidice. See discussion above under Lysidice.

Nicidion Kinberg, 1865

emended

Type species: Nicidion cincta Kinberg, 1865

Species studied in the present paper belonging to this genus: Nicidion amoureuxi (Rullier,

1974), Nicidion angeli (Carrera-Parra and Salazar-Vallejo, 1998), Nicidion cariboea

(Grube, 1856), Nicidion cincta Kinberg, 1865, Nicidion insularis (Nogueira et al., 2001),

Nicidion hentscheli (Augener, 1931), Nicidion mikeli (Carrera-Parra and Salazar-Vallejo,

1998), Nicidion mutilata (Webster, 1884) and Nicidion notata (Treadwell, 1921).

195 Diagnostic features: Three antennae (one median and two lateral) and a pair of lateral palps

on the prostomium. Pair of peristomial cirri on the posterior peristomial ring present or

absent. MxVI absent. Branchiae present or absent. Neuropodial chaetal lobe pointed from

P3 to the posterior end of the body. Inflated base of the ventral cirri round with continuous

or conspicuous tip. Notopodial aciculae present at least in the anterior end of the body.

Neurochaetae: limbate, narrow symmetric pectinate, true compound falciger bidentate,

aciculae and subacicular hook dark bidentate. Narrow pectinate dorsal to aciculae from P3

to the posterior end of the body. Darkest color shade on the subacicular hooks at the distal

half or end of the hook from P3 to the end of the body in most species. Lightest subacicular

on the most posterior region of the body with a color shade considerably different from

more anterior ones (subacicular hook yellow on most posterior chaetigers and dark on more

anterior regions).

Remarks: N. cincta is the only species in the original description of the genus that can be reliably identified to species, both other species (Nicidion gualapagensis Kinberg, 1865

and Nicidion longicirrata Kinberg, 1865) are indeterminable. Therefore we here consider

N. cincta as the type species for the genus contradicting Hartman (1959). The placement of

N. cincta on the trees resulting from the present analyses support the traditional association

of N. cariboea with Nicidion (e.g., Monro, 1930; Hartman, 1944) and the observed

similarity between the subacicular hooks of the N. cincta and N. mutilata (Fauchald,

1992a). Nicidion was originally described for abranchiate species. The diagnosis of the genus is here expanded to include branchiated species as well. Most species placed in the same clade as N. cincta bear branchiae. Branchiated species within Nicidion have from one

196 to a few branchial filaments (maximum of four in N.mutilata). Nicidion does not have any

unique feature but the combination of the characters described above is unique. It includes

species previously placed in the genera Eunice and Marphysa. In many species of Nicidion

the posterior parapodia, notopodial and ventral cirri are reduced giving a characteristic look

to the posterior end (Fig. 4.26D). All species of Nicidion that have been observed alive by

the present authors (N. cariboea, N. mikeli and N. mutilata) have a similar color pattern at

the anterior end. They are brown to magenta, with one to three white segments and white

polka dots, the latter may be inconspicuous in some specimens (Fig. 4.26A-C). Webster

(1884) observed a similar pattern in the holotype of N. mutilata even though the specimen had been fixed for several years.

CONCLUSIONS

Eunicidae is monophyletic and can be identified by two unique synapomorphies, dorsal buccal lip fused to the dorsal side of the prostomium (Fig. 4. 3A-C) and the anterior extensions of the dorsolateral fold medially connected (Figs. 4.2A, 6).

Eunice sensu lato is polyphyletic. However, Eunice sensu stricto cannot be defined because of the ambiguous placement of E. aphroditois. The genera Leodice and Nicidion are resurrected to include species of Eunice sensu lato and Marphysa sensu lato (the latter only present in Nicidion) placed in the same clades as their respective type species and diagnosed by synapomorphies supporting the clades. Nematonereis is included in Lysidice, making the latter more inclusive and the lack of palps as one of its unique synapomorphies.

Marphysa is emended to include only the species in the Marphysa sensu stricto clade,

197 inclusive N. mossambica. Nauphanta is valid but it has no diagnostic feature at this point.

Aciculomarphysa, Euniphysa, Fauchaldius, and Palola are valid genera and no changes are made to their traditional diagnostic characters.

The absence of peristomial cirri, lateral antennae and branchiae are not valid generic

characters. The coding of segmental characters for the different parts of the body yielded more phylogenetic information and morphological trees more congruent with molecular and total evidence trees than those in previous studies (e.g., Zanol et al. 2007).

198

Table 4.1: Terminal taxa examined only for morphological data.

Family Genera Species Eunicidae Eunice E. antennata (Lamarck, 1818) EILD, SEM E. aphroditois (Pallas, 1788) EILD E. cincta (Kinberg, 1865). EILD Palola Palola sp. A9Pohnpei1511,EILD Euniphysa E. aculeata Weseinber-Lund, 1949 EILD,SEM Nauphanta N. mossambica (Peters, 1854) EILD,SEM Fauchaldius F. cyrtauloni Carrera-Parra and Salazar Vallejo, 1998 EILD,SEM Aciculomarphysa A. comes Hartmann-Schroeder, 1998 EILD Onuphidae Onuphis O. eremita Audouin and Milne Edwards, 1833 EILD,SEM Dorvilleidae Dorvillea D. sociabilis (Webster, 1879) EILD, SEM 1Specimen from Schulze, 2006. EILDSpecimens excluded from ILD test. SEMSpecies examined under the SEM.

199

Table 4.2: Statistics of the maximum parsimony analyses.

Tree Number of most Data sets length parsimonious trees CI RI Morphology 71 terminal 1214 5712 0.29 0.60 73 terminal 1251 120 0.29 0.60 morphMAFFT 77 terminal 17799 180 0.24 0.42 79 terminal 17837 360 0.24 0.42 morphClustal 77 terminal 17756 110 0.24 0.42 79 terminal 17794 380 0.24 0.42

200

Fig. 4.1. Dorsal view of the posterior most chaetigers. A. Body abruptly tapering,

Marphysa sanguinea. B. Body slowly tapering, Eunice filamentosa complex CBC.

201

202

Fig. 4.2. Ventral view of the prostomium and dissection of the pharynx of the five families of Eunicida include in the present study. A. Eunice valens (Eunicidae). B. Nothria iridescens (Onuphidae). C. Arabella iricolor (Oenonidae). D. Dorvillea sociabilis

(Dorvilleidae). E. Lumbrineris latreilli (Lumbrineridae). bl, buccal lip; dbl, dorsal bl; dlf, dorsolateral fold; dlfae, dlf anterior extension; ivf, inconspicuous ventral furrow; ma, median antenna; mdf, mandible fold; mxf, maxillary fold; p, palp; phf, pharyngeal fold; pro, prostomium; vbl, ventral bl.

203

204

Fig. 4.3. Prostomium and buccal lips. A. Dorsal buccal lip inconspicuous and fused to the dorsal side of the prostomium, prostomium dorsally entire and inflated, Lysidice ninetta,

dorsal view. B. Dorsal buccal lip inconspicuous and fused to the dorsal side of the

prostomium, prostomium dimpled, Eunice filamentosa complex CBC, anterior-dorsal view.

C. Dorsal buccal lip fused to the dorsal side of the prostomium, prostomium round multilobed, Eunice aphroditois, latero-anterior view. D. Dorsal buccal lip conspicuous, free, attached to the prostomium by narrow bases, ceratophores and palpophores long and

articulated, Onuphis eremita, anterio-dorsal view. ctp, ceratophore; dbl, dorsal buccal lip;

la, lateral antennae; ma, median antenna; p, palp; plp, palpophore; pst, palpostyle; vbl,

ventral buccal lip.

205

206

Fig. 4.4. Dorsal view of the prostomium. A. Round prostomium, ceratophores and palpophores short, Eunice filamentosa complex CBC. B. Truncate deep prostomium, ceratophores and palpophores short, Eunice fucata. C. Truncate spread prostomium,

prostomium flat with a higher anterior end, ceratophores and palpophores short, Marphysa

sanguinea. D. Dorsally entire prostomium, Nematonereis unicornis. ctp, ceratophore; dbl,

dorsal buccal lip; la, lateral antennae; ma, median antenna; p, palp; plp, palpophore; pst,

palpostyle; pro, prostomium; prs, peristomium; vbl, ventral buccal lip.

207

208

Fig. 4.5. Lateral and anterior views of the prostomium. A-E. Lateral view. B-F. Anterior view. A,B. Prostomium flat with a higher anterior end, ceratophores and palpophores short,

Euniphysa aculeata. C,D. Prostomium inflated, ceratophores and palpophores short,

Marphysa hentscheli. E,F. Prostomium flat, ceratophores and palpophores short, Eunice

valens. ctp, ceratophore; dbl, dorsal buccal lip; la, lateral antennae; ma, median antenna; p,

palp; plp, palpophore; pst, palpostyle; pro, prostomium; prs, peristomium; prs-cr,

persitomial cirri; prs-vll, peristomial ventro-lateral lips; vbl, ventral buccal lip.

209

210

Fig. 4.6. Ventral view of the prostomium and of the connected dorsolateral fold anterior extension. A. Anterior notch of the dorsolateral fold anterior extension absent, Eunice

filamentosa complex CBC. B. Anterior notch of the dorsolateral fold anterior extension

present, Marphysa hentscheli. dlfae, dorsolateral fold anterior extension; prs-vll,

peristomial ventro-lateral lips; vbl, ventral buccal lip.

211

212

Fig. 4.7. Dorsal view of the prostomium showing the two types of articulations of the prostomial appendages. A. Cylindrical articulation, Eunice torquata. B. Moniliform articulation, Eunice antennata. dbl, dorsal buccal lip; la, lateral antennae; ma, median antenna; p, palp; prs, peristomium; prs-cr, persitomial cirri; vbl, ventral buccal lip.

213

214

Fig. 4.8. Eunice mikeli, dorsal view of the anterior end. Prostomial appendages irregularly

articulated; long ceratophores and palpophores; and peristomial cirri at the anterior edge of

the posterior peristomial ring. ctp, ceratophore; la, lateral antennae; ma, median antenna; p,

palp; plp, palpophore; prs, peristomium; prs-cr, persitomial cirri.

215

216

Fig. 4.9: Lateral view of the anterior end. A. Peristomial ventro-lateral lips inconspicuous,

Oenone fulgida. B. Peristomial ventro-lateral lips set off by distinct grooves, Onuphis eremita; C. Peristomial ventro-lateral lips visible only as elevated surfaces, Eunice

cariboea; D. Peristomial ventro-lateral lips medially short, about half as long as on lateral

sides of the peristomium, Dorvillea sociabilis. la, lateral antennae; p, palps; pro,

prostomium; prs, peristomium; prs-cr, peristomial cirri; prs-vll, peristomial ventro lateral

lip; vbl, ventral buccal lip.

217

218

Fig. 4.10: Dorsal view of the maxillae. A-C,E,F. MxI fang shaped; D. MxI dentate; A.

MxII teeth triangular, pointed, curved and present only on the distal half; B-D,F. MxII teeth

triangular, pointed, curved and present on the anteriormost 2/3 to 3/4 of the plate; E. MxII

teeth triangular, pointed, curved and present on the whole length of the plate, from the

anterior to the posterior end; A-C. Left MxIII arched; D. Left MxIII fang shaped with a

small proximal tooth; E. Left MxIII flat with median edged curved towards the dorsal side;

A. Left MxIV arched dorsoventrally with teeth on the whole length; B. Left MxIV arched

dorsoventrally with teeth on the dorsal quarter of the plate; C. Left MxIV arched

dorsoventrally with teeth on the dorsal half only; D. Left MxIV fang shaped; E. Left MxIV

arched anteroposteriorly; F. Left MxIV has the attachment base narrower than the distance

between the proximal and distal ends of the plate; A-C,E,F. MxV plate like, with a small

tooth; D. MxV fang shaped; A-D. Maxillary carrier shorter than half the length of MxI; E.

Maxillary carrier about half the length of MxI;

A. Marphysa brevitentaculata; B. Eunice filamentosa complexCBC; C. Eunice

violaceomaculata; D. Euniphysa aculeata; E. Diopatra dentata; F. Palola viridis. MND,

mandible; Mx, maxillae; MxC, maxillary carrier.

219

220

Fig. 4.11: Dorsal view of left MxIV. A. MxIV arched dorsoventrally with teeth on the

dorsal quarter of the plate, Eunice filamentosa complexCBC; B. MxIV arched

dorsoventrally with teeth on the dorsal half only, Eunice violaceomaculata; C. MxIV

arched dorsoventrally with teeth on dorsal half and part of the ventral half of the plate,

Marphysa sanguinea; D. MxIV arched dorsoventrally with teeth on the whole length,

Marphysa brevitentaculata. Mx, maxillae.

221

222

Fig. 4.12: Ventral vies of the inflated base of the ventral. A,B. Partially wrinkled, wrinkles on the anterior half; A. Eunice mikeli, parapodium 26 right (P2); B. Eunice filamentosa complexCBC, parapodium 38 right (P2); C. Partially wrinkled, wrinkles restricted to an anterior shield like region, Eunice fucata, parapodium 28 left (P2); D. Completely wrinkle,

Marphysa sanguinea, parapodium 33 right (P2). vcib, ventral cirri inflated base. wr, wrinkles.

223

224

Fig. 4.13: Eunice harassii, dorsolateral view of right chaetiger 113-115. br, branchiae. lbd,

lateral black dot between posterior parapodia. pd, parapodia. vc, ventral cirri.

225

226

Fig. 4.14: Pygidium. A. Pygidial ventral shield present, both ventral and dorsal pygidial cirri placed on the ventral side of the pygidium, ventrolateral view Eunice antennata; B.

Pygidial ventral shield absent, both ventral and dorsal pygidial cirri placed on the ventral side of the pygidium, lateral view Marphysa sanguinea; C. Pygidial ventral shield absent, both ventral and dorsal pygidial cirri placed on the lateral side of the pygidium, posterior view Oenone fulgida. pg, pygidium. pg-dc, pygifial dorsal cirri. pg-sh, pygidial ventral shield. pg-vc, pygidial ventral cirri.

227

228

Fig. 4.15: Parapodia. A. Notopodial cirri leaf shaped; B,I. Notopodial cirri digitiform; C.

Notopodial cirri fusiform; D,J. Notopodial cirri tapering; E. Notopodial cirri thumb

shaped; F. Notopodial cirri button shaped; G. Notopodial cirri filiform; A,H. Neuropodial

pre-chaetal lobe longer than chaetal lobe; B-F,G,J. Neuropodial pre-chaetal lobe shorter

than chaetal lobe; A,C,D,H. Neuropodial post-chaetal lobe longer than chaetal lobe;

B,F,I,J. Neuropodial post-chaetal about as long as chaetal lobe; E,G. Neuropodial post-

chaetal lobe shorter than chaetal lobe; A. Post-chaetal lobe tapering Oenone fulgida top

anterior view of parapodia 140 and 141 left (P4); B. ventral cirri thumb shaped, Eunice

antennata top anterior view of parapodia 1 to 3 left (P1); C. Post-chaetal lobe fusiform,

acicular lobe present, Onuphis eremita dorsal view of parapodium 2 right (P1); D. Post-

chaetal lobe round, ventral cirri thumb shaped, Lysidice ninetta top anterior view parapodia

2 and 3 right; E. Chaetal lobe pointed, Eunice cariboea dorsoanterior view parapodia 72

and 73 right (P4); F. Nematonereis unicornis dorsoanterior view parapodia 95 and 96 left

(P6); G.Ventral cirri bowling pin shaped, Eunice filamentosa ventral view of parapodium

176 right (P4); H. Postchaetal lobe digitiform, Lumbrineris latreille dorsal view of

parapodium 169 left (P4); I., ventral cirri digitiform, Palola viridis ventroposterior view of

parapodium 2 right (P1); J. ventral cirri thumb shaped, chaetal lobe truncate, Eunice valens

anterior view of parapodia 3 to 6 right. acl, acicular lobe. chtl, chaetal lobe. ntc, notopodial

cirri. prcl, prechaetal lobe. ptcl, postchaetal lobe. vc, ventral cirri.

229

230

Fig. 4.16: Median ridge connecting pre- and post chaetal lobes separating supra- and subacicular chaetae. A. Dorvillea sociabilis top view parapodium 5 left;. B. Lumbrineris

latreille top view parapodium 169 left (P4); C. Oenone fulgida top view view parapodium

140 left (P4). mr, median ridge. sb-hook, subacicular hook.

231

232

Fig. 4.17: Neuropodial chaetal lobe lacking acicular lobe and median ridge. A. Rounded,

Palola viridis top anterior view parapodium 14 left (P2); B. Bilobed, Marphysa mossambica parapodium 160 right (P4). chtl, chaetal lobe.

233

234

Fig. 4.18: Ventral cirri and acicular lobe. A. round inflated base, continuous tip, Eunice cariboea ventral view around parapodium 20 left (P2). B. Button shaped, Eunice cariboea ventroposterior view parapodium 75 right (P4); C. Tapering, Euniphysa aculeata

ventroanterior view parapodium 1 left (P1); D. Bottle shaped, Eunice violaceomaculata

ventral view of parapodium 143 right (P5); E. Fusiform, Onuphis eremita anterior view of

parapodium 2 right (P1); F. Cushion shaped, no tip, Onuphis eremita top view of

parapodium 17 right (P2); G. Oval, Eunice mikeli ventroanterior view of parapodium 3 left

(P1). acl, acicular lobe. vc, ventral cirri.

235

236

Fig. 4.19: Fleshy knob on the dorsal side of the neuropodium and Hayashi’s organ. A.

Eunice filamentosa complex CBC top anterior view parapodium 4 left; B. Euniphysa aculeata anterior view parapodium 2 left. fk, fleshy knob. hsh-b, Hayashi’s organ bulging.

237

238

Fig. 4.20: Aciculae. A. Blunt tipped, Eunice valens anteroventral view of parapodium 99

left (P4); B. Bidentate, Eunice filamentosa complexCBC ventral view of parapodium 101 right (P3); C. Hammer head, Eunice mikeli anteroventral view of parapodium 124 left (P4);

D. Mucronated, Onuphis eremita anterior view parapodium1 left (P1); E. Arrow shaped,

Paramphinome jeffreysii parapodium15 left (P3). ac, aciculae. bn, blunt. bd, bidentate. hm, hammer head. mc, mucronate.

239

240

Fig. 4.21: Pectinate chaetae. A. Wide and narrow pectinate chaetae, Marphysa sanguinea top view parapodium 170 right (P4); B-F,I-L. Narrow pectinate chaetae; G,H. Wide pectinate chaetae. B,C. Curved; D,F,I,J. Flat; B,D-F,L. Distal end wide; C,I,J. Distal end narrow; A-F,I,J. Symmetric; L. Asymmetric. A-E,I-L. Teeth in a tranverse row; F. Teeth

in a oblique row; B. Both outer teeth longer than inner ones; C,D,F,I,J. One of the outer

teeth longer than inner ones; E,L. Both outer teeth as long as inner ones; A-F,L. Inner teeth

with equal length; I,J. Inner teeth unequal, increasing in length from one side to the other;

G,H. Teeth wide, usually separated by gaps, tapering from the base; H. Teeth thin side by side, tapering at the distal end, almost indistinguishable as separate units at the base; B.

Marphysa sanguinea parapodium 98 left (P3); C. Eunice torquata parapodium 86 left (P4);

D. Eunice valens parapodium 20 left (P2); E. Onuphis eremita parapodium 46 left (P3); F.

Lysidice ninetta parapodium 4 right. G. Marphysa sanguinea parapodium 170 right (P4);

H. Nauphanta mossambica parapodium 224 right (P5); I,J. Marphysa disjuncta

parapodium 109 left. L. Diopatra ornata parapodium 175 right (P5). pct, pectinate. pct-n,

pct narrow. pct-wn, pct wide with narrow teeth. pct-ww, pct wide with wide teeth.

241

242

Fig. 4.22. Neuropodial compound chaetae. A. Pseudocompound chaetae; B. Fracture in between article and shaft of pseudocompound chaeta; C-M. True-compound; C,J. Junction between shaft and article completely connected, proximal end of the article directly connected to the shaft, without the presence of a socket; D,E,H,I. True-compound chaetae with the junction between shaft and article completely connected; F,G,K,L. True- compound chaetae with junction between shaft and article connected at both edges, proximal end of the article fit in a socket; A,L. Spinigerous; D,F. Distal end of the article falcigerous and bidentate, longest tooth distal; E. Distal end of the article falcigerous and tridentate, longest tooth distal; G. Distal end of the article falcigerous and bidentate, longest tooth proximal; H. Distal end of the article falcigerous and multidentate; K. Distal end of the article falcigerous and bidentate, teeth inconspicuous (appears unidentate if not very carefully examined under high magnification); M. Distal end of the article falcigerous and tridentate, longest tooth proximal. A,B. Euniphysa aculeata parapodium 3 right; C-E.

Onuphis eremita prapodia 1, 2 and 3 right (P1); F. Eunice valens parapodium 13 left (P2);

G. Eunice violaceomaculata parapodium 104 right (P4); H-J. Lumbrineris latreille parapodia 1 (P1), 6 and 5 right; K. Eunice filamentosa complexCBC parapodium 2 right

(P1); L. Marphysa sanguinea parapodium 170 right (P4); M. Eunice miurai parapodium 64 right (P6); *, distal end of a shaft missing the article.

243

244

Fig. 4.23: Subacicular hooks. A. Distal end falcate; B.Distal end tridentate; C-F. Distal end

bidentate; F. Distal end spinigerous; A,B,C,E,F. Subacicular guards solid, sheet-like; D.

Subacicular guards are disassociated fibrils; B,D,E. Subacicular guards cover the proximal

tooth only; C,F. Subacicular guards cover all teeth; A. Marphysa disjuncta parapodium 75

left (P4). B. Eunice antennata parapodium 93 left (P6); C. Nematonereis unicornis

parapodium 61 right (P4); D. Fauchaldius cyrtauloni parapodium 18 left (P4); E. Eunice

violaceomaculata parapodium 66 left (P3); F. Oenone fulgida parapodium 139 left (P4).

bd, bidentate subacicular hook. gr, subacicular hooks guards. sn, spinigerous.

245

246

Fig. 4.24: Pattern of dark color on the subacicular hooks. Columns correspond to the different regions along the body. Numbers below hooks are the hook length in µm. A, B.

Darkest color present on most of the length of the hook, but color more concentrated towards proximal end, Eunice aphroditois; C. Darkest color present near the distal end of the hook, Eunice cariboea.

247

248

Fig. 4.25: Hayashi’s (1994) sensory organ. A. Flat, Marphysa sanguinea parapodium 98 left (P3); B. Bulging, Eunice mikeli parapodium 3 left (P1); C. Slightly elevated, Lysidice ninetta parapodium 20 right (P2). hsh, Hayashi’s organ. hsh-b, hsh bulging. hsh-e, hsh slightly elevated. hsh-f, hsh flat. ntc, notopodial cirri.

249

250

Fig. 4.26: Color pattern of Eunice sensu lato species herein transferred to the resurrected genus Nicidion. A. Dorsal view of the anterior end of Eunice cariboea; B. Dorsal view of the anterior end of Eunice mikeli; C. Dorsal view of the anterior end of Eunice mutilata; D.

Lateral view of the posterior end of Eunice mutilata.

251

252

Fig. 4.27: Strict consensus tree of the morphology only most parsimonious trees, 77- terminals. Dashed gray lines represent the placement of Aciculomarphysa comes and

Fauchauldius cyrtauloni in the morphology only most parsimonious trees with 79- terminals. Numbers in parentheses are clade numbers equivalent to those in Chapter 2. * denotes differences among the current clade and the equivalent one in Chapter 2 due to the new placement of a single taxon or the inclusion of additional species not present in the analyses in Chapter 2. Additional numbers on branches are Bremer/bootstrap support values or just the Bremer support value if only one value is present.

253

254

Fig. 4.28: Strict consensus tree of the morphMAFFT most parsimonious trees, 77- terminals. Dashed gray lines represent the alternative placement of Aciculomarphysa comes and Fauchauldius cyrtauloni in the morphMAFFT most parsimonious trees with 79-

terminals. Numbers in parentheses are clade numbers equivalent to those in Chapter 2. *

denotes differences among the current clade and the equivalent one in Chapter 2 due to the

new placement of a single taxon or the inclusion of additional species not present in the

analyses in Chapter 2. Additional numbers on branches are Bremer/bootstrap support

values or just the Bremer support value if only one value is present.

255

256

Fig 4.29: Bayesian trees of the morphMAFFT data set, 77-terminals. A. Bayesian tree resulted from the bayesian analsyses with morphgene+codon partition. Dashed gray lines represent the alternative placement of Aciculomarphysa comes and Fauchauldius cyrtauloni in the morphMAFFT Bayesian tree with 79-terminals. B. The only topological difference between the morphgene+codon partition and the morphgene partition morphMAFFT Bayesian trees. Numbers in parentheses are clade numbers equivalent to those in Chapter 2. * denotes differences among the current clade and the equivalent one in

Chapter 2 due to the new placement of a single taxon or the inclusion of additional species not present in the analyses in Chapter 2. Posterior probability values are placed on the branches.

257

258

Fig. 4.30: Strict consensus tree of the morphClustal most parsimonious trees, 77-terminals.

Dashed gray lines represent the alternative placement of Aciculomarphysa comes and

Fauchauldius cyrtauloni in the morphClustal most parsimonious trees with 79-terminals.

Numbers in parentheses are clade numbers equivalent to those in Chapter 2. * denotes

differences among the current clade and the equivalent one in Chapter 2 due to the new

placement of a single taxon or the inclusion of additional species not present in the analyses

in Chapter 2. Additional numbers on branches are Bremer/bootstrap support values or just

the Bremer support value if only one value is present.

259

260

Fig 4.31: Bayesian trees of the morphClustal data set, 77-terminals. Bayesian tree resulted from the bayesian analsyses with gene+codon partition. Dashed gray lines represent the alternative placement of Aciculomarphysa comes and Fauchauldius cyrtauloni in the morphClustal Bayesian tree with 79-terminals. Numbers in parentheses are clade numbers equivalent to those in Chapter 2. * denotes differences among the current clade and the equivalent one in Chapter 2 due to the new placement of a single taxon or the inclusion of additional species not present in the analyses in Chapter 2. Posterior probability values are placed on the branches.

261

262

Chapter 5: Conclusion

Correspondence and topological similarities of the external morphology and innervation refined previous knowledge about the distinctive structures on the prostomium and in the anterior end of the pharynges of the families of the order Eunicida. This allowed us to improve hypotheses of primary homology. These hypotheses corroborated the presence of previously described and at times contradictory structures such as buccal lips, pharyngeal fold and dorsolateral fold in all families. The dorsolateral fold has an anterior

extension in all families, except Dorvilleidae, recognized and homologized for the first time

in this study. These refined and new hypotheses of homology improved morphological

phylogenetic information among and at the family level clades in Eunicida and resulted in

the only two morphological unique synapomorphies for a monophyletic Eunicidae, dorsal buccal lip fused to the dorsal side of the prostomium (Fig. 4.3A-C) and the anterior extensions of the dorsolateral fold medially connected (Figs. 4.2A, 4.6).

This study confirmed that Eunicidae is monophyletic with Onuphidae as its sistergroup. Palola and Euniphysa are monophyletic; Lysidice is paraphyletic with

Nematonereis nested within it; Eunice and Marphysa sensu lato are polyphyletic; and

Marphysa sensu stricto is paraphyletic (morphological and total evidence analyses) with

Nauphanta mossambica (Peters, 1854) nested within it. Furthermore, some clades represent

monophyletic redefinitions of current genera and the defunct Leodice and Nicidion.

Thus, Leodice and Nicidion are revived as valid genera diagnosed by synapomorphies supporting the clades including their type species, respectively, Eunice sensu lato and

Marphysa sensu lato (the latter only present in Nicidion) species placed in the same clades.

263 Lysidice includes Nematonereis, therefore the lack of palps is one of the unique

synapomorphies for this genus. Marphysa includes only the species in the Marphysa sensu

stricto clade, including N. mossambica. Nauphanta is valid but it has no diagnostic feature

at this point. Aciculomarphysa, Euniphysa, Fauchaldius, and Palola are valid genera diagnosed by the traditional characters. Eunice sensu stricto cannot be defined because of the ambiguous placement of, the type species, Eunice aphroditois. The relationships among most eunicid genera are ambiguous. However, Leodice is consitently the earliest genus to diverge from the remaining eunicids.

The absence of peristomial cirri, lateral antennae and branchiae are not valid generic characters. The novel coding of morphological characters used here, including information for variation of segmental characters along the body, improved the morphological phylogenetic signal yielding trees more resolved and congruent with molecular and total evidence trees than those in previous studies (e.g., Zanol et al. 2007).

The three genes used here (16S, COI and 18S) contain different kinds of phylogenetic information and were informative at different levels of the trees. 16S and COI were

important in recovering a monophyletic Eunicidae and the relationships within the family,

while 18S was important in the resolution of basal eunicidan relationships (i.e., in the

rooting of the tree), the monophyly of Onuphidae and the basal relationships within this

family. Thus, the combination of these three genes in a single molecular data set was

essential for more robust and congruent trees across analyses.

The thorough examination of the morphology using detailed anatomical study as

well as the incorporation of serial homologous information, in order to refine hypotheses of

homology in combination with molecular data as used here proved to be a successful

264 approach. It consistently recovered similar hypotheses of phylogeny, which supported the

monophyly of the family, the same phylogenetic status for the genera and about the same

intergeneric relationships. The inclusion of the new morphological hypotheses also

provided synapomorphies useful to circumscribe the family and the monophyletic

redefinition of its genera.

Future studies should focus in increasing the taxonomic sample of eunicid species

as well as data to resolve ambiguous intergeneric relationships. Additional taxa may have

different character combinations, which could change the recovered relationships as well as

stabilize ambiguous ones (e.g., the placement of Eunice fucata Ehlers, 1887). Stable

relationships among the genera are essential for the unambiguous interpretation of most

character evolution and future comparative studies.

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287 Appendices

Appendix 1: Saturation curves of the different genes and codon positions of the COI for the complete data set. Transition and transversion distances corrected with the model Kimura

2-parameter (K80) plotted against the total distance corrected with the same model. Dashed blues lines, transition. Full green line, transversion. S, transition. V, transversion.

288

289

Appendix 2: Saturation curves of the different genes and codon positions of the COI for

Eunicidae and Onuphidae, respectively. Transition and transversion distances corrected with the model Kimura 2-parameter (K80) plotted against the total distance corrected with the same model. Dashed blues lines, transition. Full green line, transversion. S, transition.

V, transversion.

290

291

Appendix 3: Trees from the analyses of the 16S gene partition. A,C,E. Results of the

MAFFT alignment analyses. B,D,F. Results of the hand adjusted Clustal alignment analyses. A,B. Maximum likelihood phylograms. C. Strict consensus cladogram of the

most parsimonious trees. D. Most parsimonious cladogram. E,F. Bayesian inference

phylograms. Light grey box, Onuphidae. Dark grey box, Eunicidae.

292 293

294

Appendix 4: Trees from the analyses of the COI gene partition. A. Maximum likelihood phylogram. B. Strict consensus cladogram of the most parsimonious trees. C. Bayesian inference phylogram resulted from the codon partition analysis. D. Bayesian inference phylogram resulted from the non partitioned analysis.

295

296

Appendix 5: Trees from the analyses of the 18S gene partition. A,C,E. Results of the

MAFFT alignment analyses. B,D,F. Results of the hand adjusted Clustal alignment analyses. A,B. Maximum likelihood phylograms. C,D. Strict consensus cladogram of the most parsimonious trees. E,F. Bayesian inference phylograms. Light grey box, Onuphidae.

Dark grey box, Eunicidae.

297

298 299 Appendix 6: Specimens examined to code for morphological data. #CS, number of

complete specimens examined. #IS, number of incomplete specimens examined. AM,

Australian Museum, Sidney, Australia. IBUFRJ, Departamento de Zoologia, Instituto de

Biologia, UFRJ, Rio de Janeiro, Brazil. IRSNB, Institut Royal des Sciences Naturelles de

Belgique, Brussels, Belgium. MNCN, Museo Nacional de Ciencias Naturales de Madrid,

Madrid, Spain.MR, morphological data. MRL, morphological and molecular data. MZSP,

Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil. NTM, Northern

Territory Museum, Darwin, Australia. SEM, specimen examined under the SEM. SMNH,

Swedish Museum of Natural History, Stockholm, Sweden. USNM, National Museum of

Natural History, Smithsonian Institution, Washington, DC, USA. ZMB, Museum für

Naturkunde, Berlin, Germany. ZMH, Zoologisches Museum, Hamburg, Germany.

Glyceridae

Glycera dibranchiata Ehlers, 1868. USNMXXXX (MR), 1CS.

Amphinomidae

Paramphinome jeffreysii (McIntosh, 1868). USA, North Atlantic Ocean, off New

England: USNM1602871 (MR), 1CS.

Lumbrineridae

Lumbrineris inflata Moore, 1911. USA, North Pacific Ocean, Washington, San Juan

Island: USNM40596 (MR), 1CS.

300 Lumbrineris latreille Audouin & Milne Edwards, 1834. USA, North Atlantic Ocean,

North Carolina: USNM 53018 (MR, SEM), 1CS.

Dorvilleidae

Dorvillea erucaeformis (Malmgren, 1865). USNMXXXX (MRL), 1IS.

Dorvillea sociabilis (Webster, 1879). USA, North Atlantic Ocean, Virginia: USNM

33830 (MR, SEM), 1CS.

Oenonidae

Arabella semimaculata (Moore, 1911). USA, North Pacific Ocean, California, Santa

Cruz Island: USNM 40129 (MR), 1CS.

Oenone fulgida (Savigny, 1818). USA, North Atlantic Ocean, Florida, Conch Key:

USNM 53751 (MR, SEM), 1CS.

Onuphidae

Diopatra dentata Kinberg, 1865. Australia, Indian Ocean, South Australia, Adelaide:

USNMXXXX A666.1F(MRL), 1IS.

Diopatra ornata Moore, 1911. USA, North Pacific Ocean, California, Los Angeles

Harbor: USNM 49790 (MR), 1CS.

Hyalinoecia sp. USA, North Atlantic Ocean, off Massachusetts: USNMXXXX

A584.1F (MRL), 1IS.

Mooreonuphis pallidula (Hartman, 1965). USA, North Atlantic Ocean, off

Massachusetts: USNM179507 (MR), 1CS.

301 Onuphis elegans (Johnson, 1901). USA, North Pacific Ocean, Washington, San Juan

Island: USNMXXXX A156.1F (MRL), 1IS.

Onuphis eremita Audouin and Milne Edwards, 1833. France, Mediterranean Sea,

Marseille: USNM 54462 (MR, SEM), 1IS.

Onuphis cf. iridescens (Johnson, 1901). USA, North Pacific Ocean, California:

USNMXXXX A394.1F (MRL), 1IS.

Paradiopatra quadricuspis (M. Sars, 1972). Norway, North Atlantic Ocean,

Trondheim: USNMXXXX A273-1F (MRL), 1IS.

Eunicidae

Eunice americana Hartman, 1944. USA, North Pacific Ocean, California, Los

Angeles Harbor: USNMXXXX E4F (MR), 1CS; USNMXXXX station 5B(MR), 1CS;

USNMXXXX E5F (MR), 1IS; USNMXXXX E1F (MR), 1CS.

Eunice amoureuxi Rulliers, 1974. Belize, Caribbean Sea, Carrie Bow Cay:

USNMXXXXCBCKF07/2004 (MRL), 1CS.

Eunice antarctica Baird, 1869. Antarctic Peninsula: Southern Ocean, Hugo Island,

USNMXXXX A910 (MRL), 1IS; Drake Passage, USNM 56788 (MR), 2CS; South

Shetland Island, USNM 56792 (MR), 1IS.

Eunice antennata (Lamarck, 1818). Red Sea, Gulf of Suez, Zeit Bay: USNM 96434

(MR, SEM), 3CS.

Eunice antillensis Ehlers, 1887. Belize, Caribbean Sea, Carrie Bow Cay:

USNMXXXXCBCKF07/2004 (MRL), 1CS, posterior end regenerating.

302 Eunice aphroditois (Pallas, 1788). Indonesia, South Pacific Ocean: USNM 96453

(MR), 2CS. Japan, North Pacific Ocean, Kamagawa Prefecture, USNM 100202 (MR),

1CS.

Eunice cariboea Grube, 1856. Belize, Caribbean Sea, Blue Ground Range:

USNMXXXX Anja (MR, SEM), 3CS.

Eunice cincta (Kinberg, 1865). French Polynesia, Moorea, South Pacific Ocean:

SMNH Type-418 (MR), 1IS.

Eunice filamentosa complex CBC Grube, 1856. Belize, Caribbean Sea, Carrie Bow

Cay: USNMXXXX CBCTILE-F59 (MR, SEM), 1CS; USNMXXXX CBC03-16D

(MRL), 1IS; USNMXXXX CBCTILE-F57 (MR), 1CS; USNMXXXX CBCTILE-F60

(MR), 1CS.

Eunice filamentosa complex BR Grube, 1856. Brazil, South Atlantic Ocean, Ceará:

MZSP890 (MRL), 1CS.

Eunice fucata Ehlers, 1887. Belize, Caribbean Sea, Carrie Bow Cay: USNMXXXX

CBCRERe53 (MR, SEM), 1CS; USNMXXXX EDa (MRL), 1CS; USNMXXXX CB03-

9NAnja (MR), 1CS; USNMXXXX CBC-KF1010 (MR), 1CS, posterior end regenerating.

Eunice harassii Audouin & Milne Edwards, 1833. United Kingdom, North Atlantic

Ocean: SMNH28277 (MR), 1 CS, Plymouth, IBUFRJ341 (MR), 1CS; Western Sahara,

IRSNB10910 (MR), 1CS. France, North Atlantic Ocean, close to Ile Verte,

USNMXXXX Acc2035050 (MR), 1CS.

Eunice impexa Grube, 1878. Australia: Indian Ocean, South Australia, Port Lincoln,

USNMXXXX A673 (MRL), 1IS; South Pacific Ocean, New South Wales, Port Stephens,

303 AM-W3829 (MR), 1IS; South Pacific Ocean, New South Wales, Cape Howe, AM-

W197056 (MR), 1IS.

Eunice cf. insularis Nogueira et al., 2001.Brazil, South Atlantic Ocean, Ceará:

MZSP889 (MRL), 1CS; MZSP895 (MR), 1CS.

Eunice cf. limosa Ehlers, 1868. Spain, Mediterranean Sea, Catalonia, Puerto de la

Selva: USNMXXXXEduardoLopez (MRL), 1IS.

Eunice lucei Grube, 1856. USA North Pacific Ocean, California, Los Angeles

Harbor: USNMXXXX E9F (MR), 1CS; USNMXXXX E9F (MRL), 1CS; USNMXXXX

E7F (MR), 1CS; USNMXXXX E8F (MR), 1CS.

Eunice marcusi Zanol et al., 2000. Brazil, South Atlantic Ocean: Espírito Santo,

MZSP893 (MRL), 1CS; Rio de Janeiro, IBUFRJ0004 (MR), 1CS.

Eunice mikeli Carrera-Parra & Salazar-Vallejo, 1998. Belize, Caribbean Sea, Carrie

Bow Cay: USNMXXXX CBCSBE-F28E (MR, SEM), 1CS, posterior end regenerating;

USNMXXXX CBC03-14B (MRL), 1IS; USNMXXXX CBCD1-F19 (MR), 1IS.

Eunice miurai Carrera-Parra and Slazar-Vallejo, 1998. Belize, Caribbean Sea, Carrie

Bow Cay: USNMXXXXAnja14a (MRL), 1IS; USNMXXXX CBC-KF-1007(MR), 1IS

and 1CS.

Eunice mutilata Webster, 1884. Bermuda Islands, North Atlantic Ocean:

USNM4789-holotype (MR), 1IS. Belize, Caribbean Sea, Carrie Bow Cay: USNMXXXX

CB031EAnja (MRL), 1IS; USNMXXXX CBCD4-E40 (MR), 1CS.

Eunice norvegica (Linnaeus 1767). Norway, North Atlantic Ocean, Rödberg:

IBUFRJ348 (MR), 3CS, USNMXXXX A278 (MRL), 1IS.

304 Eunice notata (Treadwell, 1921). Belize, Caribbean Sea, Carrie Bow Cay:

USNMXXXX CBC03-12F EAnja (MRL), 1CS; USNMXXXX CBC03-10C EAnja

(MR), 1CS; USNMXXXX CBC03-16C EAnja (MR), 1IS.

Eunice roussaei Quatrefages, 1866. Italy, Adriatic Sea, Muggia: USNMXXXX EB2b

(MRL), 1CS. Italy, Mediterranean Sea, Naples: MNCN 16.01/313 (MR), 1IS. MNCN

16.01/315(MR), 1IS.

Eunice rubra Grube, 1856. Brazil, South Pacific Ocean: São Paulo, IBUFRJ363

(MR), ICS, IBUFRJ365 (MR), 1CS; Espírito Santo, IBUFRJ 364 (MR), 1CS; Ceará,

MZSP888 (MRL), 1CS.

Eunice thomasiana Augener, 1922. Belize, Caribbean Sea: Blue Ground Range,

USNMXXXX EB2c (MRL), 1CS; Manatte Cay, USNMXXXX CBCMCRE-F35, 1CS,

USNMXXXX CBCMCRE-F45, 1IS.

Eunice torquata Quatrefages, 1866. Spain, Mediterranean Sea: Catalonia, Cadaques,

USNMXXXXEduardoLopezEB2a (MRL), 1IS, USNMXXXXEduardoLopez15 (MR,

SEM), 1CS; between Cabo San Antonio and Puerto Valencia, MNCN16.01/1716 (MR),

1IS, MNCN16.01/1722 (MR), 1CS.

Eunice valens (Chamberlin, 1919). USA, North Pacific Ocean, Washington, San Juan

Island: USNMXXXX1-SH68 (MR, SEM), 1CS, USNMXXXXSH67 (MR), 1CS,

USNMXXXXa (MR), 1CS, USNMXXXXb (MR), 1CS.

Eunice cf. violaceomaculata Ehlers, 1887. Belize, Caribbean Sea: Blue Ground

Range, USNMXXXX Anja (MR, SEM), 1CS; Carrie Bow Cay, USNMXXXX CB03-9

Anja (MRL), 1CS, posterior end regenerating.

305 Marphysa angeli Carrera-Parra and Salazar-Vallejo, 1998. Belize, Caribbean Sea,

Carrie Bow Cay: USNMXXXX MC2a (MRL), 1IS. Mexico, Caribbean Sea, Quintana

Roo, USNM177955 (MR), 1CS.

Marphysa bellii (Audouin and Milne Edwards, 1833). Spain: North Atlanic Ocean,

Cantabria, MNCN16.01/330 (MR), 2IS; Mediterranean Sea, between Cabo San Antonio

and Puerto Valencia, MNCN16.01/2447 (MR), 1IS.

Marphysa brevitentaculata Treadwell, 1921. Mexico, Caribbean Sea, Quintana Roo,

Xahuaycho: USNMXXXX MB2b (MRL), 1CS. USA, North Atlantic Ocean, Florida,

Tampa Bay: USNM45594 (MR), 2CS.

Marphysa californica Moore, 1909. USA, North Pacific Ocean, California: San

Diego, USNMXXXX M7 (MRL), 1CS, USNMXXXX M6 (MR), 1IS; Los Angeles,

USNM39094, 1IS

Marphysa disjuncta Hartman, 1961. USA, North Pacific Ocean, California, Los

Angeles, USNMXXXX M3F, station 9B (MRL), 1IS, USNMXXXX M5F, station 5B

(MR), 1IS, USNMXXXX M2F, station 10C (MR), 1IS.

Marphysa fallax Marion and Bobretzky, 1875. Italy, Mediterranean Sea, Sardinia:

USNMXXXX (MR, SEM), 3CS.

Marphysa cf. hentscheli Augener, 1931. Brazil, South Atlantic Ocean, Ceará:

MZUSP892 (MRL, SEM), 1CS; IBUFRJXXXX M2Pa (MR), 1IS.

Marphysa regalis Verrill, 1900. Brazil, South Atlantic Ocean, Ceará: MZSP891

(MRL), 1CS; MZSP896 (MR), 1IS; IBUFRJXXXX E7RC (MR), 1CS, posterior end regenerating.

306 Marphysa sanguinea (Montagu, 1807). United Kingdom, North Atlantic Ocean,

Plymouth Sound. IBUFRJ 377 (MR), 3CS. France, North Atlantic Ocean, North

Bretagne: USNMXXXX (MR, SEM), 1CS.

Marphysa viridis Treadwell, 1917. Brazil, South Atlantic Ocean, Ceará: MZSP887

(MRL), 1CS; MZSP894 (MR), 1CS.

Palola cf. siciliensis (Grube, 1840). Spain, Mediterranean Sea : Catalonia, Puerto de la Selva, USNMXXXX Pa, 7 (MRL), 1IS ; Melilla, Islas Chafarinas, MNCN16.01/5627

(MR), 2IS.

Palola viridis Gray, 1847. Micronesia, North Pacific Ocean, Kosrae Island: USNM

1084405 (MRL, SEM), 1IS, USNM 1084403 (MR), 1IS.

Palola sp. Pohnpei142. Micronesia, North Pacific Ocean, Pohnpei: USNM 1084377

(MRL), 1CS.

Palola sp. A3Guam102 and 103. Guam, North Pacific Ocean, Mangiloa: USNM

1084359 (MRL), 1IS, USNM 1084360 (MR), 1IS.

Palola sp. B1Bocas68, 70 and 79. Panama, Caribbean Sea, Bocas del Toro: USNM

1084323 (MR), 1IS, USNM 1084325 (MRL), 1IS, USNM 1084330 (MR), 1IS.

Palola sp. B7Bocas85. Panama, Caribbean Sea, Bocas del Toro: USNM 1084333

(MR), 1CS.

Palola sp. B5Belize34. Belize, Caribbean Sea, Carrie Bow Cay: USNM 1084314

(MR), 1CS.

Palola sp. A1Perlas57, 61 and 63. Panama, North Pacific Ocean, Las Perlas

Archipelago: USNM 1084343 (MRL), 1IS, USNM 1084347 (MR), 1CS, USNM

1084348 (MR), 1CS.

307 Palola sp. A9Pohnpei151. Micronesia, North Pacific Ocean, Pohnpei: USNM

1084380 (MR), 1IS.

Lysidice collaris Grube, 1870. Italy, Mediterranean Sea, Ischia: USNMXXXXL2105

(MR), 1IS, USNMXXXXL1106 (MR), 1IS.

Lysidice ninetta Audouin and Milne Edwards, 1833. Spain, Mediterranean Sea,

Alicante: USNMXXXX102-104 (MR, SEM), 3IS.

Lysidice sp.1. Belize, Caribbean Sea, Carrie Bow Cay: USNMXXXX CBC03-3E

(MRL), 2IS.

Lysidice sp.2. Brazil, South Atlantic Ocean, Ceará: MZSP 884 (MRL), 1CS.

Nematonereis unicornis Schmarda, 1861. Spain, Mediterranean Sea: Alicante,

USNMXXXX112 (MR, SEM), 1CS; Catalonia, Puerto de la Selva, USNMXXXX114

(MR), 1CS; Catalonia, Cadaquis, USNMXXXX115 (MR), 1IS.

Euniphysa aculeata Weseinber-Lund, 1949. Indian Ocean, Red Sea, Gulf of Suez:

USNM096436 (MR, SEM), 1CS and 1IS. Indonesia, South Pacific Ocean, Madura Strait:

USNM37717 (MR), 1IS.

Euniphysa tridontesa (Shen and Wu, 1990). Indonesia, South Pacific Ocean, Bangka

off Sumatra: NTM-WO19209 (MRL), 2IS.

Nauphanta mossambica (Peters, 1854). Mozambique, Indian Ocean: ZMB4005 (MR,

SEM), 3CS.

Fauchaldius cyrtauloni Carrera-Parra & Salazar Vallejo, 1998. Mexico, Caribbean

Sea, Quintana Roo, Punta Musquitero: USNM177944 (MR, SEM), 1CS and 1 IS.

USNM177943 (MR), 1CS.

308 Aciculomarphysa comes Hartmann-Schroeder, 1998. South Pacific Ocean, Loyalty

Island: ZMH P-22044 (MR), 1CS.

309 Appendix 7:Morphological data matrix. Letters represent polymorphic states. A=0,3.

B=0,1. C=0,2. D=1,2. E=2,6. F=0,1,2. G=2,3. H =0,4. I=1,3.

Characters 1 - 50

1 6 11 16 21 26 31 36 41 46 | | | | | | | | | | Glycera dibranchiata 000100?????-???--00------1?10-0--20-00------Paramphinome jeffreysii 0001011????-???3411010-0-1010-0--20-00------Lumbrineris inflata 00010000----00---00------0------210-11010211110303 Lumbrineris latreille 00010000----00---00------0------210-11010211110303 Dorvillea erucaeformis 0???0000----1?---0120--0-110--0-?10-1110-0?-?????? Dorvillea sociabilis 33310000----1?---0111100-1012-0-310-1110-0?-?????? Arabella semimaculata 00010000----00---00------0------010-1100-10-210404 Oenone fulgida 00010000----00---1120--0-0-0----000-1100-10-210404 Hyalinoecia sp. 0???00110---00-1311010-111010-11100-11011210210110 Mooreonuphis pallidula 200100110---00-1311010-111010-11101-11011210210111 Onuphis eremita 2???00110---00-131100--111012-11100-11011210210111 Onuphis cf. iridescens 2???00110---00-031100--11100--11101-11011210210111 Onuphis elegans 20??00110---00-131100--11100--11101-11011210110111 Diopatra dentata 20??00110---00-031100--11100--11101-11011210210111 Diopatra cf. ornata 200000110---00-0211010-111010-11101-11011210210111 Paradiopatra quadricuspis 0???00110---00-2311010-111010-11101-11011210210111 Eunice antennata 33310011112101102110111101011110211111011210110110 Eunice rubra 30010011110101112110111101011110211111011210110110 Eunice americana 0001001111000110211010-101011010211111011210110111 Eunice miurai 03310011112001102110111101011110211111011210110111 Eunice cf. limosa 11??0011111101102110110101011010211111011210110111 Eunice lucei 30010011112101112110111101011110211111011210110110 Eunice antarctica 3331001111010110211010-101011010211111011210110110 Eunice cf. antillensis 00310011112001102110110101011010211111011210110110 Eunice valens 00A10011111101102110110101011010211111011210110110 Eunice harassii 3001001111210110211010-101010-10211111011210110110 Eunice torquata 00310011112101102110110101011010211111011210110010 Eunice marcusi 00310011110101102110111101011110211111011210110010 Eunice thomasiana 00010011110101002110110101011110211111011210110010 Eunice fucata 0331001111210110211010-101010-10211011011210010010 Eunice cariboea 000B001110200111211010-101010-10211011011210110010 Eunice cf. insularis 000B001110000101211010-101010-102111110112100100B0 Eunice cincta ?????01??????????11010-101010-10?11?11?112???????? Marphysa angeli 00?1001110210111211010-1C1010-12210-11011210010010 Marphysa cf. hentscheli 0001001110010111211010-1C1010-1C210-11011210010010 Eunice amoureuxi 1330001110010111211110-101010-10211011011210010010 Eunice mutilata 0001001110200101211010-101010-10211011011210010010 Eunice notata 0111001110210101211010-121010-12211111011210010010 Eunice mikeli 0001001110210111211010-121010-12211011011210010010 Marphysa sanguinea 0220B01114300111211010-101010-10210-11011210010010 Nauphanta mossambica 1220101110310111211010-101010-10210-11011210010010 Marphysa brevitentaculata 2220101110310111211010-101010-10210-11011210010010 Marphysa disjuncta 00?0001113310101211010-101010-10210-11011210010010 Marphysa fallax 0001001113000100211010-101010-10210-1101121011001? Marphysa californica 0220001110310101211010-101010-10210-11011210010010 Marphysa viridis 02B0001114320101211010?101010?1C210-11011210010010 Marphysa regalis 020B001110010110211010?101010?10210-11011210010010 Marphysa bellii 20??001113310101211010-1C1010-1C210-11011210110110 Lysidice ninetta 03??00111300010--11110-100----0-210-11011210100010 Lysidice collaris 00??00111001010--11110-100----0-210-11011210100010 Lysidice sp.1 1????0111002010--11010-100----0-210-11011210100010 Lysidice sp.2 3A3100111001010--11110-100----0-210-11011210100010 Nematonereis unicornis 000100B0---0010--10110-1C0----0-210-11011210000010 Euniphysa aculeata 100110111431011021100--10100--10211011011210110515 Euniphysa tridontesa 10?110111431011121100--10100--1021111101120-010515 310 Eunice norvegica 03?1001112210110211010-101010-10211111011210110010 Eunice cf. violacemaculata 0031001112200111211010-101010-10211111011210110010 Eunice roussaei 0001001112200111211010-101010-10211111011210110010 Eunice aphroditois 0331B01112200111311010-101010-10211111011210110010 Eunice filamentosa complexBR 3001001110110101111010-101010-10211111011210110010 Eunice filamentosa complexCBC 1031001110210101211010-101010-10211111011210110010 Eunice impexa 33??B01110010101211010-1C1010-1C211111011210110010 Palola sp. A7Pohnpei142 3331101110200102111010-101010-10211111011210101212 Palola viridis 30??B01110200102211010-101010-10211111011210101212 Palola siciliensis 33??1011140?0102011010-1C1010-1C211111011210101212 Palola sp.A1 30?1001110220102211010-101010-10211111011210101212 Palola sp.A9 3????01110220102211010-101010-10211111011210101212 Palola sp.A3 30??0011102201?2111010?101010-10211111011210101212 Palola sp.B1 3001101110220102211010?101010-10211111011210101212 Palola sp.B7 3031001110220102211010-101010-10211111011210101212 Palola sp.B5 3?01101110??0102211010-101010-10?11111011210101212 Faulchadius cyrtauloni 00000011130001?1211110-101010-10211011011210010??? Aciculomarphysa comes 0011001??00----111101--10101--10210-11011210---?1?

Characters 51-100 51 56 61 66 71 76 81 86 91 96 | | | | | | | | | | Glycera dibranchiata ----0----10--012110---01------0011-112----11111110 Paramphinome jeffreysii ----0----13-2101110---11------0???-1------11000011 Lumbrineris inflata -100100100-----10-----01------0100------11101111 Lumbrineris latreille -100100100-----20-----01------?100------11111011 Dorvillea erucaeformis ????1010-0-----2100---01------????---?56??1?11??11 Dorvillea sociabilis ????1010-0-----1100---01------0101----100111222012 Arabella semimaculata -00-101120-----00-----01------0000------11222211 Oenone fulgida -00-101120-----110----01-101000100----222211111111 Hyalinoecia sp. 310210110????2?210????01?20???????-0??40??1?00??11 Mooreonuphis pallidula -?021011010--11210100001-1000001?1-00-400011000011 Onuphis eremita -1001011010--1?11010--01??00??????-11?450?11000?11 Onuphis cf. iridescens -1021011010--3?110????01-?0???????10??40??1?00??11 Onuphis elegans -1021011010--3?11010??01-100??????023?455?11012?11 Diopatra dentata -1021011012-010210100001-10?0?????-0-?450?11000?11 Diopatra cf. ornata -1021011012-010110100001-100010111-1--450011000011 Paradiopatra quadricuspis -1021011011021?010????01-1000?????-2??44??1?00??11 Eunice antennata 210110110111111010100001-001001111-102100111000212 Eunice rubra 210110110111011010100001-B01001111-012400011022212 Eunice americana -11110110111000010101-01-301001111-0--405011000012 Eunice miurai -10110110111C10010100001-B01001011-0--101011000012 Eunice cf. limosa -10110110111200010100001-00?0?1???-00?045?11000?12 Eunice lucei 210110110111011010100001-B01001111-11-100011000012 Eunice antarctica 21B110110111C00010100001-B0100B111-00-100011000210 Eunice cf. antillensis 211110110111210010100001-101001011-1--045011000010 Eunice valens 211110110111B00010100001-10100B1?1-1--000111000012 Eunice harassii 211110110111001010100001-B010010??-00-000011000012 Eunice torquata 21111011011120101010000010110DB1?1-11-000111200212 Eunice marcusi 2111101101B02110101000001011021011-11-100111000212 Eunice thomasiana 2111101101112010101000001011010011-11-000111000010 Eunice fucata 2111101101110100101001006011020011-1--106011200212 Eunice cariboea 210C101100-----0101000006011020001----003311000012 Eunice cf. insularis 2102101100-----010101-00E0110D0011----004011000012 Eunice cincta ?????????0-----?1010??00?01???0??????????????????? Marphysa angeli 21011011010--21010100000401?0201??-00?101?11000?11 Marphysa cf. hentscheli 1101101101B02210101000006011020101-002000111000012 Eunice amoureuxi 21011011010--100101000002111020111-01-100011000011 Eunice mutilata 1101101101112110101000002110020001-11-100411200211 Eunice notata 11011011010--210101000006011020011--1-101111000010 Eunice mikeli 11011011010--21010100000?0110?0101--0-000011000012 Marphysa sanguinea 210110110111221010101-003011000101-00-400011000011 Nauphanta mossambica 2101101101112210101000004001100101-112450311000011 Marphysa brevitentaculata 3101101101112210101100000012B00111-10-401011000011 Marphysa disjuncta 210110110111120010100000?01?0?????-0-?405?11000?11 Marphysa fallax ?1011011010--D0010100001-0010101??-11?000?11000?11 Marphysa californica 3101101101B12210101000005011000111--0-400011000011 311 Marphysa viridis 2101101101112210101100004001000101-00-000311000011 Marphysa regalis 31011011011122B010110001-001010B11--1-001111000011 Marphysa bellii 310010110111120010101-01-F110?????-0-?405?11000?11 Lysidice ninetta 1100121120-----110100000?0000?????---?004?11000?11 Lysidice collaris 1100121120-----010100000?00???????---?004?11000?12 Lysidice sp.1 210012112??--??010100000??0???????--??11??1?00??11 Lysidice sp.2 1100121120-----0101000004010020111----000011200011 Nematonereis unicornis 1100121120-----1101000007000000101----004311000011 Euniphysa aculeata -0111011011022101010000043100?0111-112440011000010 Euniphysa tridontesa -0111011011021?010100000?21?0?????-11?445?11200?11 Eunice norvegica 111110110111111010100000?01?0??B??-11?045?11000?10 Eunice cf. violacemaculata 1111101101110110101000003B11020001-112400111000?12 Eunice roussaei 1111101101110D101010000041110?0011-11-000011000211 Eunice aphroditois 1111101101110110101000000?13B00001-002456011000012 Eunice filamentosa complexBR 0111101101112210101000006010020111--11405411000012 Eunice filamentosa complexCBC 0111101101112210101000004010020111-111445011000212 Eunice impexa 0111101101B022?010100000?G1?0?????-10?450?11000?10 Palola sp. A7Pohnpei142 -1101111110--220101000004-----0???--0-000311000012 Palola viridis -1101111110--22010100000?-----0???--1?100?11000?12 Palola siciliensis -1101111110--2?010100000?-----????--1?000?11000?10 Palola sp.A1 -1101111110--220101000004-----0111--1-004011000012 Palola sp.A9 -11011111??????0101?0000?-----????--??00??1?00??12 Palola sp.A3 -1101111110--2?010100000?-----????--1?014?11000?12 Palola sp.B1 -1101111110--220101000000-----0101--1-011111200012 Palola sp.B7 -1101111110--220101000004-----01?1--1-014311200011 Palola sp.B5 -1101111110--220101000004-----0111--1-104011000012 Faulchadius cyrtauloni ???1101??0-----0100---01-204000001----334301--2?0- Aciculomarphysa comes -?0?10???0------1010--01-4000000??????????????????

Characters 101-150 101 106 111 116 121 126 131 136 141 146 | | | | | | | | | | Glycera dibranchiata 002-00-????11????00--000?-?000?000000---?0000----- Paramphinome jeffreysii 0002222000011?02?00--000????33011111100000000----- Lumbrineris inflata 00000001---11----00--00000022??11011011--0000----- Lumbrineris latreille 00010221---11----00--1000?0000010011001--0000----- Dorvillea erucaeformis 0??43??1--?11--??00--11??--?0??00?00?--??00??--??- Dorvillea sociabilis 112----1---11----00--??????0???00011?22??0000----- Arabella semimaculata 00000011-??11-???00--1000-02222111011?1110000----- Oenone fulgida 00011001---11----00--0000??000011111122220000----- Hyalinoecia sp. 0??32??010?11-2??00--01??0022??11?11?11??01??-0??- Mooreonuphis pallidula 02232--010011-?2200--000000222200011111110010--0-- Onuphis eremita 00?322?011011--2?00--101?00222?000111111?011?-00?- Onuphis cf. iridescens 0??32??010?11-1??00--00??0022??00?11?10??01??-0??- Onuphis elegans 00?322?01?011-???00--100?00?22?000111000?111?000?0 Diopatra dentata 00?322?010011--2?00--000?00?22?010111111?011?-00?- Diopatra cf. ornata 002322-010011-02200--000000222200011100010111-000- Paradiopatra quadricuspis 0??32??010?11-1??00--01??0122??00?11?11??01??-0??- Eunice antennata 121----000011111100--11110000??0001111111111100000 Eunice rubra 011?1--000011001100--1111002?1D000111111111110000? Eunice americana 122----000011001211001111000?0200011111011110000-0 Eunice miurai 222----00001101D100--111100?10D00011111111110000-0 Eunice cf. limosa 12?---?000011001?1100111?00?20?0001?11?1???1???0?? Eunice lucei 222----00001110B200--11110000110001111111111100000 Eunice antarctica 121----000011111?00--111100-?00000111111111110000? Eunice cf. antillensis 222----000011111100--11110000000001111111111100000 Eunice valens 222-?--000011111100--1111000?000001111111111100001 Eunice harassii 122----000011111111001111000C0C0001111111111100000 Eunice torquata 221?---000011111200--111100?0000001110111111100000 Eunice marcusi 111----000011111100--111100?002000111111111110000? Eunice thomasiana 122--?-000011111200--111100000000011111111110000-0 Eunice fucata 111--?-000011111100--11110000000001111111111100001 Eunice cariboea 122----000011222200--0000--0?000001110011111100000 Eunice cf. insularis 122----000011002200--1000--?00000011110111?110?00? Eunice cincta ???????????11?????????????????????11?????111?????? Marphysa angeli 02?01-?000011102?00--100?000-0?000111101?111?000?0 Marphysa cf. hentscheli 122--?-000011222200--10000-000000011100111110000-0 312 Eunice amoureuxi 02211--000011?02200--?100?0?00?000111101?111?000?1 Eunice mutilata 1212---000011002200--11000-0000000111101111110000? Eunice notata 222----000011212200--1100-0??C00001111111111100000 Eunice mikeli 122--?-000011112200--11000000120001111011111100001 Marphysa sanguinea 1210---000011002000--000011?00000011100010111-0B0- Nauphanta mossambica 2211---000011033000--000011000011111100000110-11-- Marphysa brevitentaculata 2221---000011102200--10001000000001110011111100B1? Marphysa disjuncta 02?00-?000011002?00--001?11000?000111000?011?-00?- Marphysa fallax 01?00-?000011??2?00--110??-?00?000111001?111?000?1 Marphysa californica 02201--000011002200--11000000000001110011111100B10 Marphysa viridis 1220---000011021200--000000000000011100010110-0B-- Marphysa regalis 02201--000011002200--110000?0000001111111111100000 Marphysa bellii 02?00-?000011122?00--100?01000?000111000?111?000?0 Lysidice ninetta 12?1--?000011222?00--100?--000?000111111?111?000?1 Lysidice collaris 12?---?000011002?00--000?-?000?000111110?011?-00?- Lysidice sp.1 2??1-??000?11?2??00--10??--?0??00?11?11??01??-0??- Lysidice sp.2 02211--000011002200--1100--0000000111101?111?000?1 Nematonereis unicornis 1211---000011102200--1000--000000011110010010--00- Euniphysa aculeata 222----0000110022101100000-00?200011110110010--0-- Euniphysa tridontesa 22?1--?000011022?1011100?0-010?100111100?001?-00?- Eunice norvegica 22?---?000011111?00--111?00000?000111111?111?000?? Eunice cf. violacemaculata 122---?000011102211011111-?0?0200011100111110000-0 Eunice roussaei 1111---000011121?1101111100?00?0001111011111100000 Eunice aphroditois 222----0000111112110111110000000001111111111100001 Eunice filamentosa complexBR 222----000011102211011100--1112000111111011110000? Eunice filamentosa complexCBC 122-?--000011102210011100-??11200011111111110000-0 Eunice impexa 22?---?000011001?1101111?11000?000111111?111?000?? Palola sp. A7Pohnpei142 122----000011012200--1100--?000000111111?0000----- Palola viridis 12?---?000011201?00--011?--000?000111111?000?---?- Palola siciliensis 22?--??000011122?1001100?-0000?000111110?000?---?- Palola sp.A1 222---?000011122200--1100--0000000111100?0000----- Palola sp.A9 2??--??000?1111??00--11??--00??00?11?10??00??--??- Palola sp.A3 22?---?000011112?00--110?-?000?000111100?000?---?- Palola sp.B1 222?-?-000011112200--11100-000000011110000000----- Palola sp.B7 1221---000011?22200--1100--200000011110000000----- Palola sp.B5 222----000011122200--1000--000000011110000000----- Faulchadius cyrtauloni -1?-----00000--2?00----?-----0-000000----0000----- Aciculomarphysa comes ???????????11?????????????????????11?????000?-----

Characters 151-200 151 156 161 166 171 176 181 186 191 196 | | | | | | | | | | Glycera dibranchiata ------111?111???432?2210 Paramphinome jeffreysii ------0000------0 Lumbrineris inflata ------110011--0032--11-0 Lumbrineris latreille ------10001---0-3---1--0 Dorvillea erucaeformis -??--??--??--?--??---??-??-??-??11??11??1100??11?0 Dorvillea sociabilis ------111111111100001100 Arabella semimaculata ------0000----0-4---1--0 Oenone fulgida ------0000------0 Hyalinoecia sp. 1??-0??-2??-0?-1??0-1??-??-??-??00??--??----??--?0 Mooreonuphis pallidula -1---0---?---1--1-0--1------10011--10-2--00--0 Onuphis eremita 01?-00?-22?-11-?2?0-01?------100?1--?0-D--?0--0 Onuphis cf. iridescens ???-0??-1??-1?-2??0-1??-??-??-??10??1-??0-2-??0-?0 Onuphis elegans 01???0?222?000222?0001?--?--?--?100?1--?0-2--?0--0 Diopatra dentata 00?-00?-22?-00-22?0-00?--?--?--?100?1--?0-1--?0--0 Diopatra cf. ornata 111-011-112-00-2220-111------10001---0-1---0--0 Paradiopatra quadricuspis 1??-1??-2??-??-0??0-???-??-??-??10??1-??0-D-??0-?0 Eunice antennata 01100002222000111101000------1111111111000B0000 Eunice rubra 010?0002222?00111101111------111111111110010000 Eunice americana 00-?00-?22-?00111-0?01------111111111100BB0000 Eunice miurai ??-??0-2?2-???1?1-0??1------111111111100B10000 Eunice cf. limosa ??????????????????0?????-??-??-?111??11??1?00??000 Eunice lucei 01100002221000?1110?1?0------111111111111000000 Eunice antarctica 110?0002222?00111101011------111111111111000000 Eunice cf. antillensis 01100002211000111101111------111111111111000000 Eunice valens 11100002222000111100000------111111111111000000 Eunice harassii 000?0002222?00111101110------111111111100000000 313 Eunice torquata 00000001222000111100110------111111111111000000 Eunice marcusi 001?000?222?00?1110??11------111111111111000000 Eunice thomasiana 00-000-211-000111-00??------111111111111000000 Eunice fucata 11100002212000111101111------111111111111?00000 Eunice cariboea 11100000011000111101110------111111111100000000 Eunice cf. insularis ?01??002?11??0??110??11------111111111100000000 Eunice cincta ???????????????????????------111?111?11000????0 Marphysa angeli 01?000?211?000111?0111?--?--?--?111?111?11000?0000 Marphysa cf. hentscheli 11-000-101-000011-0111------111111111100000000 Eunice amoureuxi ?1?0?0?211?0?0111?0110?--?--?--?111111111100000000 Eunice mutilata 111?000?111?00?1110??01------111111111100000000 Eunice notata ?110?0011110?0111101111------111111111100000000 Eunice mikeli 1110000211?000001101110------111111111100000000 Marphysa sanguinea 111-000-102-00-0000-110-0--1--0-111111111143220000 Nauphanta mossambica ------0----1B-00-00-0000------0 Marphysa brevitentaculata 11???1?110???0?00?0?11--00-11-?01111111111432?0000 Marphysa disjuncta 00?-00?-11?-00-00?1-00?--?--?--?111?111?1143C?0000 Marphysa fallax 11?000?111?000000?0001?--?--?--?111?111?11HA0?0000 Marphysa californica 11-001-110-000000-0?11--00-01-01111111111143220000 Marphysa viridis 11--00--10--00-00-0-01--0--1--0-1111111111432C0000 Marphysa regalis 10100001200000000000001------111111111100000000 Marphysa bellii 00?000?101?000000?1001?--?--?--?111?111?11HA0?0000 Lysidice ninetta 11?000?221?B00112?000B?------111?111?11000?0000 Lysidice collaris 11?-0??-11?-0?-?1?0-?1?--?--?--?111?111?11000?0000 Lysidice sp.1 1??-0??-1??-0?-1??0-1??-??-??-??11??11??1100??00?0 Lysidice sp.2 11?000?211?000111?0111?--?--?--?111111111100000000 Nematonereis unicornis -11--00--1?--0--000--00------111111111100000000 Euniphysa aculeata -0---0---1---0--1-0--1------11110111-143223000 Euniphysa tridontesa 00?-?0?-11?-00-?1?0-10?--?--?--?011?-11?-1-3C?-000 Eunice norvegica 00??00?222??00?11?0?11?--?--?--?111?111?11000?0000 Eunice cf. violacemaculata 00-000-111-000111-0111------111111111100000000 Eunice roussaei 000?0012111?00111100000------111111111100000000 Eunice aphroditois 111000021110001?2100000------111111111100000000 Eunice filamentosa complexBR 011?000?111?00?1110?111------111111111154000000 Eunice filamentosa complexCBC 00-000-221-000111-0111------111111111154000000 Eunice impexa 00?000?221?000111?0110?--?--?--?111?111?11540?0000 Palola sp. A7Pohnpei142 ------111?111?11000?0000 Palola viridis --?---?---?------?----?------111?111?11000?0000 Palola siciliensis --?---?---?------?----?--?--?--?111?111?11000?0000 Palola sp.A1 ------111111111100000000 Palola sp.A9 -??--??--??--?--??---??-??-??-??11??11??11?0??00?0 Palola sp.A3 --?---?---?------?----?--?--?--?111?111?11000?0000 Palola sp.B1 ------111111111100000000 Palola sp.B7 ------111111111100000000 Palola sp.B5 ------111111111100000000 Faulchadius cyrtauloni ------0000------1 Aciculomarphysa comes ------000??????????????0

Characters 201- 232 201 206 211 216 221 226 231 | | | | | | | Glycera dibranchiata 000------115051-???1 Paramphinome jeffreysii 000------00----????1 Lumbrineris inflata 000------00----????- Lumbrineris latreille 000------00----0000- Dorvillea erucaeformis 0??-??--??--??--?????1?02??????1 Dorvillea sociabilis 000------112223????1 Arabella semimaculata 000------00----????- Oenone fulgida 011-II--11--111-00---00----00001 Hyalinoecia sp. 1??1??-1??-1??10?????1?11??????0 Mooreonuphis pallidula 111111-?1?-?0?1?B0---1061--????1 Onuphis eremita 11?11?-11?-11?111?---1061-?220?? Onuphis cf. iridescens 1??1??-1??-1??1B?????1?61??????1 Onuphis elegans 11?11?-11?-11?1BB?--?1061-???1?1 Diopatra dentata 11?11?-11?-11?1BB?--?1011-???2?1 Diopatra cf. ornata 111111-?11-?111BB1---1061--????1 Paradiopatra quadricuspis 1??1??-1??-1??1B?????1?61???2??1 314 Eunice antennata 011-22--?1--?01-11---11032000001 Eunice rubra 011-22--11--111-00--?1163840???1 Eunice americana 011-22--11--111-00---116314221?1 Eunice miurai 011-22--??--??1-1?---11232222221 Eunice cf. limosa 01?-2?--??--??1-0?--?11438?1?2?1 Eunice lucei 011-22--11--111-11---11038222221 Eunice antarctica 011-11--1?--1?1-10---11234221?21 Eunice cf. antillensis 011-11--1?--0?1-11---113384?1??1 Eunice valens 011-11--11--001-00---11033000001 Eunice harassii 011-11--11--111-00---116382222?1 Eunice torquata 011-11--11--001-00110113300?2221 Eunice marcusi 011-11--11--001-00DD211038222??1 Eunice thomasiana 011-11--11--001-00D22110332212?1 Eunice fucata 011-1B--01---01-0022-11050223221 Eunice cariboea 011-11--11--001-1122-1104432?0?1 Eunice cf. insularis 011-11--1?--1?1-002C0110343????1 Eunice cincta 01?-1?????????????0????????????? Marphysa angeli 01?-1?--1?--1?1-0?00?11034?212?1 Marphysa cf. hentscheli 010-1---1---0-1-1-00-11030311101 Eunice amoureuxi 011-11--1?--0?1-002C-1133432?0?1 Eunice mutilata 011-11--??--??1-000C011034311101 Eunice notata 011-11--1?--1?1-0000-110432222?1 Eunice mikeli 011-11--??--??1-0011?11333111001 Marphysa sanguinea 011-11--1?--0?1-0?11111033500021 Nauphanta mossambica 011-11--1?--0?--0?---11044000??1 Marphysa brevitentaculata 011-00--00------0111-110373?21?1 Marphysa disjuncta 01?-B?--1?--1?1-0?11?11161?111?1 Marphysa fallax 01?-1?--??--??1-1?--?1?03??22??0 Marphysa californica 011-11--1?--0?1-0011-110330??2?1 Marphysa viridis 010-1---?---?---0----11037321221 Marphysa regalis 011-00--00------00---1103752???1 Marphysa bellii 01?-1?--1?--0?1-0?11?11344??01?1 Lysidice ninetta 11?11?-11?-?1?101?-1?11034?220?0 Lysidice collaris 11?11?-11?-?1?100?--?11033??11?0 Lysidice sp.1 1??1??-1??-1??10?????1?03???0??0 Lysidice sp.2 111111-111-111110011-110333??2?0 Nematonereis unicornis 111111-111-?111111---11431322020 Euniphysa aculeata 111111-111-?01100011?11164011121 Euniphysa tridontesa 11?11?-11?-11?100?D0?11764?211?1 Eunice norvegica 01?-1?--1?--0?1-0?11?11038?211?1 Eunice cf. violacemaculata 011-11--11--001-0?11-11063220221 Eunice roussaei 011-11--??--??1-01111116684????1 Eunice aphroditois 001--1---?---?1--0--1110465????1 Eunice filamentosa complexBR 111111-111-0001000111116634?2??1 Eunice filamentosa complexCBC 111111-111-?00101?10-11368211101 Eunice impexa 01?-1?--??--??1-0?B0?11063?22??1 Palola sp. A7Pohnpei142 000------112622211?0 Palola viridis 00?--?---?---?---?--?11264?111?0 Palola siciliensis 00?--?---?---?---?--?11067??22?0 Palola sp.A1 000------110611?2??0 Palola sp.A9 0??-??--??--??--?????1?03??2???0 Palola sp.A3 00?--?---?---?---?--?11634?0???0 Palola sp.B1 000------110622222?0 Palola sp.B7 000------110622?2??0 Palola sp.B5 000------1106122?2?0 Faulchadius cyrtauloni 111111111100000???---1107430???? Aciculomarphysa comes 111111??????????????????????????

315

Appendix 8: Characters optimized on the branches of the morphMAFFT maximum parsimony strict consensus tree using maximum parsimony criterion and the optimization mode (unambiguous, ACCTRAN or DELTRAN) that increased unique origins and secondary losses as opposing to convergences. Hash marks are character transformations, number above is the character number and below the character state it transforms to at that branch. Black circle hash mark, nonhomoplasious transformation. White square hash marks, homoplasious transformation.

316 A.

317 B.

318 C.

319 D.

320 E.

321

Appendix 9: Characters optimized on the branches of the morphClustal maximum parsimony strict consensus tree using maximum parsimony criterion and the optimization mode (unambiguous, ACCTRAN or DELTRAN) that increased unique origins and secondary losses as opposing to convergences. Hash marks are character transformations, number above is the character number and below the character state it transforms to at that branch. Black circle hash mark, nonhomoplasious transformation. White square hash marks, homoplasious transformation.

322 A.

323 B.

324 C.

325 D.

326 E.

327 Appendix 10: Key to genera of the family Eunicidae, with exception of Nauphanta.

Nauphanta is not included in the key because at this time there are known diagnostic features for the genus (see discussion for Nauphanta in the taxonomic accounts section of

Chapter 4). Features underlined are unique to the species within that genus, but may not be present in all species.

1. Palps absent. Mandibles curved, poorly calcified, anterior outer edge and carriers

from opposite sides diverging from each other, X shaped. Lysidice

Palps present (absent in juveniles). Mandibles flat or strongly curved, forming an

open scoop, anterior end strongly calcified, anterior outer edge and carriers from

opposite sides parallel to each other. 2

2. Mandibles strongly curved, forming an open scoop, plates fused anteriorly.

Subacicular hooks absent. Palola

Mandibles flat. Subacicular hooks present. 3

3. Limbate chaetae absent. Subacicular hook guards as disassociated fibrils (Fig. 23D)

Fauchaldius

Limbate chaetae present. Subacicular hook guards solid, sheet-like (Fig. 23A-

C,E,F). 4

4. Peristomial cirri absent. MxVI always absent. 5

Preristomial cirri present. MxVI present or absent. 7

5. Chaetae absent in first parapodium. Pectinate chaetae absent. Aciculomarphysa

Chaetae present in first parapodium. Pectinate chaetae present. 6

328 6. True compound chaetae falcigerous and bidentate only. Subacicular hooks dark,

bidentate, with darkest shade closest to the distal end. Wide pectinate chaetae

absent. Nicidion

True-compound chaetae either falcigerous and bidentate or spinigerous. Subacicular

hooks light or dark, falcate or bidentate. In dark subacicular hooks, darkest shade

closest to the proximal end. Wide pectinate chaetae (Fig. 21G,H) present in some

species. Marphysa

7. MxIII, IV and V fang like, with slender pointed tips. Euniphysa

MxIII and IV with short triangular pointed teeth. MxV plate like, a small tooth may

be present. 8

8. Antennae and palps regularly articulated Leodice

Antennae and palps irregularly articulated or smooth. 9

9. Aciculae light. Subacicular hooks light, bi- or tridentate. Leodice

Aciculae dark. Subacicular hooks dark, falcate or bidentate. 10

10. Lateral black dot between posterior parapodia present Leodice

Lateral black dot between posterior parapodia absent 11

11. MxVI absent Nicidion

MxVI present (Fig. 10B,C) Eunice

329