Tubificids with trifid chaetae: morphology and phylogeny of

Heterodrilus (Clitellata, Annelida)

Erica Sjölin

Department of Zoology Stockholm University

2007

Doctoral dissertation 2007 Tubificids with trifid chaetae: morphology and phylogeny of Heterodrilus (Clitellata, Annelida) Erica Sjölin Department of Zoology Stockholm University SE-106 91 Stockholm Sweden and Department of Invertebrate Zoology Swedish Museum of Natural History Box 50007 SE-104 05 Stockholm Sweden ©Erica Sjölin, Stockholm 2007 ISBN 978-91-7155-533-5 Cover illustration of Heterodrilus minisetosus by Wim Willems and Tobias Kånneby Printed in Sweden by US-AB, Stockholm 2007 Distributor: Department of Zoology, Stockholm University

To Ulf and Ninn

LIST OF ORIGINAL PUBLICATIONS AND MANUSCRIPTS This thesis is based on the following papers, referred to in the text by their Roman numerals: I. SJÖLIN, E. AND ERSÉUS, C. 2001. New species of Heterodrilus

(Oligochaeta, Tubificidae) and records of H. maiusculus from the Mediterranean Sea. Italian Journal of Zoology, 68: 223-228.

II. SJÖLIN, E., ERSÉUS, C. AND KÄLLERSJÖ, M. 2005. Phylogeny

of Tubificidae (Annelida, Clitellata) based on mitochondrial and nuclear sequence data. Molecular Phylogenetics and Evolution, 35: 431-441.

III. SJÖLIN, E. AND GUSTAVSSON, L.M. 2007. An ultrastructural

study of the cuticle in the marine Heterodrilus (Tubificidae, Clitellata). Journal of Morphology, in press.

IV. SJÖLIN, E., JOHANSSON, U.S. AND ERSÉUS, C. Tubificids with

trifid chaetae: a phylogeny of the genus Heterodrilus (Clitellata, Annelida). Manuscript.

This thesis is not to be regarded as a publication in the sense of the International Code of Zoological Nomenclature (ICZN, 1999), and scientific names mentioned in it should not be cited in any form. Parts of this thesis, including the manuscript paper, must not be reproduced without permission.

CONTENTS Introduction……………………………………………………………………...9 Setting the Stage………………………………………………………………..10 Tubificidae……………………………………………………………………...10 The Subfamilial Classification of Tubificidae……………………11 Some Basic Tubificid Morphology…………………………………………….12 Chaetae……………………………………………………………13 Epidermis and Cuticle…………………………………………….14

Muscles…………………………………………………………...14 Alimentary Canal…………………………………………………15 Nervous System and Sensory Organs…………………………….16 Peritoneum and Septa……………………………………………..16 Coelomocytes……………………………………………………..16 Vascular System…………………………………………………..17 Excretory System…………………………………………………17 Reproductive System……………………………………………..17

Heterodrilus……………………………………………………………………19

Descriptive and Taxonomic Work……………………………….19 Phylogeny of Heterodrilus……………………………………….20

The Trifid Anterior Chaetae……………………………………...21 Inter- and Intraspecific Variation?……………………………….22 Quo Vadimus?……………………………………………………23 Swedish Clitellate Scientists…………………………………………………..24 Summary of the Included Papers………………………………………………26 Acknowledgements……………………………………………………………28 References……………………………………………………………………..30 Appendix………………………………………………………………………38

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INTRODUCTION

My first memory of worms was when I was three years old. It was a rainy day and I was trying to save all the worms that crawled on the streets by picking them up and put them in a bucket. I do not remember what I did with all the worms when the rain stopped pouring, although I do remember my mother not being so keen to snuggle me, for the obvious reason that I had “saved” some of the worms by putting them in my mouth. For most people the word “worm” is used to describe a legless, crawling critter. Some of the “worms” people encounter are cabbageworms, cutworms, tomato worms and parsley worms, which are all moth or butterfly caterpillars, but also roundworms, tapeworms and ribbon worms which are interesting creatures in their own right, but maybe the quintessential “worms” to most people are earthworms, the familiar annelid of fishhooks, soils and sidewalks after rain. During the 18th century there was a great expansion of natural history knowledge, but it was first with the naturalist Carl Linnaeus that conventions for the naming of living organisms became universally accepted in the scientific world. Linnaeus attempted to describe the entire known natural world and gave every species (mineral, plant, animal) a two-part name in Latin, where the first part indicates to which more inclusive group the organism belongs and the two names together denote the species. Linnaeus also dumped all pliable, legless animals (e.g. jellyfish, corals, molluscs, snakes, polychaetes, echinoderms) into a single category, Vermes, but it became soon apparent that this potpourri of animals was not at all closely related. Linnaeus removed snakes from Vermes to a more sensible location with other vertebrates and Lamarck (1802) recognized the segmented nature of a large collection of the remaining worms, creating the taxon Annélides (Annelida). Today researchers in biological systematics group organisms according to common descent, and scientists have since long divided Vermes among various branches of the family tree of life; currently, however, great rearrangements in the classification of the Animal Kingdom are proposed due to new discoveries using DNA data. The main focus of this thesis is a group of annelids called Heterodrilus. The aim is to increase our knowledge about the morphology of this group, their systematic position, and of the phylogenetic relationships between its members. To put this taxa in a wider context I will start by introducing the more inclusive groups to which Heterodrilus belongs.

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SETTING THE STAGE The most recognizable feature of annelids, beside their shape, is the segmented nature of their bodies. The segmentation is not only an external feature, but is also seen internally in the repetitive arrangement of organs and in the partition into segments by septa. Annelida is a substantial group of animals with over 15,000 described valid species, and until recently it was split into three major groups: Polychaeta (bristleworms), Oligochaeta (earthworms etc.), and Hirudinida (leeches). The division of Annelida is currently being revised. In recent years it has become recognized that Hirudinida is nested within Oligochaeta, hence, together forming Clitellata: molecular studies (Siddall and Burreson, 1998; Siddall et al., 2001; Martin, 2001; Erséus and Källersjö, 2004) have shown that Acanthobdellida (the Arctic salmon parasite, Acanthobdella peledina) and Branchiobdellida (leech-like commensals or parasites on crayfish) share a recent common ancestor with leeches, which together form the sister group to the oligochaetous family Lumbriculidae. The monophyly of Clitellata is well supported by morphological (Purschke et al., 1993; Westheide, 1997) and molecular data (Moon et al., 1996; Kim et al., 1996; McHugh, 1997, 2000; Winnepenninckx et al., 1998; Kojima, 1998; Martin et al., 2000; Erséus et al., 2000; Martin, 2001; Siddall et al., 2001). There is increasing evidence that along with Echiura (spoon worms) and Siboglinidae (beard-worms), Clitellata may well be nested within Polychaeta (e.g., McHugh, 1997; Westheide et al., 1999; Struck et al., 2002; Bleidorn et al., 2003; Jördens et al., 2004). Oligochaetous clitellates are most familiar as terrestrial or freshwater animals; however, many marine species have been discovered and described in the past 30 years. There are about 5,000 valid species of oligochaetous clitellates, of which 1,600 are aquatic or semi-aquatic, and about 600 of the aquatic forms are marine (Erséus, 2005). The most speciose aquatic family is Tubificidae, with about 800 described limnic and marine species worldwide.

TUBIFICIDAE Aquatic tubificids occur throughout the world wherever suitable habitats exist. In freshwater habitats, most species burrow in bottom debris, a few construct tubes, and some live on aquatic plants. In marine habitats most species live interstitially among sand grains, are shallow burrowers or live beneath intertidal

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rocks. Tubificids are most abundant where the water is shallow, although some have been reported from deep-sea trenches, e.g., Bathydrilus hadalis, which is known from over 7000 m depth. Some freshwater forms, e.g. Tubifex tubifex, Limnodrilus hoffmeisteri and Branchiura sowerbyi, are highly tolerant to organic pollution, but the majority of tubificids are adapted to more specific habitats (Erséus, 2005).

THE SUBFAMILIAL CLASSIFICATION OF TUBIFICIDAE Brinkhurst and Jamieson (1971) recognized seven subfamilies within the Tubificidae: Aulodrilinae, Branchiurinae, Clitellinae, Phallodrilinae, Rhyacodrilinae, Telmatodrilinae and Tubificinae. Three of them were later merged with other subfamilies: Aulodrilinae and Clitellinae with Tubificinae (Brinkhurst and Baker, 1979; Giani et al., 1984), and Branchiurinae with Rhyacodrilinae (Baker and Brinkhurst, 1981). Limnodriloidinae was established by Erséus (1982) for a number of marine species, all characterized by morphological modifications of the oesophagus in segment IX (Gustavsson and Erséus, 1999a) and a unique arrangement of the prostatic tissue associated with the male genital ducts (Gustavsson, 2002). In 1986, Finogenova proposed Nootkadrilinae for two marine genera described by Baker (1982), but they were later considered as (morphologically derived) members of Phallodrilinae (Erséus, 1990a, 1992). Due to the limited amount of morphological characters available the subfamilial definitions deal with only three principle characters, i.e. the presence or absence of (numerous) coelomocytes, the arrangement of the prostate glands, and the presence or absence of specialized sperm aggregates, termed “spermatozeugmata” (Erséus, 1984, 1990a; Finogenova, 1986). In recent years molecular markers have been used for the assessment of internal phylogeny of Tubificidae. For instance, partial sequences of the nuclear 28S rRNA gene and the mitochondrial COI gene (Christensen and Thiesen, 1998), and the 18S rRNA gene (Erséus et al., 2000, 2002), and the 18S rRNA gene combined with the 16S rRNA gene (Paper II), have supported the hypothesis, suggested by morphological data (Erséus, 1987, 1990a: Brinkhurst, 1994; Ferraguti et al., 1999), that Naididae, another clitellate family, is nested within Tubificidae. Thus, the former Naididae was recently proposed as a sixth subfamily (Naidinae) of Tubificidae (Erséus & Gustavsson, 2002; see below).

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Classification proposed by Erséus and Gustavsson 2002:

Family TUBIFICIDAE Vejdovský, 1876 Subfamily NAIDINAE Ehrenberg, 1828 Subfamily TUBIFICINAE Vejdovský, 1876 Subfamily TELMATODRILINAE Eisen, 1885 Subfamily LIMNODRILOIDINAE Erséus, 1982 Subfamily PHALLODRILINAE Brinkhurst, 1971 Subfamily RHYACODRILINAE Hrabe, 1963

SOME BASIC TUBIFICID MORPHOLOGY Below is a section about the general morphology of a tubificid. The text is a compilation of information from Stephenson (1930), Brinkhurst and Jamieson (1971), and Jamieson (1981, 1992). Information from other sources is referred to in the text.

Figure 1. General appearance of a marine tubificid worm. (Modified from Cook & Brinkhurst

1973.)

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Tubificids are small, elongate, cylindrical aquatic worms. They vary greatly in length (1-185 mm) but are typically 4-20 mm long, an average marine species being smaller than a typical freshwater one (although there are exceptions, some freshwater representatives of Chaetogaster are less than one millimeter long). The number of body segments varies, both inter- and intra-specifically, ranging from about ten in some Naidinae to several hundreds in other tubificids. The prostomium (Fig.1) is at the anterior end of the body, followed by the first segment, the peristomium, where the mouth is located ventrally. The body then continues with more or less similar segments and ends with the pygidium, which bears the anus. A feature of sexually mature oligochaetes is the possession of a clitellum (= a glandular, epidermal structure, which develops in the shape of a ring or saddle around a specific part of the body). In tubificids, the clitellum is one cell thick and encircles the segments where the genital pores are located; its length varies between 2 to 3 segments. Other visible features in some species are pigmentation (e.g., Branchiodrilus menoni), a prolongation of the prostomium (e.g., Stylaria lacustris), a papillated body wall (e.g., Duridrilus tardus, Tubificoides benedii, and Spirosperma ferox), and possession of gills (branchiae) (e.g., Branchiura sowerbyi;) or eyes (e.g., Ripistes parasita).

CHAETAE

A distinctive feature of tubificids is stiff bristles called chaetae. Chaetae are composed of ß-chitin arranged in thin-walled cylinders, and held together by a proteinoid part (Avel, 1959; Dennell, 1949). They are produced by a microvillar border of certain invaginated epidermal cells and so can be defined as cuticular structures that develop within epidermal follicles. In tubificids the chaetae are usually symmetrically placed in two ventral and two dorsal bundles in each segment except the peristomium, which is devoid of chaetae and the genital region where the ventral chaetae may be absent or modified into genital chaetae. Each chaetal bundle may contain from one to up to twenty chaetae, and as a rule there are more chaetae in the anterior bundles than in those posterior to the clitellum. There are basically two types of chaetae: hair chaetae and sigmoid chaetae. The hair chaetae are thin, slender structures, and occur only in the dorsal bundles of some species, whereas the sigmoid chaetae are s-shaped with a nodulus in the middle part. The distal end of the sigmoid chaetae may be single-pointed, bifid, trifid or pectinate (comb-like). Sigmoid chaetae occur in both dorsal and ventral bundles of tubificids.

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EPIDERMIS AND CUTICLE

An epidermis and an overlying cuticle limit the tubificid body. The epidermis is a single-layered epithelium that is composed of several cell types: most common are supporting cells but there are also gland cells as well as sensory cells present. The clitellum is a specialization of the epidermis, which is developed in the region of the gonads at sexual maturity for production of the cocoon and secretion within this. Although the clitellum may be several times thicker than in the rest of the epidermis, it is still only one cell thick and characterized by the presence of a high proportion of glandular cells.

The cuticle is a flexible, non-cellular, outer covering of the tubificid body. It is secreted by the epidermal cells (Burke and Ross, 1975, Humphreys and Porter, 1976) and consists mainly of collagen and polysaccharides (Maser and Rice, 1962; Watson, 1958). The cuticle comprises several parts (see Paper III). Closest to the epidermis is a comparatively thick zone of collagen fibres surrounded by a matrix. The matrix continues outside the fibre zone and forms an epicuticle. The external surface of the epicuticle is covered by evenly distributed ellipsoid bodies termed epicuticular projections, with their longitudinal axis perpendicular to the cuticular surface. Different extensions occur from the epidermal cells e.g. long microvilli, which penetrate and sometimes pass completely through the cuticle, and short knobs, which anchor the cuticle to the epidermis.

MUSCLES

Beneath the epidermis there are two layers of muscles fibres; a thin outer layer consisting of fibers with their long axes concentric to the long axis of the worm, forming the circular muscle, and a thicker inner layer of longitudinal fibers (Fig. 2). In addition to the musculature of the body wall there are muscles associated with the chaetae, around the intestine, spermathecae, and genital ducts, and in the septa, and the walls of blood vessels.

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Figure 2. Tubificid anatomy, cross section through trunk. (Modified from Brown 1950.)

ALIMENTARY CANAL

The alimentary system of most tubificids is more or less a straight tube running from the mouth, situated ventrally on the first segment, through the full length of the worm to the anus on the pygidium. The alimentary tube is regionally specialized to perform several functions. The mouth leads into a dorsally thickened pharynx, which acts like a suction cup or has an adhesive surface, helping the mouth to draw in organic matter. Associated with the pharynx are the pharyngeal glands, which are chromopilic cells. Posterior to the pharynx, the gut narrows to become the thin-walled oesophagus, which widens posteriorly and becomes the intestine. The posterior intestine continues without any changes to the anus. The intestine is covered with chlorogogen tissue throughout most of its length. The anterior half of the intestine is the region that is specialized for enzyme secretion and digestion of food particles, the posterior part is primarily absorptive. There are however different deviations from this general morphology. For instance, in members of the subfamily Limnodriloidinae, a modification of the oesophagus, usually in segment IX, may occur. In this segment, the alimentary canal either has a swollen part or a pair of anteriorly directed diverticula, or is dilated, forming a barrel-shaped portion with a clear blood plexus (Gustavsson and Erséus, 1999a). There are even two marine genera (Olavius and Inanidrilus) lacking a normal alimentary system, but

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instead thriving in association with endosymbiotic chemoautotrophic bacteria (e.g., Giere, 1981; Dubilier et al., 1999).

NERVOUS SYSTEM AND SENSE ORGANS

The nervous system consists of two pairs of ganglia located one above the other, forming a brain dorsal to the buccal cavity in the first segment. A number of prostomial nerves are given off anteriorly, most of them having a sensory function. A pair of circumpharyngeal connectives exit the brain, one on each side, and extend around the pharynx to join the ventral paired nerve cord at the subpharyngeal ganglia in segment II. The ventral nerve cord extends posteriorly through all body segments and in each segment except the first two; it gives rise to four pairs of segmental nerves. Three of these pairs extend laterally into the body wall where they unite dorsally; forming complete nerve rings, and the fourth pair terminates close to the dorsal chaetae. A lateral nerve, which unites with each of the segmental nerves in a series of small ganglia located in the lateral line, was reported by Isossimoff (1926). Some fibers in these nerves are sensory in function and detect stimulation of the body surface. Other fibers are motor in function and innervate circular muscles, longitudal muscles, or chaetal muscles in the body wall. Paired pigment-cup ocelli are present at the anterior end of some Naidinaes. Ciliated sensory cells are scattered throughout the epidermis (Gustavsson, 2001). Sensory buds, consisting of a group of cilia, were first described for lumbricids (an earthworm family), but have also been reported for the tubificids, Limnodrilus hoffmeisteri and Tubifex tubifex (Smith, 1983).

PERITONEUM AND SEPTA

The peritoneum is membrane that lines the coelomic cavity. It is composed of a layer of mesothelium supported by a thin layer of connective tissue. The coelomic cavity is divided into a number of paired segmental sacs by transversely placed septa and sagitally placed mesenteries. A septum is composed of three layers: a layer of muscle fibres lies between layers of peritoneal epithelium.

COELOMOCYTES

Free cells that circulate in the coelomic fluid are collectively called coelomocytes. They originate from the peritoneum and are granular, spherical to ovoid cells. There is a clear differentiation in the structure of coelomocytes

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associated with their functions, such as phagocytosis (Dales and Kalac, 1992), wound healing (Parry, 1975), and immune reactions (Dales, 1978; Cooper, 1996). In tubificids, coelomocytes have been described from rhyacodrilines (Nomura, 1915) and naidines (Sperber, 1948, Envall et al., submitted manuscript).

VASCULAR SYSTEM

The basic plan of the vascular system is fairly uniform throughout tubificids. A large dorsal blood vessel, usually close to the gut, runs the entire length of the body. It divides anteriorly in the region of the brain, joining two ventral vessels in the first segment. In some tubificids e.g. Aktedrilus monospermathecus and Thalassodrilus prostatus, the dorsal blood vessel is replaced by a non-contractile sinus (Giere, 1983). The two ventral vessels unite in one of the pregenital segments, and the remaining vessel runs dorsal to the nerve cord (but ventral to the gut) throughout the length of the body. In each segment except the first, the dorsal and ventral vessels are connected either by connectives, or indirectly in postgenital segments by a blood vessel plexus, which consists of a fine capillary network close to the gut surface. The blood flows anteriorly in the dorsal vessel, posteriorly in the ventral vessel, dorsally through the alimentary plexus and the connectives. This general system can be modified; e.g. the connectives may be pulsatile, and secondary dorsal and ventral vessels may be present.

EXCRETORY SYSTEM

The excretory system basically consists of one pair of nephridia in each segment except a few anterior and genital segments, the nephridia may be absent or poorly developed on one side of the body. Each nephridium consist of a ciliated funnel, a short canal which penetrates a septum, and a looped or coiled tube which opens to the exterior by a small pore located near the ventral chaetae of the segment posterior to that bearing the funnel.

REPRODUCTIVE SYSTEM

Tubificids are hermaphroditic, their genital organs contain both male and female parts (Fig. 3). The male genitalia consist of a pair of testes, male funnels, vasa deferentia and atria with or without prostate glands. The testes are located ventrally on the anterior septum of their segment. The posterior septum in the same segment may posses pouches, the sperm sacs, which contain spermatogonia in different stages and mature spermatozoa. A pair of sperm

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Figure 3. Schematic, lateral view of genital organs in a tubificid worm. Segment numbers

indicated by Roman numerals. (Modified from Erséus 1980.)

funnels are located on the posterior septum of the testicular segment where they open into the coelom. Each funnel continues posteriorly as a vas deferens, passing through the septum and forming a more or less coiled tube before it connects to a pair of atria, or sometimes a single atrium. The atria are variable in size and form. In general, they are tubular structures that open to the exterior as a simple pore or via a modified copulatory organ (penis or pseudo-penis). Externally, the atrium may be covered with prostatic cells, forming diffuse prostate glands or more or less stalked, compact prostate glands. The prostate glands have been shown to have different ontogenetic origins in different taxa, the compact prostate glands in the subfamily Tubificinae have an ectodermal origin, and the diffuse prostate glands in the subfamilies Rhyacodrilinae, Naidinae and Limnodriloidinae have a mesodermal origin (Gustavsson and Erséus, 1997, 1999b; Gustavsson, 2002). The female system consists of paired ovaries that are attached to the anterior septum of their segment, and usually a pair of ducts in the posterior part of the ovarian segment. Each duct begins with a funnel and continues posteriorly as a short duct that opens to the exterior through a small pore. Egg sacs or ovisacs, similar in structure and function to the sperm sacs, are usually present in the posterior septum of the ovarian segment.

During copulation, ventral contact is established between the anterior ends of the two oppositely facing worms. Spermatozoa are transferred from one

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individual to the spermathecae of another where they are preserved until each recipient worm’s eggs are mature, and the cocoons are formed. The spermathecae are (usually) paired organs each consisting of an ampulla and a duct; in a few species the spermathecae are either single, opening ventrally or dorsally, or absent. The worm then secretes a cocoon into which it deposits its eggs and the sperm from its mate; fertilization and development of the eggs occur in the cocoon. When the young emerge they are miniatures of the adults. The cocoon is secreted by the clitellum, which is developed over a few segments in the region of the gonads, in tubificids it usually covers the second half of segment X, and the whole of XI and XII.

HETERODRILUS The exclusively marine taxon Heterodrilus Pierantoni, 1902 includes 42 described valid species. The worms are small to medium sized tubificids (3-25 mm) and occur interstitially in coarse coralline sandy sediments from the intertidal zone down to about 150 m depths. The taxon has been recorded from localities in the North-west Atlantic Ocean (including the Caribbean), the Galapagos Islands, the Mediterranean Sea, and the Indo-Pacific Region.

DESCRIPTIVE AND TAXONOMIC WORK

The taxon was established for the sublittoral Heterodrilus arenicolus Pierantoni, 1902, which was found in the Bay of Naples, Italy. Brinkhurst (1966) included H. arenicolus in Clitello Savigny, 1820, but this was questioned by Hrabe (1967) because of differences in the male genitalia, and a decade later the species was transferred back to Heterodrilus (Brinkhurst and Baker, 1979). The type material of H. arenicolus is missing and the description of the species is incomplete (Erséus, 1981). This has consequences for phylogenetic studies including this species, as many of its characters are unknown. Attempts to collect new material from the area of the type locality have failed (Brinkhurst, 1966; Erséus, 1981). Additional species of Heterodrilus were described by Pierantoni (1917), Jamieson (1977), Giere (1979) and Milligan (1987), but the most substantial contribution was made by Erséus (e.g. 1981, 1985, 1986, 1988a-b, 1990b). The original circumscription of the genus (Pierantoni, 1902, 1917) was based on characters such as anterior chaetae trifid, hair chaetae absent, male pores paired, etc. However, all species now included in Heterodrilus do not share all these features and the generic diagnosis of Heterodrilus has subsequently been modified twice (Erséus, 1981, 1990b). In 1981, Erséus looked at tubificid species with trifid chaetae, and recognized three

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separate genera: Heterodrilus Pierantoni, 1902, Heterodriloides Erséus, 1981, and Giereidrilus Erséus, 1981. The monotypic Heterodriloides was distinguished from Heterodrilus by two main features: its spermathecae are located in segment XII, i.e., in the segment immediately posterior to the one bearing the male pores, with a supplementary pair generally located in XI, and its vasa deferentia enter the ectal parts of the atria. In Heterodrilus and other tubificids the normal position of the spermathecae is in the segment immediately anterior to the male pore, and the vasa deferentia enters the apical, ental part of the atrium. Giereidrilus was established for two species which both have unpaired spermathecal and male pores, and their atria are not internally ciliated. However, in 1990, Erséus considered Giereidrilus and Heterodriloides to be derived members within Heterodrilus, since all three taxa share the following features: the anterior chaetae are trifid, the chaetal bundles are bichaetal anterior to clitellum, but unichaetal posterior to clitellum, and the prostate glands are diffuse and broadly attached to the atria.

PHYLOGENY OF HETERODRILUS The systematic position of Heterodrilus within Tubificidae has long been problematic. It was assigned to the subfamily Rhyacodrilinae based on morphological features (Erséus, 1981), but has later been suggested to be a member of Phallodrilinae based on molecular markers (Erséus et al., 2000, 2002; Siddall et al., 2001; Paper II). Not much is know about the relationships within Heterodrilus, only a few analyses spanning a range of taxa have been conducted. In 1990 Erséus assembled a data matrix with 15 morphological characters from all 24 species then known, as well as of the two supposedly closely related taxa Heterodriloides and Giereidrilus, and he used a hypothetical ancestor as the outgroup. When assigning all character transformations equal weights, the result was a high number of equally parsimonious trees with limited congruence in their branching pattern. When some of the characters were weighted, the result was three equally parsimonious trees. Based on a consensus of these, Erséus (1990b) concluded that Heterodriloides and Giereidrilus are advanced members of Heterodrilus. In Paper II, eight Heterodrilus species were included and two genes were used (18S rRNA gene and 16S rRNA gene) and the monophyly of the group was corroborated, but rather than being an advanced member of Heterodrilus, H. ersei [= Giereidrilus ersei sensu Erséus, (1981)] was placed as the sister taxon to the rest of the group. Paper IV is the most comprehensive molecular study of Heterodrilus to date, 18 species were included and three genes were utilized (18S rRNA gene, COI and 16S rRNA gene). The taxon sampling represents barely half of the described species, and it appears futile to discuss the validity or phylogenetic position of each species in detail. The positions of, in particular, H. inermis [= Giereidrilus inermis sensu

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Erséus (1981)] and two morphologically similar species, H. apparatus and H. rapidensis, as well as H. quadrithecatus [= Heterodriloides quadrithecatus sensu Erséus (1981)] are yet not known.

A B C D

Figure 4. Different types of somatic chaetae within Tubificidae. A: ordinary preclitellar

tubificid chaetae, B-D: preclitellar chaetal types in Heterodrilus.

THE TRIFID ANTERIOR CHAETAE

A majority of the species of Heterodrilus are characterized by having trifid anterior chaetae i.e., chaetae with three teeth at the distal end (Fig.4B) compared to ordinary bifid chaetae found in other tubificids (Fig 4A). However, some species assigned to Heterodrilus (H. subtilis, H. tripartitus and H. hispidus) have bifid anterior chaetae (Fig. 4D), and they have thus been regarded as having lost the third tooth secondarily (Erséus, 1990b). Other species are intermediate in the sense that they have some/all anterior chaetae with a subdistally located third tooth (“modified trifids” see Fig. 4C). When present together with other chaetal types, the lower tooth on the “modified” trifid tends to move up or down when going backwards from segment II. That is, if there are “modified” trifids in segment II, the lower tooth moves upwards in the following segments, and in segment V, or thereabout, the “typical” trifids appear. Alternatively, if there are “typical” trifids in segment II the lower tooth seems to move down in the following segments, and in segment IV there are “modified” trifids.

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In Paper IV most Heterodrilus species represented have “typical “ trifid anterior chaetae, three species (H. modestus, H. paucifascis and H. jamiesoni) have “modified” trifids. Heterodrilus ersei which is the sister group to all other Heterodrilus species has trifid anterior chaetae, and while the species with the ”modified” anterior chaetae are all phylogenetically embedded in Heterodrilus, the hypothesis that the third tooth is secondarily reduced in some lineages in the taxon seems reasonable.

INTER- AND INTRASPECIFIC VARIATION?

Within Heterodrilus species are distinguished by morphological characters in the internal organization of the male and female genitalia, but also by characters in the form and number of chaetae. Identification is based on morphology of mature worms, and in most cases a whole set of characters is necessary for the identification of a particular species of the taxon. The inter- and intraspecific variation in morphology of different Heterodrilus species has barely been studied. In Paper III, the ultrastructure of the cuticle in twelve specimens of Heterodrilus, representing four species (H. paucifascis, H. pentcheffi, H. flexuosus, H. minisetosus), was investigated with transmission electron microscopy (TEM). The result showed that there is interspecific variation in the morphology of the cuticle. In one of the studied species, H. paucifascis, there is intraspecific variation in the cuticular morphology; the other species had a high degree of uniformity between the specimens. In H. paucifascis there is a clear difference between the specimens collected in Bahamas and those collected in Belize. The intraspecific variation found could be due to that the two populations represent different forms within a complex of sibling species. There are no voucher specimens available from the H. paucifascis material used in Paper III, but when I studied other material from Bahamas more closely I could find no H. paucifascis specimens at all. However, I did find specimens that are morphologically similar to H. paucifascis but differ, e.g., in the position of the spermathecal pores. Thus, I find it possible that the material of H. paucifascis from Bahamas used in Paper III could have been misidentified (when studied alive only before being fixed for the TEM study), and that the H. paucifascis from Bahamas are in fact an undescribed new species that is deceptively similar to H. paucifascis. In a recent, preliminary molecular study of Heterodrilus using mitochondrial COI (Sjölin et al., unpublished data), five specimens comprise a particular clade (bootstrap support 99%) that is divided into two subclades (both with 100% support), one of which consists of two worms identified as H. paucifascis from Belize, the other of three worms belonging to the Bahamian form mentioned above (the one with spermathecal pores in another position). The average COI sequence distance is over 9% between, but less than 1%

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within, these two subclades. Thus, as the molecular data seem to support the hypothesis that the Bahamian “H. paucifascis” is a separate species, the ”intraspecific” variation in the cuticular ultrastructure noted for H. paucifascis in Paper III may instead be interspecific in nature.

QUO VADIMUS?

In areas where more extensive collections have been made, an astonishing diversity of species with trifid chaetae has been found. Therefore, it is likely that there are numerous Heterodrilus species yet to be described. At the same time, however, more characters are needed to improve the basis for the phylogenetic studies, and to facilitate the circumscription and identification of the individual species. The spermathecae, as well as other morphological characters not associated with the male genitalia, may provide some additional information; male duct characters (shape of atrium, prostate glands, etc) may not be independent of each other. It would also be interesting to study the ontogenetic origins of the diffuse prostate glands in Heterodrilus, since the prostate glands associated with the atrial epithelium may have different ontogenetic origins. The diffuse prostate glands in Rhyacodrilinae have been shown to have a mesodermal origin, but the prostate glands in Phallodrilinae have also not yet been studied. Furthermore, the type specimens of many species need to be re-examined, and larger samples (new individuals) of previously described species need to be collected and carefully studied, so that intraspecific variation can be more fully assessed and, ultimately, the different species much better delineated. Finally, molecular studies will probably provide crucial information, hopefully enabling us to resolve the phylogeny within Heterodrilus in a not too distant future.

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SWEDISH CLITELLATE SCIENTISTS In 1996 Sigvaldadottír presented a review of Swedish annelid scientists with emphasis on polychaetae researchers. At that time research on clitellates had just commenced, but with the introduction of molecular methods, new clitellate researchers have radiated. I now see a need to increase the taxon sampling and review the current phylogeny of the clitellate clade (Fig. 5, dashed line). The results indicated two major clades largely corresponding to geographical distribution, in one clade all researchers (Lena Gustavsson, Ida Envall, and Erica Sjölin) are from Stockholm on the east coast of Sweden, in the other clade both researchers (Pierre De Wit and Lisa Matamoros) are from Göteborg on the west coast of Sweden. The results imply that the male dominance among the polychaetologists is receding among the clitellatologists. Many of the new clitellate researchers found are more or less immature (=thesis not yet defended), and taken together with the female dominance in this clade it appears plausible that the predominant life strategy has shifted from sexual to asexual reproduction (cloning). As cloning may rapidly increase the proportion of a new genotype in a population, facilitating speciation and divergence, it is even possible we will see an even greater radiation of new clitellatologists.

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Figure 5. Cladogram showing Swedish annelid researchers. Dashed line showing clitellate

researchers.

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SUMMARY OF THE INCLUDED PAPERS Paper I. SJÖLIN, E. AND ERSÉUS, C. 2001. New species of Heterodrilus (Oligochaeta, Tubificidae) and records of H. maiusculus from the Mediterranean Sea. Italian Journal of Zoology, 68: 223-228. Two new taxa are described: Heterodrilus tripartitus and H. ursulae both found in the Mediterranean Sea. The paper also includes a redescription of H. maiusculus from the Mediterranean Sea, previously only known from the Arabian Gulf coast of Saudi Arabia. Paper II. SJÖLIN, E., ERSÉUS, C. AND KÄLLERSJÖ, M. 2005. Phylogeny of Tubificidae (Annelida, Clitellata) based on mitochondrial and nuclear sequence data. Molecular Phylogenetics and Evolution, 35: 431-441. The phylogeny of 55 representatives from five of six subfamilies of Tubificidae is assessed using sequence information from the mitochondrial 16S rRNA gene and the nuclear 18S rRNA gene. The study reveals that several associations traditionally recognized within the Tubificidae are also supported with molecular markers. The monophyly of Tubificidae (including former Naididae) is strongly supported. The results indicate that Rhyacodrilinae is polyphyletic, some of its members (Heterodrilus spp.) fall into a clade with Phallodrilinae, and two other rhyacodriline genera (Monophylephorus and Ainudrilus) is nested within Naidinae. All in all, most of the tubificid genera included are supported as monophyletic, with three exceptions: Tubifex and Limnodriloides are refuted, and Tubificoides is unresolved from other Tubificinae taxa. With five of the six subfamilies of Tubificidae represented, only two is rendered monophyletic, Limnodriloidinae and Tubificinae. Paper III. SJÖLIN, E. AND GUSTAVSSON, L.M. 2007. An ultrastructural study of the cuticle in the marine Heterodrilus (Tubificidae, Clitellata). Journal of Morphology, in press. In this paper the ultrastructure of the cuticle in twelve specimens of Heterodrilus, representing four species (H. paucifascis, H. pentcheffi, H. flexuosus, H. minisetosus), is investigated with transmission electron microscopy. The result shows that there is interspecific variation in the morphology of the cuticle. One of the studied species, H. paucifascis, shows

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intraspecific variation, which is associated with sample locality, the Bahamian specimens differs in morphology of the cuticle to the Belizean specimens. Paper IV. SJÖLIN, E., JOHANSSON, U.S. AND ERSÉUS, C. Tubificids with trifid chaetae: a phylogeny of the genus Heterodrilus (Clitellata, Annelida). Manuscript. The relationships of 18 Heterodrilus species is assessed using the nuclear 18S rRNA gene, and the mitochondrial cytochrome c oxidase subunit I (COI) and 16S rRNA gene. The study reveals that the two major clades in our tree corresponds to different geographical distributions, in one clade all species are from Western Atlantic, and in the other clade all species are from West Pacific. Some of the traditionally used morphological characters in tubificid taxonomy are optimised on our tree and all of them show a high degree of homoplasy.

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ACKNOWLEDGEMENTS I would like to express my sincere gratitude to everyone making this thesis possible. I am indebted to a lot of people, and I am sure that there are some that I will forget. If you are one of those, I am sorry, but things have been very busy at the time of writing these lines, and you have unfortunately slipped my sieve-like memory. Anyway, thank you for whatever you did for me. Those fortunate still in my mind: Many thanks to my supervisor Christer Erséus for introducing me to the fascinating world of worms, showing me how to get the critters and to identify them and for reading and editing my manuscripts. To my supervisor Mari Källersjö for introducing me to the molecular side of life, and for always being enthusiastic about my projects. To my supervisor Lena Gustavsson, among other things: thank you for having confidence in my abilities, for never giving up on me, and for getting me through to the end, sorry for the cheese and other “accidents”. Vetenskapsrådet financed most of this thesis, through a grant to Christer Erséus. I have also received funding from Lars Hiertas Minne and by Helge Ax:son Johnsons stiftelse. Many thanks to all, present and former, employees at the Department of Invertebrate Zoology. Over the years: Christine Hammar, Märith Eriksson, Kerstin Rignéus, Karin Sindemark Kronstedt, Elín Sigvaldadóttir, Ylva Lilliemarck, Anita Norrthon, Carina Svensson, Emily Dock Åkerman, Anders Warén, Jan-Erik Fäldt, Stefan Lundberg, Sabine Stöhr, Ann-Marie Sundström, Ann-Katrin Gustafsson, Sven Boström, Björn Sohlenius, Purba Pal, Ulf Jondelius, and Sara Sundqvist. Thanks also to everyone at the Molecular Systematics Laboratory for technical assistance and good advice. My red flag this summer made all the difference! Pia Eldenäs, Bodil Cronholm, Keyvan Mirbakhsh, Annika Einarsson, Mattias Myrenås, and Martin Irestedt. Other systematists that have inspired: Mikael Thollesson, Fredrik Pleijel, Tomas Pape, Rasmus Hovmöller, Livia Wanntorp, Ida Trift, Reihaneh Dehghani, Olle Israelsson, Michael Norén, Jan Ohlson, Magnus Gelang, Marianne Espeland, Åsa Söderberg, Lisa Matamoros, Pierre De Wit, Anna Ansebo.

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Thanks to the staff at the Department of Zoology, Stockholm University, especially Anette Lorents, Berit Strand and Siw Gustafsson, for always answering all my strange questions and for help with all practical things, and to Bertil Borg for constructive comments on this thesis. Despite my name I have had very little to do with things green, but I do want do give a special thanks to a special plant, Coffea arabica, which without I would not have coped. Thanks to Lena and Hanna, who suggested an alternativ title for this thesis (based on the cover figure): “Tubificids with trifid chaetae: the Candyworm- with rasberry flavour”. And for those of you who read the introduction: Q: Why do worms taste like chewing gum? A: Because they're wrigleys! A special mention goes to my fellow colleague Ida Envall. We almost never worked together due to the blips and blops of offspring, however, thank you for your support and good company during these years. We will prevail! My office mates in the “panic-room”- Karolina Larsson, wormy-girl highly adapted to bake delicious creamy cakes, if everything falls apart we will glue it together with frosting! Tobias Kånneby- gastrotrich-boy and coffee-connoisseur, one of the few people I know who can say, “do you want a banana?” without sounding fishy. Wim Willems, beardy Belgian flatworm-scientist, whom I heard a lot of swearword from. Hanna Taylor, game enthusiast, and not so innocent after all. I wish to thank my mother for endless encouragement even though she doesn’t has a clue to what I work with and is very sceptical to the use of wriggly worms. All the rest of my big family (siblings, nephews, nieces, extended family etc., you know who you are!) is greatly thanked for putting up with my strange interest. Finally, my heartfelt thanks to my fiancé Ulf who I turned to constantly for help with formatting and multitudes of problems with software and data analysis, or just plain problems. He always set the best example and pushes me to try that little bit harder and is always there for me. He supported me when at times it got tough, and showed me that there is light in the end of the tunnel, even if at times I felt like I was going backwards! I love you. And to Ninn, present in every part of my life!

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APPENDIX I

List of Heterodrilus taxa H. amplus Erséus, 1992 H. apparatus Erséus, 1993 H. arenicolus Pierantoni, 1902 H. ascensionensis Erséus, 1981 H. bulbiporus Erséus, 1981 H. carinatus Erséus & Wang, 1993 H. claviatriatus Erséus, 1981 H. chenianus Wang & Erséus, 2002 H. decipiens Erséus, 1997 H. densus Erséus, 1997 H. devexus Erséus, 1997 H. dolosus Erséus, 1997 H. eremita Erséus, 1997 H. ersei (Giere, 1979); originally as Phallodrilus ersei. H. flexuosus Erséus, 1990 H. hispidus Erséus, 1986 H. inermis (Erséus, 1981); originally as Giereidrilus inermis. H. jamiesoni Erséus, 1981 H. keenani Erséus, 1981 H. lacertosus Erséus, 1981 H. maccaini Erséus, 1985 H. maiusculus Erséus, 1988 H. mediopapillosus Takashima & Mawatari, 1997 H. minisetosus Erséus, 1981 H. modestus Erséus, 1990 H. nudus Wang & Erséus, 2002 H. obliquus Erséus, 1997 H. occidentalis Erséus, 1981 H. paucifascis Milligan, 1987 H. pentcheffi Erséus, 1981 H. perkinsi Erséus, 1986 H. quadrithecatus (Erséus, 1981); originally as Heterodriloides quadrithecatus. H. queenslandicus (Jamieson, 1977); originally as Clitellio arenicolus queenslandicus. H. uniformis Wang & Erséus, 2002 H. ursulae Sjölin & Erséus, 2001 H. rarus Erséus, 1990 H. rapidensis Erséus, 1997 H. salmonensis Erséus, 1993 H. scitus Erséus, 1981 H. subtilis (Pierantoni, 1917); originally as Clitellio subtilis. H. tripartitus Sjölin & Erséus, 2001 H. virilis Erséus, 1992

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APPENDIX II

KEY TO THE TAXA OF HETERODRILUS The following artificial key is provided to facilitate the identification of the known taxa of Heterodrilus. As new taxa are likely to be encountered in new material, the key should only be regarded as help to get an idea where a specimen may belong. A careful comparison with the complete descriptions in the literature is always essential. Anterior chaetae trifid, or bifid with parallel straight teeth in line with the chaetal shaft. Somatic chaetae two per bundle in most preclitellar segments, only one per “bundle” from about segment X and onwards; hair chaetae absent; penial chaetae present or absent. Spermathecal pores in segment X; male pores in segment XI. When alive, most species pinkish, and tightly coiled into a spiral when disturbed. No eyes. 1. Unpaired male pore-2

-Paired male pores-5 2. Modified genital chaetae present-4

-Modified genital chaetae absent-3 3. From about segment XXIX, dorsal chaetae small and bifid, ventral chaetae

large and single pointed- Heterodrilus rapidensis -All chaetae from segment XXX sharply single-pointed and of the same size dorsally and ventrally- Heterodrilus inermis

4. Modified, grooved penial chaetae present- Heterodrilus apparatus

-Only penial chaetae present- Heterodrilus ersei 5. Preclitellar chaetae bifid-6

-Preclitellar chaetae trifid, with third tooth either located close to middle teeth, or located on chaetal shaft at some distance from distal end-8

6. Penial chaetae present-7

-Penial chaetae absent- Heterodrilus subtilis 7. Spermathecal ducts with vestibules (fig)- Heterodrilus tripartitus

-Spermathecal ducts slender, without vestibules- Heterodrilus hispidus

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8. All preclitellar chaetae trifid-9

-Some preclitellar chaetae trifid and others with third tooth subdistally located, or all preclitellar chaetae with third tooth subdistal-11

9. Trifid chaetae with ligaments connecting lowermost tooth with chaetal shaft-10 -No ligaments on trifid chaetae-29

10. Spermathecae present-20

-Spermathecae absent-18 11. All preclitellar chaetae with third tooth subdistal- 12

-Some preclitellar chaetae more typically trifid- 14 12. Spermathecae present-13

-Spermathecae absent-Heterodrilus modestus 13. Spermathecal pores in line with ventral chaetae- Heterodrilus jamiesoni

-Spermathecal pores ventral to lines of ventral chaetae- Heterodrilus paucifascis -Spermathecal pores in line with lateral lines- Heterodrilus salmonensis

14. Vas deferens shorter than atrium- 15

-Vas deferens longer than atrium- 16 15. Atrial musculature thick (11-16 µm)- Heterodrilus amplus

-Atrial musculature not thick (1-2 µm)- Heterodrilus maccaini 16. Vas deferens coiled in tight spiral- Heterodrilus occidentalis

-Vas deferens not coiled in tight spiral- 17 17. Spermathecae with short triangular ducts (fig)- Heterodrilus maiusculus

-Spermathecae with long ducts (fig)- Heterodrilus ursulae 18. Penial chaetae present- 19

-Penial chaetae absent- Heterodrilus chenianus 19. Penial chaetae long (140-200 µm), 2(3) per bundle- Heterodrilus virilis

-Penial chaetae short (30-45 µm), 1 (2) per bundle- Heterodrilus flexuosus

20. Vas deferens long (>200 µm)- 21 -Vas deferens short (about 130 µm)- Heterodrilus uniformis

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21. Atrial musculature >2.4 µm thick - 22

-Atrial musculature < 2.0 µm- 24 22. Penial chaetae 90-215 µm, parallel within bundle, and with outer ends

bent- 23 -Penial chaetae 35-50 µm, arranged in V-shaped bundles- Heterodrilus decipiens

23. Penial chaetae 90-100 µm- Heterodrilus lacertosus

-Penial chaetae 175-215 µm- Heterodrilus mediopapillosus 24. Spermathecal pores located in line with ventral chaetae- 25

-Spermathecal pores located between lateral lines and lines of ventral chaetae-27

25. Atrium 210-350 µm- 26

-Atrium 135-190 µm- Heterodrilus keenani 26. Male pores on two bulbous porophores/protuberances- Heterodrilus eremita

-No porophores present- Heterodrilus claviatriatus 27. Atrium 195-260 µm- 28

-Atrium 105- 155 µm- Heterodrilus queenslandicus 28. Penial chaetae straight with nodes extal, appearing like “keels”, followed by

sharply single-pointed, slightly curved tips-Heterodrilus scitus -Penial chaetae straight and erect, not “keeled”- Heterodrilus dolosus

29. Vas deferens shorter than atrium- 30

-Vas deferens longer than atrium- 33 30. Penial chaetae minute, sigmoid, one per bundle or absent- 31

-Penial chaetae straight, 1-2 per bundle- 32 31. Spermathecae present- Heterodrilus minisetosus

-Spermathecae absent- Heterodrilus arenicolus 32. Atrium 100-120 µm- Heterodrilus ascensionensis

-Atrium 1400-3200 µm- Heterodrilus perkinsi 33. Atrium 85-150 µm- 34

-Atrium 400-3200 µm- 36

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34. Penial chaetae present- 35

-Penial chaetae absent- Heterodrilus nudus 35. Penial chaetae one per bundle- Heterodrilus rarus

-Penial chaetae > 2 per bundle- Heterodrilus quadrithecatus 36. Vas deferens tightly coiled in spiral-37

-Vas deferens irregularly coiled-38 37. Muscular copulatory bulb present- Heterodrilus bulbiporus

-Muscular copulatory bulb not present- Heterodrilus pentcheffi 38. Spermathecal pores dorsal to lines of ventral chaetae- 39

-Spermathecal pores in line with ventral chaetae- Heterodrilus carinatus 39. Atrium with prominent (3-5 µm) muscle layer- 40

-Atrium with slender (1-2 µm) muscle layer- Heterodrilus devexus 40. Spermathecal vestibules granulated- Heterodrilus obliquus

-Spermathecal vestibules not granulated- Heterodrilus densus