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Childia

phylogenies ,

In memory of my youngest brother, Robel Isaak Tekle

Papers included in this thesis

This thesis is based on the following papers, which will be referred to in the text by their Roman numerals (I-V)

I. Tekle Y. I., Raikova O. I., Ahmadzadeh A., and Jondelius U. 2005. Revision of the Childiidae (Acoela), a total evidence ap-proach in reconstructing the phylogeny of acoels with reversed muscle layers. Journal of Zoological Systematics and Evolution-ary Research 43(1): 72–90.

II. Tekle Y. I., Raikova O. I., Justine J-L., Hendelberg J. and Jonde-lius U. 2005. New sperm characters for unraveling acoel phy-logenies. Ultrastructural and immunocytochemical investigation of acoels with 9 + 1 axoneme structure, non-homologous with that of the Trepaxonemata (Platyhelminthes). Zoomorphology(accepted).

III. Raikova O. I., Tekle Y. I., Reuter M., Gustafsson M. K. S. and Jondelius U. Copulatory organ musculature in Childia (Acoela) as revealed by phalloidin fluorescence and confocal microscopy. Tissue & Cell (in review).

IV. Tekle Y. I., Raikova O. I., Justine J. -L. and Jondelius U. Ultra-structure and tubulin immunocytochemistry of the copulatory stylet in Childia species (Acoela). Journal of Morphology (in review).

V. Tekle Y. I., Raikova O. and Jondelius U. A new viviparous acoel Childia vivipara sp. n. with observations on the develop-ing embryos, sperm ultrastructure, body wall and stylet muscula-tures. Acta Zoologica (in review).

________________________________________________________Published and accepted papers are reproduced with the publishers’ kind permission.

Papers I, II, IV & V were written by the first author. Papers were planned together with the co-authors. All analyses, molecular work (except for one species), and most of the morphological work (except immunocytochemistry) were done by author of the thesis. In paper IV, the author of this thesis is responsible for most of the lab work and was also involved in writing of the paper.

Important note. This thesis is not intended for permanent scientific record in the meaning of the International Code of Zoological Nomenclature (ICZN). Thus, any nomenclatural acts proposed here are not valid. The dissertation may be cited in its own right, but it should not be cited as a source of nomenclatural statements.

Contents

Introduction.....................................................................................................9What are the Acoela? .................................................................................9Phylogenetic position of the Acoela.........................................................10Taxonomy of the Acoela ..........................................................................11

Body-wall musculature........................................................................12Spermiogenesis and sperm structure....................................................13Nervous system....................................................................................14Musculature and ultrastructure of copulatory organs ..........................15Molecules.............................................................................................15

Focus of the thesis ....................................................................................17The Childiidae .....................................................................................17Objectives ............................................................................................18

Material and Methods ...................................................................................20Taxa studied and Sampling ......................................................................20Morphological methods............................................................................21Molecular methods...................................................................................22Data preparation and Analyses.................................................................22

Results...........................................................................................................24Phylogenetic analyses (I) .........................................................................24Sperm ultrastructure (II & V)...................................................................26Musculature (III & V) and ultrastructure of the copulatory organs (IV)..28

Discussion.....................................................................................................30The utility of classical morphological characters and 18S rRNA gene in Childiidae (I-IV)...................................................................................30Revision of the Childiidae Dörjes, 1968 (I) and description of a newChildia species (V)...................................................................................32Sperm morphology at higher taxonomic levels........................................34

The Childia- Mecynostomidae clade...................................................349 + 1 axoneme: a homoplasy or homology of the Acoela and the Trepaxonemata?...................................................................................34

Conclusions...................................................................................................35Summary in Swedish ....................................................................................36Acknowledgements.......................................................................................39References.....................................................................................................41

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Introduction

What are the Acoela?

The Acoela are small acoelomate free-living invertebrates, formerly classi-fied within the Platyhelminthes (Ehlers 1985), which were always consid-ered crucial in the discussions of the origin and evolution of metazoans (Willmer 1990). Interest in this group has recently increased, since it was proposed as the sister-group of the Bilateria separate from the Platyhelmin-thes (e.g. Ruiz-Trillo et al. 1999; Jondelius et al. 2002). Acoels are among the model animals used for understanding the evolution of the nervous sys-tem in bilaterians.

The name Acoela derives from their lack of a defined digestive cavity ("A" - absence; "coela" - gut cavity). The mouth, which is mostly located in the mid-ventral region of the body, opens into the central parenchyma com-posed of a mass of syncytial tissue surrounded by cellular parenchyma (Smith and Tyler 1985). Acoels are small (0.5-10 mm) animals, mostly liv-ing in marine habitats, ranging from the littoral one, either interstitial (be-tween sand grains) or gliding on algae, to deep water (20m - 300m) on ma-rine substrates. Freshwater acoels have been described (e.g. Faubel and Ko-lasa 1978), but are quite rare. Acoels have relatively simple morphology; most often they lack hard structures. A typical acoel worm is recognized by the presence of a statocyst with a single statolith and two parietal cells, lo-cated in the anterior region of the animal. The epidermis is completely cili-ated and is underlain by layers of circular, diagonal and longitudinal mus-cles. The arrangement of the body-wall muscles differs among acoels and has important phylogenetic significance (Hooge 2001; paper I). Acoels vir-tually lack an extracellular matrix, except for the statocyst capsule. Numer-ous glands are found scattered in the epidermis. On the extreme anterior pole of the animal, several frontal glands are located. Often their necks open into a pore, thus forming the frontal organ. The frontal organ with a common pore is developed only in some acoels, the others have glands opening sepa-rately – frontal gland complex. The reproductive system is hermaphroditic. Mature animals have both female and male reproductive organs, located in

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the parenchyma. Some acoels show protandry/protogyny that is development of either male or female organ before the other to avoid self-fertilization. The female system consists of one or two pairs of ovaries and, often, female accessory structures such as the seminal bursa for storage of allosperms. The bursa can be weakly developed or armored with special structure of scle-rotized elements – the bursa nozzle. The male system constitutes a key struc-ture in the identification of the major groups of acoels (see Dörjes 1968). The male copulatory organ can be quite complex, comprising diverse muscu-lar or sclerotized elements, or weakly developed, reduced to a simple epi-dermal invagination. A seminal vesicle, for storage of mature sperms, is often present that can be strongly muscular or with weak musculature. In some species, the seminal vesicle is simply an aggregation of sperm cells with virtually no musculature. The filiform sperm cells in acoels have two incorporated axonemes in an inverted position. Asexual reproduction also sometimes occurs, through processes of architomy or paratomy. Acoels have a simple nervous system, organized as a network of longitudinal nerve cords and transverse commissures, in which a brain is formed by thickening of the foremost commissures and sometimes by a centralized mass of nerves sur-rounding the statocyst in the anterior end of the animal. Acoels display a unique mode of embryonic development, the duet spiral cleavage (e.g. Apelt 1969; Boyer et al. 1996; Henry et al. 2000), which differs from the typical quartet spiral cleavage characteristic of other Spiralians.

Phylogenetic position of the Acoela The Acoela have been traditionally classified as an Order in the Class “Tur-bellaria” of the phylum Platyhelminthes. The class “Turbellaria” was shown to be paraphyletic, and it is now considered an invalid taxon name (Ehlers 1985). Ehlers (1985) recognized three major groups of flatworms (Platy-helminthes) thought to be monophyletic: the Acoelomorpha, consisting of two groups, the Acoela and the Nemertodermatida, the Catenulida, and the Rhabditophora (most flatworm species, including the parasitic ones). The interrelationships between these three clades were not clear, and the mono-phyly of the Platyhelmintes was questioned (e.g. Smith et al. 1986). Hasz-prunar (1996), reassessed the available morphological evidence and sug-gested that the Acoelomorpha might be the most basal bilaterian offshoot, followed by the Rhabditophora, the Catenulida and the rest of the Bilateria.

The monophyly of the Acoelomorpha is supported by several morpho-logical apomorphies, including intricately interconnected ciliary rootlets, epidermal cilia with shelfed tips, presence of degenerating epidermal (pulsa-tile) bodies and lack of protonephridia (e.g. Tyler and Rieger 1977; Ax 1996; Lundin and Hendelberg 1996). However, its monophyly has been questioned (Carranza et al. 1997; Ruiz-Trillo et al. 1999). Based on 18S

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rRNA sequences, Ruiz-Trillo et al. (1999) placed the Acoela as the most basal bilaterians unrelated to the Platyhelminthes. According to this study, the sequence datum used to represent the Nemertodermatida was placed in their analysis within the Rhabditophora. However, studies based on addi-tional molecular data (including more nemertodermatid species) have indi-cated that neither the Acoela nor the Nemertodematida are members of the Platyhelminthes, both instead represent separate early branches within the Bilateria (Jondelius et al. 2002; Telford et al. 2003). Thus the taxon Acoelo-morpha was not supported in these studies. Nevertheless, a separate analysis of myosin heavy chain type II (myosin II) sequences has shown support for the monophyly of the clade Acoelomorpha (Ruiz-Trillo et al. 2002), in agreement with previous morphological studies (e.g. Ehlers 1985; Ax 1996). Therefore, the question of the monophyly or paraphyly of the Acoelomorpha still remains open.

Even though the phylogenetic position of the Acoela has been one of the hotly debated topics in metazoan evolution, its monophyly is not questioned. The monophyly of the Acoela is not only supported by several morphologi-cal characters, but also by all the molecular studies done so far (Hooge et al. 2002; Jondelius et al. 2002). Morphological apomorphies (Ehlers 1992; Smith et al. 1986) shared by acoels include an acoel-type statocyst with a single statolith, the absence of an epithelized gut, epidermal ciliary rootlets interconnected at two levels, and near absence of extracellular matrix (except for the statocyst capsule). Molecular studies involving large data sets of 18S rRNA gene sequences from acoels also support the monophyly of the Acoela (Hooge et al. 2002; Jondelius et al. 2002).

Taxonomy of the Acoela The taxonomy of the Acoela has been problematic due to the lack of easily obtainable and taxonomically useful characters and the plasticity of the clas-sical morphological characters, that is, those characters obtained from study-ing live specimens and simple histological methods at light microscopy level. Early classifications of acoels were based on the gonopore position (Graff 1911) and the presence of a seminal bursa (Luther 1912). Westblad’s (1942, 1948) classification system of the Acoela was based on the female reproductive organ and the respective positions of male and female open-ings. Based on these, Westblad (1948) recognized three tribes: Opistandro-pora-Abursalia, Proandropora-Abursalia and Proandropora-Bursalia. Thesetribes were further divided into families and genera based on characters of male copulatory organ, development of frontal organs, mouth types and ad-ditional female features (Westblad 1948). The basis of the currently accepted classification of acoels is the male copulatory organ. Accordingly, Dörjes (1968) recognized 15 families based on the characters of the male copulatory

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organ. In Dörjes’ system the interrelationship among the different families is not evident. He placed approximately half of the acoels into one family, the Convolutidae, while other families comprised only few species, or were monotypic. Subsequent taxonomic work on acoels have based the classifica-tion on the discovery of a new type of male copulatory organ (Kostenko 1989), the fine structure of epidermal glands “sagittocysts” (Kostenko and Mamkaev, 1990), and patterns of body-wall musculature (e.g. Hooge 2001, 2003; Paper I). Recent molecular studies based on 18S rRNA gene se-quences of 32 acoels (representing 10 families) have pointed out many in-consistencies in the current system (Hooge et al. 2002). This result is not surprising, since the original study (Dörjes 1968) was based on a single structure: comparison of male copulatory organs using traditional light mi-croscope techniques. This necessitates the need for more detailed studies of the male copulatory organ and the search for other characters. Some of the characters that have proven useful in the systematics of acoels include pat-terns of body-wall musculature (e.g. Tyler and Hyra 1998; Hooge and Tyler 1999; Tyler and Rieger 1999; Hooge 2001), spermiogenesis and sperm struc-ture (Raikova et al. 2001; Petrov et al. 2004; Paper II), patterns of the nerv-ous system (Raikova et al. 2004) and ultrastructure and musculature of the copulatory organ (Hooge and Tyler 2005; Papers III and IV). A short de-scription of these characters, with phylogenetic perspective, is presented below.

Body-wall musculature The body-wall musculature of several acoel taxa have been described using advanced techniques such as confocal scanning laser microscopy (CSLM) of whole-mount preparations stained by phalloidin, conjugated with a fluores-cent molecule (e.g. Tyler and Hyra 1998; Hooge and Tyler 1999; Hooge 2001; Paper I). The body-wall musculature of acoels is generally composed of circular, longitudinal, and diagonal muscles, as well as various types of accessory muscles related to openings (pore muscles) (Fig. 1A, B).

The arrangement of the body-wall muscle layers as observed in traditional histological preparations was used for species description (e.g. Steinböck 1931, Westblad 1942, 1945). Hooge (2001) used a number of variations observed in the body-wall musculature in a phylogenetic study of acoels, rabditophorans and catenulids. Some of the useful aspects of the variations observed in the body-wall musculature include the shape, number, thickness, spacing, orientation, location (arrangement) and presence/absence of the different muscle fibers making up the body-wall. Diagonal muscles vary among acoel sub-groups in shape, area of coverage, origin (of circular or longitudinal muscles) and their presence/absence on the ventral and/or dorsal sides of the body. Accessory muscles occur around mouth and genital open-ings (Fig. 1A). Some of them were described in previous study

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A B

Figure 1. A general schematic representation of ventral dorsal (A) and dorsal (B) body-wall musculature in the Acoela. Circular muscles near the mouth region are not shown in A. amla, accessory muscles going in latero-anterior direction; amlp, accessory muscles going in latero-posterior direction; cm, circular muscles; fo, fe-male opening; lm, longitudinal muscles; m, mouth; mo, male opening; A-type; B-type, U1; U2, U3; U4; U5 (see paper I); vc, ventral cross-over muscles; vd, ventral diagonal muscle.

(e.g. Hooge 2001). We have used these variations in our phylogenetic analy-ses of the Childiidae (Paper I).

Spermiogenesis and sperm structure

Spermiogenesis and sperm structure have been extensively used in taxo-nomic studies of acoels. Spermiogenesis has been studied in several species of acoels (for ref see Raikova et al. 2001). Raikova et al. (1997) reported that the spermiogenesis in Paratomella rubra differs greatly from that of other acoels studied. The taxon Paratomella has been proposed as a sister group to the other acoels, “Euacoels” (Ehlers 1992). However, there is still no evi-dence that the peculiar spermiogenesis in Paratomella represents a plesio-morphic condition and not an autapomorphy of the taxon (Raikova et al. 2001). Further investigation of acoel spermiogenesis might provide a better understanding of the ancestral conditions within acoels and other bilaterians.

Several sperm characters of acoels have been identified (see Raikova et al. 2001), and some of these were shown to corroborate the molecular phy-

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logenetic tree by Hooge et al. (2002). Acoel spermatozoa are long, filiform cells containing two incorporated axonemes in inverted orientation (with centrioles lying at the opposite end of the cell from the nucleus). Three types of sperm axoneme structure are identified in acoels: 9 + 2, 9 + 0 and 9 + 1 (Fig. 2A). The 9 + 2 axoneme structure with two central microtubules and nine peripheral microtubule doublets is probably plesiomorphic as it is common for the cilia and flagella of the Metazoa. The 9 + 0 axoneme struc-ture is a modification of the 9 + 2, where two central microtubules are ab-sent. The 9 + 1 axoneme structure has one central microtubule with an elec-tron-dense material surrounding it. Apart from the axonemes, the acoel sperm often contains cytoplasmic microtubules. The cytoplasmic micro-tubules underlying the cellular membrane are called cortical, while cyto-plasmic

Figure 2. Diagram showing types of axoneme structure (A) and cytoplasmic micro-tubule arrangement (B) on cross-sections of acoel spermatozoa.

microtubules arranged along the central axis of the cell are called axial (Fig. 2B). Other types of variations observed in acoel spermatozoa and recom-mended for phylogenetic purposes by Raikova et al. (2001) include the ratio of nuclear length to sperm length (nucleus-to-sperm-length-ratio), position of the nucleus, and the degree of overlap between flagella and nucleus. In paper II new sperm characters including types of granules and a third set of cyto-plasmic microtubules ‘distal microtubules’ are described (Fig. 2B). Use of sperm characters in phylogenetic reconstruction within the Childia species and their closest allies is also reported (paper II).

Nervous system Acoels exhibit great variability in the construction of their brain and nervous system. Despite this, they have traditionally been used as model animals to

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infer the ancestral condition of the nervous system of the Bilateria, due to the simplicity of their nervous system (e.g. Reisinger 1972; Ivanov and Mam-kaev 1973). Recent neuroanatomical studies have demonstrated that the acoel brain is non-homologous to that of other Platyhelminthes (e.g. Hasz-prunar 1996; Raikova et al. 2001; Reuter et al. 2001), which is in agreement with recent molecular studies (Ruiz-Trillo et al. 1999; Telford et al. 2003). The phylogenetic utility of the nervous system characters in a monophyletic group of acoels was demonstrated by Raikova et. al. (2004). Some of the variable characters of the nervous system that are useful in the study of the acoel phylogeny include the position and development of the brain; number, position and thickness of dorsal commissures; number and shape of lateral connectives; number of ventral commissures; and brain contact with the nerve net (see Raikova et al. 2004). Nervous system characters from Raikova et al. (2004) are used in the phylogenetic analyses in Paper I.

Musculature and ultrastructure of copulatory organs As mentioned earlier, the morphology of the male copulatory organ remains the keystone of the current taxonomy of the acoels. Although the phyloge-netic signal of male copulatory organ characters in acoels taxonomy is unde-niable, the method used to study these characters remains critical. For exam-ple, confocal microscopy coupled with fluorescence techniques and electron microscopy have generated several new characters and provided acoel sys-tematists with fine details to discern between seemingly similar (at the light microscope level) copulatory organs. Hooge and Tyler (2005) recently re-vised the family Convolutidae Dörjes, 1968, based on copulatory organ musculature. Similar revision of the Childiidae is done in Paper I. Papers IIIand IV present both muscular and ultrastructural characters of the copulatory organ and their phylogenetic significance in the taxon Childia.

MoleculesThe advent of the Polymerase Chain Reaction (PCR) has enabled molecular systematists to obtain a plethora of sequence data from different molecular markers at all taxonomic levels. Generally, molecular markers with con-served sequences (e.g. rRNA genes) are used for inferring deep phylogenies, while fast evolving markers (e.g. mitochondrial genes) are used for lower taxonomic levels. Several molecular sequences (markers) from acoels have been obtained including nuclear ribosomal genes (18S rRNA (18S) and 28S rRNA (28S)), protein coding genes such as the mitochondrial gene cyto-chrom oxidase I (COI) and the nuclear genes elongation factor one alpha (EF1), myosin II and Histone H3 (H3) as well as Hox genes. Obtaining se-quences from acoels is unusually difficult. Many of the universal primers used in Bilateria do not work in acoels. As a result one has to design specific

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primers to amplify a particular gene of interest. The small size of the worms and shortage of material that limits the amount of DNA for experimentation are additional problems in molecular acoel systematics. Molecular data are expected to contribute greatly in our understanding of acoel phylogenies owing to the limited numbers of morphological characters available. Be-sides, most of the techniques used for obtaining the morphological characters shown to be useful in acoel taxonomy are as costly as those used in obtain-ing molecular data and morphological studies generally require plenty of materials compared to molecular studies. Despite this, molecular systematics of the Acoela is at its earliest stage due to the above-mentioned technical problems and difficulty associated with collection and identification. Acoels are not only difficult to collect but also to identify, some of which requiring examination of sectioned material for the correct identification. Until now, only three studies have used molecular data to examine relationships within the Acoela: Hooge et al. 2002; Raikova et al. 2004; and the study in paper Iof this thesis. Other molecular studies related to acoels involve few se-quences and are primarily concerned in determining the phylogenetic posi-tion of the Acoela within the Bilateria (Ruiz-Trillo et al. 1999; Jondelius et al. 2002; Telford et al. 2003). The ribosomal RNA genes coding for the smaller subunit (18S) and the larger subunit (28S) are among the most com-monly used markers to infer phylogenetic relationships at all taxonomic lev-els of acoels (e.g. Ruiz-Trillo et al. 1999; Hooge et al. 2002; Telford et al. 2003; Raikova et al. 2004; paper I). Particularly, the 18S rRNA gene has become a de facto tool in invertebrate systematics. The 18S rRNA and 28S rRNA molecules are structural components of the ribosome, which are the protein synthesis factories of living cells.

In addition of ribosomal genes, Paper I presents the first use of the protein coding gene Histone H3 within the acoel phylogeny. This gene is involved in packaging and storing of DNA. Histone H3 is a nuclear gene coding for 135 amino acid residues, which is thought to be highly conserved (Thatcher and Gorovsky 1994). It has been used in evolutionary studies of many inverte-brates (e.g. Brown et al. 1999; Colgan et al. 2000; Thollesson and Norenburg 2003).

In sum, the few molecular studies carried out so far in Acoela have given us insights to an extent into the intra- and inter-relationships between some sub-groups of the Acoela. Furthermore, they have assisted us in discerning those morphological characters that could be used in resolving acoel phylog-enies. For instance, several morphological characters relating to sperm ultra-structure, musculature and nervous system characters were found to corrobo-rate molecular-generated phylogenies (Hooge et al. 2002; Raikova et al. 2004; papers I-IV).

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Focus of the thesis Advances in acoel systematics with discovery of new morphological charac-ters assisted by modern techniques and the advent of molecular data have contributed to refine and expand the number of acoel families from 15 to 21 (see Tyler et al. 2005). However, much work remains to be done. Further-more, despite the growing number of new morphological characters, their phylogenetic value to define monophyletic groups within the Acoela has not yet been assessed. This thesis focuses on one of the least known acoels fami-lies, the Childiidae sensu Dörjes, 1968, which are commonly found on the Swedish West Coast. The present thesis explores the phylogenetic signifi-cance of different morphological characters from several anatomical struc-tures and attempts to identify those characters that could be used in phyloge-netic analysis of other acoel groups. Finally, although this thesis focuses on the Childiidae sensu Dörjes, 1968 (mainly Childia and Paraphanostoma and one species representing Actinoposthiidae (see Table 2), other genera of Actinoposthiidae (see Table 1) are not included due to shortage of material), we have also studied some member of the Mecynostomidae, which recent studies have pointed out to be the closest relatives to some members of the Childiidae (Hooge et al. 2002).

The ChildiidaeThe Childiidae is one of the families of the Acoela, originally erected by Dörjes (1968). According to his definition, members of the Childiidae are characterized by a well-developed copulatory organ; a cone-shaped penis stylet built of sclerotized needles or muscular elements that is not invagi-nated into a seminal vesicle (when present); variable male pore position; and ventral mouth opening. Based on the above definition, 13 valid genera were included in the family, with a total of 45 species (Table 1; see Tyler et al. 2005). Most of the genera were monotypic, the two genera with the highest number of species were Paraphanostoma (Westblad, 1942) and Philacti-noposthia (Dörjes, 1968). Members of Paraphanostoma and Childia are common on the Swedish West coast. The Childiidae have been subjected to several taxonomic revisions. Studies of the sperm structure (Raikova et al. 2001) and patterns of the body-wall musculature (Hooge 2001) have ques-tioned the monophyly of the Childiidae. Based on differences observed in the arrangement of the body-wall muscles, Hooge erected a new family, the Actinoposthiidae Hooge, 2001, for all the known childiids, except the mono-typic genus Childia. However, a recent study using 18S sequence data and nervous system characters by Raikova et al. (2004) has questioned this rear-rangement (see also Hooge et al. 2002). The strong support from the 18S sequences and similarities in morphological characters, such as reversed body-wall musculature, patterns of nervous system, and the sperm and stylet

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structure, between Paraphanostoma species and C. groenlandica has led Raikova et al. (2004) to recommend reclassification of Paraphanostoma back to Childiidae.

Table 1. Representative genera of the Childiidae sensu Dörjes, 1968 and their cur-rent taxonomic status

Genus No. of representatives Current taxonomic status

Actinoposthia (An der Lan, 1936)

6 Actinoposthiidae Hooge, 2001

Adeloposthia (An der Lan, 1936)

- nomen nudum

Archactinoposthia (Dörjes, 1968)


Atriofronta (Dörjes, 1968) 1 Actinoposthiidae Hooge, 2001

Childia (Graff, 1910) 1 Childia (Paper I) Childianea (Faubel & Came-ron, 2001)


Microposthia (Faubel, 1974) 1 Actinoposthiidae Hooge, 2001

Monoposthia (Ehlers & Dörjes, 1979)

- Preoccupied

Paractinoposthia (Ehlers & Dörjes, 1981)


Paraphanostoma (Westblad, 1942)

11 Childia (Paper I)

Paraproporus (Westblad, 1945)


Pelophila (Dörjes, 1968) 2 Actinoposthiidae Hooge, 2001

Philactinoposthia (Dörjes, 1968)


Proactinoposthia (Dörjes, 1968)

- Original misspelling

Pseudactinoposthia (Dörjes, 1968)


Tetraposthia (Steinböck, 1931)

- nomen nudum

Objectives

1. To discover using modern techniques, new morphological charac-ters, that could be used in the taxonomy and phylogeny of the Childiidae and their closest relatives, through the exploration of dif-ferent anatomical structures: a) body wall musculature, statocyst and internal muscles (paper I); b) sperm ultrastructure (paper II); c) male copulatory organ musculature (paper III); d) stylet ultrastructure (paper IV).

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2. To generate a robust phylogenetic hypothesis of the Childiidae, us-ing molecular markers, such as 18S and 28S rRNA genes and His-tone H3 as well as morphological characters in a “total evidence” analysis

3. To use the reconstructed phylogeny for the following purposes:

a. To revise the current classification in the family Childiidae (I)b. To test the utility of the 18S rRNA gene as a phylogenetic

marker at low taxonomic levels within the Acoela. c. To assess the value, as phylogenetic markers, of classical mor-

phological characters, such as the male copulatory organ and seminal bursa, traditionally employed in the taxonomy of acoels in general and the Childiidae in particular (I)

d. To assess the phylogenetic value of the new morphological characters described in this thesis.

4. To describe a new viviparous acoel species found to belong to the taxon Childia, on the basis of the reliable characters identified in this thesis (V)

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Material and Methods

Taxa studied and Sampling Table 2 lists the species studied in papers I-V. In addition to the seven mem-bers of Childia (see Table 2), several other species were also investigated: Actinoposthia beklemischevi, a taxon suggested to share some morphological similarities with Childia and classified within the Childiidae by Dörjes (1968), Philocelis karlingi, a species with a penis stylet from the family Oto-celididae Dörjes, 1968, five taxa belonging to the Mecynostomidae Dörjes, 1968 (see Table 2), which were shown to be the closest relatives of Childiaby molecular studies (e.g. Hooge et al. 2002); and an undescribed viviparous acoel, (now Childia vivipara), which was shown to group with Childia in previous studies (e.g. Raikova et al. 2004).

Table 2. List of species investigated in this thesis (I-V).Taxon name Group Included in paper/s

Childia brachyposthium (Westblad, 1942)

Childia (Graff, 1910) I-IV

Childia macroposthium (Steinböck, 1931)


Undescribed viviparous acoel species

Childia (Graff, 1910) I-V

Childia groenlandica (Levin-sen, 1879)


Childia crassum (Westblad, 1942)


Childia submaculatum (Westblad, 1942)


Childia trianguliferum (Westblad, 1942)


Childia cycloposthium (Westblad, 1942)


Mecynostomum auritumWestblad, 1946 (from the Baltic Sea)

Mecynostomidae Dörjes, 1968

I & II

Mecynostomum sp. (from the White Sea)


II

Eumecynostomum flavescens Mecynostomidae Dörjes, II

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(Ørsted, 1845) 1968 Eumecynostomum westbladi (Dörjes, 1968)


II

Eumecynostomum altitudi Faubel and Regier, 1983


I & II

Philocelis karlingi(Westblad, 1946)

Otocelididae Westblad, 1948 I & II

Actinoposthia beklemishevi (Mamkaev, 1965)

Actinoposthiidae Hooge, 2001

I

Specimens of the seven Childia species (see Table 2), an undescribed vi-viparous acoel species and Eumecynostomum altitudi, were sampled on muddy bottoms at 30-60 m depth in the Koster and Gullmar fjord areas on the Swedish West coast in 1999-2005. Specimens of Mecynostomum auri-tum and Philocelis karlingi were collected near Torö, south of Stockholm, in August 2003. Specimens of Mecynostomum sp., very similar in appear-ance to M. auritum, and specimens of Actinoposthia beklemischevi were sampled in the vicinity of the White Sea Biological Station Kartesh (Russia), in September 2003. Specimens of Eumecynostomum flavescens and E. west-bladi were sampled in September 1999 from algae, at a depth of about 1 m, near the Tjärnö Marine Research Station at the Swedish West Coast.

Morphological methods Original morphological data were obtained by using both traditional and modern techniques. Specimens were fixed according to the requirements of each technique. For histology and transmission electron microscopy (TEM), specimens were fixed in 3% glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.2) plus 2% CaCl2 and 8% sucrose for 1 h at 4°C and post-fixed in 1% osmium tetroxide with the same buffer for 2 h at room tempera-ture. After dehydration in a series of ethanol, the specimens were embedded in Spurr epoxy resin. For phalloidin staining of muscles, animals were fixed in Stefanini's fixative (2% paraformaldehyde and 15% picric acid in 0.1 M Na-phosphate buffer) at pH 7.6.

For histological observations, serial sections of 1.5–2.5 µm thickness were cut with a LKB I Ultratome and stained with 1% aqueous toluidine blue. Digital images from the sections were recorded using a Fuji Finepix S1 pro camera. Additional morphological data were collected by studying mate-rial borrowed from the Swedish Museum of Natural History (SMNH) and from the literature.

For TEM observations, ultrathin sections were cut with an LKB 1 Ul-tracut equipped with a diamond knife, stained with an aqueous solution of uranyl acetate and lead citrate and examined with a Zeiss Supra 35-VP (Carl

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Zeiss SMT, Oberkochen, Germany) field emission SEM equipped with a STEM detector for transmission microscopy.

Staining of F-actin fibers with TRITC-labeled phalloidin (Sigma) (1:200) of whole-mount specimens was performed for 2 h at room temperature (Wahlberg 1998). After rinsing in PBS, the animals were mounted in 60% glycerol in PBS. The preparations were examined with a confocal scanning laser microscope (CSLM) LEICA TCS 4D. Specimens were viewed either from the dorsal or ventral side.

For immunocytochemistry, three monoclonal anti-tubulin antibodies (anti-alpha-tubulin, clone DM 1A, Sigma (Blose et al. 1984), anti-beta-tubulin, clone TUB 2.1, Sigma (Gozes and Barnstable 1982) and anti-acetylated-tubulin, clone 6-11B-1, Sigma (Piperno and Fuller 1985)) were used. Data on immunocytochemistry were obtained using the protocols in papers II and IV. Immunocytochemistry methods were used to either deter-mine the composition of some sub-structures in flagella and copulatory stylets or to visualize/localize the nucleus and the flagella.

Molecular methods Three molecular markers, 18S, 28S and H3, were used in paper I to inferphylogenetic relationships within the Childiidae . The H3 sequences and all 28S sequences except one were originally generated in paper I. Nine of the twelve 18S rRNA sequences and one of the 28S rRNA sequences for the species investigated were obtained from the Genbank. DNA extraction and cloning were performed using standard protocols given in manufacturers’ manual. Amplification and sequencing of 18S, 28S and H3 were performed following the protocols in Norén and Jondelius (1999), Littlewood et al. 2000 and Colgan et al. (2000), respectively.

Data preparation and Analyses DNA sequences were aligned using the computer program for automatic alignment Clustal X (Thompson et al. 1997), followed by manual re-alignment in Se-Al v2.0a10 (Müller 2002). The sensitivity of the 18S se-quence data to different alignment parameters in ClustalX was explored by means of a sensitivity analysis sensu Wheeler (1995). A total of 36 matrices were produced by a combination of gap-opening penalties (pairwise and multiple alignment) values of 5/15/30/50/75/95. Another 18S data matrix (for the analysis in Figure 4 in this thesis) was prepared using a sequence alignment program ‘hmmer’, which is based on the secondary structure of the molecule (Eddy 1998).

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Maximum parsimony analyses in PAUP* 4.0b10 (Swofford 2002) were carried out on the 18S data matrices, and on the combined data sets includ-ing the three molecular markers and 50 morphological characters in a “total evidence” approach. Branch support was evaluated in PAUP* 4.0b10 by using bootstrap pseudo-sampling. Clade support for the four data partitions was assessed using the partitioned Bremer support (Baker and DeSalle 1997; Baker et al. 1998; Bremer 1988).

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Results

Phylogenetic analyses (I)The total evidence analysis was based on three molecular markers (18S, 28S & H3) and 50 morphological characters. The morphological characters relat-ing to body-wall musculature, statocyst and internal muscles in six Childiaspecies (former Paraphanostoma) are reported in paper I. Body-wall muscu-lature of all Childia species (including C. vivipara- paper V) is characterized by the reversed arrangement of the longitudinal and circular muscle layers, by the absence of diagonal muscles on the ventral side of the body and the presence of one or two types of diagonal muscles on the dorsal side. Stato-cyst and internal muscles differ in abundance and arrangement among Childia species.

The 18S matrix aligned using the default values (15/15) of gap opening penalties in Clustal X (Thompson et al. 1997) was selected in the final com-bined analysis. The tree from analysis of this matrix contained most of the groups represented in the most parsimonious trees (MPTs) of 36 matrices produced by a combination of gap-opening penalties (pairwise and multiple alignment) values of 5/15/30/50/75/95. A total of 12 taxa (9 ingroups and 3 outgroups) were included in the combined analysis. A parsimony analysis of the combined molecular and morphological data (3734 characters) generated a single MPT of 2756 steps with consistency index (CI) excluding unin-formative characters = 0.6.

All ingroup taxa, Childia + Paraphanostoma, except Actinoposthia bek-lemischevi formed a monophylum with 100% bootstrap support. Childiagroenlandica is nested in a clade consisting of all species of Parap-hanostoma; as a sister group to the viviparous acoel + P. brachyposthium +P. macroposthium. The total evidence tree is well resolved and consists of well-supported clades (>70%) with the exception of one clade, P. trian-guliferum + P. cycloposthium, that is weakly supported (56%) (Fig.3). Mor-phological apomorphies with C.I.=1 for each of the clades in the total evi-dence tree, including other morphological characters generated in this thesis (see Table 3), are mapped in Figure 3. In addition to maximum parsimony (MP), other methods of phylogenetic inference (Bayesian inference and

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Neighbour joining) were used to infer total evidence phylogeny. Both meth-ods yielded similar result to MP (results not shown).

Figure 3. Total evidence tree (modified from paper I); bootstrap frequencies and partitioned Bremer support for 18S, 28S, H3 and morphological characters are shown above branches. Selected morphological synapomorphies listed in Table 3 are shown below branches.

Incongruence, as indicated by negative partitioned Bremer support, in the node support of the groups among the four partitions was very low in the

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total evidence tree; except for the H3 partition. Only one clade (the vivipa-rous acoel + P. brachyposthium + P. macroposthium) received a positive Bremer support by the Histone H3 partition in our total evidence tree. Exclu-sion of the H3 data set did not affect the branching order of the clades with bootstrap support above 60% in the total evidence tree. Bootstrap support increased for all sub-groups, except for one clade (the viviparous acoel + P. brachyposthium + P. macroposthium), which decreased from 88% to 67%.

Sperm ultrastructure (II & V)Sperm ultrastructure in eight Childia species and in the outgroup taxa, me-cynostomids (see Table 2) and Philocelis karlingi, has been studied.

Spermatozoa of all Childia species studied share the same basic ultra-structural characters. The axonemes have a 9 + 1 structure, with 9 peripheral microtubule doublets and a central core element, a single microtubule sur-rounded by electron dense material. When viewed in cross-section, the axonemal core element is observed to be composed of three concentric parts: an outer tubular cylinder, an intermediate zone, and a central microtubule. Immunocytochemical labeling of the sperm axonemes in two Childia species (C. groenlandica and C. cycloposthium) showed that both the 9 doublets and the central singlet microtubule are labeled by the three anti-tubulin antibod-ies used (anti-alpha-tubulin, anti-beta-tubulin and anti-acetylated-tubulin). Other common sperm characters of Childia species are the presence of 6 distal cytoplasmic microtubules in the absence of axial or cortical ones, long nucleus and extensive nucleus-flagella overlap. Distal microtubules are newly identified set of cytoplasmic microtubules lying in the distal end of the sperm cell.

The main differences observed in spermatozoa of Childia species are re-lated to types, distribution and abundance of cytoplasmic granules (dense bodies), which we classified as follows: sausage shaped granules, uniform membrane granules and loose membrane granules with either uniform core or amorphous core. Childia groenlandica has three additional types of gran-ules not found in others, which include: the lamellate inclusions, small round granules and gastrula-shaped granules.

The four species of the Mecynostomidae studied share the following sperm characters with Childia: 9 + 1 axoneme structure, six distal micro-tubules and considerable nucleus-flagella overlap. Three of the mecy-nostomid species (Mecynostomum auritum (from the Baltic Sea), Eumecy-nostomum westbladi and Eumecynostomum flavescens) similarly to the Childia species, lack axial or cortical cytoplasmic microtubules, while Me-cynostomum sp. from the White Sea, in addition to the distal microtubules, possesses two parallel lines of axial microtubules, lining a group of mito-chondria in the shaft region.

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Figure 4. MPT (tree length=5164 steps, CI=0.40) based on parsimony analysis of 18S data. In this analysis more taxa are included, particularly two ingroup taxa (in bold) and more mecynostomids compared to the total evidence tree. Bootstrap sup-port (1000 replicates/10 random additions/TBR) is shown above branches. Sperm characters, related to axoneme structure and types of cytoplasmic microtubules, shared among the taxa analyzed, are shown below nodes.

Philocelis karlingi possesses a 9 + 2 axoneme pattern (with nine periph-eral doublets and two central microtubules) and axial cytoplasmic micro-tubules arranged in two groups, 12-17 each.

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Musculature (III & V) and ultrastructure of the copulatory organs (IV)Copulatory organ musculature of seven species of Childia and the viviparous acoel, C. vivipara, was studied by phalloidin fluorescence method and con-focal microscopy of whole mount preparations. The musculature develop-ment varied greatly, from the absence of a seminal vesicle (SV) to exten-sively developed SV and several additional types of specialized muscles associated with the SV and the stylet/s. Accessory muscles associated with the SV include: muscles radiating from its walls to the body-wall, tentacle muscles, collar muscles and ring muscles located at its opening. The stylet could be stained strongly or weakly, and in some species it was observed to have ring muscles. In general the following species have well developed copulatory muscles compared to the rest: C. brachyposthium, C. macropost-hium, C. vivipara and C. groenlandica.

Stylets of Childia species at the ultrastructural level show little variation. Needles composing the stylet are intracellular differentiations. As shown both by ultrastructural and immunocytochemical methods, the stylet needles, in all species studied, are composed of long parallel microtubules, either tightly packed, or polymerized. The microtubules in some species are ob-served to polymerize forming a honeycomb-like structure in cross section. Variations of stylet ultrastructure among Childia species include: numbers and arrangement of stylet needles, shape of needles, needle compactness, microtubule polymerization, direction of stylet growth and presence/absence of different types of granules. Childia brachyposthium, C. macroposthium, C. vivipara and C. groenlandica species, generally, display more compact stylets than the other species of Childia.

Selected variable characters of the copulatory organ in Childia are given in table 3 and mapped in Figure 3.

Table 3. List of selected morphological characters.

Body-wall musculature 1. Longitudinal muscles lie outside circular muscles 2. Diagonal muscles on the ventral side absent 3. B-Type (sigmoid) dorsal diagonal muscles forming the second layer 4. U2- ventral circular muscles associated with the mouth 5. Well developed statocyst muscles 6. Statocyst muscles in the form of ladder-like grid of muscles supporting the statocyst and

the brain 7. Accessory muscles radiating from the mouth going in anterior direction 8. Abundant inner muscles 9. Diagonal muscles on the dorsal side of the body form a dense grid

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Male copulatory organ musculature

10. Proximal part of the stylet inserted into the seminal vesicle 11. Stylet needles strongly stained by phalloidin 12. Numerous tentacle muscles running from the seminal vesicle down along the stylet 13. Muscular seminal vesicle 14. Stylet rings, tightly encircling the proximal part of the stylet present

Stylet ultrastructure

15. Stylet composed of closely packed concentric layers of needles that are composed of closely packed or polymerized microtubules (more than 14 needle cells)

16. High degree of stylet compactness and microtubule polymerization 17. Flat cells lining the male antrum

Sperm characters

18. Considerable nucleus-flagella overlap 19. Absence of axial and cortical microtubules 20. Loose membrane amorphous core granules

Nervous system

21. Dorsal side of the brain more developed than the ventral part 22. Dorsal posterior commissure (dpc) thicker than the other commissures 23. Deep brain position 24. Dorsal connectives (dc) composed of many fibers, thick in dorso-ventral direction 25. Loop-shaped lateral connectives (lc) 26. Less pronounced brain contact with the nerve net 27. Extensive fiber networks associated with lateral connectives 28. Two symmetrical pairs of large GYIRFamide immunoreactive marker neurons

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Discussion

The utility of classical morphological characters and 18S rRNA gene in Childiidae (I-IV)Dörjes’ (1968) generic classification of the Childiidae was based on three features obtained using simple traditional methods: the male and female reproductive system and the arrangement of body-wall musculature. In this thesis the classical morphological characters were re-investigated, using advanced techniques. This enabled us to evaluate their value in the classifi-cation of the Childiidae, with main focus on three genera: Actinoposthia,Childia and Paraphanostoma. The total evidence analysis presented in paper I demonstrates that the classical morphological characters, particularly the seminal bursa and the male copulatory organ, do not identify monophyletic groups. The nested position of the monotypic genus, Childia in the clade consisting of all Paraphanostoma species in the total evidence tree is in con-tradiction to the previous systems by Westblad (1948) and Dörjes (1968). Westblad (1948) placed Childia in a different group (Opisthandropora-Abursalia) from Paraphanostoma (Proandropora-Bursalia) because of its lack of seminal bursa. A thorough examination of the seminal bursa in Paraphanostoma species, which include both re-examination of sections of the type specimens and the use of phalloidin staining techniques, showed a large variation concerning the size and location of the bursa, its wall muscu-lature and accessory structures. The characters related to the seminal bursa were shown to be homoplasious or autapomorphic in our total evidence phy-logeny. Dörjes (1968) placed both Childia and Paraphanostoma in the fam-ily Childiidae based on male copulatory organ. However, he assigned them into different genera mainly because he used the same criteria, lack of semi-nal bursa, as Westblad, and due to the limitations of his techniques to further reveal their resemblance in the copulatory organ structure (Papers III & IV), body-wall musculature (Hooge 2001; Paper I) and sperm (Paper II). Both ultrastructural and muscular studies of the copulatory organ show that C.groenlandica share several copulatory organ characters with members of Paraphanostoma in composition and arrangement of the stylet needles and accessory muscles associated with the stylet and the seminal vesicle (Fig. 3). Similar to all Paraphanostoma species, C. groenlandica has a reversed ar-rangement of the body-wall musculature (see Westblad 1942; Hooge 2001;

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Paper I) and sperms with 9 + 1 axoneme pattern and the same number and arrangement of distal microtubules (Paper II).

Both, the total evidence (Fig. 3) and 18S (Fig. 4) analyses show that members of the Actinoposthiidae Hooge, 2001 do not form a monophyletic group with Paraphanostoma-Childia clade (see Figs. 3, 4). Dörjes (1968) previously referred Childia, Paraphanostoma and other acoels, now placed in the Actinoposthiidae, to the Childiidae based on classical characters of the male copulatory organ. Phalloidin staining techniques (paper III) and ultra-structural study (paper IV) of the copulatory organ showed some important differences. All species within the Paraphanostoma-Childia clade have rela-tively more stylet needles that are closely packed together in a form of con-centric layers, whereas A. beklemischevi possesses a stylet made of few sepa-rate needles (Paper I). However, the main component of the stylet needles, the microtubules, was found to be the same in three of the abovementioned taxa (paper IV). The monophyly of the Childiidae sensu Dörjes (1968) is rejected not only by all the molecular markers used (Figs. 3, 4), but also by sperm structure (Raikova et al. 2001; paper II) and the patterns of body-wall musculature (Hooge 2001; paper I). The following sperm features character-ize all members of the Paraphanostoma-Childia clade: 9+1 axoneme struc-ture and six distal microtubules in absence of axial or cortical microtubule, while two members of Actinoposthiidae, with known sperm morphology, possess 9+2 axoneme structure and cortical microtubules. Sperm characters have been shown to be reliable phylogenetic characters in acoel taxonomy (Hooge et al. 2002; Paper II). Other important differences include the ar-rangement of body-wall musculature (see also Westblad 1942; Hooge 2001). As mentioned earlier, our findings strongly show that Paraphanostoma is closely related to C. groenlandica in agreement with Raikova et al. (2004) report.

The high degree of congruence, indicated by positive Bremer support (Fig. 3), among the 18S+28S+morphological data partitions indicates that 18S sequences are useful also at lower taxonomic levels. The high degree of incongruence in Histone H3 partition was likely due to homoplasy in the first and third codon positions. The incongruence observed between morpho-logical partition and 18S+28S partitions in the placement of C. vivipara in paper I was due to limited amount of data and the technique used to obtain them for that species. In paper I morphological characters for C. viviparawere only obtained using traditional methods due to shortage of material. The copulatory stylet of Childia vivipara is similar to one of the stylets of C. groenlandica when studied using only light microscopy techniques. How-ever, these similarities were found to be superficial, when we studied the stylet using TEM and phalloidin staining techniques. The stylet of C. vi-vipara shares more important characters: tentacle muscles (III) and more than one layers of concentric needles (IV), with C. macroposthium and C. brachyposthium, than with C. groenlandica (see Fig. 3 for additional simi-

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larities among C. vivipara + C. macroposthium + C. brachyposthium in sta-tocyst muscle arrangement). These similarities better reflect the phylogeny of these species in the total evidence analysis (Fig. 3). This reinforces the argument against using superficial characters of the copulatory organ as phy-logenetic markers (see above).

Revision of the Childiidae Dörjes, 1968 (I) and description of a new Childia species (V)All data partitions demonstrated that members of the Actinoposthiidae and the newly defined taxon Childia (comprising Childia and ParaphanostomaWestblad, 1942) are not close relatives. The monophyly of Childia and Paraphanostoma is strongly supported by both the 18S and 28S data parti-tions. Our study also reveals additional apomorphies uniting Childia with Paraphanostoma from body-wall musculature, statocyst muscles, male copulatory organ, nervous system and sperm ultrastructure. Since the mono-typic taxon Childia Graff, 1910 has priority over Paraphanostoma Westblad,1942, all Paraphanostoma species are transferred to Childia. The following stem-modified node-based definition of the name Childia, in accordance with the PhyloCode (Cantino & de Quieroz 2004), is provided: The name Childia is applied to the clade stemming from the most recent common an-cestor of Childia groenlandica (Levinsen, 1879) and all extant organisms that share a more recent common ancestor with Childia groenlandica than with Mecynostomum auritum (Schultze, 1851) or Actinoposthia beklemishevi Mamkaev, 1965. Photomicrograph of live or sectioned material of all known members of Childia, including a newly described viviparous acoel (paper V)and four taxa not included in our analyses are shown in Figure 5. A new viviparous acoel is placed within Childia. All molecular markers (18S, 28S and H3) and several morphological synapomorphies obtained from sperm, copulatory organ and musculature support the position of the new species in Childia. The new species can be easily distinguished from other Childiaspecies by its viviparous mode of reproduction and a single curved stylet.

In light of the present knowledge, a modified version of diagnosis for the taxon Childia (as compared to the one given in paper I) is provided here:

Diagnosis of currently known members of Childia: Well-developed sin-gle, paired, or multiple copulatory organs built of concentric layers of closely packed stylet needles composed of tightly packed or polymerized microtubules (stylet absent in Childia dubium). Proximal part of the stylet inserted into seminal vesicle if present. Body-wall musculature with longitu-dinal fibers positioned outside circular fibers. Ventral diagonal muscles ab-sent. Ventral straight longitudinal muscles between frontal pore and mouth present. Dorsal diagonal muscles present. Mouth opening ventral. Sperm

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Figure 5. Photomicrographs of the current members of Childia. A-H. Whole live specimens. I-L. Sectioned specimens of type materials at the SNHM. I-K. Longitu-dinal sections. L. Cross-section. Scale bar = 250 mm. e, egg; eg, epidermal gland agglomerations; em, embryo; fo, female opening; gc, glandular complex; gl, glass-like structure; m, mouth; mco, male copulatory organ; mo, male opening; p, penis; sb, seminal bursa; sp, sperm; st, statocyst; sv, seminal vesicle; sty, stylet.

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with 9+1 axonemes, with six distal cytoplasmic microtubules, and without axial or cortical microtubules.

Sperm morphology at higher taxonomic levels The Childia- Mecynostomidae clade The closest relative of Childia among acoels, as shown by molecular data, is the Mecynostomidae (Fig. 4). Morphological synapomorphies supporting this sister group (Childia+Mecynostomidae) relationship are only known from sperm ultrastructure, which include: 9 + 1 axoneme structure and six distal microtubules (Fig. 4). The latter sperm character is a newly described sperm feature that might have a potential phylogenetic significance in acoel systematics. Distal microtubules differ from other cytoplasmic microtubules (axial and cortical) in their localization: they are located in the extreme distal end where no other cytoplasmic inclusions are found other than the axonemes. Distal microtubules usually differ in number from axial micro-tubules and the variation observed in their number and arrangement might be used for phylogenetic studies of acoels. Absence of axial or cortical micro-tubules could be considered as an additional synapomorphy of the Childia-Mecynostomidae clade. However, two of the five mecynostomids, with known sperm morphology, are reported to have axial microtubules. The 18S phylogeny (Fig. 4) shows that the Mecynostomidae is paraphyletic and this might explain the deviations observed in this character. Little is known about the phylogeny of the Mecynostomidae; therefore, this family requires further investigation.

9 + 1 axoneme: a homoplasy or homology of the Acoela and the Trepaxonemata?The spermatozoa of Childia and Mecynostomidae show 9 + 1 axoneme con-figuration, seemingly similar to the 9 + ‘1’ axoneme pattern of the Platy-helminthes – Trepaxonemata. Paper II demonstrates, based on electron-microscope immunocytochemistry, that the 9 + 1 axoneme structure ob-served in acoels Childia and mecynostomids is not homologous to that found in the Trepaxonemata. Unlike the central cylinder of trepaxonematans, the central cylinder of the 9 + 1 axonemal pattern in acoels is immunoreactive to tubulin and contains a single central microtubule recognizable by its shape and size (25 nm). We propose to refer to the acoel central element as 1 with-out a quotation mark, as it is a normal microtubule, while the central element of the Trepaxonemata does not contain tubulin. That will permit to designate the 9 + 1 pattern in Acoela from the 9 +’1’ pattern (with quotation) in Tre-paxonemata.

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Conclusions

This thesis hitherto presents the most comprehensive systematic study within the Acoela. An acoel subtaxon, the Childiidae, has been revised based on well-corroborated phylogeny. Combined application of traditional and mod-ern techniques within a monophylum has many implications.

Firstly, it enabled us to compare and assess the reliability of the classical morphological characters that has been used in acoel taxonomy. Our findings undermine the phylogenetic use of the seminal bursa as a phylogenetic char-acter in the newly defined taxon Childia. Detailed study of the copulatory organ has allowed us to discern important homologous characters from su-perficial similarities observed at light microscopy level. The male copulatory organ bears important phylogenetic signal, however, methods used to obtain the characters are critical.

Secondly, we were able to describe several new morphological characters that could be used at all taxonomic levels within the Acoela (see Table 3). The morphological characters, shown to be useful at lower taxonomic levels within Childia, include those related to body-wall musculature, statocyst musculature, male copulatory organ musculature, copulatory stylet ultra-structure, nervous system, and sperm granule characters. Most of sperm characters: axoneme structure, the three types of cytoplasmic microtubules (axial, cortical and distal) are useful at higher taxonomic levels within the Acoela. The phylogenetic significance of these characters is determined by their congruence with other sources of data, such as molecules. The com-monly used molecular marker for inferring deep phylogenies, the 18S rRNA gene, is shown to be useful also in deciphering lower taxonomic groups within the Acoela.

Finally, the identification of phylogenetically reliable diagnostic charac-ters within a monophylum will be useful when classifying new acoel species.

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Summary in Swedish

Acoela är en grupp små (0.2 - 10 mm kroppslängd) marina maskar. Acoeler-na är enkelt byggda djur med en mun, men utan ändtarmsöppning. I framän-den finns ett starkt ljusbrytande jämviktsorgan, en sk statocyst, som är ett gott kännetecken på att det är en acoel man studerar. Namnet Acoela syftar på att dessa djur saknar tarmhålighet (a- negationsprefix, koilia -grekiska för mage). Den enkla byggnaden har gjort att många zoologer ansett acoelerna stå nära ursprungsformen för de bilateralsymmetriska djuren, en grupp som omfattar det överväldigande flertalet av dagens djurarter förutom svampdjur, nässeldjur, kammaneter och några få andra. Denna gamla uppfattning har fått förnyat stöd av moderna molekylärfylogenetiska undersökningar där acoelerna utgör den äldsta grenen bland Bilateria.

Trots den nyckelställning som acoelerna tycks ha i djurens evolution, är de inbördes släktskapsförhållandena mellan de ca 350 kända arterna av acoe-ler är mycket dåligt kända. Väl stödda hypoteser för acoelernas inbördes fylogeni saknas i stort sett. Hittillsvarande taxonomi härrör huvudsakligen från 1960-talet eller tidigare, och grundas på enkla ljusmikroskopiska studier av några få organ. Den här avhandlingen handlar om en grupp bland acoe-lerna, Childia, släktskapen mellan de ingående arterna och evolutionen av ett antal olika morfologiska egenskaper. Avhandlingen baserar sig på fem de-larbeten, vilka fortsättningsvis refereras till med de romerska siffrorna I - V.Ett syfte har varit dels att specifikt utreda fylogenin och göra en klassifice-ring av Childia baserad på denna, ett annat att beskriva nya morfologiska egenskaper som kan användas vid studiet av evolutionära mönster bland acoelerna (avhandlingsdel I). Phalloidininmärkning i kombination med kon-fokal laserskanningmiroskopi användes för att göra en detaljerad rekonstruk-tion av kroppsväggens muskulatur hos flera Childia arter. Kroppsväggsmus-kulaturen hos Childia har längsmuskulatur ytterst, vilket avviker från den normala organisationen hos acoeler liksom de flesta övriga Bilateria. Genom att tre olika gener och flera olika typer av morfologiska data använts kunde också det fylogenetiska informationsinnehållet i de olika generna och morfologiska karaktärerna utvärderas. Evolutionen av den inverterade kroppsväggsmuskulaturen inom Childia rekonstruerades. Den väl korrobore-rade fylogenin baserad både på nukleotiddata och morfologutgör grundval för en omklassificering av de studerade arterna. Den enda arten av släktet Childia är närmast besläktad med vissa arter inom Paraphanostoma. Efter-som namnet Childia är äldre, blir Paraphanostoma en yngre synonym.

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Spermier har kallats “djurens ID-kort” eftersom de ofta är av stort intres-se som fylogenetiska markörer på hög taxonomisk nivå. De är lätta att identi-fiera och studera ultrastrukturellt och homologibedömningar brukar vara oproblematiska. Hos acoelerna är spermierna mycket avvikande från den vanliga morfologin hos tex mollusker eller nässeldjur som har en fri flagell (spermiesvans) med ett 9 + 2 mönster av perifera och centrala mikrotubuli. Acoelspermierna har två flageller (spermiesvansar) som ej är fria, utan in-korporerade i spermiecellen och hos många acoeler bildar flagellens mikro-tubuli istället ett 9+1 mönster. Detta 9+1 mönster är ganska likt konfigura-tionen i de egentliga plattmaskarnas (gruppen Rhabditophora) spermiesvan-sar och har uppfattats som en homologi mellan de två grupperna. Med im-munocytokemi visas i del II att denna likhet endast är ytlig. Antikroppar mot olika tubulinepitoper binder till acoelspermiernas centrala struktur, (1:an i 9+1), men ej till motsvarande struktur hos Rhabditophora. De två varian-terna av 9+1 konfiguration är sannolikt ej homologa. Kan spermier användas om fylogenetiska markörer också bland närbesläktade arter? Detta studera-des hos åtta Childia arter och tre andra acoeler som användes som utgrupp. Childia spermierna är mycket lika varandra, men vissa variationer form stor-lek och antal hos cytoplasmakropparkunde konstateras. Vidare upptäcktes en ny typ av sk distala mikrotubuli som utgör en synapomorfi för gruppen Chil-dia + Mecynostomidae.

Om acoelernas allmänna anatomi är enkel, är könsorganen relativt kom-plext uppbyggda. Acoelerna är hermafroditer med inre befruktning. Hos många arter finns såväl hanliga som honliga kopulationsorgan och andra sekundära könsorgan. Det hanliga kopulationsorganet är av särskilt stort intresse från en taxonomisk utgångspunkt eftersom den klassiska klassifice-ringen av acoelerna till stor del grundades på utseendet hos detta. Studier av muskulaturen hos kopulationsorganet och associerade strukturer med phallo-idininmärkning kombinerat med konfokal laserskanningmikroskopi rappor-teras i del III. Muskulaturens utformning och komplexitet är mycket variabel hos de åtta Childia arter som studerades. Exempelvis saknades en sädesblåsa helt hos vissa arter, medan andra hade en sädesblåsa med mycket välutveck-lad och komplex muskulatur. Arbete IV i avhandlingen handlar om struktu-ren hos det hanliga kopulationsorganet hos Childia som studerats med transmissionselektronmikroskopi i kombination med immunohistokemiska metoder. Det visade sig att det stilettformiga kopulationsorganet, “penissti-letten”, hos Childia består av ett relativt stort antal nålar som var och en bildas intracellulärt i en särskild cell. Nålarna består av tubulin, som är ett protein med skelettfunktioner i de flesta celler. Denna byggnad hos kopula-tionsorganet skiljer sig tydligt från de på ljusmikroskopisk nivå likartade organen hos acoeler inom släktet Actinoposthia. Det hanliga kopulationsor-ganet är inte en enkel nyckel till ett fylogenetiskt acoelsystem. Det finns fylogenetisk signal i kopulationsorganets byggnad, men den måste dechiffre-

38

ras med mer sofistikerade metoder såsom konfokalmiroskopi och elektron-mikroskopi, och framför allt prövas mot annan information.

Arbete V beskriver en ny vivipar acoel. Normalt är acoeler äggläggande, men en vivipar art som är symbiont i sjöborrar är känd sedan tidigare. Här beskrivs den första frilevande vivipara acoelen under namnet Childia vivipa-ra. Nukeotidsekvenser liksom ett flertal morfologiska karaktärer Acoelernas utvecklingsbiologi är dåligt känd: endast de allra tidigaste stadierna upp till 32 celler är beskrivna från ett fåtal arter. Upptäckten av en vivipar acoel erbjuder möjligheter att beskriva den fortsatta embryonalutvecklingen, tex bildning av mesoderm och nervsystem. De första stegen mot en mer full-ständig beskrivning av acoelernas utvecklingsbiologi tas i del V då de fåtali-ga Childia vivipara embryoner som var tillgängliga beskrivs från snittserier.

39

Acknowledgements

I wish to acknowledge my sincere gratitude to all the individuals who have assisted me in different ways during my thesis work. In particular, I am es-pecially indebted to my major supervisor Ulf Jondelius for his professional advice, constructive critiques and invaluable help.

I am also thankful to Olga Raikova, for whom I cannot even begin to ex-press how grateful I am for all the help and support she has bestowed on me. You are my role model in every aspect of scientific work. Thank you for teaching me fluorescent staining techniques, for your inspirational and con-structive comments on my thesis and previous papers, and for being a super co-worker. I would also like to extend my thanks to your family: your son and mother, for their patience during long hours of work that resulted into irregular and late lunches.

I am grateful to Prof. Fredrik Ronquist for his helpful advices and discus-sions concerning my research.

I would like to thank Isabel Sanmartin (assistant supervisor) and Sylvain Razafimandimbison for interesting discussions and practical assistance throughout my study. I also appreciate your friendship and concerns. Isabel Sanmartin is also thanked for her last minute comments on my thesis.

My collaborator, Matthew Hooge, from the University of Maine is thanked for his constructive input in paper I, readiness to help and discus-sions regarding acoel systematics. Thanks also for providing whole live pho-tomicrograph of C. groenlandica in Fig. 5 of this thesis.

Many thanks to Afsaneh Ahmadzadeh for her practical help related to molecular lab work. Nahid Heidari is also thanked for sharing her molecular lab experiences in absence of Afsaneh.

Mikael Thollesson (assistant supervisor) is thanked for sharing his mo-lecular experience, discussions regarding the PhyloCode and reading my thesis.

Special thanks are due to Kristina Articus, Prof. Christine Dahl and Anne Krueger for translating German literatures. Maria Carlota Monroy is simi-larly thanked for her help in translating Portuguese literature.

The assistance of all the staff at Klubban and Kristineberg marine sta-tions, at the Swedish West Coast, during sampling seasons is greatly appre-ciated.

40

I am grateful to Stefan Gunnarsson, Annette Axén and Gary Wife for their unreserved and skillful help in microscopy and microtomy. Stefan Gunnarsson is also thanked for his tips and practical assistance in Photoshop.

I like to thank Johan Nylander and Andreas Wallberg for being there for me in dealing with my computer and analytical problems.

Thanks to all my former and present colleagues at the departments of Sys-tematic Zoology: Hege, Per, Lars, Karolina, Mattias, Andreas, Johan and all colleagues at the department of Systematic Botany for your concern and support.

I am indebted to Sven Boström from the Swedish Museum of Natural History for his assistance in facilitating the loans of specimens.

I am grateful to SIDA/SAREC for partial financial support of my PhD study. All staff members of the International science program (ISP) and Ulla Hedenquist at the Department of Systematic Zoology are thanked for admin-istrative and technical assistance in Sweden. I am also thankful to my home University in Eritrea, University of Asmara, for the opportunity given.

My colleagues – Ermias (extra tack!), Kidane A., Kidane Y., Lijam and others are thanked for friendship and interesting discussions concerning re-search and extracurricular life. I like to thank my friend Carina Crawford for reading my thesis.

All my friends in Sweden (long list…) are also thanked (Stor Tack) for their support and making my stay in Sweden wonderful.

My parents Mom- Haimanot (the one and only) and late father- Isaak, and my siblings: Freweini (& her family), Goitiom (& his family), Samson (& his family), Nighisti (& her family), Georgo (& his family), Fitsum (& his family), Aster, Helen and Solomon are greatly appreciated for their love, kindness and moral support.

Lastly, I would like to conclude my acknowledgements, especially, with two people who have been there for me since young age to help me to where I am today, Haimanot Araya and Freweini Isaak. Thanks for believing in me.

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