Contents List of Papers 2 Abstract 3 Introduction 5 Biodiversity – why should we care? 5 Marine meiofauna 6 What are Nemertodermatida 7 General morphology 8 Ultrastructure 8 Historical overview of the taxon 9 Phylogenetic position 11 Classification 12 Methods 13 Sampling 13 Infrastructure and general workflow 14 Fixation 16 Abundance and patchiness 18 Collection effort for this thesis 19 Confocal Laser Scanning Microscopy (CLSM) 20 Principal of CLSM 20 Phalloidin staining of F-actin 21 Immunolabelling of the nervous system 22 Molecular Analyses 24 DNA extraction, amplification & sequencing 25 Data properties 26 Phylogenetic analyses 27 Discussion and future perspectives 27 Literature cited 33 Acknowledgements 39

List of papers

Paper I Meyer-Wachsmuth I, Raikova OI, Jondelius U (2013): The muscular system of Nemertoderma westbladi and Meara stichopi (Nemertodermatida, Acoelomorpha). Zoomorphology 132: 239–252. doi:10.1007/s00435-013-0191-6.

Paper II Meyer-Wachsmuth I, Curini Galletti, M, Jondelius U (in press): Hyper-cryptic marine meiofauna: species complexes in Nemertodermatida. PLOS One 9: e107688. doi:10.1371/journal.pone.0107688

Paper III Meyer-Wachsmuth I, Jondelius U: A multigene molecular assessment reveals deep divergence in the phylogeny of Nemertodermatida. (Manuscript)

Paper IV Raikova OI, Meyer-Wachsmuth I, Jondelius U: Nervous system and morphology of three species of Nemertodermatida (Acoelomorpha) as revealed by immunostainings, phalloidin staining, and confocal and differential interference contrast microscopy. (Manuscript)

2 Abstract

Nemertodermatida is a group of microscopic marine worm-like animals that live as part of the marine meiofauna in sandy or muddy sediments; one species lives commensally in a holothurian. These benthic worms were thought to disperse passively with ocean currents, resulting in little speciation and thus wide or even cosmopolitan distributions. Individuals occur in low abundance and have few light microscopically available characters, which altogether may explain why only eight species had been described between the discovery of the taxon in 1930 and this thesis. We used molecular methods to address the diversity and phylogeny of this group for the first time. In a study of two nominal species with samples from all around the world, a high degree of cryptic speciation was discovered and several new species described. Diagnoses were based on molecular data complemented by morphological characters, where available. Given the patchy geographical record it can be assumed that the majority of the biodiversity of Nemertodermatida is yet to be described. A phylogenetic study including all but three known species revealed a deep divergence between the two families of Nemertodermatida but non-monophyly of the taxon was rejected by an Approximately Unbiased test. Confocal laser scanning microscopic studies of several species show that the pattern of the body-wall musculature and the nervous system are specific for different genera. The muscular system of all species consists of a basic orthogonal grid with specific diagonal musculature and specialized muscles associated with body openings. The mouth appears to be transient feature in Nemertodermatida, developing only after hatching and being reduced again in mature worms. The nervous system is highly variable with very different ground patterns between the genera, such as an epidermal net, a centralized neuropile or a commissural brain.

Keywords: Nemertodermatida, Acoelomorpha, morphology, CLSM, Phalloidin, musculature, DNA, cryptic species, species delimitation, dispersal, taxonomy, phylogenetics, IHC, nervous system

3

4 Introduction

Biodiversity – why should we care?

Biodiversity has become one of the keywords in the biological sciences since E.O. Wilson stated the “biological diversity crisis” in 1985 (Wilson 1985). Although species have become extinct at all times the rate of extinction has increased dramatically over the past few centuries, being estimated to be 100 to 1000 times higher today than the natural rate (Pimm et al. 1995; 2014). Some species have become extinct due to direct human pressure, like the dodo or the passenger pigeon. The indirect effects of human endeavour, however, are much more threating and affect even the remotest areas of the planet. The most often quoted example arguably being climate change leading to melting ice caps. This results in the decrease of habitat for example for the polar bear, and to rapidly rising sea levels threating habitats such as the mangroves. Erosion, due to mining activities or deforestation as another prime example, leads to an increase in sediment pollution in rivers and estuaries, also threatening coral reefs or mangroves. The amount of undisturbed natural environments is rapidly decreasing, which leads to the decline of many species. Yet, we know little about the biodiversity of our planet (Costello et al. 2012; 2013). Many non-biologists think that most extant species are known (Ebach et al. 2011) and that only very few new species are being described today, for example from remote places such as the deep sea; taxonomy is perceived as the archiving of knowledge, not as its generation (Kholia & Fraser-Jenkins 2011). This is, however, not true. New species are described at a high rate, arguably the highest rate ever (Costello et al. 2013). Over the past decade around 2000 species have been described per year, and not only small and conspicuous ones but also some unexpected discoveries such as a large pink iguana from the Galapagos islands (Gentile & Snell 2009) and three new species of monkey (Jones et al. 2005; Geissmann et al. 2010; Hart et al. 2012), amongst many others. The total number of species is unknown and so is the number of species yet to be described (Scheffers et al. 2012). There is a fear that many species will become extinct before the become recorded and formally described (Hawksworth & Cowie 2013; Pimm et al. 2014).

5 It is not even possible to determine the number of described species today. Firstly, there is no one authoritative repository that comprises all names described since the introduction of the binomial nomenclatural in 1753 by Linnaeus and available today. Most journals today require the registration of nomenclatural acts, especially the description of new species, in zoobank (http://zoobank.org/), a repository curated by the International Commission on Zoological Nomenclature. There are also several databases attempting to collect and curate available animal species names altogether, like the Encyclopedia of Life (http://eol.org/), or in a specialized areas, like the World Register of Marine Species (WoRMS, http://www.marinespecies.org/). But older species names have to be included into those databases from books and collections and, crucially, validated. Synonymy can range from 7% to 80% depending on the taxon, which inflates the estimates of the number of described species (Costello et al. 2012). In Mollusca, for example, the estimated number of described species ranges from 50,000 to 120,000. Most estimation methods of the total number of species on this planet, including undescribed ones, are based on these numbers and therefore vary greatly (Snelgrove 1999; Fonseca et al. 2010; Mora et al. 2011; Scheffers et al. 2012).

Marine meiofauna

Marine meiofauna is defined as all organisms fitting through a 500 µm (sometimes 1000 µm) mesh but retained by a 44 µm mesh (Giere 2009). It usually only includes benthic (associated to the bottom) and no pelagic (living in the water column) animals. Although the marine benthic zone is one of the largest habitats on earth, as oceans cover about 70% of the earth’s surface, marine meiofauna remains poorly known. Snelgrove et al. assume that only 0.07% of its species are described yet. Compared to other habitats, little research efforts have been undertaken to describe its biodiversity and understand its ecosystem functions (Snelgrove 1997). This is largely due to the limited accessibility of deep sediments, but even sandy beaches or estuaries are still not fully understood (Armonies & Reise 2000; Fonseca et al. 2010). Marine meiofauna contains species of nearly all animal phyla (Giere 2009), some phyla are even exclusively meiofaunal, such as

6 Xenacoelomorpha. However, many such animals are fragile and can only be extracted from the sediment using specialised techniques. They are poor in morphological characters and not abundant or evenly distributed. Quantitative studies are also difficult to conduct as only small samples of sediments can be processed before they spoil (Armonies & Hellwig 1986). A recent study utilized second-generation sequencing technology on environmental samples and showed that diversity and composition of species changed dramatically on a small scale (hundreds of meters) (Fonseca et al. 2010). Marine meiofauna has been thought to disperse according to the “Everything-is-everywhere” hypothesis first formulated by Baas Becking for microorganisms (1934). This hypothesis states that small organisms are distributed passively and homogenously through drifting and that their global diversity could therefore be expected to be low. This notion was later extended to include meiofaunal organisms as well (Fenchel et al. 1997; Finlay 2002). The discovery of different species compositions on a small geographical scales (Fonseca et al. 2010), as well as high levels of endemicity in some groups of meiofauna, i.e. Platyhelminthes and Acoela (Curini-Galletti et al. 2012), Polychaeta (Nygren & Pleijel 2011), Rotifera (Leasi et al. 2013) and Nemertodermatida (Paper II) show that this hypothesis is not true for all small taxa.

What are Nemertodermatida?

Nemertodermatida is one taxon of the solely meiobenthic phylum Xenacoelomorpha, which also consists of Xenoturbella and Acoela. Xenoturbella is famous among the scientific community as it contains only two species and has long defied phylogenetic placement; Acoela is the largest taxon with about 400 named species and many putative ones awaiting description. Nemertodermatida has long been the neglected taxon, with only eight described species in 84 years before this thesis. Nemertodermatids are microscopic, exclusively marine worm-like animals that live either benthic or epi-benthic in sandy or muddy sediments, respectively; one species also lives commensally in the foregut of a holothurian.

7 General morphology

Individuals of different species are between 150 µm (juveniles) and up to 1 cm long. The body shape is cylindrical, drop-shaped or filiform, usually with rather rounded tips (Fig. 1). Most species are colourless, often appearing more or less opaque due to an abundance of glands. A few species, however, can have red pigmentation either as a dorsal stripe or in the whole body. When viewed under the microscope, only few characters can be distinguished, mainly the reproductive, frontal and epidermal glands. Unique to the Nemertodermatida, and visible under the dissecting scope, is the statocyst containing two statoliths.

Fig. 1: Morphological diversity within Nemertodermatida

Ultrastructure

Ultrastructural studies of Nemertodermatida revealed a strong terminal web within the epidermal cells as in Acoela, and a distinctive ciliary rootlet type

8 (Tyler & Rieger 1977; Lundin & Hendelberg 1995). A ciliary rootlet is roughly as wide as the cilium it bears and is anchored in the terminal web at a steep angle. At about half its length it forms a shallow bend to the anterior. Posteriolateral microtubule bundles, sometimes also called posteriolateral rootlets, protruding from the basal bodies of the anteriolateral cilia, connect to this knee. The cilia themselves are “shelf-like”, a character state shared with Acoela and Xenoturbella. At the base they show the typical 9+2 axoneme structure of motile cilia, but towards the distal end the doublet microtubules number 4-7 (numbering according to (Afzelius 2008), based on the relative positions of the microtubules to the central microtubules) terminate while the microtubules 8 and 9 become singlets. This results in an anterior step-like thinning of the cilia, hence the name “shelf-like” (Tyler & Rieger 1977). The epidermis is multi-layered with the nuclei not sunken under the muscle layer similar to Xenoturbella. As in Acoela and some species of Catenulida they do have a extracellular matrix (Ehlers, 1985). Gland necks always exit between epidermal cells a feature shared by Acoela and some Platyhelminthes. Epidermal cells are replaced by neoblasts migrating from the parenchyma while the old cells degenerate; a feature that is shared with Acoela and Xenoturbella. On top of the epidermis associated with microvilli is a strong glycocalyx. The frontal glands open at the anterior with many or a single gland neck, but no connection to sensory elements. Nemertodermatida have uniflagellate spermatozoa with a 9+2 axoneme (Tyler & Rieger 1975), as opposed to Acoela and other species of “Turbellaria”. The ultrastructure of the spermatozoa, however, also differ between Nemertodermatidae and Ascopariidae, the two families of Nemertodermatida (Boone et al. 2011).

Historical overview of the taxon

Between 1930 and 1998 eight species of Nemertodermatida have been described. The discovery of the first nemertodermatid, an “odd [eigenartig] turbellarian out of the depths of Disco Bay” (Steinböck 1930) (translation by I M-W), caused a small sensation. There was only one specimen of this “odd” worm caught during this expedition to Greenland and it was different from other taxa. Its size and appearance made it look like a flatworm, the epithelium

9 resembled that of a nemertean and bilithophoric statocysts were known from some multilithophoric catenulids; which, however, appeared different. Steinböck (1930) classified the new species as a new genus of Acoela within Platyhelminthes and called it Nemertoderma bathycola, after the nemertean resemblance and the depth it was found in. A heated debate ensued when Westblad found several specimens of a similar looking worm at the Swedish and Norwegian west coasts. He argued that Steinböck’s worm 1) was not yet completely mature, 2) belonged to a different ecotype of Westblad’s worm that was overall smaller due the colder climate and reduced abundance of food and 3) that Steinböck’s interpretation of the morphology and anatomy were completely wrong (Westblad 1937). The debate was set to rest, although not settled, by naming the Scandinavian species Nemertoderma westbladi after its discoverer (Steinböck 1938) and retaining the name Nemertoderma bathycola for the Greenland worm found by Steinböck. Shortly afterwards Nemertodermatida was assigned the same rank (suborder) as Acoela in a major reclassification (Karling 1940). In the following about 50 years five new species and four new genera of Nemertodermatida were described.

The first phylogenetic and therefore evolutionary informative study including all subgroups of Platyhelminthes was published in the mid-eighties (Ehlers 1985). In this system Acoela and Nemertodermatida were recovered as sistergroups, forming the Acoelomorpha (Fig. 2a). This was based on ultrastructural characters of the epidermal cilia and ciliary rootlets. The only comprehensive taxonomic revision of Nemertodermatida was published in the late nineties with data collected over more than 30 years (Sterrer 1998). It includes the formation of a second family and the description of two new species, each based on only a few specimens, bringing the species count to eight. The taxonomic revision was followed by a cladistic analysis of the taxon based mainly on ultrastructural characters, resulting in the creation of a new genus (Lundin 2000). By the beginning of the new millennium, Nemertodermatida consisted of eight species in six genera and two families.

10 Phylogenetic position

After decades of morphological and histological studies, convincing synapomorphies connecting the whole taxon were still missing (Ehlers 1986; Rieger et al. 1991). When molecular methods became more commonly available and efficiently applicable even on small samples of tissue (s. methods section) they were used on Platyhelminthes as well. Initially, these methods were used to answer the question whether the Platyhelminthes are in fact monophyletic (Smith et al. 1986). Initial molecular studies supported the paraphyly of Platyhelminthes (Carranza et al. 1997) and indeed Acoelomorpha. First, Acoela were reclassified as an early bilaterian lineage, based on the small ribosomal subunit (SSU) gene, embryonic cleavage

Fig. 2: Different hypotheses of the phylogenetic position of Nemertodermatida in the animal tree of life (a-c) and of the relationships of the genera within Nemertodermatida. a) Nemertodermatida as part of the platyhelminthes; this position is generally accepted to be wrong today (Ehlers, 1985). b) Nemertodermatida as early Bilaerian lineage (a.o. Hejnol et al. 2009) and c) as sister group to Ambulacraria (Philippe et al. 2011), the two hypotheses currently debated. d) Phylogenetic hypothesis for the inner relationships of Nemertodermatida formulated by Lundin (2000) based on mainly ultrstructural characters supporting the two families Nemertodermatidae and Ascopariidae of the classification and supported in Paper III, and e) the hypothesis out forward by Wallberg et al. (2007) based on ribosomal sequence data, which is considered to be an artifact due to chimeric sequences (Paper III).

11 patterns and the anatomy of the nervous system (Ruiz-Trillo et al. 1999). Then Nemertodermatida were reclassified as a separate early bilaterian lineage based on SSU gene data (Jondelius et al. 2002). Myosin heavy chain II sequence data on the other hand suggested a monophyletic Acoelomorpha as an early bilaterian lineage (Ruiz-Trillo et al. 2002), which was later corroborated by mitochondrial genome data (Ruiz-Trillo et al. 2004). Since then it has been dismissed based on the analyses of the large and small ribosomal subunit genes (Wallberg et al. 2007), several protein coding genes (Paps et al. 2009) and Hox gene clusters (Sempere et al. 2007) but supported in two recent phylogenomic studies (Hejnol et al. 2009; Philippe et al. 2011). The monophyly of Acoelomorpha is still highly disputed, along with its position in the animal tree of life. Hejnol et al. (2009) recovered a monophyletic Acoelomorpha that together with the elusive Xenoturbella formed a sister group to the remaining Bilateria (Fig. 2b). The same cluster, named Xenacoelomorpha, was retrieved by Philippe et al. (2011) but within Deuterostomia as the sister group to Ambulacraria (Fig. 2c).

Classification

Including the results of this thesis there are currently 18 (raised from eight in Paper II) nominal species of Nemertodermatida and also ten (raised from 2 in Paper II) putative species that have not yet been delimited well enough to warrant formal description.

Nemertodermatida Karling, 1940 Nemertodermatidae Steinböck, 1930 Nemertoderma Steinböck, 1930 bathycola Steinböck, 1930 westbladi Steinböck, 1938 Meara Westblad (Karling), 1949 stichopi Westblad, 1949 sp. Lundin, 1998 Sterreria Lundin, 2000 psammicola Sterrer, 1970 rubra (Faubel, 1976)

12 boucheti Meyer-Wachsmuth et al., 2014 lundini Meyer-Wachsmuth et al., 2014 martindalei Meyer-Wachsmuth et al., 2014 monoloithes Meyer-Wachsmuth et al., 2014 papuensis Meyer-Wachsmuth et al., 2014 variabilis Meyer-Wachsmuth et al., 2014 ylvae Meyer-Wachsmuth et al., 2014 sp. S2 Meyer-Wachsmuth et al., 2014 sp. S3 Meyer-Wachsmuth et al., 2014 sp. S7 Meyer-Wachsmuth et al., 2014 sp. P3 Meyer-Wachsmuth et al., 2014 Nemertinoides Riser, 1987 elongatus Riser, 1987 glandulosum Meyer-Wachsmuth et al., 2014 wolfgangi Meyer-Wachsmuth et al., 2014 sp. N1 Meyer-Wachsmuth et al., 2014 sp. N2 Meyer-Wachsmuth et al., 2014 sp. N3 Meyer-Wachsmuth et al., 2014 sp. N4 Meyer-Wachsmuth et al., 2014 Ascopariidae Sterrer, 1998 Flagellophora Faubel, 1978 apelti Faubel, 1978 Ascoparia Sterrer, 1998 neglecta Sterrer, 1998 secunda Sterrer, 1998 sp. Sterrer, 1998

Methods

Sampling

Sampling is often the biggest challenge and limiting factor in the study of marine meiofauna, especially in cases of as scarce and fragile taxa as Nemertodermatida. This is not only due to the small size of the animals but also because different habitats require different collection methods, and

13 different taxa different extraction techniques. Nemertodermatids are very fragile and require gentle handling. Rougher methods such as fixing sediment samples in formalin prior to sorting, the seawater-ice method or the bubble- and-blot method destroy basically all soft-bodied specimens. Species identification in these taxa is only possible in live animals under high magnification, preferably with a camera to take photographs.

In order to sample marine meiofauna efficiently, trained specialists have to go to the field with access to boats equipped with various types of sampling gear or SCUBA divers, many buckets or deep trays and a place to store them undisturbed (which is more often a limiting factor than one might think), possibly with climate control, dissecting microscopes and high quality microscopes with mounted camera, a fridge and a freezer to store fixatives and fixed samples, and possibly a fume hood.

Fig. 4: a) Light microscope with differential interference contrast with mounted camera. b) Storage room with mud samples and taps with flowing seawater. c) Buckets with samples of different sandy sediments.

Infrastructure and general workflow

Nemertodermatids live in the oxygenated upper few centimetres of sediment in various depths, which require different methods of sediment collection.

14 Intertidal samples can therefore be taken easily by wading or snorkelling and scooping up the upper layer of sediment by hand. Deeper sediments require either SCUBA divers or boats with special equipment such as for example van-Veen grabs (Fig. 3a). Small grabs can be deployed by hand, whereas larger ones require winches with according lengths of cable. Larger depths or very muddy sediments necessitate the use of sediment sledges such as Warén- or Ockelman-dredges (Fig. 3b, 3c) but if those are unavailable small box- corers (Fig. 3d) or triangular dredges can be used as well. Sediment is collected in buckets or large trays and left standing undisturbed in temperatures similar to that of the sediment’s origin, this may require temperature-controlled rooms (Fig. 4b,c). During this time, the sample deoxygenates and the animals rise to the surface; this acts as a concentration step. If the sample consists of mainly fine silt and organic matter, the surface will be carefully siphoned off with a hose and the water led through a fine sieve of about 125µm mesh size, in which the animals will be caught. The contents of the sieve are then washed into a petri dish.

Many interstitial species have adhesive organs or swim actively down to stay between the sand grains, which necessitates the anesthetization of the animals. For this the upper few centimetres of the samples, into which the worms have migrated during storage, are mixed gently with two to three times the volume of the Magnesium chloride or -sulphate solution of the same osmolarity as the seawater of the sampling locality. If MgCl2 is unavailable it can be replaced by Epsom salts. After ten to fifteen minutes the animals are anesthetized and the MgCl2-solution with the sediment are stirred up. The heavier sand grains settle quicker than the anesthetized animals, which stay suspended in the water column. The water containing the floating animals is then decanted through a sieve with 100µm to 125µm mesh size to catch the animals. The sieve is then set into a petri dish with normal seawater. After about 30 minutes the meiofaunal animals wake up and migrate through the mesh into the petri dish. Sterrer (1968) was the first to use the magnesium chloride extraction, which led to a higher recovery rate for fragile animals such as Acoela and Nemertodermatida than other widely used but rougher extraction methods for marine meiofauna. Both methods, siphoning and MgCl2-decantation, capture

15 a variety of meiofauna including diverse worm-like animals, snails, bivalves, crustaceans, foraminiferans and gastrotrichs. Targeted taxa have to be sorted out under a dissecting scope and species identified using a microscope with high magnification and phase or differential interference contrast (Fig. 4a). However, squeezing between microscope slide and coverslip, as well as repeated pipetting, often damages the specimens (Fig. 5). Especially the application and subsequent removal of the coverslip are always delicate tasks during which the quick, and by naked eye barely visible, worms are often severely damaged or even lost. Injured worms can disintegrate within seconds so that no cohesive peace of tissue can be recovered, making even molecular studies impossible.

High-quality photographs taken preferably with a remotely released camera mounted on the microscope are necessary for later reference in molecular studies, as whole specimens need to be used for DNA extraction, leaving photographs, sketches and notes of specimens as their only morphological record and therefore voucher/type material. Worms are generally fragile and deteriorate within hours in a sorting dish and seldom last longer than a few minutes under a cover slip. Photographing, identification and decisions as to the fixation method have to be taken quickly in order to not to lose samples. Specimens intended for further morphological studies cannot be viewed under the microscope in greater detail, due to the fragility of the animals, but have to be fixed directly. Depending on the fixatives used, certain appliances are needed.

Fixation

For this thesis, a few specimens were fixed for histological sectioning or electron microscopy but none were eventually used. Both methods are immensely useful and have the potential to reveal evolutionary informative characters in Nemertodermatida but we refrained from applying them here due to problems in unequivocal species identification of material in the field. Furthermore, did our studies result in the description of nine new species within two nominal species, as they turned out to be in fact hyper-cryptic species complexes (Paper II). Species delimitation, however, is based

16 primarily on molecular data, with little or weak morphological characters descriptions. Because it is not possible to identify worms to species level before fixation and specimens cannot be split and one part fixed for DNA another part for histology, morphology and genotype cannot be connected unequivocally for know. But detailed histological data is only useful, if it is connected to a biological name. As specimens fixed for DNA can be studied in greater detail under the microscope and photographs can be taken of details of the morphology, like e.g. the structure of the frontal glands, light microscopically available characters may be documented over time, allowing for species identification in life animals, and more detailed histological studies.

Fig. 5: Microphotographs of Nemertodermatids in squeeze preparations of Nemertodermatids a) Healthy squeezed specimen of Flagellophora apelti. b) Damaged specimens of Sterreria rubra. c) Dissolving specimen of Flagellophora apelti.

For this thesis we decided to focus on collecting DNA samples (Paper II & III) and take additional worms for IHC/phalloidin fluorescent staining for the study of the muscular and nervous systems, that are usually studied on taxonomic levels higher than species (Paper I & IV).

Samples for DNA extraction were usually fixed in 95% to 99% Ethanol and then stored at -20°C. A somewhat better but more expensive alternative is the commercially available buffer RNAlater® that can be stored, at least temporarily, at room temperature.

For fluorescent labelling, several different fixation methods are available. We used 2% and 4% paraformaldehyde in filtered seawater or 0.1 M Na-

17 phosphate buffer (PBS) as well as Stefanini’s fixative (2% paraformaldehyde and 15% picric acid in 0.1M Na-phosphate buffer) at pH7.6 (Stefanini et al. 1967). Worms were fixed for 1h or 1.5hrs at 4°C in order to avoid overfixing. After fixation they were washed in PBS and stored in 0.01M PBS with 0.1M

NaN3. Specimens can stay in this solution for several weeks before washing in PBS containing 10% sucrose prior to staining. Both Stefanini’s fixative and paraformaldehyde have to be kept refrigerated and solutions should not be stored for longer than a few months. Furthermore, both alternatives include poisonous components and may require the use of a fume hood, secure waste collection and other measures of chemical safety. An easier to handle alternative is the commercially available Histochoice®, which can be stored at room temperature and according to the manufacturer’s facts sheet can be disposed of through the drain. It works well with immunolabelling but not Phalloidin, possibly because it does not preserve the structure of filamentous actin that Phalloidin binds to.

Abundance and patchiness

The amount of meiofauna extracted from any given sample can be quite different. Some extractions appear devoid of life, others are so full of animals that the dishes appear to move by themselves. Nemertodermatida, however, are generally found in low numbers. Ten litres of sand may contain only a handful of specimens. Moreover, of two samples filled only metres away from each other, one may contain many specimens and the other not a single worm, even though the sediment appeared very similar. This patchiness on a small geographic scale has been shown for other meiofaunal taxa as well (Fonseca et al. 2010). The perceived patchiness of their distribution may well be caused by biotic factors, i.e. abundance of food or scarcity of predators in certain patches, or abiotic factors, i.e grain size, oxygen content, recent disturbance.

There are two species of Nemertodermatida that can be reliably found in greater numbers, Nemertoderma westbladi and Meara stichopi. N. westbladi lives in the upper most layer of very fine mud. Mud is sampled with for example the Ockelman-sledge, which is being dredged over hundreds of meters, accumulating several tens of litres of surface layer mud in one dredge.

18 This large amount of sediment, furthermore, comprises the surface layer of hundreds of metres of seafloor. In sandy areas more often manual sampling or van-Veen grabs are used, which result in much smaller sediment samples and add to the perceived bias in species abundance.

Collection effort for this thesis

Collection of Nemertodermatida requires work on site with the live animals, thus this thesis is based on lots of fieldwork. Between 2010 and 2014 I have been on twelve sampling campaigns from the Swedish West coast to Papua New Guinea (Fig. 6), accumulating to about 21 weeks field work. This thesis is based on the worms collected during those trips and about 13 weeks of

Fig. 6: Upper left: The most successful sampling site in Bermuda, John Smith's Bay, in the tropical storm that prevented us from sampling for several days. Upper right: View near the Sven Lovén Centre for marine Sciences in Tjärnö. Lower left: The entrance of Diskofjord, Greenland, the site with the most promising mud (if unfortunately without nemertodermatids). Lower right: Wanad Island in Papua New Guinea, a fantastic place, not only for its beauty but also in terms of species diversity. At least four new species were found in the samples of this beach.

19 sampling by Prof. Ulf Jondelius between 2007 and 2010, before I started my thesis, and some worms collected by co-workers. Overall, about 250 worms were collected over seven years in 18 sampling trips, together 35 weeks of sampling. I was even able to go to the type locality of N. bathycola near Disko Island, Greenland, during a two-week field course, but despite our best efforts and broad sampling, no specimens were obtained.

Confocal Laser Scanning Microscopy (CLSM)

CLSM is a useful tool to investigate the presence of certain proteins in situ, either in cells, tissues or whole-mounts of microscopic animals. It utilizes antibodies labelled with fluorescent dyes that are later captured on high- resolution images. These images are used for comparative studies of otherwise invisible morphologies. The nervous system, for example, consists of different proteins that can be made visible and compared between different taxa or different life stages in the context of their surrounding tissue.

Prinicipal of CLSM

Confocal Laser Scanning microscopy is the combination of several elements, hence the complicated name. Confocality means that several pictures of different layers within an object can be acquired in focus, and later stacked digitally to create a high-resolution optical image with depth selectivity. This optical sectioning makes CLSM particularly useful for thicker whole-mounts of specimens and allows the creation of 3D pictures. Lasers allow the use of fluorescent dyes. Fluorochromes, such as TRITC (Tetramethylrhodamine) and FITC (Fluoresceinisothiocyanate), are excited by lasers of specific wavelengths and emit light of slightly longer wavelengths. The emitted light is then caught be a light detector. The laser scans the object so that only a small area in a certain depths is exposed to light at any given time. This reduces noise in the detectors and bleaching (loss of fluorescence) of the object. The picture is created by the computer based on the intensity of light of a certain wavelength detected. Scanning also allows for several ways of quality control, including the speed and number of scans that are added together to create the

20 final image, as well as the number and thickness of levels that are being scanned. Beam splitters and filters allow the use of more then one fluorescent dye in one object, as long their excitation and emission curves do not overlap. The light detector only senses the intensity of light, not its wavelength; the colours shown in CLSM images are always arbitrarily chosen and added by the computer.

The preparations in this thesis were examined with the confocal scanning laser microscopes LEICA TCS 4D, Zeiss LSM510meta and LSM780. Usually, 20– 30 optical sections of 0.5–2µm thickness were obtained. The pictures were subsequently processed with the programs specific to the platforms: Leica LAS AF lite, LSM Image Browser v. 4.2. (Zeiss), Zen 2011 (Zeiss) and the general graphical softwares Graphic Converter v. 7.6, Adobe PhotoShop v. 7.0 and Pixelmator v.3.1..

Phalloidin staining of filamentous actin (F-actin)

The protein actin is common and present in most eukaryotic cells. It exists mainly in two forms: globular (G-actin) and filamentous (F-actin). G-actin is mostly present in the cytoskeleton whereas the filamentous molecule is an integral part of muscle fibres. Body-wall musculature has been shown to be evolutionary informative in meiofaunal taxa (Hooge 2001) and studied in this thesis (Paper I & IV). Phalloidin, a toxin of the mushroom Amanita phalloides (Death Cap), binds F-actin. The phalloidin molecule is usually labelled directly with a fluorescent dye. Using this labelled Phalloidin, muscle fibres can be made visible, showing the complete pattern of the body wall musculature in situ in in whole mounts.

Specimens fixed in Stefanini’s were rinsed for 24-48h in PBS containing 20% sucrose while specimens stored in PBS with 0.1M NaN3 were washed three times five minutes in PBS only. After washing, specimens were treated with Triton X in PBS. Triton X partially dissolves biological membranes and thus makes them permeable, e.g. for fluorescent labelled phalloidin or antibodies. The concentration of Triton depends on the size and type of the animal: too high concentrations or long exposures may damage or eventually dissolve the

21 whole animal. Phalloidin was diluted to a working solution in PBS and the worms incubated in it for one or two hours at room temperature. After washing out excess Phalloidin with PBS the animals were mounted in a 1:1 PBS-Glycerol solution and stored in the dark at -20°C. Viewing under the CLSM should be done soon in order to avoid bleaching. Commercially available mounting media such as e.g. VectaShield® can also be used to prevent bleaching.

Fig. 7: CLSM images of stained whole-mounts. a) Nemertoderma westbladi stained with TRITC-labelled Phalloidin showing the body-wall musculature in situ. The anterior points in the upper right hand corner. b) Anterior or a specimen of Flagellophphora apelti stained with anti-alpha-tubulin (FITC, green) and anti-FMRFamide (TRITC, red) showing the nervous system and the the broom organ.

Immunolabelling of the nervous system

In Nemertodermatida as well as many other taxa the nervous system is thought to yield valuable insights into the evolution of the animal tree of life. Immunolabelling is based on the response of the immune system to produce antibodies specific to certain amino acid sequences and the resulting folding pattern, the so-called antigens, of the infecting agent. In a normal immune response, the B cells of the “infected” immune system recognize the antibody attached to the infectious antigen and lyse the complex. This system has been adapted for laboratory use to make certain proteins in fixed cells and tissues

22 visible. Antibodies can be produced in animals. These are “infected” in our case with a protein that naturally occurs in the nervous system. The “infected” animal produces antibodies against this protein that will bind to this specific protein but to nothing else. In contrast to the above mentioned Phalloidin, immunolabelling is usually indirect, i.e. two antibodies are used: a primary antibody that will bind to the protein but is not labelled and a secondary antibody that will bind to the primary and carry the fluorescent label. As the primary antibody is produced in an animal, say a rabbit, it carries antigens specific to rabbit as well. Therefore, a secondary antibody binding specifically to antigens of in this case rabbits can be used to detect the primary antibody, as long as it was used in any other animal than rabbit. Secondary antibodies are produced in the same way as primary ones. If serum of a rabbit, to stick to the example, is injected into a different animal, say a goat, the goat will produce antibodies against rabbit. These “goat-anti-rabbit” secondary antibodies are then labelled with different fluorescent dyes and can be used to label any primary antibodies produced in rabbit.

In our experiments (Paper II & IV) specimens were washed and treated with Triton X in the same way as for Phalloidin staining. Specimens fixed in Histochoice® were washed in PBS prior to permeabilization. Primary antibodies were applied in working dilutions (according to the distributors recommendations and own testing) for 24-72hrs at around 10°C on a shaker. In some cases non-specific antigens were blocked with 1.5% Bovine Serum Albumin (BSA) in PBS-T to reduce background noise. After the incubation, specimens were thoroughly rinsed in PBS-T and the corresponding secondary antibodies were applied. Incubation with secondary antibodies was usually 1- 2hrs at room temperature on a shaker, before washing in PBS and mounting in Glycerol-PBS or VectaShield. Finished mounts were stored at -20°C until microscopy.

Some specimens were double stained with two antibody pairs and others double stained with one pair of antibodies and Phalloidin. In case of double stainings, the primary antibodies were mixed prior to application and the secondary antibodies applied one after the other (Coons et al. 1955).

23 Counterstained animals were treated with primary and secondary antibody before incubation with Phalloidin and mounting.

Molecular analyses

Traditionally, biological classification and phylogenetic analyses were based on morphological characters. Species delimiting or even evolutionary informative characters may, however, be difficult to find (and to define). DNA sequencing has therefore become a major objective of research in the sixties and seventies of the last century, not only in its own right but because it allows the use of millions of characters for phylogenetic inference and species delimitation. The DNA of an organism is the accumulation of all evolutionary changes in its phylogenetic history. The implementation and steady development of dideoxy-chain-determination sequencing (Sanger et al. 1977) made thousands of putatively informative positions available to systematists. With new sequencing approaches made available over the past decade or so, the number of available positions and specimens (i.e. taxa) has increased even faster and will keep doing so (Glenn 2011).

For efficient phylogenetic inference the choice of markers is important. Highly conserved markers are incapable of resolving recent splitting events, underestimating diversity, while variable ones show only recent events and may suffer from saturation on broader taxonomic scales, showing incorrect relationships. The availability of markers, however, is restricted by many factors and is a trade-off between the scope of the present study, the practicality in obtaining sequences and the comparability of the new data to older data.

The use of markers used before has the advantage of the results being comparable to those from previous or similar studies. Furthermore, for many markers that have been proven useful in phylogenetic analyses, reliable primer pairs (short sequences of DNA defining a region that will be amplified and subsequently sequenced) are available. However, as primers are specific to certain DNA sequences they may not work, or perform poorly, when applied

24 to other taxa. New, better fitting, primers can be designed based on known sequences.

Before this thesis there were 33 sequences of Nemertodermatida publicly available on genbank covering nine different markers. The large (LSU) and small (SSU) ribosomal subunit genes had been used before in several studies including Nemertodermatida and Acoela (Halanych et al. 1995; Carranza et al. 1997; Wirz et al. 1999; Giribet et al. 2000; Mallatt et al. 2004; Wallberg et al. 2007). Therefore, fitting primers and protocols were available. Throughout the work for this thesis, however, we discovered that Nemertodermatida consists of more species than previously thought and that those are genetically quite distinct, affecting primer binding (Paper II & III). Due to the microscopic size of the worms the yield of DNA per specimen after extraction is low, leaving little room for extensive development and optimization of primers and PCR protocols. For some species the original primers worked just fine, for others new primers and protocols based on the known primers and some successful sequencing attempts could be designed. For others, the sequencing success remained unreliable. We intended to use mitochondrial markers also and tests with the cytochrome oxidase subunit 1 (COI), the cytochrome oxidase B (Cytb), the large (16S) and small (12S) mitochondrial ribosomal subunit genes were conducted. Of these, only COI could be sequenced with any reliability using the original barcoding primers LCO1490 and HCO2198 (Folmer et al. 1994) but sequencing success was still less than 40%. Often the PCRs already failed or sequences were unreadable or actually belonged to other species.

We were able to amplify and sequence 3650bp of the LSU gene in three consecutive parts and 1800bp of the SSU gene in two consecutive parts, as well as 328bp of the nuclear protein coding Histone 3 (H3) gene (Colgan et al. 1998). H3 is a nuclear protein coding gene that has high rates of evolution and can resolve species as well as higher level relationships.

DNA extraction, amplification and sequencing

DNA was extracted using the Qiagen Micro Tissue Kit with a six hour lysis

25 step. All markers were amplified and sequenced using several different primer combinations, and, in the case of 18S, a nested PCR approach. Two microlitres of the DNA extract were used with the illustra PuReTaq Ready- To-Go PCR Beads in a 25 µl PCR reaction with a 10 µM primer concentration. PCR products were run on 1% agarose gels with GelRed™. When several bands were present, mostly in case of the Histone 3 gene, the Qiagen Gel Extraction Kit was used to extract and clean the band with the PCR product of the correct length. PCR products were cleaned using the ExoFap protocol, which removes primers and dNTP’s. Depending on the strength of the correct band, one to several microliters of the cleaned PCR product were being added to a 20 µl cycle sequencing reaction with the BigDye® Terminator v.3.1 Cycle Sequencing Kit and 1.6 µM primer concentration. The cycle sequencing reactions were cleaned using the DyeEx spin column kit. The cleaned cycle sequencing reactions were then analysed in an ABI 3130 capillary analyser.

Data properties

The chromatograms created by the 3130 Genetic Analyzer were uploaded to the Geneious package (different versions throughout analyses of Paper II & III) available from biomatters.com and edited. Alignments were conducted within the package using the MAFFT algorithm (Katoh & al 2002) and checked by eye. H3 could be aligned without any ambiguity due to its protein coding nature and its short size. Protein coding DNA sequences are first transcribed into messenger RNA in the nucleus and then in the cell translated into sequences of amino acids, which fold and form functional proteins. The termination of the translation is signed by so-called stop codons, ending the translation. It has been shown that protein coding genes especially of mitochondria can be incorporated into the nuclear DNA, where they are not translated and therefore are not subjected to selection (Vanin 1985). These pseudogenes have very similar sequences, as they are copies of the functioning version, but have accumulated mutations over time. Sequences of supposedly protein coding genes that show stop codons are therefore likely to be pseudogenes or belong to taxa that use a different genetic code. We

26 translated H3 sequences into amino acids and discarded those showing stop codons. SSU is conserved and has few indels (insertion-deletion) even across the large taxonomic dataset of Paper III and aligned nicely. The first 1kb of the LSU gene is very variable already in the taxonomically more limited dataset of Paper II. However, although aligning this region was generally more difficult it proved to be non-random and its exclusion did not improve support values throughout the analyses.

Phylogenetic analyses

The single gene or concatenated datasets were analysed using Maximum Likelihood (ML) as well as Bayesian inference methods, as described in Paper II and Paper III. We chose to use two different inference methods despite the fact that conflicting topologies would result in two equally valid phylogenetic hypotheses. It is generally accepted to do both analyses and if both show the same topology, it may support the validity of the dataset and model used for inference. Furthermore, the program used for ML analyses, RAxML (Stamatakis 2006) is much quicker than the Bayesian program MrBayes (Ronquist et al. 2012), allowing for more time-efficient preliminary analyses of the datasets.

Discussion and future perspectives

Nemertodermatida has always been neglected as a taxon, and although this thesis sheds more light on it, it still remains largely unknown.

To date (August 2014) the phylogenetic position of Nemertodermatida is unresolved. Two widely diverging hypotheses are proposed, each with a plethora of inherent assumptions about the evolution of Bilateria, which encompass the vast majority of extant animal species. I hope, that the results of this thesis will help to solve this question in a larger framework.

Part of this project was the resolution of the inner phylogeny of Nemertodermatida with molecular methods. We could confirm the monophyly of the two families, Ascopariidae and Nemertodermatidae. Proof for them

27 being sister taxa, and therefore Nemertodermatida being monophyletic, is limited, based mainly on the bilithophoric statocysts. Although I do not doubt the monophyly of Nemertodermatida, it certainly needs to be further supported. For this, the focus should be aimed on the family Ascopariidae, which is sorely understudied, taxonomically, morphologically and molecularly.

There was little known about the true biodiversity of Nemertodermatida before this thesis, to a great extent due to the scarcity of the material, but also because of the assumption that in these small animals had cosmopolitan distributions with little speciation (Baas Becking 1934; Sterrer 1998). The second publication in this thesis raised the species count from eight nominal and two putative species to 18 nominal and ten putative species. In Paper II we could show that many species of Nemertodermatida do not have cosmopolitan distributions but have more restricted ranges. These new species counts, however, are still based on very limited geographic sampling and it can be assumed that increased geographic sampling would amount to increased taxon sampling, i.e. the description of many more species of Nemertodermatida. This is particularly true outside European waters and the north Atlantic, to which records are ultimately restricted. But even here we could describe new species (Paper II) and find evidence that N. westbladi, known from hundreds of specimens for nearly 80 years, might be more than one species (Paper III).

We know that Nemertodermatida live in warm and shallow as well as in cold and deep habitats, in relatively coarse to very fine sand, with no or a very high amount of organic content, in silty mud, in summary: basically everywhere. They also live in the littoral of geologically young islands such as Bermuda or Hawaii, which they must have colonized by dispersal. It is reasonable to assume that species of Nemertodermatida are living in all geographical regions, possible even below 600m, the deepest record so far. However, apart from the North Atlantic and its adjacent seas there are only few records of Nemertodermatida. In the Pacific they have been reported from a handful of localities and in the whole Indian Ocean they are known only from one locality. In two locations, Hawaii and Papua New Guinea, two and three new

28 species were found within less than 10km distance (Paper II).

As mentioned above, Nemertodermatida have been recorded from geologically and evolutionary young islands such as O’ahu (Hawaii) in the Pacific, and Bermuda in the Atlantic. Especially the existence of three different lineages of Nemertodermatida on O’ahu indicates that the island has been colonized more than one time (Paper II). Yet, we still do not know how these fragile interstitial worms without dormant eggs or larval stage can disperse over longer distances. The biology of nemertodermatids and their interactions with the biotic and abiotic environment is still largely a mystery. Feeding has never been observed, although in the guts of some specimens of M. stichopi diatoms and copepods have been found (Westblad 1949) and we found diatoms in the guts of some specimens of S. variabilis (Paper II). We could also show that F. apelti indeed has a mouth at least during some stages of its life (Paper III). The development of the mouth after hatching and its subsequent loss has not been described from Acoel species and appears to be a synapomorphy for Nemertodermatida.

Most species descriptions until the 1990ies include detailed morphological data. Most of them, however, are based only on a few, often not fully mature, specimens. In the presence of cryptic and sympatric species it is not possible to connect this morphological data, and therefore the type specimens, unequivocally with the genotypes derived in Paper II (see 2.1.2.). I hope that through detailed study and documentation of live specimens and subsequent molecular delimitation of species, characters can be defined a posteriori that will make species identification in the field possible. Confidence in species identity will then allow detailed histological or electron microscopic studies of species, and subsequently possibly a morphological species delimitation. And as the morphology of an animal is borne by its function, this may also lead to valuable insight into the biology of these taxa.

Some morphological properties do not show differences between species, as much as between higher level taxa. The pattern of the body-wall musculature, for example, has been shown to be evolutionary informative and distinguish between genera in Platyhelminthes and Acoelomorpha (Hooge 2001; Todt 2009). Before this thesis, the pattern of the body-wall musculature had been

29 described based on fluorescent-labelled phalloidin only for S. psammicola (Hooge 2001), the description is extended in Paper IV. In Paper I we describe the pattern of the body-wall musculature for Nemertoderma westbladi and Meara stichopi, including the development of musculature in different life stages after hatching for the former. Recently, the development of the body-wall musculature of M. stichopi has been published (Børve & Hejnol 2014). All three species belong to the same family, Nemertodermatidae, but represent different ecological principals: S. psammicola lives interstitially in shallow waters, N. westbladi lives free on muddy sediments in deeper waters and M. stichopi lives commensally in a holothurian. All three species show different adaptations of the body-wall musculature, probably due to their different body forms and life styles. Data on more species in different genera could help, for example, to reconcile currently discussed conflicting species trees inferred from morphologically and genetically inferred. Lundin (2000) recovered Meara and Nemertoderma as sister groups based on ultrastructural data while genetic data clusters Meara with Sterreria and Nemertinoides (Paper III). Meara and Sterreria both show u-shaped musculature coming from the anterior and bending posteriorly around the mouth, a pattern lacking in Nemertoderma. If Nemertinoides showed the pattern of musculature with the u-shaped muscles around the mouth, it would support the close relationship between these three genera, to the exclusion of Nemertoderma, as suggested by genetic data (Paper III). The body-wall musculature is not known for any ascopariid species, although these are the only nemertodermatids with a female opening and bursa, and the body- wall pattern can therefore be expected to be different. Also, the mouth appears to be a transient feature in at least N. westbladi (Paper I) and M. stichopi (Børve & Hejnol 2014) but possibly also F. apelti (Faubel & Dörjes 1978) but see Paper IV) and some Sterreria species (Faubel & Dörjes 1978; Hooge 2001). Its ontogenetic development would be an interesting character to trace through Nemertodermatida and compare them to other Xenacoelomorpha.

The nervous system has been considered a valuable evolutionary informative character for a long time (Ax 1961; Karling 1974; Ehlers 1985). Recently, the nervous system of the acoel Isodiametra pulchra (Smith & Bush, 1991) has

30 been described in detail and its evolution discussed in the framework of Acoelomorpha (Achatz & Martinez 2012). In this study N. westbladi, a member of the family Nemertodermatidae, was used as the representative of Nemertodermatida. Its nervous system consists mainly of a basiepidermal brain ring with two ventrolateral nerve cords drawing caudad (Raikova et al. 2000; 2004). The second species ever studied with immunolabelling of the nervous system was M. stichopi, in which no brain could be detected, but merely a basiepidermal nerve net and two dorsolateral nerve bundles (Raikova et al. 2000). In Paper IV we describe the nervous systems of Sterreria sp. and F. apelti, the first ascopariid species, and add detailed information to our knowledge of the nervous system of N. westbladi and the muscular system of Sterreria sp.. We could visualise the brain of Sterreria, composed out of longitudinal and transverse fibres, reminiscent of the commissural brains found in some acoels. F. apelti on the other hand has a large neuropile brain with brain cells mainly anterior to that and extra innervation of the broom organ, as well as an epidermal nerve net. The nervous systems of these four species are so different from each other that no comprehensive hypothesis about nervous system evolution in Nemertodermatida and with respect to Acoelomorpha can be formulated. The observed nervous systems appear primarily simple and show plesiomorphic characters. It is obvious, that more detailed studies of the nervous system with different sets of antibodies, and counterstaining with Phalloidin in order to define its position with respect to the body-wall musculature, may reveal many potentially informative characters, which could give valuable information about how the nervous system evolved in Nemertodermatida, Xenacoelomorpha and possibly early Bilateria.

Our knowledge of Nemertodermatida can only be accumulated in small steps over time as the material is so scarce. Species and species trees must truly be treated as the hypotheses they are. During the assembly of the dataset for PAPER II, each addition of new sequences helped to resolve the tree. New data will further change some hypotheses, for example resolve the relationships within Sterreria and Nemertinoides better (PAPER II & PAPER III).

31 Integrative taxonomy, i.e. the use of various data to delimit species, has been proposed (Dayrat 2005; Will et al. 2005) and is increasingly be applied (Puillandre et al. 2009; Casu et al. 2009; Fujita et al. 2012; Riedel et al. 2013; López et al. 2013). Dayrat in his plea (2005), however, went further and proposed to not to name any new species unless a number of conditions have been met, amongst others that a “recent taxonomic revision has dealt with the totality of the names available for the group”, that any “set of specimens differing in some regard from existing species can be described with the abbreviation ‘sp.’ (for ‘species’) and not with a real species name regulated by the codes of nomenclature” and that ”Ideally, names should only be created for species that are supported by broad biological evidence (morphology, genealogical concordance, ecology, behaviour, etc.)” (Dayrat, 2005). All of his seven guidelines are sensible and should be held as the “gold-standard” in taxonomy. However, especially in cases of cryptic or rare species, these high expectations may lead to valuable but incomplete data locked and forgotten in desk drawers or to an overwhelming number of unnamed but somewhat recognized species (GenusA sp.1, GenusA sp.2, etc.) (Lim et al. 2011). Handling numbered putative species, however, is cumbersome and has the potential for one putative species being studied by different authors under different “nicknames” or OTU definition, obscuring rather than streamlining taxonomy. This is already the case even under slightly more liberal rules as shown in PAPER II. We introduced as many putative species as formally described new species based on thorough species delimitation. However, it is difficult to assess thoroughly the intra- and interspecific variation, another condition proposed by Dayrat (2005) in animals of which only few specimens are ever found.

Furthermore are names important. Sequences not assigned to a proper species name are often not considered reliable in genbank and possibly not used as reference. However, the assessment of biodiversity based on metagenetic studies of environmental samples will become more important with the increasing quality of new sequencing methods, especially in such work intensive fields as meiofauna (see 2.1.1) (Fonseca et al. 2010; Bik et al. 2011). For this a populated database with molecularly well-delimited and named species is necessary.

32

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38 Acknowledgements

I would like to thank my supervisor Prof. Ulf Jondelius, who made this work possible. He introduced me to the world of meiofauna and tried to teach to me to express myself concise and precise. He also introduced me to the world of wittertainment that helped me through endless hours of sorting said meiofauna.

I am most grateful to Dr. Olga Raikova who taught me immunohistochemistry and Phalloidin staining. I am deeply impressed by her knowledge of comparative morphology in flatworms. Thank you for all the good discussions.

I would like to thank St.-Petersburg State University and the Zoological Institute of the Russian Academy of Sciences for hosting me during my work for paper iv. Immunostainings and microscopic observations were carried out at the Research Resource Center “Molecular and Cell Technologies” of St.- Petersburg State University. The staff was most helpful but I would like to thank particularly Nikolai A. Kostin, specialist in confocal microscopy, for his support.

Dr. Diego Fontaneto left Stockholm shortly after I started my PhD but I am grateful for his help in species delimitation and phylogenetic inference and his general support.

Many thanks go to Prof. Marco Curini Galletti who was good company in the field and found so many of my worms.

Special thanks go to Dr. Wolfgang Sterrer and Dr. Robbie Smith, who hosted me at the Bermuda Aquarium, Museum and Zoo. Robbie, thank you again for entering the cold ocean for me in November – none of the worms from Bermuda would have been found without your diving efforts.

One of the most exceptional fieldwork experiences was the participation in the biodiversity expedition 2012/2013 to Papua New Guinea, “merci infiniment” to Philippe Bouchet. I am also most grateful to Bastian Brenzinger and Timea Neusser, who generously shared equipment and samples with me. Thanks also to all the others who made this experience so positive. To be surrounded by so many passionate scientists was an inspiration and the memory will give me strength and courage to go through the rough patches of a scientist’s life.

2 For all the lab-work, short notice and “asap” sequencing I would like to thank Keyvan Mirbakhsh. Without your immeasurable knowledge, patience and accuracy, this PhD would not have been possible.

Johan Nylander made me loose my fear of the command line and taught me that nothing will blow up if I make a typo.

Sabine Stöhr gave me a particularly warm welcome in the department and was most helpful not only in all things administrative and IT. Ich werde unsere Lunchs vermissen.

I would also like to thank Anders Warén for sharing all these absolutely stunning pictures and interesting stories about his work; I can understand why one would like to become a malacologist (although the pictures of the crustaceans are as fabulous). I would also like to thank Carina Svensson, who did not only keep the histology lab in such good order but was always approachable for questions and requests. I also enjoyed our talks, which made me feel part of the invertebrate department. Many thanks also to Sven Boström and Emily Dock-Åkerman, who with their friendliness and humour were often the nicest persons I would meet the day. And Lena Gustavsson, who made sure that I did not get lost in the jungle of bureaucracy, thank you for that! Knowing that I could always drop in and get answers was a great support.

And then there are all the guys from the Naturhistoriska riksmuseet. Tobias Kånneby – thank you for all the advice and hugs, it made especially my beginning so much easier. Karin Nilsson – the first field trip would have broken my neck if it had not been for you. Michel Clément – I really enjoyed our discussions about work and all other matters, I missed you the last year. Rasa Bukontaite, Tobias Malm, Marika Källsen, Joie Cannon Seraina Klopfstein, George Sangster, Marianne Espeland and Alexander Alsén for all the fun, talks and help over the years.

Julia Stigenberg I would like to mention particularly: thanks for all the Badminton matches (I will miss those), talks and sharing your piece of heaven near Norrtälje with us.

Ein grosser Dank auch an meine Freunde in Bremen, die mir immer das Gefühl

3 geben, dort nach so vielen Jahren noch ein zu Hause zu haben. Besonders, aber nicht nur, Sarah Marquardt und Elke Brandt mit Leon. Auch danke ich meiner “zweiten Familie”, der Turngruppe from ATS Buntentor, die mich in all dieser Zeit nicht vergessen hat.

Der größte Dank natürlich aber meiner Familie, die mein Interesse an der Natur geweckt und gefördert hat. Danke, daß Ihr mich habt meinen Weg gehen lassen. Ohne Eure Unterstützung wären die vielen Jahre Studium nicht möglich gewesen.

And Pavel; who would have imagined, all those years ago, on a moonlit jetty in Pigeon Bay on the other side of the world, shared with dogs and zombies, that we stick through all this, me accomplishing my dream and you about to embark on yours? You have been a huge support and I admire your endless patience with me. I hope I can do the same for you. Miluji tě!

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