Dick

Monique Van Dijk

Ilse Ceelie

Prof D Tibboel
email: d.tibboel@erasmusmc.nl

Part of TEDDY supplement Endpoints in pediatric pain studies

Keywords: pain, assessment, pharmacokinetics, pharmacodynamics, long-term outcome

HAL is

Introduction 1. Neurobiology of pediatric pain: essentials for drugs-related studies

The neonatal stage of life is characterized by high sensitivity to pain and great vulnerability to neuronal cell death [1]. Anecdotal reports have shown prolonged allodynia and hyperalgesia after pain and tissue damage within the first weeks of life, extending beyond the period associated with tissue healing [2][3][START_REF] Peters | Does neonatal surgery lead to increased pain sensitivity in later childhood?[END_REF][START_REF] Taddio | Effect of neonatal circumcision on pain response during subsequent routine vaccination[END_REF][START_REF] Andrews | Abdominal sensitivity in the first year of life: comparison of infants with and without prenatally diagnosed unilateral hydronephrosis[END_REF][START_REF] Buskila | Pain sensitivity in prematurely born adolescents[END_REF]. For example, 4-6 months-old term infants who had undergone circumcision responded more intensely to immunization than did their uncircumcised peers [START_REF] Taddio | Effect of neonatal circumcision on pain response during subsequent routine vaccination[END_REF]. On the other hand, children who had undergone major surgery in combination with preemptive analgesia within the first months of life did at 14 and 45 months of age not show different behavioural pain responses and saliva cortisol concentrations when exposed to vaccinations compared to age matched controls. [START_REF] Peters | Major surgery within the first 3 months of life and subsequent biobehavioral pain responses to immunization at later age: a case comparison study[END_REF]. Thus, the question is whether preemptive administration of analgesics indeed prevents from possible long-term consequences of neonatal pain. There are some clues from animal experiments. Neonatal nerve ligation in rodents led to long-term hyperalgesia, which was not attenuated when local anesthetics were administered [START_REF] Lima | Sensory hyperinnervation after neonatal skin wounding: effect of bupivacaine sciatic nerve block[END_REF]. Neonatal exposure to carrageen or Freud adjuvants CFA led to hyposensitivity or no alterations, but to hypersensitivity when adult animals were re-exposed to inflammatory pain [START_REF] Alvarez | Modelling the prolonged effects of neonatal pain[END_REF][START_REF] Ruda | Altered nociceptive neuronal circuits after neonatal peripheral inflammation[END_REF][12][START_REF] Tachibana | Increased Fos induction in adult rats that experienced neonatal peripheral inflammation[END_REF][14]. In contrast, formalin injections or laparotomy in newborn rats led to thermal hyposensitivity at adult age [START_REF] Tachibana | Increased Fos induction in adult rats that experienced neonatal peripheral inflammation[END_REF][14][15], which was attenuated by morphine administration [15]. Tissue damage and neonatal pain disturb normal development of the nociceptive neural circuits, as expressed by structural and functional neuroanatomical changes both peripherally [9, 16] [17] and at spinal cord level [START_REF] Ruda | Altered nociceptive neuronal circuits after neonatal peripheral inflammation[END_REF]18]. Moreover, changes in spinal gene expression involved in the transmission of nociception have been documented [START_REF] Tachibana | Increased Fos induction in adult rats that experienced neonatal peripheral inflammation[END_REF]. These animal experiments may provide an explanation for the long-term effects found in human children.

In summary, we do not know whether adequate analgesia prevents the development of long-term alterations in pain sensitivity. And, if alterations occur, will they be restricted to the dermatome of tissue injury (spinal changes) or be generalized all over the body (supraspinal changes) [START_REF] Peters | Does neonatal surgery lead to increased pain sensitivity in later childhood?[END_REF].

Exposing neonates to pain or tissue damage is developmentally inappropriate and analgesics may not prevent them from developing subsequent pain hypersensitivity. A next question is whether this pain hyposensitivity. The few studies on this issue provide no or little information of the total analgesic dosages during hospital stay [START_REF] Hohmeister | Responses to pain in school-aged children with experience in a neonatal intensive care unit: cognitive aspects and maternal influences[END_REF][START_REF] Hermann | Long-term alteration of pain sensitivity in school-aged children with early pain experiences[END_REF]. It would seem, therefore, that we need to perform more randomized controlled analgesic trials (RCT) in children and perform follow-up studies in these same patients through childhood and adolescence to gain insight in the long term effects of neonatal pain and neonatal analgesia.

Endpoints in clinical trials

A clinical trial endpoint is a measure that allows us to decide whether the null hypothesis of a clinical trial should be accepted or rejected [START_REF] Bakhai | Endpoints[END_REF]. Possible endpoints in pediatric analgesic trials are: pain intensity, time to first (rescue) analgesia, total analgesic consumption, adverse effects and long term effects [START_REF] Mcgrath | Core outcome domains and measures for pediatric acute and chronic/recurrent pain clinical trials: PedIMMPACT recommendations[END_REF]23]. RCTs may have more than one endpoint, in which case it is customary to differentiate between primary and secondary outcomes.

Assessing pain intensity in (preverbal) children is more difficult than in adults. Adults' self report of pain is generally accepted as the gold standard (ref IASP) of pain assessment. The discussion merely limits itself to the question which of the available self report scales is most appropriate in a given situation. Pain intensity in young children can be assessed with validated observational pain assessment instruments or multidimensional pain assessment instruments that include both behavioural and physiological parameters. Self report is feasible from the age of 4 to 5 years. Because observational pain instruments provide subjective outcomes, it is crucial that observers are well trained and that interrater reliability has been tested and proven good. Establishing cutoff points that differentiate between different levels of pain intensity is an important requirement, because rescue medication is given when scores exceed specific values.

An important reference article is the one from the Pediatric Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (Ped-IMMPACT), in which core domains and measures for clinical pain trials have been defined [START_REF] Mcgrath | Core outcome domains and measures for pediatric acute and chronic/recurrent pain clinical trials: PedIMMPACT recommendations[END_REF].

The type of outcome measure also depends on the type of pain under study. Physiological parameters, for example, are more promising for acute painful procedures such as heel lances or venipunctures than for chronic pain. In the next paragraph the different types of endpoints will be presented with a focus on postoperative pain.

Endpoint Pain intensity: a. Behavioral assessments

The International Association for the Study of Pain (IASP) underlines that the inability to communicate verbally does not negate the possibility that an individual is experiencing pain and is in need of appropriate pain-relieving treatment [START_REF] Merskey | Classification of chronic pain: description of chronic pain syndromes and definitions of pain terms[END_REF]. Based on this note, behavioral-based pain observation instruments have been developed. The Children's Hospital Eastern Ontario Pain Scale (CHEOPS) [START_REF] Mcgrath | Core outcome domains and measures for pediatric acute and chronic/recurrent pain clinical trials: PedIMMPACT recommendations[END_REF][START_REF] Suraseranivongse | Cross-validation of a composite pain scale for preschool children within 24 hours of surgery[END_REF], and the Faces, Legs, Arms, Cry and Consolability (FLACC) [26,27] have been validated for postoperative pain in 1 to 7-year-old children. The COMFORT-behavior scale has been validated to that aim in 0 to 3-year-old children in the intensive care setting [START_REF] Van Dijk | The reliability and validity of the COMFORT scale as a postoperative pain instrument in 0 to 3-year-old infants[END_REF]. These scales have several items in common; facial expression, cry and body movements.

Children with severe intellectual disabilities may show idiosyncratic behavior when they are in pain. It may be advised against, therefore, to apply pain scales developed for children without intellectual disabilities to these children [START_REF] Van Dijk | Children with intellectual disabilities and pain perception: A review and suggestions for future assessment protocols[END_REF]30]. At least four validated postoperative pain instruments for children with intellectual disabilities have been developed. One is the revised FLACC, which allows for individualized behavior added to each of the 5 items of the scale. It has been validated for postoperative pain [31] and proved to have a high degree of clinical utility [START_REF] Voepel-Lewis | A comparison of the clinical utility of pain assessment tools for children with cognitive impairment[END_REF]. The second scale is the Paediatric Pain Profile (PPP), a 20-item scale that has been validated for postoperative pain [START_REF] Hunt | Clinical validation of the paediatric pain profile[END_REF][START_REF] Hunt | Development of the paediatric pain profile: role of video analysis and saliva cortisol in validating a tool to assess pain in children with severe neurological disability[END_REF]. The PPP consists of three sets of recordings: two retrospective parent ratings of the child's behaviour -i.e. when the child was at his or her best and during painful episodes -and a prospective rating by the nurse, e.g. postoperatively. Although completing the PPP may take up more time than the FLACC, it may be well worthwhile for research purposes. The third scale is the non-communicating children's pain checklist (NCCPC) [START_REF] Breau | Psychometric properties of the noncommunicating children's pain checklist-revised[END_REF] of which the postoperative version (NCCPC-PV) [START_REF] Breau | Validation of the Non-communicating Children's Pain Checklist-Postoperative Version[END_REF] includes 27 items and requires a 10 minutes observation. A fourth scale is the Checklist Pain Behaviour (CPB), which has been validated for postoperative pain and reduced without loss of information to a 10 item version [37,38]. In addition, a recent study described the use of an

c. Physiological parameters

As behavior-based assessment instruments remain subjective, researchers continue to search for neurobiological-based and more 'objective' parameters of pain intensity [START_REF] Berde | Pain measurement and Beecher's challenge: 50 years later[END_REF]. Several instruments indeed go some way in this direction by including physiological items as well, such as the PIPP and the COMFORT scale [START_REF] Stevens | Premature Infant Pain Profile: development and initial validation[END_REF][START_REF] Ambuel | Assessing distress in pediatric intensive care environments: the COMFORT scale[END_REF]. However, heart rate and blood pressure proved not been sensitive enough for postoperative pain assessment, probably because treatment, blood loss, fever and other conditions will influence these parameters, [50,[START_REF] Van Dijk | The association between physiological and behavioral pain measures in 0-to 3-year-old infants after major surgery[END_REF].

New methods, such as near-infrared spectroscopy (NIRS) and skin conductance, may help to objectify pain or stress in nonverbal humans. NIRS measures regional changes in oxygenated and deoxygenated hemoglobin concentration. The technique is based on the assumption that increased tissue oxygenation represents a greater regional cerebral blood flow. This, in its turn, is associated with higher neuronal activity as seen in noxious events (encoded by frequency of firing and number of activated neurons) [START_REF] Logothetis | Neurophysiological investigation of the basis of the fMRI signal[END_REF]. The use of NIRS in pediatrics has been limited to acute pain in neonates [START_REF] Bartocci | Pain activates cortical areas in the preterm newborn brain[END_REF][START_REF] Slater | How well do clinical pain assessment tools reflect pain in infants?[END_REF][START_REF] Slater | Cortical pain responses in human infants[END_REF]. One study compared NIRS measurements during 33 heel lance procedures in 12 stable newborns with facial expression. Brain activity in most of the newborns was related to facial expression. Some newborns did not show a The measurement of stress by skin conductance is based on neurophysiologic arousal with increased activity in the sympathetic nervous system, leading to sweating in the palms of the hand and the foot soles. The level of increase may serve as a surrogate measure of stress and not of pain per se [START_REF] Eriksson | Skin conductance compared to a combined behavioural and physiological pain measure in newborn infants[END_REF].

d. Biomarkers

Hormonal stress markers such as salivary cortisol and (nor) epinephrine may have additional value in the context of analgesia trials [57]. Nevertheless, because stress and pain are correlated but difficult to distinguish, these hormone levels should not be considered as primary endpoints of pain studies. Agedependent differences in hormonal levels as well as age-dependent differences in circadian rhythm are important confounders. Especially in postoperative patients the extent and duration of the so-called hormonal stress response are highly determined by age [57] However, salivary cortisol could be a substitute marker for pain or stress in severely cognitively impaired children. Remarkably, RCTs in this vulnerable patient group have not yet been performed despite the fact that co-medication, such as anticonvulsants, could influence opioid use during surgery, as reported in a single study from 1990, which so far has never been replicated [START_REF] Tempelhoff | Anticonvulsant therapy increases fentanyl requirements during anaesthesia for craniotomy[END_REF].

e. Brain activity related parameters

Experimental approaches involving the use of fMRI and PET scans have been tested in the research setting only [START_REF] Rainville | Pain affect encoded in human anterior cingulate but not somatosensory cortex[END_REF][START_REF] Peyron | Functional imaging of brain responses to pain. A review and meta-analysis[END_REF]. PET scans performed solely for pediatric research reasons may, however, meet with ethical and practical obstacles as they involve administration of radioactive labeled drugs. As a non invasive procedure, fMRI is more promising for (semi)clinical evaluation and can be combined with quantitative sensory testing [START_REF] Hohmeister | Cerebral processing of pain in school-aged children with neonatal nociceptive input: an exploratory fMRI study[END_REF].

Neurophysiological measurements such as EEG and somatosensory response have so far not identified a specific pain signal that could be useful in daily clinical practice. There is direct EEG evidence of specific noxious-evoked neural activity in the infant brain [START_REF] Slater | Evoked potentials generated by noxious stimulation in the human infant brain[END_REF]. Somato-sensory responses have been demonstrated in young infants but as yet cannot serve as endpoints; we first need to establish normal values of voltage, frequency and duration.

f. Time to first (rescue) analgesia and analgesic consumption

As many postoperative patients will receive preemptive analgesic drugs, time to first rescue analgesia may serve as a clinical endpoint together with the total analgesic consumption over the first 12, 24 or 48 hours. Consumption should be expressed in microgram or mg per kg per hour or per 24 hours so as to enable comparison. Ideally these endpoints should be combined with scores obtained from validated pain assessment instruments.

g. Safety/adverse effects

Documentation of drug safety is highly important, especially in pediatric drug trials. There is some debate on whether it is better to have a pre-defined list of possible adverse events to be taken into account, or to resort to an unstructured approach in which researchers, parents or other individuals report any suspected adverse events [START_REF] Mcgrath | Core outcome domains and measures for pediatric acute and chronic/recurrent pain clinical trials: PedIMMPACT recommendations[END_REF]. This latter approach may carry the risk of underreporting of adverse events. Safe and effective pain treatment in neonates and young infants requires thorough understanding of various developmental aspects of drug disposition and metabolism. In general, the phenotypic variation in drug disposition and metabolism is based on constitutional, genetic and environmental factors. The clearance rate of most drugs is lower in neonates than in adults and older children.

Neonates still show immature renal function, i.e. decreased glomerular filtration rate and less effective tubular reabsorption and/or excretion. Moreover, they have a lower capacity of drug metabolizing enzymes (39-44). Furthermore, as reviewed by both Weinshillboum (45) and Evans and McLeod [START_REF] Huguet | Measurement of self-reported pain intensity in children and adolescents[END_REF], the disposition and action of many drugs are polygenetic determined events. Polymorphisms in drug metabolizing enzymes, transporters and receptors determine to a larger extent the spectrum of drug response (i.e., ranging from no effect to drug toxicity).

h. Long-term effects of analgesic treatment

The short and long-term consequences of prolonged opioid use in newborns and infants are largely unknown. Studies in animals suggest potential adverse long term effects of morphine. Morphine administration to neonatal rats produces long-term changes in behavior and brain function [63] and impairs the adult cognitive functioning [64], in particular spatial recognition memory [65]. Basic science has shown that the opioid system modulates neural proliferation in vivo [START_REF] Sargeant | Opioidergic regulation of astroglial/neuronal proliferation: where are we now?[END_REF]. Thus, it may well be that morphine treatment harms the neurogenesis of newborn babies. Mechanistically, morphine induces apoptosis of human microglial cells [START_REF] Hu | Morphine induces apoptosis of human microglia and neurons[END_REF] and stimulates red neuron degeneration in the rat brain, which may lead to cerebral dysfunction [START_REF] Atici | Opioid neurotoxicity: comparison of morphine and tramadol in an experimental rat model[END_REF]. Boasen et al recently showed in rodents that either neonatal stress and morphine treatment produced long-lasting behavioral effects to a degree sufficient to alter learning, while the combination of neonatal stress and morphine did not [START_REF] Boasen | Neonatal Stress or Morphine Treatment Alters Adult Mouse Conditioned Place Preference[END_REF].

Endpoints in human studies should therefore include cognition, neuropsychological tests, a chronic pain questionnaire, and pain and detection thresholds. The latter thresholds may be assessed with quantitative sensory testing (QST), for which normal values are available [START_REF] Blankenburg | Reference values for quantitative sensory testing in children and adolescents: developmental and gender differences of somatosensory perception[END_REF].

Finally, we should realize behavioral assessment instruments reveal other aspects of the phenomenon pain than do neurophysiological evaluation or the use of biomarkers. Moreover, no single parameter covers the whole spectrum from a nociceptive stimulus to behavior. It would seem essential, therefore, to also evaluate the fate of drugs in the body (pharmacokinetics) as well as the response of the body (pharmacodynamics).

Pharmacokinetics of the parent drug and (active) metabolites in relation to pharmacodynamics.

In summary

Tools to measure pain are being used as primary endpoints in studies on the effects of analgesic drugs.

Nevertheless, further research is needed to develop more objective pain measurements, to identify causes of variation in pain intensity and responses to pain treatment (both non-pharmacological and pharmacological PK-PD), and to develop age and disease specific pain treatment protocols.

  supplement hypersensitivity will still exist 15 years after tissue injury or has recovered or reverted to

  individualized Numeric Rating Scale based solely on the child's individual pain indicators as Part of TEDDY supplement described by parents and caregivers[39].The psychometric properties of this scale are promising; nevertheless, the essential involvement of the parents may be a drawback especially when the scale is used for research purposes[START_REF] Breau | The science of pain measurement and the frustration of clinical pain assessment: does an individualized numerical rating scale bridge the gap for children with intellectual disabilities?[END_REF].b. Self reportExamples of self report tools for 2 to 3 year old toddlers are the Poker Chip Tool[START_REF] Hester | Measurement of pain in children: Generalizability and validity of the pain ladder and poker chip tool[END_REF] and the Faces Pain Scale-Revised, recommended for research purposes in over 4-year-olds [42, 43]. The Numeric Rating Scale pain (NRS-11) [44] and Visual Analogue Scale pain (VAS) [45] should preferably not be used in children below the age of 8 years because it requires a certain cognitive level of development to translate pain intensity into numbers or distances on a 10 cm ruler. The Poker Chip Tool, Faces Pain Scale-revised and VAS have also been suggested by the Pediatric Initiative on Methods, Measurements and Pain Assessment in Clinical Trials (PedIMMPACT) and also came up as valid in two reviews [22] [43, 46].

  supplement change in facial expression, however, although NIRS readings revealed increased cortical activity during the procedure[START_REF] Slater | How well do clinical pain assessment tools reflect pain in infants?[END_REF].

  It has become easier to measure plasma levels of drugs in children. Sophisticated analytical methods (e.g. LC-MS-MS) and statistical analyses (e.g. population pharmacokinetics-pharmacodynamics, such as NONMEM) require smaller and fewer samples [71]. A possible relationship between therapeutic plasma ranges and pharmacodynamic parameters has not yet been found. Mutation analysis can provide answers to individual aberrant responses although tailoring of analgesic dosing is still a long way to go [72]. Efforts to improve pain therapy, for example by means of RCTs, should be developed within the context of regulatory initiatives. American legislation ('Food and Drug Administration Modernization Act' in 1997, 'Best Pharmaceuticals for Children Act' in 2002 and 'Pediatric Research Equity Act' in 2003) has come into force to promote drug development and authorization of medicines for use in pediatric patients. Similar legislation was introduced in the European Union in January 2007 ('The Pediatric Regulation') (Full text on www.fda.gov and www.emea.europe.eu). These legislations and supplement clinical trial registers (http://clinicaltrials.gov) provide essential information on ongoing studies in other centers and prevent duplication of studies in this vulnerable age group.

  2005) The development of nociceptive circuits. Nat Rev Neurosci 6 (7): 507-520 2 Grunau RVE, Whitfield MF, Petrie JH (1998) Children's judgements about pain at age 8-10 years: do extremely low birthweight (< 1000g) children differ from full birthweight peers? Journal Child Psychology Psychiatry 39 (4): 587-594 Anseloni V, Zou SP, Wade EB, Novikova SI, Ennis M, Traub RJ, Gold MS, Dubner R, Lidow MS (2004) Characterization of basal and re-inflammation-associated long-term alteration in pain responsivity following short-lasting neonatal local inflammatory insult. Pain 110 (3): 588-596 Tibboel D, Koot HM, van Dijk M, Peters JW (2006) Pain assessment in profound cognitive impaired children using the Checklist Pain Behavior; is item reduction valid? Pain 126 (1Koot HM, de Boer JB, Tibboel D (2003) Measuring pain in children with cognitive impairment: pain response to surgical procedures. Pain 103 (1-2): 187-198.

European Journal of Clinical Pharmacology

Part of TEDDY supplement

Acknowledgements

This contribution originates from the Task-force in Europe for Drug Development for the Young (TEDDY) Network of Excellence supported by the European Commission's Sixth Framework Program (Contract n. 0005216 LSHBCT02005-005126)


Laura ; Martin

Phillip C Watts

Laura E Martin

Susan E Kimmance

David J S Montagnes

Chris D Lowe

The distribution of Oxyrrhis marina -a global disperser or poorly characterised endemic?

Keywords: dispersal, speciation, cosmopolitan species, biogeography

It is often argued that microscopic organisms typically have cosmopolitan distributions. This argument has been criticised as it fails to acknowledge the many protists that have limited geographic distributions. Oxyrrhis marina, a common heterotrophic dinoflagellate in many intertidal and coastal habitats, is regarded as globally-distributed, yet despite its prominent role as an experimental planktonic organism there are few explicit studies of this species' geographic range. An analysis of 846 water samples globally confirmed the wide geographic distribution of O. marina and extends this morphospecies' range into the southern hemisphere. We did not find O. marina beyond 63º N latitude, implying that it is rare, or even absent, in Polar seas. Despite being rare in open water, O. marina inhabits the coastal waters of remote islands, such as Hawaii and the Azores. Beneath the apparent broad spatial range are some emerging patterns on the distribution of distinct genetic clades that form two divergent lineages that may represent different species. Two of the clades have discrete, non-overlapping distributions (one in the European Atlantic and western Mediterranean, and the second in the eastern Mediterranean Sea): one has a broad distribution (both coasts of North America and also in the eastern Mediterranean Sea), and one is limited to culture collections from the Baltic Sea and Red Sea. Thus, the Mediterranean Basin appears to be a diversity hotspot for Oxyrrhis, and there is a mixture of wide distributions and endemicity. These issues are discussed with respect to protist biogeography.

INTRODUCTION

Oxyrrhis marina, a common heterotrophic dinoflagellate in many intertidal and coastal habitats, is typically regarded as cosmopolitan or globally-distributed (e.g. see AlgaeBasehttp://www.algaebase.org/ -date accessed 15 October, 2009). It is surprising, therefore, that despite extensive research using O. marina as a model marine protist (Montagnes et al., this issue-a), there are few explicit studies of this species' natural distribution. Thus, the apparent global occurrence of O. marina is poorly-characterised. As a consequence, we lack the basic data that underpin an understanding of the ecological and evolutionary processes that generate present patterns of diversity, and this inevitably creates problems when studies attempt to interpret patterns of divergence (e.g. functional or genetic) between isolates of O. marina.

Below, we first provide an indication of why it is important to assess model-species distributions in marine environments. We then indicate the scope of knowledge regarding the distribution of O. marina, indicate why this species can act as a model, in this respect, and finally suggest which data are required to improve our use of O. marina as a model.

DISTRIBUTIONS OF MARINE SPECIES

Most macroscopic species (defined here as organisms greater than 10 mm in size) have relatively restricted geographic distributions that presumably reflect a combination of their ecological requirements, evolution, and historical contingency (see [START_REF] Finlay | Cosmopolitan metapopulations of free-living microbial eukaryotes[END_REF]. The relative influences of these processes depend upon the taxon, but there remains some debate about the general importance of such processes in generating observed biogeographic patterns. For example, a contrast may be made between 1) taxa that evolve at a point and subsequently disperse outwards to realise their geographic distribution and 2) species whose evolution is driven by vicariance or allopatry who are characterised by no obvious centre of origin [START_REF] Heads | Towards a panbiogeography of the seas[END_REF]. An issue that remains central to any debate about biogeographic patterns is a species' ability to disperse across heterogeneous landscapes and, therefore, it's potential to colonise and exploit new habitats, and to maintain gene flow over wide areas.

As an extreme biogeographic pattern, cosmopolitanism (organisms that occur globally or quasi-globally) nonetheless has been described for a substantial number of large species, particularly marine taxa which may be distributed by currents or allowed to disperse due to the connectivity of the oceans (e.g. [START_REF] Graves | Molecular insights into the population structures of cosmopolitan marine fishes[END_REF][START_REF] Watts | Natural and anthropogenic dispersal mechanisms in the marine environment: a study using cheilostome Bryozoa[END_REF]. On the one hand, the pelagic marine environment is perceived to be relatively more stable and typically lacking in barriers to dispersal, as compared with terrestrial landscapes or even benthic marine systems [START_REF] Graves | Molecular insights into the population structures of cosmopolitan marine fishes[END_REF][START_REF] Palumbi | Genetic divergence, reproductive isolation, and marine speciation[END_REF][START_REF] Sexton | Dispersal and biogeography of marine plankton: longdistance dispersal of the foraminifer Truncorotalia truncatulinoides[END_REF]. For species with life-history traits suitable for wide dispersal, particularly free-living pelagic species, such uniformity potentially permits broad geographic distributions. On the other hand, however, this picture is an overly simplistic, and somewhat outdated, view of a marine environment that is now recognised to be heterogeneous. Indeed, numerous studies document effective barriers to dispersal (e.g. oceanic fronts), and concomitant genetic divergence, in a broad range of marine taxa [START_REF] Darling | Molecular evidence links cryptic diversification in polar planktonic protists to Quaternary climate dymanics[END_REF][START_REF] Gallindo | Seascape genetics: a coupled oceanographic-genetic model predicts population structure of Caribbean corals[END_REF][START_REF] Hansen | Landscape genetics goes to sea[END_REF][START_REF] Patarnello | Pillars of Hercules: is the Atlantic-Mediterranean transition a phylogeographical break?[END_REF]. Nevertheless, there is also contemporary evidence for extensive and cosmopolitan distributions for a broad range of marine organisms. With this comparison in mind, it is somewhat surprising that the underlying biology of "cosmopolitan" species, such as O. marina, is often overlooked, as they should possess interesting life-histories.

One possible reason for a relative lack of recent interest in the biology of cosmopolitan species appears to be a degree of scepticism towards this phenomenon. An extensive geographic range, for example, may be an artefact of inadequate taxonomic resolution, which combined with undersampling in the marine environment, could lead to unrecognised cryptic species being amalgamated into a morphospecies with artificially large distributions (Knowlton, 1993;[START_REF] Klautau | Does cosmopolitanism result from overconservative systematics? A case study using the marine sponge Chondrilla nucula[END_REF]. With the increasing number of molecular-genetic studies that have identified cryptic species in apparently widely-distributed taxa [START_REF] Todaro | Are meiofaunal species cosmopolitan? Morphological and molecular analysis of Xenotrichula intermedia (Gastrotricha: Chaetonotida)[END_REF][START_REF] Klautau | Does cosmopolitanism result from overconservative systematics? A case study using the marine sponge Chondrilla nucula[END_REF][START_REF] Westheide | Cosmopolitan versus cryptic meiofaunal polychaete species: an approach to a molecular taxonomy[END_REF][START_REF] Fukami | Conventional taxonomy obscures deep divergence between Pacific and Atlantic corals[END_REF]Deryke et al. 2008;Lowe et al., this issue-a, but cf. Aitken et al. 2007), reports of cosmopolitanism are typically regarded as more apparent than actual and the debate appears to have reduced.

MICROBIAL DISTRIBUTIONS

For microbial species, by contrast, controversy has developed with conflicting theories and predictions about the extent and distribution of (free-living) microbial diversity, and ultimately whether microbial biogeography is fundamentally different to that of macroorganisms. On the one hand it has been argued that microscopic or "small" organisms (<1 mm) typically have a cosmopolitan distribution in terrestrial, freshwater and marine environments [START_REF] Finlay | Apparent global ubiquity of species in the protist genus Paraphysomonas[END_REF][START_REF] Finlay | Global dispersal of free-living microbial eukaryote species[END_REF]Fenchel andFinlay, 2003, 2004;[START_REF] Finlay | Cosmopolitan metapopulations of free-living microbial eukaryotes[END_REF]. These authors argue that wide geographic ranges are a consequence of huge absolute population sizes (rather than inherent properties of certain taxa), which drive wide dispersal potential and maintain a low probability of local population extinction. Since endemism largely generates patterns of species diversity of large organisms, there is an apparent effect of scale, with the diversity of small species exceeding that of larger organisms at local scales, while the feature is reversed at larger scales (e.g. global at the extreme). The corollary is that the distribution of small organisms is less dependent on historical contingency and that free-living microbial species are less likely to have biogeographies.

Clearly, the above arguments are relative, being based on the size of the organism, its dispersal rate, and the size of the spatial niche that it occupies, but some support for the notion of very wide or cosmopolitan distributions follows from a number of studies on eukaryotic microbial taxa, such as diatoms, foraminiferans, and diplonemids [START_REF] Hasle | Are most of the domoic acid-producing species of the diatom genus Pseudonitzschia cosmopolites?[END_REF]Castelyn et al., 2008;[START_REF] Kooistra | Global diversity and biogeography of Skeletonema species (Bacillariophyta)[END_REF][START_REF] Lara | Pan-oceanic distribution of new highly diverse clades of deep-sea diplonemids[END_REF]; in some cases these imply that certain protist species are capable of global gene flow [START_REF] Darling | Molecular evidence for genetic mixing of Arctic and Antarctic subpolar populations of planktonic foraminferans[END_REF]Pawlowski et al., 2007).

Several flaws in the "ubiquity model" have been highlighted, including poor taxonomic resolution and under-sampling [START_REF] Heger | The curse of taxonomic uncertainty in biogeographical studies of free-living terrestrial protists: a case study of testate amoebae from Amsterdam Island[END_REF]reviewed by Dolan, 2005;[START_REF] Foissner | Biogeography and dispersal of micro-organisms: a review emphasizing protists[END_REF][START_REF] Foissner | Protist diversity and distribution: some basic considerations[END_REF], with perhaps the most serious being an apparent failure to acknowledge the many studies that demonstrate many protists have limited geographic distributions [START_REF] Wilkinson | What is the upper size limit for cosmopolitan distribution in free-living microorganisms?[END_REF][START_REF] Chao | A statistical approach to estimate soil ciliate diversity and distribution based on data from five continents[END_REF][START_REF] Smith | Not all free-living microorganisms have cosmopolitan distributions -the case of Nebela (Apodera) vas Certes (Protozoa: Amoebozoa: Arcellinida)[END_REF]reviewed by Foissner, 2006reviewed by Foissner, , 2008;;[START_REF] Vanormelingen | The diversity and distribution of diatoms: from cosmopolitanism to narrow endemism[END_REF].

While it is reasonable to assume that biogeographic and evolutionary concepts used in macroorganismal biology are applicable to free-living microbes, it is important recognise that the majority of free-living microbes tend to have larger geographic ranges than do macroorganisms.

Accordingly, a "moderate endemicity" counter argument has been proposed [START_REF] Foissner | Biogeography and dispersal of micro-organisms: a review emphasizing protists[END_REF][START_REF] Foissner | Protist diversity and distribution: some basic considerations[END_REF], whereby protists, compared to prokaryotic microbes, generally have low abundances, and low rates of dispersal that result in moderate extinction rates and moderate (~30%) levels of endemism.

Anecdotally, the literature indicates O. marina to be cosmopolitan, isolates of which have been collected from throughout the northern Atlantic and Pacific Oceans, as well as the Mediterranean, Baltic, and Red Seas. The older literature , reviewed by [START_REF] Kofoid | The Free-Living Unarmored Dinoflagellata[END_REF], indicates a wide distribution of O. marina, including: Kiel (Germany), Plymouth (England), Genoa harbour (Italy), Jersey, Marseilles (France), Palingbrug (Belgium), Tengeriek (New Guinea), more broadly in the Mediterranean and White Seas and, most unusually, in a salt lake in Kharkoff (Russia). [START_REF] Kofoid | The Free-Living Unarmored Dinoflagellata[END_REF] also noted that O. marina is a regular inhabitant of marine aquaria around Europe: Frankfort, Helgoland and Heidelberg (Germany), Naples (Italy), Roscoff and Villefranche (France). Thus, even as early as the turn of last century, O. marina was recognised to have a wide distribution. One problem with these data is the increasingly numerous examples of morphologically simple protist 'species' that have been shown, using molecular-genetic techniques, to harbour extensive cryptic diversity [START_REF] De Vargas | Molecular evidence of cryptic speciation in planktonic foraminifers and their relation to oceanic provinces[END_REF][START_REF] Darling | Molecular evidence links cryptic diversification in polar planktonic protists to Quaternary climate dymanics[END_REF][START_REF] Slapeta | Global dispersal and ancient cryptic species in the smallest marine eukaryotes[END_REF][START_REF] Schekenbach | Molecular comparisons of freshwater and marine isolates of the same morphospecies of heterotrophic flagellates[END_REF]. Oxyrrhis marina is no exception, as it possesses few easily observable cytological structures (see Lowe et al., this issue-a, for taxonomic assessment), yet recent work (Lowe et al., 2005a[START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF] has identified several divergent lineages within what is described as "Oxyrrhis marina", culminating in support for the notion of distinct species [START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF] issue-a). The apparent wide distribution of "O. marina" may be a simple consequence of overconservative taxonomy. We, therefore, argue that it is difficult to interpret a range of experimental studies on protists that have uncovered extensive ecophysiological variation between isolates (Lowe et al., 2005a;[START_REF] Weisse | Pronounced ecophysiological clonal differences of two common freshwater ciliates, Coleps spetai (Prostomatida) and Rimostrombidium lacustris (Oligotrichida), challenge the morphospecies concept[END_REF], as it is not known whether this variation reflects adaptation within a single eurytopic species (i.e. one that is able to tolerate a wide range of environments) or has simply described a number of species-specific responses.

In the following sections, we highlight the current extent of our understanding of O. marina spatial and temporal distributions. In particular, we stress that explicit studies of O. marina distributions are scarce and that our current knowledge of distributions is almost entirely derived from a synthesis of methodological observations from the literature. Further, in light of recent studies describing high levels of functional and genetic variation within the O. marina morphospecies (Lowe et al., 2005a(Lowe et al., , 2010, this issue-a), this issue-a), we indicate that the majority of studies on O. marina are confined to few isolates that likely belong to disparate phylogenetic groups. We emphasise that far from assuming cosmopolitanism on the part of O. marina, future studies must address the distributions of potentially multiple, cryptic Oxyrrhis species. Finally, it is apparent, that in a biogeographic context, O. marina has the potential to be an important model organism to assess demographic parameters determining the processes that drive species distributions and geographic patterns of functional and genetic diversity in free-living protists.

"OXYRRHIS MARINA" IS BROADLY DISTRIBUTED AND LOCALLY ABUNDANT

Oxyrrhis marina was described over 150 years ago from a salt marsh habitat in Belgium (Fig. 1), and early work mentioned above characterised its putative wide distribution; typically it is now widely-regarded as a single, broadly-distributed species. A citation search using the keyword "Oxyrrhis" in Web of Knowledge (http://wok.mimas.ac.uk/) returned 114 papers published between 1938 and 2009 (date of search September 2009) that reported 38 isolates/sampling locations (in 64 cases no details of the strains were provided, or a reference only was provided, and 19 papers could not be accessed by the authors). Based on these published data, the O. marina morphospecies is best described as broadly distributed, inhabiting areas of the Atlantic and Pacific coasts of the US, the Gulf of Mexico, the Atlantic coasts of Europe, the Mediterranean and Baltic Seas, Persian Gulf, the Indian Ocean, and the western Pacific (Fig. 1). Throughout this distribution, however, O. marina has been isolated only from coastal areas, and predominantly from intertidal pools. This is potentially a sampling bias as a result of the particular ease with which littoral environments can be accessed (compared with open waters). Oxyrrhis marina is unlikely to be an exclusively intertidal species, since it has been identified from plankton surveys of neritic environments, estuaries and coastal embayments (Quevedo and Anadon, 2000;[START_REF] Johnson | Microzooplankton grazing on Prorocentrum minimum and Karlodinium micrum in Chesapeake Bay[END_REF][START_REF] Begun | A "bloom" in the water of Amursky Bay (Sea of Japan) caused by the dinoflagellate Oxyrrhis marina Dujardin, 1841[END_REF][START_REF] Jeong | Feeding by heterotrophic dinoflagellates on the common marine heterotrophic nanoflagellate Cafeteria sp[END_REF][START_REF] Orlova | Flora of planktonic microalgae of Amursky Bay, Sea of Japan[END_REF]. Despite these observations, O. marina does not appear to be common in neritic habitats as other, direct comparisons of lagoon and coastal sites have not reported its presence [START_REF] Lopez-Flores | Comparative composition and dynamics of harmful dinoflagellates in Mediterranean salt marshes and nearby external marine waters[END_REF]. Indeed, in a summary of the distribution of dinoflagellates in UK waters, [START_REF] Dodge | Marine Dinoflagellates of the British Isles. Her Majesty's Stationary Office[END_REF] classifies O. marina as a eurytopic species that inhabits pools, estuaries, and marshes, rather than open water. Given its distinctive morphology (see Lowe Thus, it is unlikely that it is a common component of the coastal and oceanic plankton community, although periodically it may form red tides in shallow embayments [START_REF] Begun | A "bloom" in the water of Amursky Bay (Sea of Japan) caused by the dinoflagellate Oxyrrhis marina Dujardin, 1841[END_REF].

Clearly, the observations made above are more anecdotal than systematic; crucially, we lack data about locations where O. marina appears to be absent, or at least rare. As part of our research into levels of genetic and ecophysiological variation of O. marina throughout its range, we have acquired 846 samples from 40 countries. The distribution of samples that yielded O. marina reinforces the observations made above and also extends this morphospecies' range into the southern hemisphere, into Australia, South Africa, and Brazil (Fig. 2). We did not find O. marina beyond 63º N latitude (e.g. northern Norway, Iceland), implying that it is rare, or even absent, in Polar seas. However, it has to be stressed that relatively few (<20) samples were collected from these areas; thus we may simply lack power to detect O. marina in these locations (see Lowe et al., this issue-b). Indeed, some reports of O. marina at northern latitudes (e.g. White Sea, and Tromsø, Norway; [START_REF] Hansson | South Scandinavian Marine Protoctista. Provisional check-list compiled at the Tjärnö Marine Biological Laboratory[END_REF], suggest that O. marina may occur in Polar seas. A second feature of this species' apparent distribution is that, despite being rare in open water, it inhabits the coastal waters of remote islands, such as Hawaii and the Azores (Fig. 2). Given an apparent rarity in open water, speculation about dispersal mechanisms highlights our ignorance of this basic process; for example, clonal reproduction could allow even just a few cells of O. marina to persist for relatively long periods (and therefore travel large distances, e.g. by ocean currents), with colonisation success simply an inevitable outcome of continued, but low frequency, dispersal from neritic areas over evolutionary time. Alternatively, O. marina may be associated with, and thus dispersed by, macroalgal rafts. Finally, it is possible that anthropogenic dispersal (such as transport in ballast water) has extended the natural distribution of this species. Indeed, genetic data provide evidence for trans-Atlantic transport of O. marina, possibly associated with shipping routes (Lowe et [START_REF] Burkholder | Phytoplankton and bacterial assemblages in ballast water of U.S. military ships as a function of port of origin, voyage time, and ocean exchange practices[END_REF]. Given the long history of human global movements using ships, exploring this possibility raises a broader issue of disentangling natural patterns (i.e. currents) and processes from anthropogenically enhanced distributions.

Despite the preceding discussion, this assessment of the literature and our own sampling support the notion that "O. marina" is broadly distributed and common in coastal environments and thus occupies a range of contrasting tropical and temperate environments (Fig. 2), which is perhaps not surprising given its tolerance to estuarine/intertidal pool habitats (for details of physiological tolerances see Lowe et al., this issue-b; Montagnes et al., this issue-b). One intriguing, feature of our recent sampling, however, is that we have not yet isolated O. marina from samples taken from the Baltic Sea (the positive sample in Fig. 2 and 3 is a strain maintained in a culture collection [CCAP1133/5], which was isolated from coastal waters in Finland in 1951). [START_REF] Droop | On the ecology of flagellates from some brackish and fresh water rockpools of Finland[END_REF] reported O. marina to be a common inhabitant of intertidal pools from the area surrounding Tvärminne, Finland, and [START_REF] Hansson | South Scandinavian Marine Protoctista. Provisional check-list compiled at the Tjärnö Marine Biological Laboratory[END_REF] reported O. marina from the Gulf of Finland and the south and west Baltic. Interpreting negative results is problematic, however, as our failure to isolate O. marina from the Baltic Sea could occur for a number of reasons. For example, our samples (n = 27) from the Baltic Sea were collected between April and July, and the population growth of O. marina may occur during early spring or in the late summer. Counter to this argument is the onset of the spring phytoplankton bloom in the southern Baltic Sea during April [START_REF] Wasmund | Phytoplankton spring blooms in the southern Baltic Sea-spatio-temporal development and long-term trends[END_REF] More widely, the present records of redlisted protists (http://www.iucnredlist.org/ date accessed 18 October, 2009) are limited to 15 species of phaeophycea. A confirmed decline or loss of O. marina from such a broad geographic area would be the first example of a marine protist neoextinction from a relatively wide area. Crucially, this work emphasises basic gaps in our knowledge about protist distributions and that this deficiency will continue to hamper any serious efforts to conserve protist biodiversity (see also discussion by [START_REF] Cotterill | Conservation of protists: is it needed at all? Biodiv[END_REF]. As more investigations expand our catalogue of protist species (e.g. [START_REF] Gross | Untapped bounty: sampling the seas to survey microbial biodiversity[END_REF][START_REF] Foissner | Protist diversity and distribution: some basic considerations[END_REF], the question remains: what have we lost?

Beneath the relatively broad geographic patterns mentioned above, a small number of specific studies have quantified the spatial and temporal distribution of O. marina within a shore, although they are restricted in scope. For example, Jonsson (1994) associated O. marina abundance (described as Oxyrrhis sp.) with tidal cycle and flushing. Subsequently, [START_REF] Johnson | Physical control of plankton population abundance and dynamics in intertidal rock pools[END_REF] identified a positive correlation between tidal height and abundance of O. marina on the Isle of Man, with greater numbers in the summer than in winter. Also relevant to our failure to find O. marina in some samples, particularly from the Baltic Sea, is work by [START_REF] Kimmance | The interactive effects of temperature and food concentration on growth responses of aquatic protists, with particular reference to the heterotrophic dinoflagellate Oxyrrhis marina[END_REF] who recorded seasonal variation in the abundance of O. marina in intertidal pools (also on the Isle of Man), with peaks in abundance during late June and also between late July and September (Fig. 4).

Prominent in our analysis of the literature is the large number of O. marina isolates that are reported in only a single study. Almost certainly this reflects the relative ease with which isolates can be newly derived from the environment (see Lowe et al., this issue-b, for discussion on this issue). In addition, there is also a bias for experimental work on just one or few strains that are kept in culture. The two most commonly studied isolates are CCAP1133/5 isolated from Finland (reported in 17 studies) and an isolate from Villefranche-sur-Mer (reported in 11 studies). Given

CRYPTIC DIVERSITY AND THE OCCURRENCE OF RESTRICTED DISTRIBUTION

Underlying the apparent broad spatial range there are some emerging patterns on the distribution of distinct genetic clades that have implications for experimental studies on a "model organism".

Most genetic characterisation of Oxyrrhis has been directed towards uncovering its phylogenetic position relative to the dinoflagellates and apicomplexans (e.g. Saldarriaga et al., 2004 who sequenced SSU rRNA, actin, α-tubulin and β-tubulin); however the pattern of sequence variation at part of the SSU rDNA did uncover two lineages of ''O. marina''. Unfortunately, the level of genetic diversity within Oxyrrhis at this genetic marker is relatively low which limits power to detect additional clades. More importantly, since only a small number of isolates were studied (n=2, 3, 11 for Saldarriaga et al., 2003;[START_REF] Cavalier-Smith | Protalveolate phylogeny and systematic and the origins of Sporozoa and dinoflagellates (phylum Myzozoa nom. nov.)[END_REF]Lowe et al., 2005, respectively) the geographic distributions of divergent lineages could not be determined. Thus, the most extensive spatial study completed to date [START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF] used sequence data from cytochrome oxidase I (COX1) and a more polymorphic marker, the 5.8S internal transcribed spacer 1 and 2 rDNA (5.8S ITS rDNA), to determine the genetic relationships of 58 O. marina isolates; this study identified four distinct clades that formed two highly divergent lineages. Two of the O. marina clades had discrete, non-overlapping distributions, one with a broad distribution and one with few samples (summarised in Fig. 5): clade 1 is European Atlantic and western Mediterranean in distribution, while clade 2 occurs off both coasts of North America and was also found in Clade 4 was taken from culture collections and occurs in the Baltic Sea and Red Sea. The Mediterranean Basin appears to be a diversity hotspot for Oxyrrhis (and also the clades/species defined by Lowe et al., 2010, this issue-a) , as it is for many taxa [START_REF] Myers | Biodiversity hostspots for conservation priorities[END_REF]. Thus, there is a mixture of wide distributions and endemicity, albeit at a regional scale (e.g. clade 3 is endemic to the Mediterranean) , a pattern reported in some other recent research into protist biogeography [START_REF] Kooistra | Global diversity and biogeography of Skeletonema species (Bacillariophyta)[END_REF].

Despite the broad scale of the study by [START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF], it remains unclear how broadlydistributed (or local) the O. marina clades are, as there are some conspicuous gaps and areas without replicates in sampling. For example, both clades 1 and 2 were represented by individual isolates from Korea and Japan, respectively. Whether these potentially broad distributions are driven by wide tolerance to varying conditions, high natural dispersal potential by prevailing currents and/or anthropogenically enhanced transport (likely in ballast water, as mentioned above) remains to be determined. Nonetheless, within the O. marina morphospecies, at least one of the genetic lineages appears to be restricted to quite limited regions suggesting that allopatric divergence may occur in eukaryotic microbial species, and ubiquity is not a logical corollary of small size: a point previously emphasised in arguments [START_REF] Foissner | Biogeography and dispersal of micro-organisms: a review emphasizing protists[END_REF][START_REF] Foissner | Protist diversity and distribution: some basic considerations[END_REF][START_REF] Vanormelingen | The diversity and distribution of diatoms: from cosmopolitanism to narrow endemism[END_REF] against the ubiquity model of microbial biogeography.

CURRENT AND FUTURE BIOGEOGRAPHIC RESEARCH

It is clear that our knowledge of the distribution of O. marina has improved substantially over the past few years but remains limited. Unlike other protists, such as diatoms and foraminiferans, O. marina has limited morphological characters that may be used to delineate species (see Lowe et al., this issue-a), and thus detailed biogeographic studies on species (will) rely heavily on molecular techniques, potentially limiting the range of laboratories that will expand the known distribution of O. marina clades. Indeed, the majority of genetic-based studies that have identified cryptic speciation in protists (e.g. [START_REF] Moreira | The molecular ecology of microbial eukaryotes unveils a hidden world[END_REF][START_REF] Lundholm | A study of the Pseudo-nitzschia pseudodelicatissima/cuspidata complex (Bacillariophyceae): what is P-pseudodelicatissima?[END_REF]Lundholm et al., , 2006;;[START_REF] Darling | Molecular evidence links cryptic diversification in polar planktonic protists to Quaternary climate dymanics[END_REF]Lowe et al., 2005a[START_REF] Lowe | Patterns of genetic diversity in the marine heterotrophic flagellate Oxyrrhis marina (Alveolata: Dinophyceae)[END_REF][START_REF] Slapeta | Global dispersal and ancient cryptic species in the smallest marine eukaryotes[END_REF][START_REF] Schekenbach | Molecular comparisons of freshwater and marine isolates of the same morphospecies of heterotrophic flagellates[END_REF][START_REF] Amato | Reproductive isolation among sympatric cryptic species in marine diatoms[END_REF] have not extended this work to quantify levels of endemism or cosmopolitanism in these new clades/species, likely because this would require a huge sampling effort (but see e.g. [START_REF] Kooistra | Global diversity and biogeography of Skeletonema species (Bacillariophyta)[END_REF]. Indeed, under-sampling (at both local and global scales), combined with taxonomic uncertainties, continue to hamper our understanding microbial biogeography [START_REF] Heger | The curse of taxonomic uncertainty in biogeographical studies of free-living terrestrial protists: a case study of testate amoebae from Amsterdam Island[END_REF].

Thus, despite the impact of the discussion recently stimulated by Finlay and co-workers [START_REF] Finlay | Apparent global ubiquity of species in the protist genus Paraphysomonas[END_REF]Fenchel andFinlay, 2003, 2004;[START_REF] Finlay | Cosmopolitan metapopulations of free-living microbial eukaryotes[END_REF], there are still too few studies to make broad generalisations on the processes that drive adaptive divergence and speciation in free-living aquatic protists (see also Dolan 2005 for review). For example, combined breeding experiments and genetic data support the idea that some species are globally distributed [START_REF] Casteleyn | Pseudo-nitzschia pungens (Bacillariophyceae): A cosmopolitan diatom species?[END_REF], while a combination of ecological processes and allopatric distributions have been implicated in driving divergence of planktonic foraminifera [START_REF] Darling | Molecular evidence links cryptic diversification in polar planktonic protists to Quaternary climate dymanics[END_REF]; yet, other studies have argued for isolating mechanisms linked to behaviour rather than allopatry [START_REF] De Vargas | Molecular evidence of cryptic speciation in planktonic foraminifers and their relation to oceanic provinces[END_REF][START_REF] Sexton | Dispersal and biogeography of marine plankton: longdistance dispersal of the foraminifer Truncorotalia truncatulinoides[END_REF]. We have uncovered a mixture of widely-distributed and endemic clades of the Oxyrrhis morphospecies, and support their separation into (at least) two distinct species (Lowe et al., 2010, this issue-a). At a basic level we argue that researchers must know which species of Oxyrrhis they are working on if their experimental work is to be placed in an evolutionary context; ideally, all future studies should genetically characterise their strain to provide this context. Beyond this, recognition of cryptic species raises questions about the mechanisms that permit or prevent their coexistence [START_REF] Ortells | Coexistance of cryptic rotifer species: ecological and genetic characterisation of Brachionus plicatilis[END_REF]. Accumulation of such data on a global scale is an essential way forward to resolve the demographic (e.g. migration, population size fluctuations) and evolutionary drivers that are responsible for the present patterns of diversity contained within the genus Oxyrrhis; understanding the distribution of this species complex will be important to guide future Oxyrrhis research and the implications of experimental and genome studies (Slamovits and Keeling, this issue).

One key issue is the relative role of sexual processes for the creation of adaptive divergence -and the scale over which adaptive differences are evident. As an inhabitant of intertidal pools and estuaries, certain strains (or species) of O. marina are clearly eurytopic. Indeed, ecophysiological data indicate that O. marina inhabits waters that vary in a number of ways, including 5 and 60 PSU and 10 and 30 °C (see Lowe et al., this issue-b; Montagnes et al., this issue-b). Indeed, a mixture of early and recent work has highlighted extensive ecophysiological variation within and among clones of Oxyrrhis [START_REF] Droop | A note on some physical conditions for cultivating Oxyrrhis marina[END_REF]Lowe et al., 2005a), and this appears typical of aquatic protists [START_REF] Weisse | The significance of inter-and intraspecific variation in bacterivorous and herbivorous protists[END_REF][START_REF] Weisse | Pelagic microbes -protozoa and the microbial food web[END_REF][START_REF] Weisse | Pronounced ecophysiological clonal differences of two common freshwater ciliates, Coleps spetai (Prostomatida) and Rimostrombidium lacustris (Oligotrichida), challenge the morphospecies concept[END_REF]. We know virtually nothing about the sexual ability of O. marina (see Montagnes et al., this issue-b). Interestingly, analysis of the genetic diversity of Oxyrrhis marina on a small spatial scale (i.e. <2 km) failed to uncover extensive clonal reproduction, with just 6 pairs (out of 81 isolates) of identical multilocus genotypes (Lowe et al., submitted) which raises questions about the extent of clonal reproduction at this scale. More generally, eurytopy, combined with high dispersal ability and large population sizes, is a key trait for acquiring a wide geographic range and geologic duration (cf. [START_REF] Jackson | Biogeographic consequences of eurytopy and stenotopy among marine bivalves and their evolutionary significance[END_REF]. Another key issue is to determine the relative impacts of putative ecological and physiological processes that determine absence (or likely rarity, since, as discussed above, earlier work reported O. marina from Tromsø and the White Sea) of O. marina from Polar regions. An obvious explanation at present is that our Arctic samples have been collected outside the spring bloom, and O. marina abundance was too low. Alternatively, our sampling methods (see Lowe et al., this issue-b) may be inappropriate for establishing cold-adapted strains. Clearly, patterns of presence and absence, and the Baltic Sea and tropical areas that are biodiverse (e.g. Philippines, Wallacea, [START_REF] Myers | Biodiversity hostspots for conservation priorities[END_REF].

Finally, there is some indication that O. marina may form cysts, although it is not clear if these are resting cysts (Montagnes et al., this issue-b); appreciating the factors that stimulate cyst production may aid in our understanding of dispersal. Not only will such data highlight centres of origin, biodiversity hotspots, and opportunity for ecological specialisation, but they will also provide the crucial baseline to assess the impact of climate change and altered distributions. Studies that couple patterns of functional genetic diversity with ecophysiological data will provide an interesting model to test which clades make the most significant changes in their distribution; for instance it might be appropriate to focus on genes associated with osmoregulation when examining salinity responses (e.g. Lowe et al., 2005b).

Finally, human-mediated introductions have been commonly reported for relatively large marine species that have obvious ecosystem impacts, such as the ctenophore Mnemiopsis leidyi [START_REF] Oguz | Invasion dynamics of the alien ctenophore Mnemiopsis leidyi and its impact on anchovy collapse in the Black Sea[END_REF][START_REF] Javidpour | Seasonal changes and population dynamics of the ctenophore Mnemiopsis leidyi after its first year of invasion in the Kiel Fjord, Western Baltic Sea[END_REF]. Numerous microscopic taxa have certainly had their ranges enhanced by anthropogenic transport (e.g. diatoms, [START_REF] Vanormelingen | The diversity and distribution of diatoms: from cosmopolitanism to narrow endemism[END_REF]nematodes, Deryke et al., 2009), with recent studies of ballast water highlighting the diversity of protists and potential for introduction of non-native species by shipping in particular [START_REF] Burkholder | Phytoplankton and bacterial assemblages in ballast water of U.S. military ships as a function of port of origin, voyage time, and ocean exchange practices[END_REF].

Better descriptions of clade distributions of protist species will highlight allopatric populations that may, for example, be linked with shipping routes; with this in mind, studies of the distribution of clades and species within Oxyrrhis would seem ideally placed to examine the affect of shipping on organism distributions in the marine environment.

Beyond this consideration the broad distribution, high diversity and wide ecophysiological tolerance of the O. marina 'complex' makes it a useful model to examine demographic processes amongst free-living protists. This genus, Oxyrrhis, is widespread, but there are differences between clades that need to be clarified by future sampling, particularly away from European seas. Such work will form the foundation to identify relative roles of, for example, Quaternary climate fluctuations and allopatric processes, as opposed to putative sympatric mechanisms, that drive adaptive divergence and speciation in aquatic free-living protists (e.g. see [START_REF] De Vargas | Molecular evidence of cryptic speciation in planktonic foraminifers and their relation to oceanic provinces[END_REF][START_REF] Darling | Molecular evidence links cryptic diversification in polar planktonic protists to Quaternary climate dymanics[END_REF][START_REF] Sexton | Dispersal and biogeography of marine plankton: longdistance dispersal of the foraminifer Truncorotalia truncatulinoides[END_REF]. Studies that use O. marina appear ideally-placed to provide these data, and thus a wider appreciation of the roles that protist biodiversity play in ecosystem function.

  al., this issue-a), it seems unlikely that O. marina has been overlooked during plankton surveys.

  and studies in the UK (Fig.4,[START_REF] Kimmance | The interactive effects of temperature and food concentration on growth responses of aquatic protists, with particular reference to the heterotrophic dinoflagellate Oxyrrhis marina[END_REF]) that demonstrate O. marina is more abundant during the summer.Given that misidentification of O. marina by Droop and others is extremely unlikely, if future, rigorous sampling continues to fail to yield O. marina, it has become rare or even gone extinct. Possibly, as the Baltic Sea has experienced a range of environmental changes associated with pollution during the past decades, O. marina may have been eliminated. If supported, this important addition to the loss of the sturgeon Acipenser sturio from the Baltic.

  the apparent rarity (or even absence) of O. marina from the Baltic Sea, this raises the issues of: 1) "what" is the scientific community actually working on? and 2) can experimental data on one strain be extrapolated to understand behavioural, demographic and evolutionary responses of O. marina elsewhere? In another paper in this issueLowe et al. (this issue-a) provide guidance on this problem.

  eastern Mediterranean Sea. Clade 3 only occurs in the eastern Mediterranean Sea.

  of O. marina need to be investigated more thoroughly for other regions, particularly

Fig. 1 .

 1 FIGURE LEGENDS

Fig. 2 .

 2 Fig. 2. Summary distribution of global sampling effort for Oxyrrhis marina, indicating areas that

Fig. 3 .

 3 Fig. 3. Detailed distribution of sampling effort for Oxyrrhis marina in European coastal waters,

Fig. 4 .

 4 Fig. 4. Seasonal variation in temperature (open circles) and abundance of O. marina (filled circles)

Fig. 5 .

 5 Fig. 5. Distribution of the four O. marina phylogenetic clades that have been identified to date.

  Fig. 4

  al., Watts et al. 1. Distribution of Oxyrrhis marina 10 2010), and O. marina is present in ballast water

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ACKNOWLEDGEMENTS

The Authors would like to thank all of the people who supplied us with samples and Emily Roberts and Claudio Slamovits for their constructive comments on this manuscript. We would also like to thank the anonymous reviewers for their suggestions.

FUNDING

This work was supported by a NERC grant (NE/F005237/1) awarded to PCW, CDL, and DJSM. Page 17 of 31 http://mc.manuscriptcentral.com/jplankt Journal of Plankton Research