Universidade de Lisboa
Faculdade de Ciências
Departamento de Biologia Animal
Tardigrada: a study on integrative taxonomy,
impacts on biodiversity and concerns with
conservation
Filipe José de Amorim Vicente
Doutoramento em Biologia
(Biodiversidade)
2012
Universidade de Lisboa
Faculdade de Ciências
Departamento de Biologia Animal
Tardigrada: a study on integrative taxonomy,
impacts on biodiversity and concerns with
conservation
Filipe José de Amorim Vicente
Tese especialmente elaborada para a obtenção do grau de doutor em
Biologia (Biodiversidade)
Orientação:
Prof. Roberto Bertolani (Università degli studi di Modena e Reggio Emilia)
Prof. Doutor Artur Serrano
2012
The present thesis was financed by Fundaçãopara a Ciência e a Tecnologia
(BD/39234/2007) and is an aggregate of scientific papers. Formatting of such
papers has been altered for a uniform look of the thesis. The author declares to have
participated in data collecting and analysis and in writing of all manuscripts used.
A presente tese doutoral foi financiada pela Fundação para a Ciência e a Tecnologia
(BD/39234/2007) e resulta da agregação de um conjunto de artigos científicos,
tendo a formatação dos mesmos sido alterada para efeitos de uma apresentação
uniformizada. O autor declara que participou na recolha de dados, sua análise e
escrita dos vários manuscritos apresentados.
Para o meu filho Tomás.
Tardigrada: a study on integrative taxonomy, impacts on biodiversity and concerns with conservation
Index Acknowledgments (English and Portuguese).______________________________________6 Summary (English and Portuguese).________________________________________________8 General introduction.________________________________________________________________10 Paper 1 -­‐ Micro-­‐invertebrates conservation: forgotten biodiversity.____________15 Paper 2 -­‐ The impact of fire on terrestrial tardigrade biodiversity: a case-­‐study from Portugal._________________________________________________________________________29 Paper 3 – Considerations on the taxonomy of the Phylum Tardigrada.______________45 Paper 4 -­‐ Integrative taxonomy allows the identification of synonymous species and a new genus of Tardigrada Echiniscidae (Heterotardigrada)._______________53 Paper 5 -­‐ Observations on Pyxidium tardigradum (Ciliophora), a protozoan living on Eutardigrada: infestation, morphology and feeding behaviour.____79 Paper 6 -­‐ A phylogenetic study on Pyxidium tardigradum (Peritrichia, Operculariida), an epizoic protozoan on eutardigrades.__________________________101 Concluding remarks and future perspectives.________________________________________116 5 Filipe Vicente – Doutoramento em Biologia Acknowledgements I would like to start by thanking my parents, without whose support I would not have been able to pursue in academics, to my Inês for her support and constantly pushing me forward, and to all family members, friends, colleagues and teachers that have somehow helped shape the path the has brought me here. Thank you both to the deceased professor Maria José Boavida, and to professor Artur Serrano for having accepted the co-­‐supervision of this thesis, for welcoming me in their labs, for the suggestions, comments and advises. An additional thanks to Zé for her initial help in dealing with the insatiable bureaucratic beast; in this mater, a word of appreciation to professor Leonel Gordo is also due. I also thank my colleagues, friends and collaborators at the University of Modena, Michele Cesari, Trevor Marchioro, Roberto Guidetti, Lorena Rebecchi e Tiziana Altiero for their relentless support, both personally and professionally, in every single one of my many visits. I feel that I will always be a member of your team. Finally, I want to thank professor Roberto Bertolani, main supervisor of my thesis, without whom it would not have become a reality. Thank you for opening the doors of your institution, for welcoming me within your work group since my first stay, in 2006, when I was just an exchange student under the Leonardo da Vinci programme and had nothing more to offer than my will to learn about the animal group of his expertise. Thank you very much for the love, concern and patience, personal and professional support and for all the times that you were more than just a supervisor. Thank you for, together with your team members, always making feel at home. 6 Tardigrada: a study on integrative taxonomy, impacts on biodiversity and concerns with conservation Agradecimentos Quero começar por agradecer aos meus pais, sem o apoio dos quais não teria conseguido construir o meu percurso académico, à minha Inês pelo apoio e incentivo constante e a todos os familiares, amigos, colegas e professores que, de algum modo, me ajudaram a moldar o percurso que me trouxe até aqui. Agradeço à professora Maria-­‐José Boavida, já falecida, e ao professor Artur Serrano, por terem aceite a co-­‐orientação dos meus trabalhos doutorais, por me terem acolhido nos seus laboratórios, pelas sugestões, comentários e conselhos. Agradeço à Zé pelo apoio inicial a lidar com o insaciável monstro burocrático; neste ponto, uma palavra de apreço também para o professor Leonel Gordo. Agradeço aos meus colegas, amigos e colaboradores na Universidade de Modena, Michele Cesari, Trevor Marchioro, Roberto Guidetti, Lorena Rebecchi e Tiziana Altiero pelo incansável apoio pessoal e profissional que me concederam em todas as minhas visitas. Sinto que serei sempre um membro da vossa equipa. Por fim, quero agradecer ao professor Roberto Bertolani, principal orientador desta tese, sem o qual ela não seria hoje uma realidade. Agradeço por me ter aberto as portas da sua instituição, e acolhido no seio do grupo de trabalho que dirige desde que, em 2006, eu era apenas um aluno de intercâmbio pelo programa Leonardo da Vinci e nada mais tinha para oferecer do que vontade de aprender sobre os animais em que é especialista. Muito obrigado pelo carinho, pela preocupação, pela paciência, pelo apoio profissional e pessoal, por ter sido muitas vezes mais do que apenas um orientador. Obrigado por, conjuntamente com a sua equipa, sempre me terem feito sentir em casa. 7 Filipe Vicente – Doutoramento em Biologia Summary This thesis presents a series of papers on some understudied aspects of the biology of tardigrades. Paper 1 is an essay on how biodiversity conservation strategies have long neglected and disregarded microscopic fauna in favour of macro fauna, for non-­‐
objective reasons; this may have devastating effects for groups of small animals, since they are rarely granted with any type of study on species evaluation status, let alone consequential protective measures. Paper 2 is a pioneer study into possible effects of habitat destruction caused by forestal fires over populations of tardigrades, looking both at levels of taxonomic and genetic richness. Paper 3 is an analysis of the current state of tardigrade taxonomy, a critical look into the traditional way of describing new taxa, and a proposal for an update of taxonomic work methodologies. Paper 4 sets an example for the type of work method advocated in Paper 3; it is the review of the systematic positioning of two synonymous species based on the integration of morphological observations with genetic analysis. Paper 5 is a study about the unknown biology of the eutardigrade epizoic protozoan Pyxidium tardigradum, analysing its morphology, reproductive and feeding strategies, and also the nature of the host-­‐colonizer relationship. Paper 6 goes further on the previous topic and, offering the first DNA data of P. tardigradum, establishes the phylogenetic position of the species and analyses genetic distances between two European populations. 8 Tardigrada: a study on integrative taxonomy, impacts on biodiversity and concerns with conservation Resumo A presente tese compreende um conjunto de artigos sobre alguns aspectos pouco estudados da biologia dos tardígrados. O Artigo 1 é um ensaio sobre como os animais microscópicos vêm sendo negligenciados face à macro fauna, de forma pouco objectiva, no que refere a estratégias de biologia da conservação. Tal poderá implicar efeitos devastadores para a micro fauna, visto que esta raramente é agraciada com estudos de avaliação do estatutos de conservação das espécies, muito menos com medidas de protecção. O Artigo 2 é um estudo pioneiro sobre os efeitos, em populações de tardígrados, da destruição de habitat causada por fogos florestais, analisando os níveis de diversidade genética e taxonómica. O Artigo 3 oferece uma análise sobre o actual estado da taxonomia dos Tardigrada, com um olhar crítico sobre a forma tradicional de descrever novos taxa, e propondo uma actualização de metodologia nestes estudos taxonómicos. O Artigo 4 estabelece um exemplo do tipo de metodologia defendida no artigo anterior: revê a posição sistemática de duas espécies sinónimas, com base na integração de estudos morfológicos com análises genéticas. O Artigo 5 apresenta um estudo sobre vários aspectos desconhecidos da biologia do protozoário epizóico de eutardígrados Pyxidium tardigradum, analisando a sua morfologia, as estratégias de reprodução e alimentação, e ainda a natureza da interação hospedeiro-­‐colonizador. O Artigo 6 aprofunda o tema anterior e, oferecendo os primeiros dados genéticos de P. tardigradum, estabelece a posição filogenética da espécie, analisando ainda a distância genética entre duas populações Europeias. 9 General Introduction General introduction More than 300 years have passed since Anton van Leeuwenhoek first observed and described microscopic life. Regardless of this, in some scientific disciplines, our knowledge of biological life appears to grow proportionally to its physical size. In Conservation Biology, not only do we know very little about microscopic life, but we also give it very little credit and importance. This is the starting point for the present thesis. The Phylum Tardigrada represents a good example of such an ill-­‐known group of organisms. These are microscopic animals present across Earth’s habitats: from dwelling in marine sediments, where they are often a significant component of meiofauna; in freshwater environments, or in terrestrial micro habitats with permanent or temporary water retention, such as leaf litter, mosses or lichens, which they usually inhabit together with rotifers and nematodes, all of which possess cryptobiotic capacities (Ramazzotti & Maucci, 1983). The most studied cases of cryptobiosis are anhydrobiosis and cryobiosis. Anhydrobiosis means that these animals are capable of dramatically reducing body water volume, suspending all metabolism for up to a few years period, them rehydrating and regaining activity once environmental water returns (Keilin, 1959). This can be performed at any stage of development and has long captured scientists’ attention since it appears to be a form of paused animal life. If such biotechnology were to be mastered, it would mean that we could ‘click a stop button’ on living organisms without inducing death; instead we would have the possibility to ‘click play’ at a future time of choice. In this state of ‘suspended life’, tardigrades are impressively resistant to extreme external conditions of temperature, pressure or radiation (Altiero et al., 2011). This has justified recent experiments in the planet’s orbit, under unnatural conditions (Jönsson et al., 2008; Persson et al., 2011; Rebecchi et al., 2011). Cryobiosis is represented by the ability to resist to freezing, in this case also for several years (Wright, 2001). This allows them to colonize very cold lands and mountains (Bertolani et al., 2004). 10 Tardigrada: a study on integrative taxonomy, impacts on biodiversity and concerns with conservation
Even tough we now start to understand how tardigrades respond to being outside the planet, we still know very little about their importance on Earth’s ecological systems. To date, no study exists addressing global ecological importance, or species’ conservation status. This is, in my opinion, a severe gap in our current knowledge, not only regarding tardigrades, but all micro fauna in general. Microscopic animals play important roles in regulating water, air and nutrient cycles; in the control of otherwise destructive plagues and infections; or in climate regulation (Price, 1987; Commission of the European Communities, 2006). In an attempt to start mitigating this scientific gap, I have studied how this specific slice of biodiversity is affected by habitat destruction. In order to better understand how micro fauna responds to environmental degradation, we first need to raise and clarify our knowledge levels of the real values of biodiversity. To accomplish this goal, it is of the utmost importance that we update the ‘business-­‐as-­‐usual’ species describing protocol, traditionally limited to morphological observation, by integrating other sources of independent and complementary data, particularly by bringing genetic work into play. Genetics can help to determine the evolutionary and phylogeographic meaning behind morphological character differences, as well as shed some light on the barriers separating intraspecific from interspecific variability. I think that tardigrade taxonomy is in urgent need of this contribution, aiming for a much-­‐needed revision and also for future strongly fundamented species descriptions. An example is set in the present thesis. Another important gap on the available knowledge of tardigrades is the interactions with other species. A few studies exist already on the topics of feeding habits and predator-­‐prey interactions of terrestrial species, e.g., Doncaster & Hooper (1961); Hohberg & Traunspurger (2005); Sánchez-­‐Moreno et al. (2008). The protozoan Pyxidium tardigradum places a series of different questions, as here tardigrades are neither the predator nor the prey. Pyxidium tardigradum is a symphoriont species that specifically targets eutardigrades, one about which we knew very little, apart from the generic original description by Van der Land (1964) and a few occasional registers of occurrence (Iharos, 1966; Morgan, 1976; Hallas, 1977; Wright, 1991; Marley and Wright, 1994). It was important, however, 11 General Introduction to seek a greater understanding about the nature of this animal-­‐protozoan relationship, about the life cycle of the latter, and about the positioning of the protozoan in the evolutionary tree of life. These questions are answered in this thesis. References Altiero, T., Guidetti, R., Caselli, V., Cesari, M. & Rebecchi, L. (2011) Ultraviolet radiation tolerance in hydrated and desiccated eutardigrades. Journal of Zoological Systematics and Evolutionary Research 49(S1): 104-­‐110. Bertolani, R., Guidetti, R., Jönsson, K. I., Altiero, T., Boschini, D. & Rebecchi, L. (2004) Experiences with dormancy in tardigrades. Journal of Limnology 63: 16-­‐25. Commission of the European Communities (2006) Halting the loss of biodiversity by 2010 – and beyond; Sustaining ecosystem services for human well-­‐being. Brussels. Doncaster, C. C. & Hooper, D. J. (1961) Nematodes attacked by protozoa and tardigrades. Nematologica 6: 333-­‐335. Hallas, T. E. (1977) Survey of the tardigrades of Finland. Annales Zoologici Fennici 14: 173-­‐183. Hohberg, K. & Traunspurger, W. (2005) Predator–prey interaction in soil food web: functional response, size-­‐dependent foraging efficiency, and the influence of soil texture. Biology and Fertility of Soils 41: 419-­‐427. Iharos, G. (1966) A Bakony-­‐hegyseg Tardigrada-­‐faunaja. III. Különlenyomat az Állattani Közlemények 53: 69-­‐78. Jönsson, I. K., Rabbow, E., Schill, R. O., Harms-­‐Ringdahl, M. & Rettberg, P. (2008) Tardigrades survives exposure to space in low Earth orbit. Current Biology 18: 729-­‐731. Keilin, D. (1959) The Leeuwenhoek lecture -­‐ the problem of anabiosis or latent life -­‐ History and current concept. Proceedings of the Royal Society of London Series B-­‐ Biological Sciences, 150: 149-­‐191. 12 Tardigrada: a study on integrative taxonomy, impacts on biodiversity and concerns with conservation
Marley, N. J., Wright, D. E. (1994) Pyxidium tardigradum van der Land, a rarely recorded symphoriant on waterbears (Tardigrada). Quekett Journal of Microscopy 37: 232-­‐233. Morgan, C. I. (1976) Studies on the British tardigrade fauna. Some zoogeographical and ecological notes. Journal of Natural History 10: 607-­‐623. Persson, D., Halberg, K. A., Jorgensen, A., Ricci, C., Mobjerg, N. & Kristensen, R. M. (2011) Extreme stress tolerance in tardigrades: surviving space conditions in low earth orbit. Journal of Zoological Systematics and Evolutionary Research 49(S1): 90-­‐97. Price, P. W. (1987) The role of natural enemies in insect populations. In: Barbosa P, Schultz JC (eds) Insect outbreaks. Academic Press, Inc. London. 287-­‐312 pp. Ramazzotti, G. & Maucci, W. (1983) Il phylum Tardigrada. Memorie dell’Istituto Italiano di Idrobiologia 41: 1-­‐1012. Rebecchi, L., Altiero, T., Cesari, M., Bertolani, R., Rizzo, A. M., Corsetto, P.A. & Guidetti, R. (2011) Resistance of the anhydrobiotic eutardigrade Paramacrobiotus richtersi to space flight (LIFE–TARSE mission on FOTON-­‐
M3). Journal of Zoological Systematics and Evolutionary Research 49(S1): 98-­‐
103 Sánchez-­‐Moreno, S., Ferris, H. & Guil, N. (2008) Role of tardigrades in the suppressive service of a soil food web. Agriculture, Ecosystems and Environment 124: 187-­‐192. Van der Land, J. (1964) A new peritrichous ciliate as a symphoriont on a tardigrade. Zoologische Mededelingen 39: 85-­‐88. Wright, J. C. (1991) The significance of four xeric parameters in the ecology of terrestrial Tardigrada. Journal of Zoology 224: 59-­‐77. Wright, J. C. Cryptobiosis (2001) 300 Years on from van Leeuwenhoek: what have we learned about tardigrades? Zoologischer Anzeiger 240: 563-­‐582. 13 Paper 1 Micro-­‐invertebrates conservation: forgotten biodiversity. Filipe Vicente (2010) Biodiversity Conservation 19: 3629-­‐3634. M icro-­‐invertebrates c onservation Abstract The concern about the preservation of biodiversity is due, in part, to a great level of media coverage granted in the last few years to global warming and consequential climatic changes. However, there are still considerably large gaps in scientific knowledge regarding the ecological status of many species, which results in an absence of conservation strategy for most of Earth’s biodiversity in need of it. The extinction of many animal and plant species can have catastrophic consequences on the ecosystems’ balance and also in human well-­‐being, resultant from the break of ecological services. To exemplify how a specific group of microscopic animals can be endangered, I have analyzed the case of the phylum Tardigrada. Tardigrades are microscopic animals that inhabit most environments: terrestrial, freshwater and marine. Even though many species are widespread and the terrestrial ones granted with cryptobiotic skills, they are adapted to each habitat type and, additionally, to local environmental patterns. This means that these tiny metazoans can be under significant environmental pressure in the various habitat types they are found in. The potential need of protective and compensatory measures aiming for appropriate conservation of these life forms is discussed, as is the need of studying for their objective elaboration. Keywords Biodiversity conservation, conservation status, micro-­‐invertebrates, Tardigrada, preventive and protective measures. 17 Paper 1 Biodiversity conservation has been a worldwide issue in government agendas at least since the United Nations’ Earth Summit held in Rio de Janeiro, Brazil, in 1992, where world leaders agreed on a common strategy for “sustainable development”. The key pact achieved at the Summit resulted in the Convention on Biological Diversity, a document which stresses conservation of biological diversity as a global goal, as well as its sustainable use and the sharing of benefits arising from the exploration of genetic resources. The European Community has ever since been looking to be in the lead of friendly biodiversity policy-­‐making. Examples of such concern are the Natura 2000 Network of protected areas, LIFE projects and management plans as financial instruments supporting nature conservation projects. The most relevant of the latest political endeavors in this field was known as Countdown 2010, an agreement achieved in 2001 by EU governments towards sustaining biodiversity loss and recovering natural habitats by 2010, which around 130 other world leaders joined in 2002. Meritorious as these efforts are, there are still great gaps in knowledge regarding poorly known taxonomic groups such as invertebrates, plants, tropical biota and all aquatic and subterranean habitats (Millennium Ecosystem Assessment, 2005). Lévêque et al (2005) estimated that there are around 100,000 known freshwater animal species today, half of which are insects. However, many freshwater biodiversity assessment studies tend to focus on better-­‐known groups such as fish and/or on endemic or keystone species. Also, they claim, official species richness indexes should be severely underestimated in lesser studied groups, such as protozoans, annelids or nematodes. Concerning the Protozoa, for instance, much of our knowledge of the group’s biodiversity is tightly linked to clinical disease in vertebrates, mainly mammals (Adlard and O’Donoghue 1998). There is, however, a whole new world of diversity to be unveiled in the Protozoa alone, regarding those associated with invertebrates (i.e., Vicente et al. 2008) as well as all other free living species. The IUCN’s Red List of Threatened Species includes 44,838 species with assessed conservation statuses in its 2008 update. This number has been increasing each year and undoubtedly reflects the work of many, yet it still only represents 2.73% of all described species to date. Moreover, a quick analysis allows for a view of really how biased these assessments are towards some taxonomic groups. Considering the better studied ones, 18 M icro-­‐invertebrates c onservation mammals and birds, 100% of the currently described species have been evaluated for their conservation statuses and, out of these, 21% out of 5,488 mammal species and 12% out of 9990 bird species are considered to be endangered. Turning our attention to one of the lesser studied groups, we see that only 0.13% out of all the described insect species have an evaluated status, 50% of which are endangered. This means that half of the few insect species whose conservation statuses have been assessed were classified as threatened, yet extremely few out of the 950,000 calculated species known to science have been graced with conservational study. Let me highlight that this last number does not include an estimate of the insect species that are yet to be described (surely many more than birds or mammals), which means that considering insects alone, the actual number of threatened species could easily surpass that of the sum of all existing vertebrates. A similar scenario is shared by the rest of invertebrates, plants, algae, lichens and mushrooms: very few known species have been evaluated for their threatened statuses, with few exceptions. Therefore, it appears necessary to enrich the Red List of Threatened Species with many invertebrate species endemic and/or living in specific habitats easily endangered (caves, small lakes, small rivers). Additionally, I think that we still take biodiversity conservation under a prejudice of scale, neglecting living organisms to an extensively greater degree the smaller they get, even when knowledge is available. Stork et al. (2008) show evidence of this problem, studying canopy beetles. If this is true for small macroscopic animals, the more truthful it becomes for microscopic ones. In other words, when we talk about preserving biodiversity, we should not disregard microscopic organisms since their existence is of a crucial nature for the maintenance of a sustainable balance in all of Earth’s ecosystems. In order to illustrate how a specific group of microscopic organisms can be endangered, let’s consider the Tardigrada phylum. Tardigrades, commonly known as water bears, are microscopic metazoans, usually much less than 1 mm in length that can be found in most environments, terrestrial, freshwater and marine. On terrestrial environments, their preferential living substrates are mosses, lichens and leaf litter. Regardless of their ability to disperse with ease and high abundance, tardigrades are habitat-­‐dependent in a similar way to larger animals (Guil et al. 2009). Many limno-­‐
terrestrial species are ecologically specialized and able to survive only in particular micro-­‐
19 Paper 1 environmental conditions. This is particularly true for parthenogenetic taxa with low individual variability (Pilato 1979: Pilato & Binda 2001), and recent studies demonstrate that the number of endemic species is higher than traditionally believed (Pilato 1979; Pilato & Binda 2001). Hence, the destruction of these micro-­‐habitats, due to e.g. the humanization of natural areas, causes obvious reduction of population effectives and may cause similar results in the phylum’s biodiversity, with the extinction of some species even before they were known to science. Other causes behind habitat reduction are, for instance, air pollution, as this is known to inhibit lichen growth (Jovan 2008). Moreover, pollution can directly cause a reduction in tardigrade species and specimen number (Vargha et al. 2002). A contemporary example of the effect air pollution has on these animals comes from China, were acidic rain appears to be behind the disappearing of tardigrades from most areas where air pollution is stronger (Miller, pers. comm.). Forest fires are another obvious menace yet, ironically, some fire prevention procedures may end up being an even bigger one. Quartau (2008) pinpoints how mandatory forestall vegetation clearance methodologies have been carried out in Portugal and how much they represent a serious threat to biodiversity. These methods involve the complete removal of all potential burning materials, including bushes, herbaceous plants and grasses, pines, branches and leaf litter. Since these organic materials will usually be burnt for energy production, the outcome is clearly catastrophic for animal groups inhabiting those substrates, including ground fauna, entomofauna and other macro and micro invertebrates, as well as for all the inferior plants that are removed. Considering just the fauna, mass extinctions can take place, resulting in the loss of an unprecedented number of endemic species, before they were even known to science (Quartau 2008). Additionally, we should also consider the ecological consequences both for humankind, with the breaking of ecological services, as well as for all other fauna to some extent dependent on the lost biodiversity. Among such ecological services are the maintenance of the nutrient cycle and soil fertility, the production of food, fuel and medicines, the regulation of hydric resources, air and climate (Commission of the European Communities 2006), and the control of pests or diseases (Price 1987). These roles played by the natural systems highlight how important biodiversity is for sustainable development and general human well-­‐being. Returning to the example of tardigrades, global warming poses the greatest menace to the freshwater species. Rebecchi et al. (2009) recently demonstrated that the limnic species 20 M icro-­‐invertebrates c onservation Borealibius zetlandicus is intolerant to desiccation. In the case of this limitation being shared by other limnic species, they can become extinct in temperate areas such as Southern Europe, where future higher temperatures may turn permanent rivers, ponds and lagoons into temporary ones. The eventual verification that strictly freshwater species are desiccation intolerant should not come as a surprise since the ability to undergo anhydrobiosis is an adaptation of the terrestrial tardigrades and most marine tardigrades are known to be desiccation intolerant (Ramazzotti & Maucci 1983). That does not mean, however, that the terrestrial species cannot be endangered by the climatic changes, since their desiccation tolerances have been proved to differ from one climatic region to another (Horikawa and Higashi 2004), and local adaptation to current climatic patterns is a decisive factor in the current geographic distribution of tardigrades (Faurby et al. 2008; Pilato, 1979; Pilato & Binda 2001). In marine environments, tardigrades can be found anywhere, from deep sea floors to beaches, dwelling in the sediments. However being one of the main groups comprising meiofauna, their ecological importance is still poorly understood. On beaches, species distribution follows a tide influenced gradient (Kinchin 1992; Morgan and Lampard 1986). Considering the expected rising of the sea level as yet another consequence of global warming, the species distribution pattern can be totally disrupted along worldwide shores, wherever beaches become permanently flooded. This could mean the loss of immense habitat areas that are vital for the survival of this and other faunal groups. Adrianov (2004) estimates meiofauna to be composed of 20 to 30 million species, so it is not difficult to imagine how a swift change in the sea level would affect many animal species inhabiting the current tidal zone. Aquatic pollution from all types of sources may also have an impact on marine tardigrades, but no studies exist hitherto on this subject. Pollution has, however, been proved to negatively correlate with nematode population structure in an estuarine environment (Gyedu-­‐Ababio et al. 1999). Hence, the assumption of a negative effect from water pollution on marine tardigrades should not strike us as being too far-­‐fetched. Facing any of the previously referred cases of potential harm to the diversity of tardigrades, one could argue that given the great colonization capabilities these animals have, it would allow them to re-­‐populate any given habitat, once the threat disappears. True as it may be for some ubiquitous species, it will not be so for all others that are endemic. We should also keep in mind that the event of a re-­‐colonization does not exclude 21 Paper 1 the hypothesis of considerable genetic diversity loss. Malmström et al. (2009) found that five years after a fire the number of tardigrades had reached 52% of those found in the unburnt area. Nevertheless, this study did not include any species identification procedures, so it is impossible to infer on how effective re-­‐colonizations can be in restoring the original biodiversity levels. The destruction of a microhabitat to which an endemic species is uniquely linked produces a marked reduction of genetic diversity or even the extinction of that species. More studies on this matter are required, since our limited knowledge prevents us from reaching the understanding on whether or not preventive measures are required to protect micro-­‐fauna, as well as on which they should be. Lack of knowledge should not, however, be reason enough to prevent the taking up of protective measures, general as they may be. This is stated in the Convention on Biological Diversity: “(…) where there is a threat of significant reduction or loss of biological diversity, lack of full scientific certainty should not be used as a reason for postponing measures to avoid or minimize such a threat.” Increasing our understanding of biodiversity and the ecosystem’s services is today a critical need and also a scientific challenge in order to perfect future political response (Commission of the European Communities 2006). Considering the absolute inexistence of studies regarding tardigrade diversity from a conservational point of view, I believe that these animals, and others, could benefit from some preventive and compensatory measures, in order to counter-­‐act current threats. I hereby suggest a few, divided into general and specific ones. Generally all micro-­‐invertebrate populations would benefit from: a) A reduction in all forms of environmental pollution; b) An immediate cutback in greenhouse-­‐effect gas emissions, in order to prevent short-­‐
term climatic changes; c) A decrease in the current rate of habitat destruction resulting from human activities. An example of how habitat conversion for human usage could be compensated would be achieved by a more frequent adoption of what is known as “Green roofs”. This architectural practice is common, for instance, in some northern European regions and consists of creating gardens or other green areas in roof tops, thus ‘giving back’ a certain percentage of the soil surface that was ‘robbed’ by the construction; 22 M icro-­‐invertebrates c onservation On a specific level, this particular taxon could benefit from: d) Forestall clearance methodologies that took micro-­‐fauna into consideration. These would include the removal of only the strictly necessary amount of biomass from woods, roads, paths or forestall corridors. Additionally, the removed materials should not be burned or destroyed in any other way in order to preserve all the live-­‐
forms contained there. As an alternative, they could be translocated to a nearby area where the risk of fire would be inferior or virtually inexistent; e) Ex-­‐situ preservation projects. These could be conducted in public or private gardens or green houses and would act as genetic banks, in a similar way to the part played by zoos and aquariums today; f) Beaches partially or totally closed to humans. This would protect coastal/marine life from the great pressure imposed by people during summer months, and could be achieved by implementing coastal protected areas. g) An extension of taxonomic and biological studies. Particularly useful appears the recent genetic work: Tardigrade Barcoding Project (Schill 2009), TABAR (Guidetti et al. 2009b), TardiBASE (Blaxter 2008), Kumamushi Genome Project (Kunieda et al. 2008), MoDNA (Cesari et al. 2009; Guidetti et al. 2009a). This would not only inflate our level of knowledge but would potentially help create new lines of research where water-­‐bears have not yet been used. It would also help draw media attention to the taxon, important leverage for a successful conservation strategy. All of these suggestions are being made a priori and, even though some of them could prove to be somewhat correct, they would have to be refined in order to accurately provide protection for the Tardigrade biodiversity. Obviously, such perfectioning of any given conservational methodology can only arise from previous studying. These pioneer studies shall hopefully come true in a near future, for they are critically necessary not only to help us protect a vast animal taxon whose full ecological importance still eludes our understanding; but also, and more importantly, to help bring about a more generalized discussion on the conservation of all of those taxonomic groups thus far neglected. 23 Paper 1 Acknowledgements I wish to thank Professor Roberto Bertolani, University of Modena and Reggio Emilia, Italy, and Professor Artur Serrano, University of Lisbon, Portugal, for valuable comments and suggestions. I also wish to thank Dr. Timothy Bancroft-­‐Hinchey at the Oxford School of Languages, Lisbon, for reviewing the English manuscript. This work was supported by the Fundação para a Ciência e a Tecnologia, Portugal, and was partially presented at the 11th International Symposium on Tardigrada held in Tübingen, Germany, August 3-­‐6 2009. References Adlard RD, O’Donoghue PJ (1998) Perspectives on the biodiversity of parasitic protozoa in Australia. International Journal for Parasitology 28: 776-­‐786. Adrianov AV (2004) Current problems in marine biodiversity studies. Russian Journal of Marine Biology 30(1): 1-­‐16. Blaxter M (2008) TardiBASE – The home of the Edinburg Tardigrade Project. Available at: http://xyala.cap.ed.ac.uk/tardigrades/tardibase.html (Accessed 26 June 2009). Cesari M, Bertolani R, Rebecchi L, Guidetti R (2009) DNA barcoding in Tardigrada: the first case study on Macrobiotus macrocalix Bertolani & Rebecchi 1993 (Eutardigrada, Macrobiotidae). Molecular Ecology Resources 9: 699-­‐706. Commission of the European Communities (2006) Halting the loss of biodiversity by 2010 – and beyond; Sustaining ecosystem services for human well-­‐being. Brussels. Convention on Biological Diversity (2001) 2010 Biodiversity Target. Available at: http://www.biodiv.org/2010-­‐target/default.asp (Accessed 15 July 2009). Faurby S, Jönson KI, Rebecchi L, Funch P (2008) Variation in anhydrobiotic survival of two eutardigrade morphospecies: a story of cryptic species and their dispersal. Journal of Zoology 275: 139-­‐145. Guidetti R, Schill R, Bertolani R, Dandekar T, Wolf M (2009a) New molecular data for tardigrade phylogeny, with the erection of Paramacrobiotus gen. nov. Journal of Zoological Systematics and Evolutionary Research 47(4): 315-­‐321. Guidetti R, Rebecchi L, Bertolani R, Cesari M, Jorgensen A (2009b) Tardigrada Barcoding – TABAR. Available at: http://www.barcodinglife.org (Accessed 20 October 2009). 24 M icro-­‐invertebrates c onservation Guil N, Sánchez-­‐Moreno S, Machordom A (2009) Local biodiversity patterns in micrometazoans: Are tardigrades everywhere? Systematics and Biodiversity, 7(3): 259-­‐
268. Gyedu-­‐Ababio TK, Furstenberg JP, Baird D, Vanreusel A (1999) Nematodes as indicators of pollution: a case study from the Swartkops River system, South Africa. Hydrobiologia 397: 155-­‐169. Horikawa DD, Higashi S (2004) Desiccation tolerance of the tardigrade Milnesium tardigradum collected in Sapporo, Japan, and Bogor, Indonesia. Zoological Science 21: 813-­‐816. Jovan S (2008) Lichen bioindication of biodiversity, air quality, and climate: baseline results from monitoring in Washington, Oregon, and California. Gen. Tech. Rep. PNW-­‐
GTR-­‐737. Portland, OR: U.S. Department of Agriculture, Forest Service, Pacific Northwest Research Station. 115 pp. Kinchin IM (1992) An introduction to the invertebrate microfauna associated with mosses and lichens with observations from maritime lichens on the West coast of the British Isles. Microscopy 36: 721-­‐731. Kunieda T, Katayama T, Toyoda A, Horikawa D, Arakawa K, Kuwahara H, Yamaguchi A, Aizu T, Abe W (2008) Kumamushi Genome Project. Available at: http://kumamushi.org (Accessed 20 July 2009). Lévêque C, Balian EV, Martens K (2005) An assessment of animal species diversity in continental waters. Hydrobiologia 542: 39-­‐67. Malmström A, Person T, Ahlström K, Gongalsky KB, Bengtsson J (2009) Dynamics of soil meso-­‐ and macrofauna during a 5-­‐year period after clear-­‐cutburning in a boreal forest. Aplied Soil Ecology 43: 61-­‐74. Millennium Ecosystem Assessment (2005). Ecosystems and Human Well-­‐being: Biodiversity Synthesis. World Resources Institute, Washington, DC. 86 pp. Morgan CI, Lampard DJ (1986) Supralittoral lichens as a habitat for tardigrades. Glasgow Naturalist 21: 127-­‐138. Pilato G (1979) Correlations between cryptobiosis and other biological characteristics in some soil animals. Bollettino di Zoologia, 46: 319-­‐332. Pilato G, Binda MG (2001) Biogeography and limno-­‐terrestrial Tardigrades: are they truly incompatible binomials? Zoologischer Anzeiger 240: 511-­‐516. 25 Paper 1 Price PW (1987) The role of natural enemies in insect populations. In: Barbosa P, Schultz JC (eds) Insect outbreaks. Academic Press, Inc. London. 287-­‐312 pp. Quartau JA (2008) Preventative fire procedures in Mediterranean woods are destroying their insect Biodiversity: a plea to the EU Governments. Journal Insect Conservation 13: 267-­‐270. Ramazzotti G, Maucci W (1983) Il phylum Tardigrada. Memorie dell’Istituto Italiano di Idrobiologia, 41: 1-­‐1012. Rebecchi L, Boschini D, Cesari M, Lencioni V, Bertolani R, Guidetti R (2009) Stress response of a boreo-­‐alpine species of tardigrade, Borealibius zetlandicus (Eutardigrada, Hypsibiidae). Journal of Limnology 68(1): 64-­‐70. Schill R (2009) Tardigrade Barcoding Project. Available at: http://tardigradebarcoding.org (Accessed 20 July 2009). Stork NE, Grimbacher PS, Storey RI, Oberprieler RG, Reid CAM, Slipinski SA (2008) What determines whether a species of insect is described? Evidence from the study of tropical forest beetles. Insect Conservation and Diversity 1(2): 114-­‐119. United Nations (1993) Multilateral Convention on Biological Diversity (with annexes): concluded at Rio de Janeiro on 5 Juno 1992. Treaty series. 1760(30619): 142-­‐382. Vargha B, Ötvös E, Tuba Z (2002) Investigations on ecological effects of heavy metal pollution in Hungary by moss-­‐dwelling water bears (Tardigrada) as bioindicators. Ann agric Environ Med. 9: 141-­‐146. Vicente F, Michalczyk L, Kaczmarek L, Boavida MJ (2008) Observations on Pyxidium tardigradum (Ciliophora), a protozoan living on Eutardigrada: infestation, morphology and feeding behavior. Parasitology Research 103: 1323-­‐1331. Vié J-­‐C, Hilton-­‐Taylor C, Stuart SN (eds.) (2009) Wildlife in a Changing World – An Analysis of the 2008 IUCN Red List of Threatened Species. Gland, Switzerland: IUCN. 180 pp. 26 Paper 2 The impact of fire on terrestrial tardigrade biodiversity: a case-­‐study from Portugal Filipe Vicente, Michele Cesari, Artur Serrano & Roberto Bertolani Journal of Limnology (submitted) The impact of fire on terrestrial tardigrade biodiversity Abstract Currently, loss of habitat is the greatest threat to biodiversity, yet little is known about its effect on microscopic animal taxa, such as Tardigrada. One of the causes behind habitat destruction is forestall fire, both naturally occurring and caused by man. The latter type is a very common method used in agriculture, as a way for killing insect plagues or for soil preparation, as well as in conservation, being used for creating habitat mosaics. In Portugal, 42% of fire frequency is due to human activities. The impact of fires in biodiversity is not consensual, with studies pointing towards different conclusions. Different methodologies and target taxonomic study groups may partly explain this paradigm. This study is a first approach to possible effects caused by habitat destruction on tardigrade populations. For this we have analyzed the taxonomic and genetic variations of tardigrades from a fire affected location in a Portuguese natural park. Sampling was performed during a 10 year period, from 2000 to 2010. The location was affected by a small fire in 1998 and a big fire in 2003. A total of 11 species from nine separate genera were recorded, and 19 cox1 haplotypes were found. Our data show a pattern which suggests a negative effect of a forestall fire on tardigrade populations. Taxonomic and genetic richness, as well as animal abundance show lower levels in the years after a fire, when compared with the years that preceded it. Additionally, the population recovered visibly faster after the small fire than after the bigger one. This is consistent with the fact that larger fires destroy larger forestall areas, leaving fewer animals at a farther distance available for re-­‐colonization. Most species found before the main fire are also found after it, indicating a high capability to re-­‐colonize by these tardigrades. However, only three of all recorded haplotypes were found both pre and post the main fire, which indicates genetic diversity loss by direct consequence of fire. Therefore, we conclude that habitat destruction by means of forestall fire has a detrimental effect over tardigrade biodiversity, and may have similar effects on other small animals. Keywords Fire impact, Tardigrada, cox1, Portugal, biodiversity, re-­‐colonization. 31 Paper 2 Introduction Loss of habitat is currently the single greatest threat to biodiversity (Millennium Ecosystem Assessment, 2005). However, for many taxonomic groups there is still no clue or evidence of how habitat loss affects their intrinsic biodiversity and/or population structure (Andersen and Müller 2000, Vicente 2010). One of such groups is the phylum Tardigrada. These are microscopic metazoans that inhabit most of the planets environments: terrestrial, freshwater and marine. On terrestrial environment, they are commonly found on aquatic microcosms where water films or microwater bodies are unpredictably added and temporarily retained, such as mosses and lichens. On temperate climates, these micro-­‐habitats are usually inactive during the warmer months due to desiccation, and in winter when frozen. Mosses and lichens recover their activity with the return of moisture, together with the fauna that usually inhabits them: tardigrades, rotifers and nematodes, all of which are capable of undergoing anhydrobiosis. During these periods of inactivity, these terrestrial micro habitats are particularly exposed to environmental threats such as forestall fires. Apart from the obvious directly destructive effect that fires have on biodiversity, their impact on ecosystems is very important, e.g., by the destruction of riparian flora supporting freshwater systems (New et al. 2010), by destroying soil grass and thus accelerating soil erosion (Naveh 1998), or altering mammal’s foraging behavior (Wallace and Crosthwaite 2005). Fires have long been known to reduce populations of small fauna, as they have historically been used to attack populations of agricultural damaging insects, either by direct kill or by habitat destruction (McCullough et al., 1998). Other common reasons behind the use of deliberate fires are the preparation of land for agriculture (Sim-­‐Sim et al., 2004) or the reduction of organic fuel levels, in order to minimize the impact of high magnitude fires (Andrew et al., 2000; York, 2000). According to Simorangkir (2007) the biggest reason behind the widespread use of fire for land clearing is its low economic cost, mainly in large areas. For small forest areas, zero-­‐burning alternatives can be as low cost-­‐effective as burning. The extent of fire induced damages is dependent on several factors, such as its type and intensity, environmental variables or organism adaptation to water loss (Araújo and Ribeiro, 2005). Additionally, global warming is another source of higher fire frequency. 32 The impact of fire on terrestrial tardigrade biodiversity In the Portuguese case, human activities explain 42% of changes in fire frequency and temperature anomalies explain 43% of the area burnt (Costa et al. 2011). Nevertheless, the effect of fire on biodiversity is not consensual. Some studies suggest positive fire consequences, such as the maintenance of habitat mosaics of different succession stages (Ghandi et al., 2001). That is, according to Parr and Andersen (2006), an increasingly popular theory amongst conservation management agencies worldwide, which has resulted in patch mosaic burning being a commonly used biodiversity conservation strategy these days. However, other studies suggest a negative outcome, by destroying endemic and low dispersing species (Yanovsky and Kiselev 1996; Quartau 2009). This apparent incongruence of conclusions could result from the different conditions in which individual studies were conducted, such as the fire regimes, the pre and post-­‐fire ecology of the region, or the taxa in focus (Moretti et al., 2004; Parr and Andersen, 2006). According to New et al. (2010), pre and post fire data follow up from burning sites are a common gap in such studies. To date no such studies exist with a specific focus on tardigrades or other micro-­‐
invertebrate groups. In a broad spectrum study, Malmström et al. (2009) found that, five years after a fire, tardigrade abundance had reached 52% in comparison with the unburnt area. However, at this time no data exist regarding effects on tardigrade species, population or genetic pool diversity. In this study we are trying to understand, for the first time, how limno-­‐terrestrial tardigrade populations respond to a situation of habitat loss caused by forestall fire. To do so, we analyzed consecutive samples from one small geographic mountain area in Portugal, and focused both on changes in population dynamics as well as on their respective levels of genetic diversity. 33 Paper 2 Material and Metods Samples of the moss species Orthotrichum striatum Hedwig, 1801 were collected at Carvalhal da Moita do Conqueiro, Serra da Estrela’s Natural Park, Portugal, 1529 m above sea level, from an area of roughly 10 m2 (Fig. 1). One sample per year was collected in 2000, 2003, 2005, 2006, 2007 and 2010. A big fire occurred in 2003, about one month after sampling; a smaller fire had taken place previously, in 1998. Even though the sampling site was visited in 2004, no sample was collected then since new mosses were still starting to get established. Figure 1 – Sampling site (Carvalhal da Moita do Conqueiro, Serra da Estrela, Portugal). General (left) and detailed (right) maps of Serra da Estrela’s Natural Park. Arrow points to sampling site. Source: www.icnf.pt. Samples were left to air dry at room temperature, weighed and tested for animals by soaking for at least 30 min and washed through consecutive 500 µm and 38 µm sieves. All animals and eggs present in sieved sample were manually selected under a stereomicroscope. 34 The impact of fire on terrestrial tardigrade biodiversity Voucher specimens were photographed in vivo and then used for DNA analysis, following the protocol described in Cesari et al. (2011). The other animals and eggs were mounted on slides using Faure-­‐Berlese fluid. In vivo and mounted specimens were observed using a light microscope Leitz DM RB, always at 100x oil objective for the mounted specimens and eggs, at 40x or 100x oil for the in vivo specimens. Species richness (d), diversity Shannon-­‐
Wiener index (H’) and dominance Simpson index (λ’) have been calculated using Primer 5.2.9 (PRIMER-­‐E Ltd.), considering all animals found in the six years. DNA extraction from single specimens and PCR amplification of a fragment of the cox1 gene was carried out following Cesari et al. (2009) protocol, using LCO-­‐1490 (5’-­‐GGT CAA CAA ATC ATA AAG ATA TTG G-­‐3′; Folmer et al. 1994) and HCO-­‐2198 (5’-­‐TAA ACT TCA GGG TGA CCA AAA AAT CA-­‐3’; Folmer et al. 1994) as primers. The amplified products were gel purified using the Wizard Gel and PCR cleaning (Promega) kit. For assurance, both strands were sequenced using an ABI Prism 3100 sequencer (Applera). Sequences were translated to amino acids by using the invertebrate mitochondrial code implemented in MEGA version 5 (Tamura et al., 2011) in order to check for the presence of stop codons and therefore of pseudogenes. Nucleotide sequences were aligned with the Clustal algorithm implemented in MEGA5 (pairwise and multiple alignment parameters: Gap opening penalty: 15, Gap extension penalty: 6.66) and checked by visual inspection. Nucleotide sequences of the newly analysed specimens were submitted to GenBank (Accession Numbers JX683810-­‐ JX683833). Specimens pertaining to Diploechiniscus oihonnae (GenBank A.N. JX676191-­‐4) were already analyzed in Vicente et al. (in preparation) and they were also included in the present analysis. Minimum spanning network analysis between haplotypes was performed by using Arlequin 3.1 (Excoffier et al., 2005) and visualized by using HapStar (Teacher and Griffiths 2011). 35 Paper 2 Results A total of 276 animals (eggs were not considered) representing 11 species and nine genera were extracted from six samples, covering a 10 year time span. Sampling was not conducted in the unrepresented years. Abundances are depicted in Table 1, together with species richness (d), Shannon index (H’) for diversity and Simpson index (λ’) for dominance in each sample (year). Table 1 – Specimens and species abundances over the sampling years. d: species richness; H’(loge): Shannon index for diversity; λ’: Simpson index for dominance.
Species Number of found specimens 2000 2003 2005 2006 2007 2010 Overall 1 15 8 46 2 8 80 Pseudechiniscus facettalis 19 0 0 3 51 2 75 Echiniscus blumi 0 0 0 0 0 61 61 Diploechiniscus oihonnae 0 2 2 2 10 0 16 Milnesium cf. tardigradum 1 5 5 2 0 2 15 Macrobiotus vladimiri 3 1 1 4 0 1 10 Minibiotus furcatus 0 6 0 0 3 0 9 Bryodelphax parvulus 0 4 1 0 0 0 5 Hypsibius pallidus 0 2 0 0 0 0 2 Ramazzottius cf. oberhaeuseri 0 1 0 0 1 0 2 Echiniscus quadrispinosus 1 0 0 0 0 0 1 Total specimens 25 36 17 57 67 74 276 d 1.243 1.953 1.412 0.989 0.951 0.929 H’ (loge) 0.849 1.702 1.300 0.750 0.798 0.653 λ’ 0.580 0.219 0.287 0.655 0.599 0.689 Macrobiotus cf. macrocalix Macrobiotus cf. macrocalix is the most commonly found species overall, the richest in specimen numbers and the only one present in all sampling years. Those specimens differ from the type material of Macrobiotus macrocalix Bertolani and Rebecchi, 1993 by having a higher pit number on the egg shell and sometimes a crenulated distal disc of the egg processes, as well as for their different haplotype. In terms of abundances, it is followed by Pseudechiniscus facettalis Petersen, 1951 and then by Echiniscus blumi Richters, 1903. In terms of continuity, M. cf. macrocalix is followed by Milnesium cf. tardigradum, Macrobiotus vladimiri Bertolani, Biserov, Rebecchi and Cesari, 2011 (both not found only in 2007) and then by P. facettalis (not found in 2003 and 2005) and Diploechiniscus oihonnae (Richters, 36 The impact of fire on terrestrial tardigrade biodiversity 1903) (not found in 2000 and 2010). The remaining species show much lower abundances and are present only in one (Hypsibius pallidus Thulin, 1911, Echiniscus quadrispinosus Richters, 1902, E. blumi) or two (Minibiotus furcatus Ehrenberg, 1859), Bryodelphax parvulus Thulin 1928, Ramazzottius cf. oberhaeuseri) of the six sampling years. Species richness peaks in the year 2003, also with the highest diversity index (Shannon index H’) present in the same year, in correspondence of the lowest dominance index (Simpson index λ’) just a few months before the biggest fire. In regard to the molecular analysis, 19 cox1 haplotypes were identified (Table 2) in all considered years. DNA has been extracted from specimens of every identified species but in some cases it was not possible to determine the haplotype. Figure 2 depicts a minimum spanning network for all scored haplotypes, their presence in each sample (year) and the intraspecific distances, in terms of substitutions. Table 2 – Distinct haplotypes registered in each year, for the different species. Each species is assigned a unique haplotype letter tag. (-­‐) – Species not registered in a given year, or DNA extracted but haplotypes not determined. Species 2000 2003 2005 2006 2007 2010 Macrobiotus cf. macrocalix -­‐ A1, A2, A3 A2 A2, A4 A2 A2 Pseudechiniscus facettalis -­‐ -­‐ -­‐ B1 B2 -­‐ Echiniscus blumi -­‐ -­‐ -­‐ -­‐ -­‐ C1, C2 Diploechiniscus oihonnae -­‐ D1 D2 D3 D2 -­‐ Milnesium cf. tardigradum -­‐ E1 E2 E2, E3 -­‐ -­‐ F1 -­‐ -­‐ -­‐ -­‐ F1 Minibiotus furcatus -­‐ G1 -­‐ -­‐ G2 -­‐ Bryodelphax parvulus -­‐ H1 H1 -­‐ -­‐ -­‐ Hypsibius pallidus -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ Ramazzottius cf. oberhaeuseri -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ Echiniscus quadrispinosus I1 -­‐ -­‐ -­‐ -­‐ -­‐ Macrobiotus vladimiri The spanning network (Fig. 2) shows a reduced number of substitutions amongst most haplotypes, with the exception of D. oihonnae, where two haplotypes are separated by 22 substitutions. With four haplotypes each, Macrobiotus cf. macrocalix and D. oihonnae are the species with the highest genetic diversity. Milnesium cf. tardigradum follows with three haplotypes. Remaining species exhibit only one or two haplotypes. Macrobiotus cf. macrocalix also exhibits the only haplotype present in 100% of the species’ samplings (haplotype A2). 37 Paper 2 Figure 2 -­‐ Minimum spanning network. Haplotypes are represented by circles with the area being proportional to their frequency of occurrence. Lines represent single mutational events, while small half filled squares denote missing/ideal haplotypes. Different shades/patterns denote different sampling years. Letters and numbers indicate haplotypes as in table 2. 38 The impact of fire on terrestrial tardigrade biodiversity Discussion Macrobiotus cf. macrocalix is the only species present in all sampling years, other than those most abundant. Morphological and genetic differences of these specimens with the type material and other population of M. macrocalix are not a subject of this work and will be discussed in a future paper (Bertolani et al., in preparation). Haplotype A2 of this species is present in every sample and is largely dominant with respect to the other three haplotypes found for the same species. The highest species richness and diversity index are present in 2003, just a few months before the biggest fire. Most species found in the sampling area have been found in that sample. In 2005 the H’ is relatively high but lower than in the previous sampling year, while in the following three samplings, when the highest numbers of specimens have been found, the H’ values are the lowest. In the last three samplings, it is evident that the high number of specimens is due to the presence of a dominant species, different in each year. The pre-­‐fire sample from 2003 shows the highest number not only of species, but also of haplotypes. This is in accordance with a similar moss and lichen successional study conducted in the same area, where post-­‐fire biodiversity levels significantly decreased in burnt areas in opposition to unburnt ones, with the differences fading as the years passed by (Sim-­‐Sim et al., 2004). However, it should be noted that it was not a pristine population, regardless of being the ‘richest’ one of all. Occurrence of a previous smaller fire in 1998 (see introduction), was the reason why the 2000 sample presented considerably lower scores throughout the analysis, in comparison with those from the year 2003. The fact that the main fire, in 2003, was one of greatest proportions is quite evident when we consider not only the unmatched levels of 2003’s population richness, but also the fact that recovery was clearly delayed by that fire. This should be so, because a fire of considerable proportions destroys a larger forestall area than a smaller fire, thus killing more animals as well as their live substrates. Therefore, fewer animals are left alive and at a longer distance, making the process of re-­‐colonization less likely to occur. An example of how the destruction of living substrates can affect invertebrate biodiversity comes from Diniz et al. (2011) who, while studying caterpillars, found that the occurrence of these animals in their plant hosts was between 2.4 and 5.2 times higher in unburnt areas than in burnt ones. 39 Paper 2 The lower number of species found after the fires matches the results of York (1999), who showed that areas subjected to frequent low-­‐intensity fires had significantly lower levels of several macro invertebrate taxa. In York (1998) the loss of morphospecies as a consequence of frequent fires reached 46%, in comparison with control unburnt areas. Most species found before the main fire are also found after it, indicating a high capability to re-­‐colonize by tardigrades. The only exceptions are Hypsibius pallidus and Echiniscus quadrispinosus, but they only represent 1.09% of the overall population. Looking at the genetic data, the spanning network shows a reduced number of substitutions among most haplotypes, with the exception of D. oihonnae, where two haplotypes are separated by 22 substitutions. This accounts for a Kimura 2-­‐parameters distance of 4% (Vicente et al., in preparation), still within the single-­‐species limits. Only haplotypes A2, F1 and H1 are represented both pre and post main fire. That means that even though most species were able to return to the destroyed location, almost 90% of the haplotypes are unique to either one of the situations: pre or post-­‐fire destruction. However, this does not imply a massive genetic diversity loss as a sole consequence of fire, since the verified haplotypes continue to shift within the same species after the fire. A good example is Diploechiniscus with a different haplotype in every sampling year, indicating a very dynamic genetic change in the genetic structure of the population. Being the number of data limited, we cannot exclude that some haplotypes not found in 2000-­‐2003 were already present in those years. Nonetheless, the high numbers of newly scored haplotypes let us to hypothesize that at least some of them come from areas where the passive transport is a possibility. It is evident that the more common haplotypes were able to survive in the same or in nearby non-­‐burned habitats and then re-­‐colonize the original spot. Haplotype A2 clearly states it. The fact that all other post main fire haplotypes besides A2, F1 and H1 are not only new but also inconsistent in presence, suggests that the original genetic pool has generically been replaced and may have been lost. This suggests that the community will need many years to reach a new balance. Original biodiversity indexes were far from being reached seven years after the main fire and it is not possible to predict when and if they will ever be restored. However, in the hypothesis of a longer study, we should consider that a longer term study could constitute a very difficult goal to set: in the case of Australian lowland savannah only 3% of the landscapes remain unburnt for more than 5 years (Andersen et al., 2005). 40 The impact of fire on terrestrial tardigrade biodiversity In our opinion, an implementation of the molecular approach, other than morphological, should be considered into the elaboration of future forestall and other natural areas’ management strategies as well as biodiversity conservation policy making, particularly in what concerns induced fire management. We conclude that even though terrestrial tardigrades are a taxonomic group that can re-­‐
colonize a given destroyed habitat with considerable ease, significant biodiversity richness is lost in a destructive event such as forestall fire. The amount of biodiversity loss in such an event is, at least in part, determined by the magnitude of the fire. Even though our data cannot be extrapolated to other taxonomic groups, they could serve as reference for co-­‐
existing taxa such as nematodes or rotifers. Acknowlodegments The authors wish to thank Dr. César Garcia (Botanical Garden, Lisbon) for providing the moss samples used in this study. We are also very grateful to Dr. Juliana Hinton (McNeese State University, USA) for her kindness in revising the English. This work was partially funded by the Portuguese Fundação para a Ciência e a Tecnologia with a grant (SFRH/BD/39234/2007) to the first author. The research is also part of the project MoDNA supported by Fondazione Cassa di Risparmio di Modena (Italy) and the University of Modena and Reggio Emilia (Modena, Italy). References Andersen AL, Cook GD, Corbett LK, Douglas MM, Eager RW, Russell-­‐Smith J, Setterfield SA, Williams RJ, Woinarski JCZ, 2005. Fire frequency and biodiversity conservation in Australian tropical savannas: implications from the Kapalga fire experiment. Austral Ecol. 30:155-­‐167. Andersen AL, Müller WJ, 2000. Arthropod responses to experimental fire regimes in an Australian tropical savannah: ordinal-­‐level analysis. Austral Ecol. 25:199-­‐209. Andrew N, Rodgerson L, York A, 2000.Frequent fuel-­‐reduction burning: the role of logs and associated leaf litter in the conservation of ant biodiversity. Austral Ecol. 25:99-­‐107. Araújo EA, Ribeiro GA, 2005. Fire Impacts on the Entomofauna of the Soil in Forest 41 Paper 2 Ecosystems. Nat. Desenvolvimento 1:75-­‐85. Cesari M, Bertolani R, Rebecchi L, Guidetti R, 2009. DNA barcoding in Tardigrada: the first case study on Macrobiotus macrocalix Bertolani and Rebecchi 1993 (Eutardigrada, Macrobiotidae). Mol. Ecol. Res. 9:699-­‐706. Cesari M, Giovannini I, Bertolani R, Rebecchi L, 2011. An example of problems associated with DNA barcoding in tardigrades: a novel method for obtaining voucher specimens. Zootaxa 3104:42-­‐51. Costa L, Thonicke K, Poulder B, Badeck FW, 2011. Sensitivity of Portuguese forest fires to climatic, human, and landscape variables: subnational differences between fire drivers in extreme fire years and decadal averages. Reg. Environ. Change 11:531-­‐551. Diniz IR, Higgins B, Morais HC, 2011. How do frequent fires in the Cerrado alter the lepidopteran community? Biodivers. Conserv.20:1415-­‐1426. Excoffier, L, Laval G, Schneider S, 2005. Arlequin ver. 3.0: An integrated software package for population genetics data analysis. Evol. Bioinform. Online 1:47-­‐50. Folmer O, Black M, Hoeh W, Lutz R. Vrijenhoek R,1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Bio. Biotechnol. 3:294-­‐299. Gandhi KJK, Spence JR, Langor, DW, Morgantini LE, 2001. Fire residuals as habitat reserves for epigaeic beetles (Coleoptera: Carabidae and Staphylinidae). Biol. Conserv. 102:131-­‐141. Malmström A, Person T, Ahlström K, Gongalsky KB, Bengtsson J, 2009. Dynamics of soil meso-­‐ and macrofauna during a 5-­‐year period after clear-­‐cutburning in a boreal forest. App. Soil Ecol. 43:61-­‐74. McCullough DG, Werner RA, Neumann D, 1998. Fire and insects in northern and boreal forest ecosystems of North America. Annu. Rev. Entomol. 43:107-­‐127. Millennium Ecosystem Assessment (2005) Ecosystems and Human Well-­‐being: Biodiversity Synthesis. World Resources Institute, Washington, DC: 86 pp. Moretti M, Obrist MK, Duelli P, 2004. Arthropod biodiversity after forest fires: winners and losers in the winter fire regime of the southern Alps. Ecography. 27:173-­‐186. Naveh Z, 1998. From Biodiversity to Holistic conservation of the biological and cultural diversity of Mediterranean landscapes. In: Rundel P, Montenegro G and Jaksic FM (eds.), Landscape disturbance and biodiversity in Mediterranean-­‐type ecosystems. Ecological studies. Vol. 136 Springer, Berlin, pp. 23-­‐50. 42 The impact of fire on terrestrial tardigrade biodiversity New TR, Yen AL, Sands DPA, Greenslade P, Neville PJ, York A, Collet NG, 2010. Planned fires and invertebrate conservation in south east Australia. J. Insect Conserv. 14:567-­‐574. Parr CL, Andersen AN, 2006. Patch Mosaic Burning for Biodiversity Conservation: a Critique of the Pyrodiversity Paradigm. Conserv. Biol. 20:1610-­‐1619. Quartau JA, 2009. Preventative fire procedures in Mediterranean woods are destroying their insect Biodiversity: a plea to the EU Governments. J. Insect Conserv. 13:267-­‐270. Sim-­‐Sim M, Carvalho P, Sérgio C, Garcia C, Rego F, 2004. Recolonisation and changes in bryophyte and lichen biodiversity in burned areas from the Serra da Estrela (Portugal). Cryptogam. Bryol. 25:279-­‐296. Simorangkir D, 2007. Fire use: Is it really the cheaper land preparation method for large-­‐
scale plantations? Mitig. Adapt. Strat. Glob. Change 12:147-­‐164. Tamura K, Peterson D, Peterson N, Stechler G, Nei, M, Kumar S, 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28:2731-­‐2739. Teacher AGF, Griffiths DJ, 2011. HapStar: automated haplotypes network layout and visualization. Mol. Ecol. Res. 11:151–153. Vicente F,2010. Micro-­‐invertebrates conservation: forgotten biodiversity. Biodivers. Conserv. 19:3629-­‐3634. Wallace LL, Crosthwaite KA, 2005.The effect of fire spatial scale on Bison grazing intensity. Landscape Ecol. 20:337-­‐349. Yanovski VM, Kiselev VV, 1996. Response of the endemic insect fauna to fire damage in forest ecosystems. pp 409-­‐413. In: Goldammer JG and Furyaev VV. (eds.), Fire in ecosystems of boreal Eurasia. Kluwer Academic Publishers. York A, 1998. Managing for biodiversity: What are the long-­‐term implications of frequent fuel-­‐reduction burning for the conservation of forest invertebrates? Third International Conference on Forest Fire Research. Proceedings Vol. II, pp. 1435-­‐1445. 16-­‐20 November 1998. Luso, Portugal. York A, 1999. Long-­‐Term Effects of Frequent Low-­‐Intensity Burning on the Abundance of Litter-­‐Dwelling Invertebrates in Coastal Blackbutt Forests of Southeastern Australia. J. Insect Conserv. 3:191-­‐199. York A, 2000. Long-­‐term effects of frequent low-­‐intensity burning on ant communities in costal blackbutt forests of southeastern Australia. Austral Ecol. 25:83-­‐98. 43 Paper 3 Considerations on the taxonomy of the Phylum Tardigrada Filipe Vicente & Roberto Bertolani Zootaxa (submitted) Considerations on the taxonomy of the Phylum Tardigrada Macrobiotus hufelandi Schultze1834 is the founding species of the Phylum Tardigrada and the group’s taxonomic list is constantly receiving new members, with several new species being added every year. In order to provide a single and complete database for all known species of tardigrades, as well as standardizing the description criteria, a checklist was created by Guidetti & Bertolani (2005). However, this effort calls for constant attention to keep an updated list that registers all the new species descriptions, which is evidenced by the fact that the currently available checklist is already in its 21st version (Degma et al., 2009-­‐2012). Here we can find a total of 1167 species representing 114 genera (Table I), 12 subfamilies, 24 families and four super families, four orders and three classes; one of which (Mesotardigrada) is represented by a single species (Thermozodium esakii) and is quite controversial (Nelson, 2002). Other uncertainties are noted in the positioning of the families of Beornidae and Necopinatidae, each containing only one species, the genus Apodibius, or the veracity of Oreella vilucensis (nomen dubium). The flood of new descriptions, recently published or in press, for new species (e.g. Kaczmarek et al. a,b, in press ; Miller et al., 2012a; Pilato et al., 2012; Zawierucha et al., in press) and new genera (Miller et al., 2012b; Vicente et al., submitted), continues unabated. A sign that tardigrade biodiversity still has a great deal of richness to reveal and that we might have only seen the tip of the iceberg. Table I lists tardigrade genera and we can see that some are substantially richer than others. The genus Echiniscus is the most speciose, with 163 species (the average is 10.33 species per genus), closely followed by Macrobiotus, with 153 species. These two genera alone contain 27.08% of all known tardigrade species and combined with Isohypsibius, Diphascon and Minibiotus, nearly half (49.36%) of the known tardigrade taxa. However, this list is more than just a portrait of the actual Tardigrada biodiversity; it also reflects a curious hidden bias. Sampling is far more abundantly performed on terrestrial environments than marine. The relatively few numbers of described marine species, therefore, could be related to this fact. Only at ninth place do we find a marine genus, Batillipes, with 27 species. At the generic level, marine tardigrades are, nevertheless, richer in terms of diversity (Appeltans et al., 2012). It is incredibly simple to sample terrestrial habitats for tardigrades, since we find them on virtually any piece of moss or lichen, anywhere. Thus, terrestrial tardigrades have been a preferential target by all of those willing to study these animals. In time, this has led to the strong terrestrial bias and the 47 Paper 3 limited number of marine species that are registered today. We therefore call for more effort to be put into the study of marine tardigrades, in order to provide a clearer picture of the Phylum’s biodiversity. Table I -­‐ Number of species per genus, following version 21 of the “Actual checklist of Tardigrada species” (Degma, Bertolani & Guidetti, 2009-­‐2012). Genus # of species Echiniscus 163 Macrobiotus 153 Isohypsibius 131 Diphascon 82 Minibiotus 47 Pseudechiniscus 43 Hypsibius 42 Doryphoribius 34 Batillipes, Paramacrobiotus 27 Ramazzottius 26 Milnesium 19 Bryodelphax 18 Dactylobiotus 16 Echiniscoides 15 Florarctus, Styraconyx, Tenuibiotus 13 Tanarctus, Itaquascon 11 Halechiniscus, Calcarobiotus 10 Cornechiniscus 9 Mixibius 8 Coronarctus, Angursa, Bertolanius, Pseudobiotus, Murrayon 7 Stygarctus, Antechiniscus, Hypechiniscus, Hexapodibius, Platicrista 6 Actinarctus, Megastygarctides, Parastygarctus, Pseudostygarctus, Mopsechiniscus, 5 Calohypsibius, Parhexapodibius, Thulinius Testechiniscus, Astatumen, Microhypsibius, Insuetifurca 4 Archechiniscus, Dipodarctus, Wingstrandarctus, Raiarctus, Rhomboarctus, Anisonyches, Oreella, 3 Bryochoerus, Auteruseus, Hebesuncus, Halobiotus, Ramajendas, Xerobiotus, Apodibius Euclavarctus, Parmursa, Chrysoarctus, Orzeliscus, Parechiniscus, Eohypsibius, 2 Haplomacrobiotus, Mesocrista, Parascon, Eremobiotus Trogloarctus, Clavarctus, Exoclavarctus, Moebjergarctus, Proclavarctus, Ligiarctus, 1 Paradoxipus, Opydorscus, Bathyechiniscus, Lepoarctus, Paratanarctus, Pleocola, Tetrakentron, Tholoarctus, Zioella, Neoarctus, Neostygarctus, Renaudarctus, Carphania, Novechiniscus, Proechiniscus, Thermozodium, Bergtrollus, Limmenius, Milnesioides, Haplohexapodibius, Bindius, Paradiphascon, Acutuncus, Borealibius, Fractonotus, Thalerius, Adorybiotus, Biserovus, Famelobiotus, Minilentus, Pseudodiphascon, Pseudohexapodibius, Richtersius, Schusterius, Macroversum, Beorn, Necopinatum Until now, species are usually only described on morphological and morphometric data based on a limited number of characters. To date, only one species, Macrobiotus vladimiri Bertolani, Biserov, Rebecchi and Cesari, 2011, has been described on combined morphological and molecular information, i.e. integrative taxonomy (Pardial et al., 2010). A few other species have also been considered using integrative taxonomy and barcoded with mtDNA cox1 gene (Cesari et al., 2009, 2011; Bertolani et al., 2010, 2011a,b; Vicente et 48 Considerations on the taxonomy of the Phylum Tardigrada al., submitted). Combine this limited use of integrated taxonomy with a small number of specialized taxonomists and we have the potential for a lot of incorrect descriptions. At times, species descriptions have been supported by minor differences that new, independent data have shown to be no more than intraspecific morphological variability (see Vicente et al., submitted). Based on experience, it is our strong belief that the tardigrade taxonomic list contains many examples of incorrect or limited taxonomic descriptions, which are in need of revision (such as the 68 subspecies on the checklist). We therefore urge taxonomists to make an effort towards using an integrative taxonomic approach in their future work. This can be achieved by incorporating the study of genetics, ecology, feeding behaviour, reproductive strategies and other available sources of independent data, and integrating these results with traditional morphological analysis. This should to make sure new species are described with stronger foundations and prevent the creation of new synonyms. It would also help speed the revision of older, past errors, thus ensuring the official tardigrade taxonomic species list reflects, as closely as possible, the true biodiversity richness of this animal group. Acknowledgements We thank Professor Artur Serrano (Faculty of Sciences, University of Lisbon, Portugal) for valuable comments on the manuscript. We also wish to thank Sandra McInnes, of the British Antarctic Survey, for her critical support and the English revision. This work was funded by the Portuguese Fundação para a Ciência e a Tecnologia with a grant (SFRH/BD/39234/2007) to the first author. References Appeltans, W., Ahyong, S.T., Anderson, G., Angel, M.V., Artois, T., Bailly, N., Bamber, R., Barber, A., Bartsch, I., Berta, A., Błazėwicz-­‐Paszkowycz, M., Bock, P., Boxshall, G., Boyko, C.B., Brandão, S.N., Bray, R.A., Bruce, L.N., Cairns, S.D., Chan, T.Y., Cheng, L., Collins, A.G., Cribb, T., Curini-­‐Galletti, M., Dahdouh-­‐Guebas, F., Davie, P.J.F., Dawson, M.N., De Clerck, O., Decock, W., De Grave, S., de Voogd, N.J., Domning, D.P., Emig, C.C., Erséus, C., Eschmeyer, W., Fauchald, K., Fautin, D.G., Feist, S.W., Fransen, C.H.J.M., Furuya, H., Garcia-­‐
Alvarez, O., Gerken, S., Gibson, D., Gittenberger, A., Gofas, S., Gómez-­‐Daglio, L., Gordon, 49 Paper 3 D.P., Guiry, M.D., Hernandez, F., Hoeksema, B.W., Hopcroft, R.R., Jaume, D., Kirk, P., Koedam, N., Koenemann, S., Kolb, J.B., Kristensen, R.M., Kroh, A., Lambert, G., Lazarus, D.B., Lemaitre, R., Longshaw, M., Lowry, J., Macpherson, E., Madin, L.P., Mah, C., Mapstone, G., McLaughlin, P.A., Mees, J., Meland, K., Messing, CG., Mills, C.E., Molodtsova, T.N., Mooi, R., Neuhaus, B., Ng, P.K.L., Nielsen, C., Norenburg, J., Opresko, D.M., Osawa, M., Paulay, G., Perrin, W., Pilger, J.F., Poore, G.C.B., Pugh, P., Read, G.B., Reimer, J.D., Rius, M., Rocha, R.M., Saiz-­‐Salinas, J.I., Scarabino, V., Schierwater, B., Schmidt-­‐Rhaesa, A., Schnabel, K.E., Schotte, M., Schuchert, P., Schwabe, E., Segers, H., Self-­‐Sullivan, C., Shenkar, N., Siegel, V., Sterrer, W., Stöhr, S., Swalla, B., Tasker, M.L., Thuesen, E.V., Timm, T., Todaro, M.A., Turon, X., Tyler, S., Uetz, P., van der Land, J., Vanhoorne, B., van Ofwegen, L.P., van Soest, R.W.M., Vanaverbeke, J., Walker-­‐Smith, G., Walter, T.C., Warren, A., Williams, G.C., Wilson, S.P. & Costello M. (2012) The Magnitude of Global Marine Species Diversity. Current Biology, 22(23), 123–456. Bertolani, R., Rebecchi, L. & Cesari, M. (2010) A model study for tardigrade identification. In: P.L. Nimis & R. Vignes Lebbe (Eds.), Tools for Identifying Biodiversity: Progress and Problems, EUT, Trieste, Italy, pp. 333–339. Bertolani, R., Biserov, V., Rebecchi, L. & Cesari, M. (2011a) Taxonomy and biogeography of tardigrades using an integrated approach: new results on species of the Macrobiotus hufelandi group. Invertebrate Zoology, 8, 23–36. Bertolani, R., Rebecchi, L., Giovannini, I. & Cesari, M. (2011b) DNA barcoding and integrative taxonomy of Macrobiotus hufelandi C.A.S. Schultze 1834, the first tardigrade species to be described, and some related species. Zootaxa, 2997, 19–36. Cesari, M., Bertolani, R., Rebecchi, L. & Guidetti, R. (2009) DNA barcoding in Tardigrada: the first case study on Macrobiotus macrocalix Bertolani & Rebecchi 1993 (Eutardigrada, Macrobiotidae). Molecular Ecology Resources, 9, 699–706. Cesari, M., Giovannini, I., Bertolani, R. & Rebecchi, L. (2011) An example of problems associated with DNA barcoding in tardigrades: a novel method for obtaining voucher specimens. Zootaxa 3104: 42–51. Degma, P., Bertolani, R. & Guidetti, R. (2009-­‐2012) Actual checklist of Tardigrada species. Ver. 21: 30-­‐06-­‐2012. http://www.tardigrada.modena.unimo.it/miscellanea/Actual%20 checklist%20of%20Tardigrada.pdf, pp. 36. Guidetti, R. & Bertolani, R. (2005) Tardigrade taxonomy: an updated check list of the taxa and a list of characters for their identification. Zootaxa, 845, 1–46. 50 Considerations on the taxonomy of the Phylum Tardigrada Kaczmarek, Ł., Jakubowska, N., & Michalczyk, Ł. (in press a) Current knowledge on Turkish tardigrades with a description of Milnesium beasleyi sp. nov. (Eutardigrada: Apochela: Milnesiidae, the granulatum group). Zootaxa. Kaczmarek, Ł., Schabetsberger, R., Litwin, N. & Michalczyk, Ł. (in press b) Dactylobiotus vulcanus, a new freshwater eutardigrade from Fiji and Vanuatu (Oceania), with a key and remarks on dubious species of the genus Dactylobiotus. New Zealand Journal of Zoology. Meyer, H.A & Hinton, J.G. (in press) Tardigrades (Phylum Tardigrada) of the Island of Barbados in the West Indies, with the Description of Milnesium barbadosense sp. n. (Eutardigrada: Milnesiidae). Caribbean Journal of Science. Miller, W.R, Clark, T. & Miller, C. (2012a) Tardigrades of North America: Archechiniscus biscaynei, nov.sp. (Arthrotardigrada: Archechiniscidae), a Marine Tardigrade from Biscayne National Park, Florida. Southeastern Naturalist, 11(2), 279–286. Miller, W.R., Schulte, R. & Johansson, C. (2012b) Tardigrades of North America: further description of the genus Multipseudechiniscus Schulte & Miller, 2011 (Heterotardigrada: Echiniscoidea: Echiniscidae) from California. Proceedings of the Biological Society of Washington, 125(2), 153–164. Nelson, D. (2002) Current Status of the Tardigrada: Evolution and Ecology. Integrative and Comparative Biology, 42, 652–659. Pardial, J.M., Miralles, A., De la Riva, I. & Vences, M. (2010) The integrative future of taxonomy. Frontiers in Zoology, 7(16), 1–14. Pilato, G., McInnes, S.J. & Lisi, O. (2012) Hebesuncus mollispinus (Eutardigrada, Hypsibiidae), a new species from maritime Antarctica. Zootaxa, 3446, 60–68. Vicente, F., Fontoura, P., Cesari, M., Rebecchi, L., Guidetti, R., Serrano, A. & Bertolani, R. (submitted) Integrative taxonomy allows the identification of synonymous species and a new genus of Tardigrada Echiniscidae (Heterotardigrada). Zootaxa. Schultze, C.A.S. (1834) Macrobiotus Hufelandii animal e crustaceorum classe novum, reviviscendi post diuturnam asphyxiam et ariditatem potens, etc. 8 Seiten, 1 tab. C. Curths, Berlin, 6 pp, I Table. Zawierucha, K., Michalczyk, Ł. & Kaczmarek, Ł. (in press) The first record of Tardigrada from the Republic of Zambia, with a description of Doryphoribius niedbalaisp. nov. (Eutardigrada: Hypsibiidae, the evelinae group). African Zoology. 51 Paper 4 Integrative taxonomy allows the identification of synonymous species and a new genus of Tardigrada Echiniscidae (Heterotardigrada). Filipe Vicente, Paulo Fontoura, Michele Cesari, Lorena Rebecchi, Roberto Guidetti, Artur Serrano & Roberto Bertolani Zootaxa (submitted) Paper 4 Abstract: The taxonomy of tardigrades is challenging as they demonstrate a limited number of useful morphological characters, therefore several species descriptions are supported by only minor differences. For example, Echiniscus oihonnae and Echiniscus mutispinosus are separated exclusively by the absence or presence of dorsal spines at position Bd. Doubts were raised on the validity of these two species, which were often sampled together. Using an integrative approach, based on genetic and in-­‐depth morphology, we studied two new Portuguese populations, and compared these with archived collections. We have determined that the two species must be considered synonymous with Echiniscus oihonnae the senior synonym. Our study showed generally low genetic distances of cox1 gene (with a maximum of 4.1%), with specimens displaying both morphologies sharing the same haplotype, and revealed character Bd to be variable. Additionally, a more in-­‐depth morphological and phylogenetic study based on the 18S gene uncovered in a new evolutionary line within the Echiniscidae, which justified the erection of Diploechiniscus gen. nov. The new genus is in a sister group relationship with Echiniscus and is, for the moment, composed of a single species. Keywords Diploechiniscus gen. nov., Diploechiniscus oihonnae comb. nov., DNA barcoding, 18S, phylogeny, morphology 55 Integrative taxonomy allows the identification of synonymous species Introduction Currently the phylum Tardigrada comprises c. 1000 described species (Guidetti & Bertolani, 2005), with regular new additions. These microscopic metazoans have a limited number of taxonomically useful morphological characters. As a consequence, species descriptions are sometimes based on minor differences that are not always easy to confirm. Only recently has α-­‐taxonomy been combined with genetic data (Guidetti et al., 2005, 2009; Møbjerg et al., 2007; Jørgensen et al., 2007, 2011; Cesari et al., 2009, 2011; Guil & Giribet, 2009; Schill et al., 2010; Bertolani et al., 2010, 2011a, 2011b). An example of one of the minor morphological differences that has separated two species can be found in the absence or presence of a spine at position Bd, which differentiates Echiniscus oihonnae Richters, 1903 from Echiniscus multispinosus Cunha, 1944b within the heterotardigrade genus Echiniscus (for the classification of dorsal plates, spines and filaments see Ramazzotti & Maucci, 1983 and Kristensen, 1987). In describing E. multispinosus, Cunha (1944b) also noted a difference in size, i.e. slightly smaller dimensions with respect to E. oihonnae. Moreover, specimens have been reported with spine Bd on only one side (found in Norway moss) together with numerous specimens attributed to regular E. multispinosus and very similar specimens attributed to E. oihonnae (Ramazzotti & Maucci, 1983). It is interesting to note that several authors have found both species at the same localities, (e.g. Cunha 1944a, 1944b (Viseu and Coimbra, Portugal); Fontoura 1981 (Amarante, Portugal), and Dudichev & Biserov 2000 (Iturup Island, Kuril Islands, Russia)). In addition, both species have black eyes, when Echiniscus eyes are normally red or absent (Kristensen, 1987). These facts described above raised the question of whether these are two valid tardigrade species, or simply variants of a single species (Ramazzotti & Maucci, 1983; Maucci & Durante, 1984; Dudichev & Biserov, 2000), and also whether these (or this) species really belong to the genus Echiniscus. For this paper we carried out an integrative taxonomy study with a more in depth morphological analysis and added molecular analysis on two Portuguese populations of E. oihonnae and E. multispinosus using mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear 18S rDNA gene markers. The former, using the DNA barcoding approach, allowed a better species description, while the latter was used to identify the generic and phylogenetic position of the specimens. 56 Paper 4 Material and Methods Fresh moss and lichen samples were collected at Moita do Conqueiro in Serra da Estrela’s Natural Park (40° 23′ 50″ N, 7° 38′ 4″ W) and at Castro Laboreiro in Peneda-­‐Gerês National Park (42° 2′ 17″ N, 8°11′ 45″ W), both in Portugal (Table 1). Animals were extracted from samples by soaking in tap water for at least 30 min and washed through consecutive 500 µm and 38 µm sieves. Individual samples were manually selected under stereomicroscope observation. Voucher specimens (Table 1) were photographed in vivo and then used for DNA analysis following the protocol described in Cesari et al., (2011). Seventy-­‐seven specimens were permanently mounted with Hoyer’s or Faure fluid and observed under a Nomarski Differential Interference Contrast Microscope (DIC) and/or at Phase Contrast (PhC). Six more were prepared for Scanning Electronic Microscopy following the protocol described by Bertolani et al. (2011a). These specimens were examined under a Philips SEM XL 40, available at the ‘Centro Interdipartimentale Grandi Strumenti’ at the University of Modena and Reggio Emilia (Italy). For morphological comparisons, specimens identified as E. oihonnae or E. multispinosus from the Maucci collection, Museo Civico di Storia Naturale di Verona, Verona, Italy, were examined. These included: 15 specimens from Forså (Norway), three specimens from Sierra de Urbion (Spain), three specimens from Caldas das Taipas and 13 specimens from Vilar Formoso (Portugal) (mounting media not specified). A total of 122 specimens pertaining to the two Echiniscus species were inspected and analyzed for this paper: 104 from Portugal, 15 from Norway and three from Spain. Unfortunately, type specimens of E. oihonnae (from Merok, Norway) and E. multispinosus (from Viseu, Portugal) were not available. To place E. oihonnae and E. multispinosus within the Echiniscidae group both morphological and molecular results were extended to other taxa: Testechiniscus spitsbergensis (Scourfield, 1897) from Lingmark Glacier, Disko Island, Greenland, Bryodelphax tatrensis Węglarska, 1959 and Bryodelphax parvulus Thulin, 1928 from Slovensky Ray, Muránska Planina National Park, Slovakia (see Table 1). Specimens from these samples were photographed in vivo and used for DNA analysis or considered for the morphological analysis. Additional samples included paratypes of Bryodelphax johannis Bertolani, Guidi & Rebecchi, 1995 from the Bertolani collection (Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy) and specimens from the Maucci collection: holotype and paratypes of Bryodelphax amphoterus Durante Pasa & Maucci, 1975, 57 Integrative taxonomy allows the identification of synonymous species specimens of B. parvulus from Monte Spaccato, Trieste, Italy, T. spitsbergensis from Gran San Bernardo, Italy and Testechiniscus spinuloides (Murray, 1907b) from Oren, Norway. Table 1 – Sampling sites, taxonomic classification and GenBank references for cox1 and 18S sequences of all utilized specimen. NA: not available. Sample Specimen Current attribution Locality Substrate GenBank A.N. cox1 18S C3039 Et.01 Echiniscus oihonnae Moita do Conqueiro Moss JX676191 JX676181 C3040 V03 Echiniscus oihonnae Moita do Conqueiro Moss JX676192 JX676182 C3041 V02 Echiniscus oihonnae Moita do Conqueiro Moss JX676193 JX676183 C3042 V07 Echiniscus oihonnae Moita do Conqueiro Moss JX676194 JX676184 C3250 V01 Echiniscus oihonnae Castro Laboreiro Lichen JX676195 NA C3250 V04 Echiniscus oihonnae Castro Laboreiro Lichen JX676196 JX676185 C3250 V08 Echiniscus oihonnae Castro Laboreiro Lichen JX676197 NA C3250 V11 Echiniscus multispinosus Castro Laboreiro Lichen JX676198 JX676186 C2257 V03 Testechiniscus spitsbergensis Disko Island Moss JX676199 JX676187 C3019 V01 Bryodelphax tatrensis Slovensky Ray Moss NA JX676188 C3019 V02 Bryodelphax parvulus Slovensky Ray Moss NA JX676189 C3020 V01 Bryodelphax tatrensis Slovensky Ray Moss NA JX676190 Molecular analysis involved DNA was extracted from single specimens by using a modified rapid salt and ethanol precipitation method (Cesari et al., 2009). PCR amplification of a portion of the mtDNA cox1 gene was carried out as described in Cesari et al. (2009), using as primers LCO-­‐1490 (5’-­‐GGT CAA CAA ATC ATA AAG ATA TTG G-­‐3′; Folmer et al., 1994) and HCO-­‐2198 (5’-­‐TAA ACT TCA GGG TGA CCA AAA AAT CA-­‐3’; Folmer et al. 1994). A region of the nuclear ribosomal small subunit gene (18S rDNA) was amplified with the primer combination 18S a2.0 (5’-­‐ATG GTT GCA AAG CTG AAA-­‐3’; Whiting et al.,1997) and 18S 9R (3’-­‐GAT CCT TCC GCA GGT TCA CCT AC-­‐5’; Giribet et al., 1996), using the following protocol: 35 cycles with 30 sec at 94 °C, 30 sec at 48 °C and one min at 72 °C, with a final elongation step at 72 °C for 10 min. The amplified products were gel purified using the Wizard Gel and PCR cleaning (Promega) kit. Both strands were subjected to sequencing reactions by using the Big Dye Terminator 1.1 kit (Applied Biosystems) and sequenced using an ABI Prism 3100 sequencer (Applied Biosystems). Nucleotide sequences of the newly analyzed specimens were submitted to GenBank (accession numbers: JX676181-­‐99; Table 1). For cox1 gene analysis, chromatograms obtained were checked for presence of ambiguous bases: sequences were translated to amino acids by using the invertebrate mitochondrial code implemented in MEGA5 (Tamura et al., 2011) in order to check for the presence of 58 Paper 4 stop codons and therefore of pseudogenes. Nucleotide sequences were aligned with the Clustal algorithm implemented in MEGA5 (pairwise and multiple alignment parameters: Gap opening penalty: 15, Gap extend penalty: 6.66) and checked by visual inspection. For appropriate molecular comparisons, we included in our analysis cox1 sequences from GenBank identified as T. spitsbergensis and several species of Echiniscus (see Table 2). Intraspecific, interspecific, and overall mean Kimura 2-­‐parameters (K2P) distances between scored haplotypes were determined using MEGA5. Table 2 – Sequences and related specimens from GenBank. Species Genbank A.N. cox1 Testechiniscus spitsbergensis (Scourfield, HM193419 1897) Echiniscus spiniger Richters, 1904b HM193408 Echiniscus wendti Richters, 1903 GU329528 Echiniscus merokensis Richters, 1904a FJ435813 Echiniscus bigranulatus Richters, 1908 HM193406 Echiniscus viridissimus Péterfi, 1956 HM193409 Echiniscus trisetosus Cuénot, 1932 FJ435815 Echiniscus canadensis Murray, 1910 FJ435814 Echiniscus testudo (Doyère, 1840) EF620368-­‐81, EU244601 Echiniscus blumi Richters, 1903 EF620382, EU046090, EU046098, EU046168, EU046197-­‐8, HM193407 Echiniscus jenningsi Dastych, 1984 Echiniscus granulatus (Doyère, 1840) Echiniscus sp. EF620367 Bryodelphax parvulus Thulin, 1928 Bryodelphax sp. Cornechiniscus lobatus (Ramazzotti, 1943) Mopsechiniscus granulosus Mihelčič, 1967 Hypechiniscus exarmatus (Murray, 1907a) Hypechiniscus gladiator (Murray, 1905) Parechiniscus chitonides Cuénot, 1926 Proechiniscus hanneae (Petersen, 1951) Pseudechiniscus islandicus (Richters, 1904c) Pseudechiniscus facettalis Petersen, 1951 Pseudechiniscus sp. Milnesium tardigradum Doyère, 1840 18S HM193385, EU266967-­‐8 HM193376 FJ435719 HM193373 AF056024 FJ435718 GQ849022 HM193375, EU049476, EU049482, EU049486 EU266969 DQ839606 EF632453, EF632457, EF632458, EU266964, EU266971, EU266974, EU266976 HM193371 EF632434 EU038077, EU038079, HM193372 HM193379 HM193377 HM193378 HM193380 HM193381 HM193383 FJ435720, HM193382 EU266965 GQ925696 59 Integrative taxonomy allows the identification of synonymous species For 18S gene analysis, nucleotide sequences were aligned with the Muscle algorithm, using default parameters implemented in MEGA5. A sequence identified as Milnesium tardigradum Doyère, 1840 was used as outgroup. Other Heterotardigrada sequences from GenBank were also included in the analysis for appropriate comparisons (see Table 2). A Bayesian inference dendrogram was computed using the program MrBayes 3.2 (Ronquist et al., 2012). Best fitting model evaluations were performed taking into account Akaike Information Criterion (AIC) and Bayes Information Criterion (BIC) (jModeltest 0.0.1; Posada, 2008), which identified the GTR+G model to be most suitable. Two independent runs, each of four Metropolis-­‐coupled Markov chains Montecarlo (MCMC), were launched for 7 x 106 generations, trees were sampled every 100 generations and the first 17500 were discarded. The analyses were run three times, all of which resulted in identical topologies. Figure 1 -­‐ Echiniscus oihonnae, specimen in vivo. Note the black eyes and the orange body color (DIC; bar = 10 µm). 60 Paper 4 Results Morphological data The first evidence obtained from all the newly collected Portuguese specimens attributable to E. oihonnae or E multispinosus is that they have black eyes (Fig. 1, 4A) and a double dorsal sculpture in the cuticular plates (Fig. 2A, B, 4D). We also found that all the specimens from the Maucci collection, atributed to these two species, had black eyes and double sculpture. In both species, the buccal tube is relatively long, narrow and the presence of stylet supports is very often recognizable (Fig. 3). Figure 2 -­‐ Echiniscus oihonnae. A: general view of the dorsal sculpture. B: Detail of the dorsal sculpture . C: Sculpture of the terminal plate IV. D: Dorsal sculpture of the scapular pate. (A: DIC; B-­‐C: PhC; D: SEM; Bar = 10 µm). 61 Integrative taxonomy allows the identification of synonymous species Using phase contrast this sculpture appears as dark, regularly shaped polygonal grains, each normally surrounded by six other grains and separated by a thin white region. Above this layer are more widely dispersed white circular grains of irregular size (Fig. 2B). The quantity of these white grains is variable in the different specimens, but always more numerous on the terminal plate IV (Fig. 2C). Using SEM, only the second type of sculpture (white circular grains) is visible as irregularly dispersed pits on the surface of the plates (Figs 2D and 5B, C). Figure 3 -­‐ Echiniscus oihonnae. Buccal-­‐pharyngeal apparatus (PhC); note the stylet supports (arrowheads). (Bar = 10 µm). Sculpture on the dorsal cephalic plate begins with the fine lower dark grains, which is followed by larger double sculpture that shows an anterior median depression (visible only on well extended specimens; Fig. 4A). The dorsal segmental plates I-­‐IV conform to the Echiniscus pattern. All median dorsal plates with double sculpture; median dorsal plates m1 and m2 are transversally divided (Fig. 4B), median plate m3 present and entire (Fig. 4C). Lateral plates are at both sides of the scapular plate (Fig. 4D). Ventral cuticular plates are always present (Fig. 4E, F), though several are often weak and sometimes difficult to identify and number. Regarding the cuticular appendages, filaments A, B, C, spine D and filament E are always present (Fig. 5A), as are filament Cd and the spine Dd. Adult animals with variable Bd morphologies were observed in the newly sampled Portuguese populations, as well as both Portuguese and Norwegian specimens in the Maucci collection. We found that from 71 specimens of the newly collected population from Castro Laboreiro the typical E. oihonnae 62 Paper 4 (without Bd) morphology was dominant (54 specimens, 78.3%) over the typical E. multispinosus (with Bd) morphology (eight specimens, 11.6%) and intermediate forms (with Bd on only one side) (seven specimens, 10.1%), and two specimens where it was not possible to see the appendages clearly. Figure 4 -­‐ Echiniscus oihonnae. A: Cephalic (cp) and neck (np) plates. B: Median plates m1 and m2 (arrowheads). C: median plate m3 (arrowhead). D; Scapular (I) and lateral (lp, arrowhead) plates. E: anterior ventral plates (arrowheads). F: Posterior ventral plate (arrowhead). (A-­‐E: PhC; F: DIC; bar = 10 µm). 63 Integrative taxonomy allows the identification of synonymous species In addition, a two clawed larva has been found, with all appendages except Bd. Barbed filaments and spines were also common (Fig. 6A-­‐C), and small dorsal-­‐lateral hooked spines B’, C’, D’ and E’ have been observed on dorsal plates. Spines E’ can be simple or double (Fig. 5B). The dentate collar is always present on the fourth pair of legs (Fig. 5C), and lateral leg plates are present, characterized by simple sculpture (uniform black grains under phase contrast; Fig. 5D). Figure 5 -­‐ Morphological features of Echiniscus oihonnae/multispinosus. A: Lateral view with discriminated spines and filaments (see text). B: Terminal plate with two spines E’ (arrowhead). C: Leg of the fourth pair with indented collar (arrowhead). D: Leg plates (arrowheads). (A: DIC; B-­‐C: SEM; D: PhC; bar = 10 µm). Internal claws of all legs with a robust, basal hooked spur. External claws usually smooth but occasionally the external claws of leg IV have one or two thin, right-­‐angled, short spurs. The shape of the gonopore reveals the presence of both females and males (Fig. 7A, B), though unfortunately this was not visible in slides of the Maucci collection. Among the 71 adults sampled from Castro Laboreiro seven were confirmed as males and 28 as females. Morphometric details of this material is provided in Table 3. The presence of stylet 64 Paper 4 supports and ventral plates, observed in fresh specimens but not always visible in the older mounted specimens, are used for morphological comparison. Other details are referred in the species re-­‐description (see below). Figure 6 -­‐ Barbed filaments and spines (A, B: PhC; C: DIC; bar = 10 µm). For comparison, we examined the morphological characters of specimens belonging to Bryodelphax and Testechiniscus (see Material and Methods). All Bryodelphax species were characterized by dorsal plates with a double sculpture that appear under phase contrast as dark and white grains, and ventral plates are present but not on all species (i.e. B. parvulus). In contrast, the sculpture of the dorsal plate in T. spitsbergensis appear as a quite different, single layer and ventral plates are more evident. Figure 7 -­‐ Echiniscus oihonnae: gonopores. A: female gonopore (arrowhead). B: male gonopore (arrowhead). (PhC; bar = 10 µm). 65 Integrative taxonomy allows the identification of synonymous species Table 3 – Measurements of specimens attributable to E. oihonnae and E. multispinosus (values in µm). Structures Standard Min Max Number of deviation specimens Body length 231.4 40.0 147.1 295.1 36 Scapular plate length 53.4 10.2 35.6 70.9 35 Internal cephalic cirrus 19.8 3.94 10.3 25.8 34 External cephalic cirrus 21.9 4.1 14.1 28.0 38 Cephalic papilla 8.3 1.6 4.9 11.4 37 Clava 6.0 1.0 4.1 8.4 34 Appendage A 71.8 15.2 39.8 101.5 35 Appendage B 44.4 15.5 13.1 75.6 38 Appendage C 80.9 25.9 31.3 122.8 38 Appendage D 31.8 7.4 17.3 44.7 38 Appendage E 113.4 44.4 38.5 186.2 34 Appendage Bd* 17.4 11.2 2.0 39.3 12 Appendage Cd 50.6 15.1 25.0 78.1 38 Appendage Dd 7.8 2.6 0.5 12.4 38 Spine leg I 4.6 0.8 2.9 5.9 31 Internal Claws II/III 14.2 2.9 8.7 18.1 38 External Claws II/III 13.2 2.8 7.6 17.1 38 Internal Claws IV 16.8 3.4 10.1 21.8 26 External Claws IV 14.9 3.3 8.9 19.2 28 Papilla leg IV 4.1 0.7 2.0 5.1 23 *Measured only in specimens of the E. multispinosus type having an evident Bd appendage. Mean Molecular data Molecular analysis was carried out on 603 bp of cox1 mtDNA gene. Six haplotypes were found in the two Portuguese populations, with genetic distances ranging from 0% to a maximum of 4.1% (Table 4). Only one specimen with the typical E. multispinosus morphology (C3250 – V11) was available for molecular analysis and it shares the same haplotype with the morphologically identified E. oihonnae (C3250 – V08). We analyzed a single T. spitsbergensis specimen with very similar results to the specimen identified in GenBank (0.6%), while it was very well differentiated from all specimens attributed to E. oihonnae (19.6-­‐20.2%). Table 4 – Kimura 2-­‐parameter distances computed among all specimens. The analysis was carried out on 603bp of cox1 gene. 1 2 3 4 5 6 7 8 9 10 66 C3039 Et.01 E. oihonnae C3040 V03 E. oihonnae C3041 V02 E. oihonnae C3042 V07 E. oihonnae C3250 V01 E. oihonnae C3250 V04 E. oihonnae C3250 V08 E. oihonnae C3250 V11 E. multispinosus C2257 V03 T. spitsbergensis HM193419 T. spitsbergensis 1 2 3 4 5 0.034 0.000 0.012 0.007 0.000 0.019 0.018 0.197 0.192 0.034 0.040 0.041 0.034 0.040 0.038 0.195 0.189 0.012 0.007 0.000 0.019 0.018 0.197 0.192 0.014 0.012 0.024 0.022 0.202 0.194 0.007 0.024 0.022 0.196 0.191 6 7 8 9 0.019 0.018 0.000 0.197 0.199 0.196 0.192 0.196 0.195 0.006 Paper 4 Comparisons between Echiniscus and Testechiniscus taxa (Table 5) showed that individuals attributed to E. oihonnae-­‐multispinosus were very well differentiated with respect to all other taxa (18.0-­‐21.3%), with values comparable to the other interspecific and intergeneric distance scores. Table 5 – Kimura 2-­‐parameter distances computed among (on the diagonal) and within (column D) taxa. All haplotypes are included in the analysis, which was carried out on 603bp of cox1 gene. NP = not possible; only one available sequence. 1 2 3 4 5 6 7 8 9 10 11 E. oihonnae-­‐multispinosus T. spitsbergensis E. spiniger E. wendti E. merokensis E. blumi-­‐canadensis E. bigranulatus E. viridissimus E. trisetosus E. testudo Echiniscus n. sp. 1 2 3 4 5 6 7 0.195 0.196 0.208 0.213 0.199 0.201 0.180 0.196 0.200 0.208 0.197 0.221 0.206 0.191 0.222 0.208 0.217 0.187 0.192 0.218 0.222 0.191 0.190 0.182 0.185 0.177 0.184 0.229 0.200 0.192 0.163 0.191 0.210 0.189 0.194 0.215 0.249 0.180 0.182 0.208 0.198 0.189 0.088 0.194 0.184 0.189 0.190 0.192 0.182 8 9 10 D 0.019 0.006 NP NP NP 0.134 NP NP 0.190 NP 0.188 0.200 0.007 0.188 0.189 0.081 NP The phylogenetic tree computed from 18S sequences (Fig. 8) shows Bryodelphax species in basal position, though the next node is ill-­‐supported (0.72 posterior probability). Inside this second cluster, Parechiniscus chitonides Cuénot, 1926 is in a sister group relationship with the remaining species, which are further divided in three main clusters, with very high posterior probability values: a) Proechiniscus + Cornechiniscus + Pseudechiniscus islandicus (Richters, 1904c); b) Mopsechiniscus + Pseudechiniscus facettalis Petersen, 1951 + a sequence attributed to Echiniscus; c) Hypechiscus in a sister group relationship with a cluster grouping Testechiniscus and Echiniscus. Inside this latter group, the phylogenetic relationships are well defined and supported with the specimens attributed to E. oihonnae and E.multispinosus grouped together and in a sister group relationship, well differentiated from the other Echiniscus taxa. 67 Integrative taxonomy allows the identification of synonymous species Discussion Our new morphological observations together with molecular analysis lead to some significant results. Firstly, the problem of species validity; where the comments from other authors (e.g. Ramazzotti & Maucci, 1983; Maucci & Durante, 1984; Dudichev & Biserov, 2000) raised our doubts about whether E. oihonnae and E. multispinosus were two species, or simply variants. Apart from Cunha’s (1944b) note that E. multispinosus individuals were smaller, only the dorsal spine Bd defined the two species. Previous works, studying the Echiniscus blumi-­‐canadensis series and utilising integrative taxonomy (molecular (cox1) and morphometrics), have demonstrated that cuticular filaments and spines can vary greatly within the same species (Guil 2008; Guil & Giribet 2009). In our study we verified that all the morphological features were shared by E. oihonnae and E. multispinosus, and represented very little variability. The most variability occurred in spines Bd (present or absent), E’ (simple or double) and spurs on external claws IV (present or absent). We also registered new characters that had not previously been noted, such as the presence of black-­‐brownish eyes, stylet supports, ventral plates and double sculpture on the dorsal plates. In the Echiniscus line (Kristensen, 1987), the presence of stylet supports is shared with Testechiniscus, Bryodelphax and Bryochoerus, ventral plates with Testechiniscus and some Bryodelphax, black eyes only with Testechiniscus and the double sculpture with Bryodelphax and a few Echiniscus species. The analysis of the cox1 sequences clearly showed all the animals were very closely related. Specimens with exact matching morphologies produced cox1 gene genetic distances ranging from 3.4 to 4.1%, considered within limits for populations of the same species (Cesari, et al. 2011). This result was further supported by two specimens, attributed by morphology to the two different species, sharing the same haplotype. We therefore consider all the specimens of E. oihonnae and E. multispinosus belonging to the same species. We have no genetic data from Norwegian (type locality) populations to analyse, and it would be interesting to confirm the Norwegian and Portuguese population species-­‐
relationships. Nevertheless, the morphological data we obtained were consistent and strong enough to report a one-­‐species diagnosis, with the conclusion that E. multispinosus should be considered the junior synonym of E. oihonnae. Reports of these species from outside Europe will require revision before the geographical distribution of E. oihonnae could be defined. 68 Paper 4 Our study of the morphological and 18S rDNA data offered the opportunity to review and revise the taxonomic position of E. oihonnae. Using morphology we found the presence of black eyes and ventral plates, are all characters absent or not visible in Echiniscus. Normally, also dorsal-­‐lateral supernumerary spines (C’, D’, etc.) are absent in Echiniscus. For this analysis we also examined (Bertolani & Guidetti, unpublished) two Echiniscus species exhibiting dorsal-­‐lateral supernumerary spines: Echiniscus menzeli Heinis, 1917 (Valle d’Aosta, Italy) and Echiniscus melanophtalmus Bartoš, 1936 (Istria, Croatia) (from the Maucci collection). These two species display Testechiniscus-­‐type dorsal plate sculpture, median plates, ventral plates and, in Echiniscus menzeli, dark eyes. Unfortunately, the stylet supports were not detectable due to the conservation state and the age of the slides. However, in our opinion, both Echiniscus menzeli and Echiniscus melanophtalmus should be attributed Testechiniscus and thus expanding this genus to six species. Comparing Bryodelphax characters, we found the double sculpture of the dorsal plates, transversally divided median plates m1 and m2 and an undivided plate m3, plus the ventral plates (not present in all species), were all shared with E. oihonnae. We were not able to include Antechiniscus, Novechiniscus, and Bryochoerus in our 18S analysis, due to partial or total lack of molecular information. According to Jørgensen et al. (2011), Antechiniscus belongs to the same clade of Proechiniscus, Cornechiniscus and Pseudechiniscus islandicus, therefore in morphology and evolutionary lines very distinct from E. oihonnae. The shape of the dorsal plates in Novechiniscus is very peculiar and very different from all other Echiniscidae (Rebecchi et al., 2008). The genus Bryochoerus is distinguished from E. oihonnae by the presence of red eyes (when present) and a transversally divided median plate 3, and the absence of a double sculpture, ventral plates and supernumerary dorsal-­‐lateral spines. There is no single morphological autapomorphy characterizing E. oihonnae, but a combination of characters, which does not match the known genera of Echiniscidae. This was confirmed by our 18S analysis, where E. oihonnae formed a distinct and well supported clade within the same evolutionary line of the Echiniscidae family that included Echiniscus and Testechiniscus (Fig. 8). Bryodelphax, which forms from a more basal node, was even further removed from E. oihonnae. 69 Integrative taxonomy allows the identification of synonymous species In conclusion, based on the clade supported by 18S analysis and the combination of morphological characters described, we propose the erection of a new taxon Diploechiniscus gen. nov for Echiniscus oihonnae (and its junior synonim E. multispinosus). Figure 8 -­‐ Bayesian inference dendrogram computed on 18S sequences. Numbers near nodes indicate posterior probability. Newly analyzed specimens are shown in bold. Grey area denotes specimens previously attributed to Echiniscus oihonnae and Echiniscus multispinosus. 70 Paper 4 Taxonomic account Diploechiniscus gen. nov. Diagnosis. Echiniscids with dorsal plates I, II, III, IV (II and III paired), transversally subdivided median plates m1 and m2 and undivided plate m3 present; double sculpture in the dorsal plates, represented (under phase contrast) by dark polygonal and white circular grains; ventral plates present, especially evident in the anterior, head region and around the gonopore; supernumerary dorsal-­‐lateral spines present; buccal tube long and narrow, with stylet supports. Orange body, dark-­‐brown eyes. Type species: Echiniscus oihonnae Richters, 1903 Composition: Diploechiniscus oihonnae (Richters, 1903) comb. nov., to date the only species attributable to the new genus. Junior synonym: Echiniscus multispinosus Etymology: from the Greek δίπλόος (diplóos) = double, composed of two parts; referring to the cuticle sculpture, and Echiniscus, the first of the echiniscid genera to be described. Remarks. The echiniscid genera most similar to Diploechiniscus are Testechiniscus, Echiniscus and Bryodelphax. Diploechiniscus is differentiated from Testechiniscus by the presence of double sculpture in the dorsal plates, subdivided dorsal plate m2 and dorsal plate m3. It is differentiated from Echiniscus by the presence of black eyes, subdivided dorsal plates m1 and m2, double sculpture in the dorsal plates, supernumerary dorsal-­‐
lateral spines, ventral plates and evident stylet supports. From Bryodelphax, Diploechiniscus is differentiated by the presence of black eyes, supernumerary dorsal-­‐lateral spines, dorsal and lateral filaments or spines (apart filament A), terminal plate notched, and the adults are much larger. The juxtoposition of the four genera into different evolutionary lines within the Echiniscidae was confirmed by 18S sequences. 71 Integrative taxonomy allows the identification of synonymous species Diploechiniscus oihonnae (Richters, 1903) comb. nov. Type locality: Merok, Norway Diagnosis Body colour reddish-­‐brown. Dark brown eye spots. Stylet supports present. Long filaments A, B, C, D and E. Short hooked dorsal-­‐lateral spines B’, C’, D’ and E’. Long filaments Cd and short spines Dd. Spine Bd present or absent. Dorsal plates present: I, paired II and III, and IV, transversally subdivided median plates m1 and m2 and median plate m3 entire. Terminal plate (IV) notched. Double sculpture of the dorsal plates observed under light microscopy. Faint ventral plates present, with those at the anterior and posterior more clearly visible. Sensory spine on leg I and papilla on leg IV, present. Lateral leg plates, present. Dentate collar on leg IV, present. Females and males present, with gonopores typical of the echiniscid form. Re-­‐description of the species (from the original description and from re-­‐examined specimens collected in Forså, Norway; Sierra de Urbion, Spain; Caldas das Taipas, Vilar Formoso, Castro Laboreiro and Moita do Conqueiro, Portugal). Body colour orange. Eye spots simple and dark brown. Buccal cirri long, clavae large. Stylet supports present (sometimes difficult to observe in older slides). Dorsal plates present, all (except neck plate) characterized by double sculpture, which appears as dark, regular polygonal grains under white circular grains when viewed with phase contrast. Dark grains are separated by thin, white region from neighbours (normally groups of six); white grains of various sizes, never overlaping dark grains, and irregularly distributed. Cephalic plate unpaired, with median depression to the anterior margin; fine anterior sculpture and larger posterior double sculpture. Neck plate, a long transverse and relatively thin band, anterior and posterior region unsculptured and fine, dark grains in the middle. Dorsal segmental plates: plate I (or scapular plate) entire, with two sculptured small lateral plates exhibiting fine, dark grains; plates II and III paired and characterised by an unsculptured transverse band, and plate IV (or terminal plate), entire but faceted and notched (not obvious in older specimens). Median intersegmental plates: plate m1, transversally subdivided, anterior region formed of a large, flat and thin rectangle not always obvious due to overlapping scapular plate; plate m2, transversally subdivided and with an unsculptured transverse band, plate appears as two obtuse angle isosceles triangles joined 72 Paper 4 by their larger side; and m3, entire, small and not obvious but with double sculpture. Lateral intersegmental plates are difficult to identify, though unsculptured spaces exist at la2 and la3. Long filaments A, B, C, D and E, sometimes barbed. Short hooked dorsal-­‐lateral spines B’, C’, D’ and E’. Lateral spine E’, simple or double. Bd variable as long spine, very short spur, or absent and can be present on one or both sides of plate II. Long filaments Cd and short spines Dd. Ventral sculpture present as fine granulation, with clearly visible head plate and posterior plates beside gonopore. Leg plates present laterally, with dark granular sculpture. Spiniform papilla present on leg I; papilla on leg IV with rounded tip. Hooked spurs an all internal claws, external claws I-­‐III smooth, occasionally one or two short right-­‐
angled spurs on the leg IV. Dentate collar variable, comprised of six to 13 triangular teeth, some irregularly bifurcated. Gonopore; a short tube in the males, and rosette in the females. The geographical distribution of E. oihonnae includes: Portugal, Switzerland, Northern Europe (including polar islands), U.S.A., Canada, Australia (Ramazzotti & Maucci 1983); Japan (Mathews, 1937); Kuril Islands, Far East Russia (Dudichev & Biserov 2000). Most of the non-­‐Eurpean citations require confirmation, as for example, Murray (1910) was doubtful about his identification of Australian and Canadian specimens, and the Californian specimens, initially assigned to E. oihonnae, were revised as T. laterculus (Schuster, Grigarick & Toftner, 1980). Acknowledgements This study was partially supported by the Fundação para a Ciência e a Tecnologia, Portugal, with a grant (BD/39234/2007) to the first author and by the program Pest-­‐
OE/MAR/UI0331/2011 to the research of the second author, and also by the European Distributed Institute of Taxonomy (EDIT) within the program ATBI: All Taxa Biodiversity Inventories in the Gemer Area, Slovakia. The research is also part of the project MoDNA supported by Fondazione Cassa di Risparmio di Modena (Italy) and the University of Modena and Reggio Emilia (Modena, Italy). The authors wish to thank Dr. César Garcia (Botanical Garden, Lisbon) for providing the moss samples from the Portuguese locality of Moita do Conqueiro, and Museo Civico di Storia Naturale di Verona for the availability of the slides of the Maucci collection. They also wish to thank Sandra McInnes, of the British Antarctic Survey, for her critical support and the English revision. 73 Integrative taxonomy allows the identification of synonymous species References Bartoš, E. (1936) Neue Tardigraden-­‐Arten aus dem unterkarpatischen Ruβland. Zoologischer Anzeiger, 114(1/2), 45-­‐48. Bertolani, R., Guidi, A. & Rebecchi, L. (1995) Tardigradi della Sardegna e di alcune piccole isole circum-­‐sarde. Biogeographia, 18, 229-­‐247. Bertolani, R., Rebecchi, L. & Cesari, M. (2010) A model study for tardigrade identification. In: P.L. Nimis & R. Vignes Lebbe (Eds.), Tools for Identifying Biodiversity: Progress and Problems, EUT, Trieste, Italy, pp. 333–339. Bertolani, R., Biserov, V., Rebecchi, L. & Cesari, M. (2011a) Taxonomy and biogeography of tardigrades using an inegrated approach: new results on species of the Macrobiotus hufelandi group. Invertebrate Zoology, 8, 23-­‐36. Bertolani, R., Rebecchi, L., Giovannini, I., & Cesari, M. (2011b) DNA barcoding and integrative taxonomy of Macrobiotus hufelandi C.A.S. Schultze 1834, the first tardigrade species to be described, and some related species. Zootaxa, 2997, 19-­‐36. Cesari, M., Bertolani, R., Rebecchi, L. & Guidetti, R. (2009) DNA barcoding in Tardigrada: the first case study on Macrobiotus macrocalix Bertolani & Rebecchi 1993 (Eutardigrada, Macrobiotidae). Molecular Ecology Resources, 9, 699-­‐706. Cesari, M., Giovannini, I., Bertolani, R. & Rebecchi, L. (2011) An example of problems associated with DNA barcoding in tardigrades: a novel method for obtaining voucher specimens. Zootaxa, 3104, 42-­‐51. Cuénot, L. (1926) Description d’un Tardigrade nouveau de la faune française. Comptes Rendus de l’Académie des Sciences de Paris, 182, 744. Cuénot, L. (1932) Tardigrades. In: Faune de France, 24, 1-­‐96. Ed. Paul Lechevalier, Paris. Cunha, A.X. (1944a) Tardigrados da Fauna Portuguesa. II. Memórias e Estudos do Museu Zoológico da Universidade de Coimbra, 155, 1-­‐12. Cunha, A.X. (1944b). Echiniscus multispinosus sp. n., un tardigrade nouveau de la fauna portugaise. Memórias e estudos do Museu Zoológico da Universidade de Coimbra, 159, 1-­‐4. Dastych, H. (1984) The Tardigrada from Antarctic with descriptions of new species. Acta Zoologica Cracoviensia, 27, 377-­‐436. Doyère, L.M. (1840) Mémoire sur les Tardigrades. I. Annales des Sciences Naturelles, Paris, Série 2, 14, 269-­‐362. 74 Paper 4 Dudichev, A.L., Biserov, V.I. (2000) Tardigrada from Iturup and Paramushir Islands (The Kuril Islands). Zoologichesky Zhurnal, 79, 771-­‐778 (in Russian). Durante Pasa, M.V. & Maucci, W. (1975) Tardigradi muscicoli dell Istria com descrizione di due specie nuove. In R.P. Higgins (ed). International Symposium on Tardigrades. Memorie dell’Istituto Italiano di Idrobiologia, 32 (Suppl.), 69-­‐91. Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3, 294–299. Fontoura, P. (1981) Contribution pour l'étude des Tardigrades terrestres du Portugal. Avec la description d'une nouvelle espèce du genre Macrobiotus. Publicações do Instituto de Zoologia "Dr. Augusto Nobre", Faculdade Ciências do Porto, 62, 157-­‐178. Giribet, G., Carranza, S., Baguña, J., Riutort, M. & Ribera, C. (1996) First molecular evidence of a Tardigrada + Arthropoda clade. Molecular Biology and Evolution, 13, 76-­‐84. Guidetti, R. & Bertolani, R. (2005) Tardigrade taxonomy: an updated check list of the taxa and a list of characters for their identification. Zootaxa, 845, 1-­‐46. Guidetti, R., Gandolfi, A., Rossi, V. & Bertolani, R. (2005) Phylogenetic analysis in Macrobiotidae (Eutardigrada, Parachela): a combined morphological and molecular approach. Zoologica Scripta, 34, 235-­‐244. Guidetti, R., Schill, R.O., Bertolani, R., Dandekar, T. & Wolf, M. (2009) New molecular data for tardigrade phylogeny, with the erection of Paramacrobiotus gen. n., Journal of Zoological Systematics and Evolutionary Research, 4, 315-­‐321. Guil, N. (2008) New records and within-­‐species variability of Iberian tardigrades (Tardigrada), with comments on the species from the Echiniscus blumi-­‐canadensis series. Zootaxa, 1757, 1-­‐30. Guil, N. & Giribet, G. (2009) Fine scale population structure in the Echiniscus blumi-­‐
canadensis series (Heterotardigrada, Tardigrada) in an Iberian mountain range—When morphology fails to explain genetic structure. Molecular Phylogenetics and Evolution, 51, 606-­‐613. Heinis, F. (1917) Tardigradenaus der _Umgebung von Triest. Zoologischer Anzeiger, 49, 94–
96. Jørgensen, A., Møbjerg, N., & Kristensen, R.M. (2007) A molecular study of the tardigrade Echiniscus testudo (Echiniscidae) reveals low DNA sequence diversity over a large geographical area. Journal of Limnology, 66 (Suppl. 1), 77-­‐83. 75 Integrative taxonomy allows the identification of synonymous species Jørgensen, A., Møbjerg, N., & Kristensen, R.M. (2011) Phylogeny and evolution of the Echiniscidae (Echiniscoidea, Tardigrada) – an investigation of the congruence between molecules and morphology. Journal of Zoological Systematics and Evolutionary Research, 49 (Suppl. 1), 6-­‐16. Kristensen, R.M. (1987) Generic revision of the Echiniscidae (Heterotardigrada), with a discussion of the origin of the family. In: R. Bertolani (ed.). Biology of Tardigrades. Selected Symposia and Monographs U.Z.I.. 1. Mucchi. Modena. pp. 261-­‐335. Mathews, G.B. (1937) Tardigrada from Japan. Peking Natural History Bulletin, 11, 411-­‐412. Maucci, W. & Durante, M.V. (1984) I Tardigradi della Penisola Iberica. Miscellanea Zoologica, 8, 67-­‐80. Mihelčič F. (1967) Ein Beitrag zur Kenntnis der Tardigraden Argentiniens. Verhandlungen der Zoologische-­‐Botanischen Gesellschaft in Wien 107, 43-­‐56. Møbjerg, N., Jørgensen, A., Eibye-­‐Jacobsen, J., Agerlin Halberg, K., Persson, D. & Kristensen, R.M. (2007) New records on cyclomorphosis in the marine eutardigrade Halobiotus crispae (Eutardigrada: Hypsibiidae). J. Limnol., 66 (Suppl. 1), 132-­‐140. Murray, J. (1905) The Tardigrada of the Scottish lochs. Transactions of the Royal Society of Edinburgh, 41, 3, 27, 677-­‐698. Murray, J. (1907a) Scottish Tardigrades collected by the Lake Survey . Transactions of the Royal Society of Edinburgh, 45, 3, 24, 641-­‐668. Murray, J. (1907b) Arctic Tardigrada, collected by Wm S. Bruce. Transactions of the Royal Society of Edinburg, 45, 3, 669-­‐681. Murray, J. (1910) Tardigrada. Reports of the Scientific Investigations of the British Antarctic Expedition. 1907-­‐1909. London, 1, 5, 81-­‐185. Péterfi, F. (1956) Contributioni la cunoasterea Tardigradelor din R. P. R. Studii si Cercetari de Biologia. Accademia R. P. R., Filiola Cluj, 7, 149-­‐155. Petersen, B. (1951) The Tardigrade Fauna of Greenland. A faunistiuc study with some few ecological remarks. Meddelelser om Grønland, 150, 5, 5-­‐94. Posada, D. (2008). jModelTest: Phylogenetic Model Averaging. Molecular Biology and Evolution, 25, 1253-­‐1256. Ramazzotti, G. (1943) Nuova varietà del Tardigrado Pseudechiniscus cornutos. Rivista di Scienze Naturali “Natura” di Milano, 34, 89-­‐90. Ramazzotti, G, Maucci, W. (1983) Il Phylum Tardigrada. III edizione riveduta e aggiornata. Memorie dell’Istituto Italiano di Idrobiologia Dott. Marco De Marchi, 41, 1–1012. 76 Paper 4 Rebecchi, L., Altiero, T., Eibye-­‐Jacobsen, J., Bertolani, R. & Kristensen, R.M. (2008) A new discovery of Novechiniscus armadilloides (Schuster, 1975) (Tardigrada, Echiniscidae) from Utah, USA with considerations on non-­‐marine Heterotardigrada phylogeny and biogeography. Organisms, Diversity & Evolution, 8, 58–65. Richters, F. (1903) Nordische Tardigraden. Zoologisher Anzeiger, 28, 5, 168-­‐172. Richters, F. (1904a) Arktische Tardigraden. Fauna Arctica, 3, 493-­‐508. Richters, F.(1904b) Beitrag zur Verbreitung der Tardigraden im südlichen Skandinavien und an der mecklenburgischen Küste. Zoologischer Anzeiger, 28, 347–352. Richters, F.(1904c) Islandische Tardigraden Zoologischer Anzeiger, 28 (10), 373–377. Richters, F. (1908) Moosbewohner. Wissenschaftliche Ergebnisse der Schwedischen Südpolar Expedition (1901-­‐1903), 5, 2, 1-­‐16. Ronquist, F., Teslenko, M. van der Mark P., Ayres D., Darling A., Höhna S., Larget B., Liu L., Suchard M.A., & Huelsenbeck J.P. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61(3), 539-­‐
542. Schuster, R. O., Grigarick, A.A. & Toftner, E. C. (1980). A new species of Echiniscus from California (Tardigrada: Echiniscidae). Pan-­‐Pacific Entomologist, 56(4), 265-­‐267. Schill, R.O., Förster, F., Dandekar, T. & Wolf, M. (2010) Using compensatory base change analysis of internal transcribed spacer 2 secondary structures to identify three new species in Paramacrobiotus (Tardigrada). Organisms Diversity & Evolution, 10, 286-­‐296. Scourfield, D.J. (1897) Contributions to the non-­‐marine Fauna of Spitbergen. Tardigrada. Proceedings of the Zoological Society of London. 790-­‐791. Tamura, K., Peterson, D., Peterson, N., Stechler, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28(10), 2731-­‐2739. Thulin, G. (1928) Über die Phylogenie und das System der Tardigraden. Hereditas, 11, 207-­‐
266. Węglarska, B. (1959) Die Tardigraden (Tardigrada) Polens. I. Tardigraden der Woiwodschaft Krakow. Acta Zoologica Cracovioensia, 2, 699-­‐745. Whiting, M.F., Carpenter, J.M., Wheeler, Q.D. & Wheeler, W.C. (1997). The Strepsitera problem: phylogeny of the holometabolous insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology. Systematic Biology, 46, 1-­‐68. 77 Paper 5 Observations on Pyxidium tardigradum (Ciliophora), a protozoan living on Eutardigrada: infestation, morphology and feeding behaviour. Filipe Vicente, Łukasz Michalczyk, Łukasz Kaczmarek & Maria-­‐José Boavida (2008) Parasitology Research 103: 1323-­‐1331. Observations o n P yxidium t ardigradum Abstract Pyxidium tardigradum is a protozoan that has been reported on a few occasions as an epizoan symphoriont living on eutardigrades. We report here the first records of this species from Kirghizia (the first Asian record), Poland and Portugal. The Portuguese population revealed the largest P. tardigradum infestation ever described in terms of both the whole tardigrade population, with 60% affected animals, as well as a single host, with 35 attached protozoan. The first ever SEM photomicrographs and pictures of live P. tardigradum are also given. No considerable ultrastructural variability was detected within or between the populations, suggesting that P. tardigradum may be a true cosmopolitan species. Given that the ciliate imposed significant extra volumes on infested tardigrades (from 1% to as much as 136%), we also discuss possible negative effects of the protozoan on the fitness of the host and suggest that P. tardigradum should probably be considered as a eutardigrade parasite. Furthermore, some hypotheses about the life history strategies of the ciliate are proposed. Keywords Pyxidium tardigradum, stalked epizoan, Tardigrada symphoriont, parasitism, life history, feeding behaviour, ultrastructure, SEM. 81 Paper 5 Introduction In some Protozoa at least a part of the life cycle is sessile, i.e. individuals live attached to organisms, such as other Protozoa, plants, animals as well as to organic debris. Pyxidium tardigradum is a ciliate protozoan (Ciliophora: Peritricha: Epistylidae) described in 1964 by Van der Land as an epizoic symphoriont of a tardigrade Ramazzottius oberhaeuseri (originally Hypsibius oberhaeuseri). It is a sessile species that has been identified on a few occasions as a eutardigrade symphoriont. Given that a symphoriont is defined as an organism that is carried and often also dispersed by its host, this term does not describe the character of the relationship in terms of benefits and costs received/paid by the host (Corliss, 2002). In other words, from the host’s point of view a symphoriont can be a symbiont, a commensal or a parasite. The observations of P. tardigradum are scarce. Iharos (1966), Hallas (1977), Wright (1991) and Marley & Wright (1994) reported low or moderate infestations on the following host tardigrade species: Isohypsibius undulatus (originally Hypsibius undulatus), Macrobiotus hufelandi, Milnesium tardigradum, Minibiotus intermedius and R. oberhaeuseri. Only one major infestation has been reported by Morgan (1976), who observed some R. oberhaeuseri populations with over 50% affected animals, carrying up to eight P. tardigradum individuals each. He also reported the ciliate attached to M. tardigradum. All these observations were based on tardigrades collected exclusively from European locations. However, a low number of epizoan Peritricha attached to Ramajendas frigidus (originally Hypsibius renaudi) from the Antarctic have been reported both by Jennings (1976) and Dastych (1984), although the Protozoan species identification was not given by the authors. All of these host species are eutardigrades; no P. tardigradum has ever been observed attached to a heterotardigrade. Sessile Peritricha, like P. tardigradum, reproduce by longitudinal cell division with reference to the stalk, thus theoretically it is possible for a few colonial cells to share a common stalk (Westphal, 1976). However, no evidence of such colonies was found so far. In this paper we illustrate and compare detailed morphology of P. tardigradum within and between the Portuguese, Polish and Kirghizian populations. We also describe some aspects of the Portuguese population structure. Moreover, we attempt to estimate the magnitude of costs imposed on tardigrades by P. tardigradum and hypothesise about the life history strategies of the ciliate. We also provide the first photographs of live P. tardigradum and the first Scanning Electron Microscope (SEM) photomicrographs of the peritrichid. 82 Observations o n P yxidium t ardigradum Material and Methods Kirghizian tardigrades were extracted from a moss sample collected from rocks in the Tien-­‐
shan Mountains, near the Issyk-­‐Kul Lake and the Karakul City, ca.1600 m above sea level, in 2002, leg. Ł. Kaczmarek. Tardigrades from Poland were found in a moss sample collected from rocks in the Ojcowski National Park, Chełmowa Mountain, near the Łokietek Cave, ca. 500 m above sea level, in 2003, leg. Ł. Kaczmarek. Except for one specimen, all Polish tardigrades were mounted on microscope slides in Hoyer’s medium and examined and photographed using Phase Contrast Microscope (PCM). The remaining specimen and all Kirghizian specimens were prepared for SEM, by being subjected to ethanol/acetone series (30, 40, 50, 60, 70, 80, 90, 100% ethanol, then 33, 67, 100% acetone, each step 5 min long, performed at room temperature), followed by CO2 critical point drying and Pt coating). Portuguese tardigrades were collected on the 14.01.2008., after a few rainy days from a moist lichen sample growing on a lemon tree (Citrus limon L.) in the locality of Quinta do Conde, about 20 km south of Lisbon, ca. 54 m above sea level (leg. F. Vicente). The lichen sample was sieved consecutively through 500 µm and 32 µm pore size mesh, and tardigrades were collected under a stereomicroscope (65-­‐400×). Live animals were examined and filmed under a Leica TCS co-­‐focal microscope (CF) (100-­‐1000×). All animals were then fixed with 4% paraformaldehyde in PBS. Some were prepared for SEM observations by being subjected to alcohol series (50, 60, 70, 80, 90, 95 and 99,5% methanol, 10 min each, at 4ºC), followed by critical point drying in CO2. These specimens were coated with Au and examined under a JEOL USM 5200 LU SEM. The remaining animals were mounted in Neo-­‐Mount medium (Merck) and observed, counted and photographed under a Nomarski Differential Interference Contrast Microscope (DIC) (40-­‐1000×). Ten random Pyxidium cells (mounted, each from a different tardigrade specimen), from the Portuguese population were measured (excluding stalks). Since the Pyxidium cell is approximately a prolate ellipsoid, the volume v can be easily calculated if cell length l and 2
4
l ⎛ w ⎞
width w are known: v = π ⋅ ⋅ ⎜ ⎟ . We also estimated volumes of eighty three random fixed 3 2 ⎝ 2 ⎠
Portuguese R. cf. oberhaeuseri specimens of which 61% were infested (the proportion of infested individuals in the sample did not differ from the whole population, p=0.88, χ12 =0.04). 83 Paper 5 As the shape of a eutardigrade body is also close to a prolate ellipsoid, we measured body length and width and calculated the volume using the equation provided above. The maximal body width was measured (between legs II and III) and body length measures excluded the hind legs. In order to assess the extra relative volumes imposed on infested tardigrades we multiplied the number of Pyxidia by the mean P. tardigradum cell volume and then divided the obtained value by the tardigrade body volume. In other words, the extra relative volume is a percentage of the host’s body volume. Figure 1 -­‐ Observed quantities of Pyxidium tardigradum attached to the Portuguese Ramazzottius cf. oberhaeuseri. Since the population consisted of a 100 tardigrades, the values can be read both as the numbers of individuals and percentages. Given that one stalk can theoretically hold more than one cell and the number of protozoans attached to a tardigrade may change over time, empty stalks were not counted (i.e. we were interested in the current population structure only). In order to establish whether Pyxidia are selective when attaching to tardigrades of different sizes we compared volumes of infested and non-­‐infested R. cf. oberhaeuseri from the Portuguese population using a two-­‐tailed independent samples Mann-­‐Whitney U-­‐test. To establish whether there is a relationship between the magnitude of infestation and host’s body volume we used a two-­‐tailed Spearman’s correlation. 84 Observations o n P yxidium t ardigradum Cortical ribs between the anterior end of the cell and the anlage of the ciliary wreath, from the ciliary wreath to the stalk and the total number of ribs were counted using SEM. Given the technical difficulties, the number of cortical ribs was determined only in three specimens of P. tardigradum from the Asian population and in five from each of the European populations. Means were compared using Kruskal-­‐Wallis tests with exact significance. The Benjamini-­‐
Hochberg correction was applied to the α-­‐level to control the overall Type I error rate in multiple tests (Benjamini & Hochberg 1995). All statistics were computed using SPSS 14.0 licensed to the University of East Anglia (Norwich, UK). Figure 2 -­‐ Non-­‐infested specimens of Portuguese R. cf. oberhaeuseri were statistically significantly smaller than infested individuals (p=0.02, means and standard errors for body volume, the dashed line represents the population mean). 85 Paper 5 Results Infestation Fifteen P. tardigradum specimens on three Macrobiotus cf. hufelandi were found in the Polish sample, whereas in the Kirghizian sample three protozoans were attached to two Ramazzottius cf. oberhaeuseri. The Portuguese population consisted of 111 tardigrades. Ramazzottius cf. oberhaeuseri was the dominant species with 100 individuals, of which 60% were affected by the protozoan. A quantitative distribution of P. tardigradum affecting these R. cf. oberhaeuseri is depicted in Fig. 1. Note that even though the most heavily infested tardigrade carried thirty-­‐five protozoans (see also Fig. 4), the majority of hosts (52%) had only one to six ciliates attached. The average number of P. tardigradum per tardigrade was 5.7. A 50% infestation affected four Milnesium cf. tardigradum, and seven Macrobiotus sp. had no P. tardigradum on them. This amounted to the total of 55.9% tardigrades affected by P. tardigradum. The number of Pyxidia attached to tardigrades varied considerably, from only a single ciliate to as many as 35 protozoans (Fig. 4). On average infested tardigrades had 5.7 ± 0.9 ciliates attached (mean ± standard error). Also, the relative extra volume imposed on tardigrades ranged from only 0.8% to as much as 135.9%. On average infested tardigrades carried extra 13.7 ± 3.5% of their volume (mean ± standard error). In addition to the reported protozoan numbers, some tardigrades exhibited also empty stalks (43 stalks attached to 17 animals total). All observed ciliates occupied the dorsal and the dorso-­‐lateral portions of the animals (i.e. no protozoans were found attached to the ventral cuticle). Most Pyxidia concentrated on the posterior parts of the animal bodies (Figs 5-­‐7). Infested individuals of the Portuguese R. cf. oberhaeuseri were statistically significantly larger than non-­‐infested specimens (means ± standard errors: 9.3 ± 1.1 × 105 µm3 (non-­‐infested) and 12.4 ± 0.9 × 105 µm3 (infested), p=0.02, U=568, N=83), see also Fig. 2. The difference was even more significant when instead of volume we used the tardigrade body length as the body size estimator (p<0.001, U=385, N=83). However, the magnitude of infestation (among infested individuals) was not correlated with the host’s body volume (p=0.865, ρ=0.25 (ρ2=0.06), N=50), see also Fig. 3. 86 Observations o n P yxidium t ardigradum Figure 3 -­‐ Among infested R. cf. oberhaeuseri the number of P. tardigradum per tardigrade was not correlated with the tardigrade body volume (p=0.88, linear regression with 95% confidence intervals). In the Portuguese population the average number of Pyxidia attached to infested tardigrades was 5.7, which translated into a volume of 1.19 × 105 µm3 (we assumed the volume of an average P. tardigradum cell, see Table 1). We calculated relative extra volumes for a hypothetical population of tardigrades with a range of body volumes similar to the Portuguese R. cf. oberhaeuseri (i.e. 1.5-­‐27.5 × 105 µm3, see Fig. 27). Our modelled population consisted therefore of 27 tardigrades (with the body volume increase = 1 × 105 µm3 per individual), each carrying 5.7 average size ciliates. The result of our simulation is depicted with the red solid curve with red data points. The relative additional volume imposed on hosts is exponentially negatively correlated with the tardigrade body size (i.e. the relationship has an asymptotic character). To recognise how profoundly the extra relative volume imposed by P. tardigradum changes with the host’s body size, lets consider three tardigrades (indicated by open circles on the graph): a small one (1.5 × 105 µm3), a medium sized one (14.5 × 105 µm3) and a large one (27.5 × 105 µm3). In our hypothetical population 5.7 average size ciliates impose as much as 87 Paper 5 79% relative extra volume on the small tardigrade, but only 8% on the medium and 4% on the large tardigrade. The difference in body volume between the small and medium and the medium and large tardigrade is exactly the same (i.e. 13 × 105 µm3), but the difference in extra relative volume imposed on the small and medium tardigrade is nearly 18 times bigger than the difference between the medium sized and large tardigrade (71% vs. 4% of the difference, respectively). Figures 4-­‐6 -­‐ Pyxidia attached to tardigrades – 4 – R. cf. oberhaeuseri from Portugal with 35 protozoans (= 53% extra relative volume), 5-­‐6 – Macrobiotus sp. from Poland with 10 ciliates (5 – dorsal view, 6 – later view, arrowheads indicate empty feet and stalks to which P. tardigradum cells were previously attached). Note that ciliates are attached dorso-­‐laterally with higher densities on the caudal part of the tardigrade body. (4 – DIC, 5-­‐6 – SEM; scale bars in µm) 88 Observations o n P yxidium t ardigradum P. tardigradum cells were all oval and did not vary significantly in size and shape either within or between analysed populations (Figs 4-­‐8, 14, 18-­‐26). Measurements of ten randomly chosen Portuguese cells are given in Table 1. Given that peristomes were contracted on all protozoans prepared for SEM, both the cytostome and the oral cilia were not visible in SEM. The pellicle is ribbed perpendicularly to the stalk axis (Fig. 8) with uniformly distributed pores (Figs 9-­‐11). Since the pores were only ca. 0.06 µm in diameter, it was not possible to observe them under Light Microscope. The number of cortical ribs was similar in all three populations. The number of ribs between the anterior end of the cell and the anlage of the ciliary wreath: Kirghizia: 69-­‐
70, Poland: 65-­‐70, Portugal: 64-­‐68 (p=0.079, H2=4.952, N=13). Ribs from the ciliary wreath to the stalk: Kirghizia: 12-­‐14, Poland: 10-­‐13, Portugal: 10-­‐12 (p=0.087, H2=4.718, N=13). Totals: Kirghizia: 82-­‐84, Poland: 75-­‐83, Portugal: 74-­‐80 (p=0.015, H2=7.060, N=13). Thus, only the total number of ribs differed significantly between the populations (at adjusted pBH<0.017). Moreover, the significance was driven by the difference between the Asian and both European samples. However, given the low sample sizes, the data do not allow to determine whether the difference has a biological meaning. Cilia were restricted to the peristome only. A distinct, C-­‐
shaped macronucleus was visible in all live and permanently mounted specimens (Fig. 12), however micronuclei were not detected in any of the observed ciliates. TABLE 1 - Basic statistics for lengths, widths and volumes of 10 random P. tardigradum cells (stalks excluded) from
the Portuguese population (MIN and MAX = the lowest and the highest measurements among all individuals, SE =
standard error of the mean).
Cell dimension
MIN
MAX
MEAN
SE
Length (height) [µm]
40.0
52.5
46.7
1.4
Width [µm]
24.3
32.2
28.9
0.7
Volume [× 104 µm3]
1.24
2.85
2.08
0.15
Morphology Stalks were wrinkled (visible in SEM only), usually shorter than the cell length and somewhat flexible (Fig. 13-­‐17). Even though stalks seemed to be non-­‐contractile, the ciliates were able to squat by contracting the cell base and pulling the stalk inside (compare the same live individual on Figs 20 and 21). Most stalks bared only single cells, however we also observed branched stalks with two cells (Fig. 13) and stalks with three attachment points (Fig. 14). The squatting is probably reduced in branched ciliates, since none of the clonal cells is able to pull the colonial stalk inside. 89 Paper 5 Feet are round and do not seemed to penetrate tardigrade cuticles (Figs. 15-­‐17), however Transmission Electron Microscopy observations are needed to reveal the nature of the attachment (i.e. how the foot is attached to the host’s cuticle, and if the cuticle is damaged by the ciliate). Figures 7-­‐12 -­‐ 7 – A closer look at the caudal part of the dorso-­‐lateral cuticle of the Polish tardigrade shown on Figs 5-­‐6; 8 – a single P. tardigradum cell (Kirghizia); 9 – contracted peristome with a cytostome (Kirghizia); 10 – topical pellicle (Poland); 11 – lateral pellicle (Kirghizia); 12 – macronucleus of a Portuguese P. tardigradum. Arrowheads on Fig. 7 indicate empty feet to which P. tardigradum cells were attached and arrows on Figs 8-­‐11 indicate pores in the pellicle. (7-­‐11 – SEM, 12 – DIC; scale bars in µm) 90 Observations o n P yxidium t ardigradum Observations of live specimens and feeding behaviour All live protozoans showed similar behaviour. Cilia were, at times, retracted by peristome contraction and then extended outside the cell, causing a water flow by a rotatory movement (Figs 18-­‐21) and, in the effect, bringing food to the cytostome. Food (most likely bacteria and maybe also some organic particles) passed through the cytopharynx, a funnel-­‐like structure connected to a forming vacuole (Figs 20-­‐21). When a vacuole reached a certain volume, it circulated within the cell (Figs 22-­‐26). A new food vacuole soon started to form. At least five such vacuoles were seen simultaneously in a single cell. Figures 13-­‐17 -­‐ 13-­‐14 – Branched stalks: 13 – a stalk with two cells (Kirghizia), 14 – a stalk with currently one cell attached, but note additional attachment points (arrowheads) where two other Pyxidium cells used to be (Portugal); 15-­‐17 – Pyxidium feet: 15-­‐16 – external appearance (Kirgizia), 17 – mid-­‐section showing no evidence of that the ciliate penetrates tardigrade cuticle, c – tardigrade cuticle surface, f – Pyxidium foot, p – Pyxidium pellicle surface, s – Pyxidium stalk, t – tardigrade body cavity (Poland). (13, 15-­‐16 – SEM, 14 – DIC, 17 – PCM; scale bars in µm) 91 Paper 5 Figures 18-­‐26 -­‐ P. tardigradum feeding behaviour.– 18-­‐21 – arrows indicate the process of extending the cilia and initiating the rotatory movement (18 – cilia are almost completely retracted and the stalk is pulled inside the cell, 21 – peristome with cilia is fully craned and the cell is floating freely on the flexible stalk), 20-­‐26 – arrowheads indicate the formation (20-­‐21) and migration (22-­‐26) of a food vacuole. Time (seconds : centiseconds) is shown in the bottom-­‐left corner of each photograph, scale bars in µm. Food available to the protozoans consisted of bacteria and organic debris present in the lichen sample. No additional food source was provided. (Portugal, CF) 92 Observations o n P yxidium t ardigradum Discussion The dorsal and dorso-­‐lateral positioning of most Peritrichids observed in this study is in accordance with previous literature (Morgan, 1976; Marley & Wright, 1994). A few ciliates were however attached to an anterior area, but never beyond the level of the first pair of legs. Such pattern exists probably because P. tardigradum attached too close to the host’s mouth would negatively affect the tardigrade ability to feed, and eventually induce death. It is also possible that Pyxidia that attached randomly and happened to be present on the anterior cuticle were removed by the host or were lost as a consequence of host’s movements in dense moss or lichen environment. In this case the selection would also favour ciliates with a preference to attach caudally. It is worth noting, however, that five Pyxidium cells were observed attached to an exuvium. This, though, may be simply because a tardigrade had just moulted and Pyxidia can survive some time with no locomotion provided by the host or maybe non-­‐mobility does not shorten the lifespan of ciliates at all, affecting (negatively) the cell division rate only. It would be interesting to observe ciliates attached to exuvia and establish whether they can detach from the cuticle and find a new host or once attached they are permanently associated with a specific animal. The latter would imply a very short lifespan of P. tardigradum, since tardigrades moult every few days, e.g. in M. tardigradum first and second moults occur at intervals of 4-­‐5 days and subsequent moults occur at intervals of 6-­‐10 days when the life cycle is not elongated by anabiosis (Suzuki, 2003). Given that P. tardigradum has always been considered simply a eutardigrade symphoriont, the nature of the ciliate-­‐tardigrade relationship has never been defined in terms of benefits/costs received/paid by the host. Since the protozoan neither does seem to penetrate the host’s cuticle nor it does feed on the host, it could be concluded that it does not inflict a direct harm. Therefore, the ciliate-­‐tardigrade relationship could be described as commensalism (we assume that phoresy is advantageous to the peritrichid). However, it is worth considering that P. tardigradum may decrease the host’s fitness in other ways than by cuticle damage or by feeding on tardigrades. In the Portuguese population, infested tardigrades carried 6 Pyxidium cells on average which translated to extra 14% of the host’s volume. Thus, it seems reasonable to expect that for a tardigrade even a few protozoans could be a significant load that imposes considerable extra energy costs related to locomotion. Also, locomotion itself could be impaired making it more difficult for tardigrades to forage as well as to avoid predators. Locomotion impairment can be caused not only by increased body weight and difficulties in 93 Paper 5 moving in dense moss/lichen cushions but it can be also generated by rotary cilia movements that may pull the host’s body in many directions. Moreover, in case of bisexual tardigrade populations, infestation may have a negative effect on sexual attractiveness of an infested animal as well as it can be a physical obstacle for mating. Therefore, in our opinion, P. tardigradum should probably be considered as a parasite, as it appears possible that it may decrease the fitness of its host in many ways. Even though there is no strong empirical evidence to support this, parasitism seems much more likely than commensalism or symbiosis when considering the tardigrade-­‐Pyxidium relationship. If the assumption that the number of protozoans attached to the host negatively affects the host’s fitness is correct, we can expect that Pyxidia should do better when a single or a low number of parasites are attached to a tardigrade. We observed such pattern in the Portuguese population, where the highest number of infested tardigrades had a single Pyxidium attached (29% of all affected individuals). Over a half (52%) of infested individuals carried only 1-­‐3 ciliates (see Fig. 1) and further 23% 4-­‐6 protozoans, leaving only 26% of tardigrades with 7 or more Pyxidia attached. However, this pattern can also be explained by an early stage of infestation in the observed population. This simple explanation could be satisfactory, given that the short inter-­‐moulting time in live tardigrades forces the protozoans to find a new host before they manage to multiply to high numbers and become lethal to the host (i.e. their host will stop moving anyway – it will either moult or die). However, the mere presence of Pyxidia attached to an animal may elongate the inter-­‐moulting time by impairing locomotion and therefore causing a lower food intake and a slower growth of the host. The implications of such relationship could be two-­‐fold for Pyxidium. Elongating the inter-­‐moulting time (but not killing the host) would increase the fitness of P. tardigradum, but it would also allow more time for other ciliates to attach and as a consequence induce the host’s death when it could still provide locomotion. This may lead to some kind of inter-­‐specific competition between Pyxidia if cells on a single host are not clonal and/or they are not capable of kin-­‐recognition. The costs imposed on the invertebrate by P. tardigradum decreases rapidly when small and medium size tardigrades are compared. However, when we compare extra relative volumes imposed on medium and big tardigrades the differences become much smaller (Fig. 27). Such asymptotic relationship between the imposed cost and body size of the host means also that an additional Pyxidium cell is going to ballast a small host significantly, whereas it is not going to have a considerable effect on a big or even a medium size tardigrade. This principle may be 94 Observations o n P yxidium t ardigradum very important from the ciliate’s point of view. If Pyxidia replicate more frequently than tardigrades moult, and if daughter cells stay on the same host even for a limited time (branched stalks found in our study suggest that it is so), it would be beneficial for the protozoan (assuming that the tardigrade fitness is positively correlated with the symphoriont’s fitness) to be attached to a big host, since an increase in Pyxidium colony size would not reduce the invertebrate fitness considerably. Thus, it should be expected that P. tardigradum would attach to bigger tardigrades more often than to smaller animals. Indeed, we found such pattern (Fig. 2) which seems to support our prediction. It can be argued though, that the same pattern could be simply a result of Pyxidia attaching to tardigrades randomly. In other words, ciliates would be more likely to attach to bigger tardigrades given that their cuticle surface is greater. However, if this was true, we should also expect a positive correlation between the number of ciliates and the host size among infested tardigrades. Yet we did not find such relationship (Fig. 3). Therefore, we conclude that P. tardigradum prefers bigger tardigrades over smaller ones. Random attachment to hosts above a size threshold can be easily explained if we assume that the ciliate fitness increases asymptotically with host size, i.e. when the relationship between the protozoan fitness and tardigrade body size is described by a mirror image of the imposed costs asymptote (Fig. 27). Thus, the ciliate can gain a lot when choosing the medium sized tardigrade over the small one, but not when choosing between the medium and the large tardigrade. In other words, above a certain host size the fitness gain is not significant and therefore it is not advantageous for the peritrichid to be choosy to any further extent. The threshold may depend on a number of factors, one of them could be the invertebrate population size. In a small tardigrade population the choice of hosts is limited and there are not many larger tardigrades available, thus the threshold should be low. In a large population, however, ciliates can afford to be more selective and the threshold is expected to be high. Another factor that may influence the threshold could be the P. tardigradum population size. In this case, though, the threshold should be negatively correlated with the ciliate population size, e.g. with time, when Pyxidia multiplied the threshold should be lowered as the result of a higher competition between protozoans (i.e. less non-­‐infested tardigrades available). The proximate mechanism of how P. tardigradum could differentiate between small and medium-­‐big tardigrades is unknown. However, given that P. tardigradum seems to be host-­‐
specific, we should expect a tight co-­‐evolutionary parasite-­‐host race that could result in sophisticated adaptations in the ciliate and tardigrades. 95 Paper 5 Both tardigrade-­‐ciliate and ciliate-­‐ciliate interactions may be very complex and more observations, measurements and possibly also laboratory experiments are required for further testing of our hypotheses described above. Lackey (1938) stated that despite the apparent cosmopolitan distribution of some protozoans, their occurrence should be determined by ecological factors such as associations with other organisms. It seems that the occurrence of P. tardigradum can be explained by this kind of ecological association, since its presence appears to be correlated with the occurrence of eutardigrades. Moreover, no P. tardigradum were ever found attached to any other moss-­‐
/lichen-­‐dwelling animal taxa such as rotifers, nematodes or acari, even though rotifers were present in much greater numbers than tardigrades in the Portuguese sample. This strongly suggests that P. tardigradum is indeed a specific tardigrade symphoriont. It is possible that hosts specificity is caused by Pyxidium requirements regarding both the host’s cuticle and locomotion type. The first requirement could explain the lack of records on heterotardigrades, the latter would explain no observations of the ciliate on rotifers. Figure 27 -­‐ The relative additional volume imposed on infested invertebrates depends on both the total volume of ciliates attached to the tardigrade and the host’s body size (the solid red curve). The dashed blue curve (with the small, medium and large tardigrades indicated) shows a hypothetical fitness curve of P. tardigradum when it is ideally negatively correlated with the costs imposed on the host (i.e. when y = –x). 96 Observations o n P yxidium t ardigradum The first ever SEM observations of P. tardigradum revealing some aspects of the external ultrastructure are described in this study. Observed feeding behaviour is consistent with the one described by Van der Land (1964). Cell measures are also consistent with previous reports (Van der Land, 1964; Dastych, 1984; Marley & Wright, 1994). No morphological differences were found between European and Asian populations even though they are as much as ca. 7000 km apart. However, in order to establish whether they belong to one species, more SEM studies and preferably also molecular analyses are needed. Contrary to earlier predictions by Van der Land (1964) and Kudo (1966) we observed many branched, colonial stalks (mainly in the Portuguese population). The vast quantity of ‘empty’ stalks may suggest, as predicted by Westphal (1976), the existence of a swarmer form that detaches from the stalk in search for a new host. Acknowledgements The kind help of Dr. Gabriel Martins (University of Lisbon) in obtaining pictures of live specimens is greatly appreciated. We are also grateful to Dr. Matt Gage (University of East Anglia) for the valuable comments on the manuscript. The study was partially supported by a grant to ŁM & ŁK from the European Commission’s (FP 6) Integrated Infrastructure Initiative programme SYNTHESYS (grant no. DK-­‐TAF-­‐2576). Parts of this paper describing the first SEM observations of P. tardigradum were presented by ŁM & ŁK at the 10th International Symposium on Tardigrada in Catania, Italy, 18th-­‐23rd June 2006. References Benjamini, Y. & Hochberg, Y. (1995) Controlling the False Discovery Rate: a Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society B, 57: 289-­‐
300. Corliss, J. O. (2002) Biodiversity and biocomplexity of the protists and an overview of their significant roles in maintenance of our biosphere. Acta Protozoologica, 41: 199-­‐219. Dastych, H. (1984) The tardigrade from Antarctic with description of several new species. Acta Zoologica Cracoviensia, 27: 377-­‐436. 97 Paper 5 Hallas, T. E. (1977) Survey of the tardigrades of Finland. Annales Zoologici Fennici, 14: 173-­‐
183. Iharos, G. (1966) A Bakony-­‐hegyseg Tardigrada-­‐faunaja. III. Állattani Közlemények, 53: 69-­‐78. Jennings, P. (1976) The Tardigrada of Signy Island, South Orkney Islands, with a note on the Rotifera. British Antarctic Survey Bulletin, 44: 1-­‐25. Kudo, R. R. (1966) Protozoology. Fifth edition. Thomas, USA, 1174 pp. Lackey, J. B. (1938) A study of some ecological factors affecting the distribution of protozoa. Ecological Monographs, 8(4): 501-­‐528. Marley, N. J. & Wright, D. E. (1994) Pyxidium tardigradum van der Land, a rarely recorded symphoriant on waterbears (Tardigrada). Quekett Journal of Microscopy, 37: 232-­‐233. Morgan, C. I. (1976) Studies on the British tardigrade fauna. Some zoogeographical and ecological notes. Journal of Natural History, 10: 607-­‐623. Suzuki, A. C. (2003) Life history of Milnesium tardigradum Doyère (Tardigrada) under a Rearing Environment. Zoological Science, 20: 49-­‐57. Van der Land, J. (1964) A new peritrichous ciliate as a symphoriont on a tardigrade. Zoologiche Mededeelingen, 39: 85-­‐88. Westphal, A. (1976) Protozoa. Blackie, First edition, UK, 325 pp. Wright, J. C. (1991) The significance of four xeric parameters in the ecology of terrestrial Tardigrada. Journal of Zoology, 224: 59-­‐77. 98 !
!
!
!
!
!
!
!
"#$%&!'!
!
(! $)*+,-%.%/01! 2/34*! ,.! !"#$%$&'( )*+%$,+*%&'! 5"%&0/&01)0#6!
7$%&13+#&004#86!#.!%$09,01!$&,/,9,#.!,.!%3/#&40-&#4%2!
!
:0+0$%!;01%./%6!<01)%+%!=%2#&06!(&/3&!>%&&#.,6!?,@%&/,!A%&/,+#.0!
B)%!C,3&.#+!,D!E3F#&*,/01!<01&,@0,+,-*!50.!$&%$#&#/0,.8!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
-(./"01,232)$4(5)&%"(13(!"#$%$&'()*+%$,+*%&' !
!(
"#$%&'()$*&#!
!"#$%$&'( )*+%$,+*%&'! ;#.! 4%&! G#.46! HI'J! 02! #! 10+0#/%! $&,/,9,#.! /)#/! +0K%2! #//#1)%4! /,!
%3/#&40-&#4%2L! M/! 02! #! N%N@%&! ,D! /)%! 23@1+#22! "%&0/&01)0#L! "%&0/&01)042! #&%! 4%D0.%4! @*! )#K0.-!
O%++! 4%K%+,$%4! ,&#+! 10+0#! #.4! #! 2,N#/01! 20+0#/3&%! &%431%4! /,! #! /%+,/&,1)#+! @#.4! 5<0#,( 2)( *0L6!
PQQJR! S/9! 2)( *0L6! PQHQ8L! T,! !6( )*+%$,+*%&'( )#2! %K%&! @%%.! &%$,&/%4! #//#1)%4! /,! #.!
)%/%&,/#&40-&#4%R!/)02!02!$&,@#@+*!43%!/,!/)%!%U02/%.1%!,D!4,&2#+!$+#/%2!O0/)!0&&%-3+#&!23&D#1%6!
D,+42!#.4!.,/1)%26!#.4!#+2,!,D!4,&2#+!2$0.%2!#.4!D0+#N%./2!/)#/!N#F%!#//#1)N%./!0N$,220@+%!D,&!
/)%!$&,/,9,#.V2!@#2#+!4021L!>0.1%!%3/#&40-&#4%2!)#K%!2N,,/)!4,&2#+!13/01+%6!/)%*!)#K%!@%1,N%!
/)%!04%#+!),2/L!A0.40.-!02!$&%4,N0.#./+*!4,.%!,.!#.0N#+V2!&%#&!4,&2#+!#&%#!5:0-L!H86!#2!#DD%1/0.-!
/)%!#./%&0,&!,&!K%./&#+!$#&/2!O,3+4!)#K%!.%-#/0K%!0N$#1/2!0.!/#&40-&#4%V2!1#$#10/0%2!/,!D%%4!#.4!
N,K%6! O)01)! O,3+4! 3+/0N#/%+*! 0.431%! 4%#/)L! (11,&40.-! /,! ;01%./%! 2)( *0L! 5PQQW8! 0.D%2/#/0,.!
+%K%+2!1#.!&%#1)!3$!/,!HX'Y!,D!%U/&#!@,4*!O%0-)/6!O)01)!0N$+0%2!20-.0D01#./!4%/&0N%./#+!%DD%1/2!
,K%&!#.0N#+V2!D0/.%226!/)32!23--%2/0.-!!6()*+%$,+*%&'(/,!@%!#!%3/#&40-&#4%!$#&#20/%L!
!
!
+*,(%-!.!/!76(486(7*'*991))$&5!0.D%2/%4!O0/)!!6()*+%$,+*%&'(5@#&Z[Q\N8L!!
!
B)%!N#],&0/*!,D!$&,/,9,#.2!#&%!20.-+%!2/#+F%46!@3/!1,+,.0%2!)#K%!@%%.!,@2%&K%4!5;01%./%!2)(*0L6!
PQQW8L! B)%&%! #&%! D%O! &%1,&42! ,D! !6( )*+%$,+*%&'( #22,10#/%4! /,! /#&40-&#4%2! D&,N! E3&,$%#.!
,&0-0.2!5M)#&,26!HI''R!<,&-#.6!HI^'R!_#++#26!HI^^R!`&0-)/6!HIIH!#.4!<#&+%*!#.4!`&0-)/6!HIIJ8!
,&! D&,N! @,/)! E3&,$%#.! #.4! (20#.! ,&0-0.2! 5;01%./%! 2)( *0L6! PQQW8L! (440/0,.#++*6! @,/)! C%..0.-2!
5HI^'8!#.4!a#2/*1)!5HIWJ8!)#K%!&%$,&/%4!20N0+#&!%$09,01!$&,/,9,#!#//#1)%4!/,!%3/#&40-&#4%26!
0.!/)%!(./#&1/016!@3/!,.!.%0/)%&!1#2%!)#2!/)%!2$%10%2!@%%.!04%./0D0%4!#2!!6()*+%$,+*%&'L!!
!
"#$!
!*.2+(: !
!!
(.#+*20.-! /)%! $&,/,9,#.V2! N,&$),+,-*6! ;01%./%! 2)( *0L! 5PQQW8! D,3.4! .,! $)*201#+! 4021&%$#.10%2!
#N,.-! (20#.! #.4! E3&,$%#.! $,$3+#/0,.2! .,&! @%/O%%.! /)%! /O,! E3&,$%#.! $,$3+#/0,.2! 2/340%46!
O)01)! 23--%2/! /)#/! !6( )*+%$,+*%&'! 02! #! 1,2N,$,+0/#.! 2$%10%2L! _,O%K%&6! 0.4%$%.4%./! 4#/#!
O,3+4!@%!&%b30&%4!/,!1,.D0&N!/)02L!B,!4#/%6!/)%&%!#&%!.,!-%.%/01!4#/#!#K#0+#@+%!,.!/)%!2$%10%2L!
`%! $&%2%./! #! $)*+,-%.%/01! $,20/0,.0.-! D,&! !6( )*+%$,+*%&'! #2! O%++! #2! #! 2/34*! ,.! /)%! -%.%/01!
402/#.1%2!#N,.-!/O,!$,$3+#/0,.26!@#2%4!,.!.%O+*!,@/#0.%4!aT(!4#/#L!!
!
01$-%*12!1#'!0-$3&'4!
;*'.0$3,(
<,22!#.4!+01)%.!2#N$+%2!O%&%!1,++%1/%4!#/!c30./#!4,!=,.4%6(",&/3-#+!5XWd!XJV!HIVV!TR!Id!PV!J[VV!
`86!<,4%.#6!M/#+*!5JJd!X^V!PPVV!TR!HQd!['V!XPVV!E8!#.4!=+#&%!M2+#.46!M&%+#.4!5[Xd!PWV![WVV!TR!Id!XJV!
WVV8L! >#N$+%2! O%&%! +%D/! /,! #0&! 4&*! #/! &,,N! /%N$%&#/3&%6! O%0-)%4! #.4! /%2/%4! D,&! #.0N#+2! @*!
2,#F0.-! D,&! #/! +%#2/! XQ! N0.! #.4! O#2)%4! /)&,3-)! 1,.2%13/0K%! [QQ! eN! #.4! XW! eN! 20%K%2L! (++!
#.0N#+2!$&%2%./!0.!20%K%4!2#N$+%2!O%&%!N#.3#++*!2%+%1/%4!3.4%&!#!2/%&%,N01&,21,$%L!(DD%1/%4!
#.0N#+2!O%&%!2%$#&#/%4!#.4!0.40K043#+!$&,/,9,#!O%&%!4%/#1)%4!D&,N!),2/2!@*!320.-!/3.-2/%.!
.%%4+%2L!!
!
<=-(2#)+*4)$13(*3%(52>&234$3,!
aT(!%U/&#1/0,.!D&,N!20.-+%!2$%10N%.2!O#2!1#&&0%4!,3/!D,++,O0.-!=%2#&0!2)(*06(5PQQI8!$&,/,1,+L!
B)%! HW>! /#&-%/! 2%b3%.1%! O#2! #N$+0D0%4! 320.-! 3.0K%&2#+! $&0N%&2! E3F(! 5[Vf((=! =Bg! gBB! g(B!
==B! g==! (gBfXh8! #.4! E3FA! 5[VfBg(! B==! BB=! Bg=! (gg! BB=! (==! B(=fXV8L! =*1+0.-! $#&#N%/%&2!
O%&%!#4#$/%4!D&,N!T,&D!i!:,022.%&!5PQHQ8j!H!1*1+%!5P!N0.!#/!I[!d=8R!XQ!1*1+%2!5XQ!2!#/!I[!d=R!
XQ! 2! #/! [P! d=R! J[2! #/! ^P! d! =8R! #.4! H! 1*1+%! 5'! N0.! #/! ^P! d=8L! B)%! MB>Hf[LW>fMB>P! &%-0,.! O#2!
#N$+0D0%4!320.-!/)%!D,++,O0.-!$&0N%&2j!MB>f:!5[VfgBB!===!=BB!g((!=g(!gg(!(BB!=fXV8!#.4!MB>f
?!5[VfB(=!Bg(!B(B!g=B!B((!gBB!=(g!=ggfXV8!#.4!1*1+0.-!$#&#N%/%&!#4#$/%4!D&,N!>3.!2)(*0L!5!
PQHQ8j!H!1*1+%!5[!N0.!#/!IJ!d=8R!XQ!1*1+%2!5H[!2!#/!IJ!d=R!XQ!2!#/!J'!d=R!H!N0.!#/!^P!d=8R!#.4!H!
1*1+%!5[!N0.!#/!^Pd=8L!B)%!#N$+0D0%4!$&,431/2!O%&%!-%+!$3&0D0%4!320.-!/)%!`09#&4!g%+!#.4!"=?!
1+%#.0.-! 5"&,N%-#8! F0/L! T31+%,/04%! 2%b3%.1%2! O%&%! #+0-.%4! O0/)! /)%! <321+%! #+-,&0/)N!
0N$+%N%./%4!0.!<Eg([!K%&20,.![!5B#N3&#!2)(*0L6!PQHH8!320.-!4%D#3+/!$#&#N%/%&2!#.4!1)%1F%4!
@*!K023#+!0.2$%1/0,.L!!
"#%!
!
-(./"01,232)$4(5)&%"(13(!"#$%$&'()*+%$,+*%&' !
(
(
!/"01,232)$4(*3*0"5$5(
B)%!@,3.4#&0%2!@%/O%%.![LW>!#.4!MB>H!#.4!MB>P!&%-0,.2!O%&%!4%/%&N0.%4!@*!1,N$#&02,.!O0/)!
,/)%&! 2%b3%.1%2! #K#0+#@+%! D&,N! g%.A#.F! #.4! @*! 320.-! /)%! ?D#N! O%@/,,+! #K#0+#@+%! D&,N!
)//$jkk&D#NL2#.-%&L#1L3Fk! 5g&0DD0/)2fC,.%2! %/! #+L! PQQ[8L! (440/0,.#++*! /,! /)%! .%O! HW>! #.4! [LW>!
2%b3%.1%2! ,@/#0.%4! @*! 32! 5B#@+%! H86! #++! ,/)%&! #.#+,-,32! 2%b3%.1%2! #K#0+#@+%! 0.! g%.A#.F6!
$%&/#0.0.-! /,! "%&0/&01)0426! O%&%! 0.1+34%4! 0.! /)%! #.#+*202! 52%%! #11%220,.! .3N@%&2! 0.!
4%.4&,-&#N28L!
:,&! HW>! -%.%! #.#+*2026! @%2/! D0//0.-! N,4%+! %K#+3#/0,.2! O%&%! $%&D,&N%4! /#F0.-! 0./,! #11,3./!
(F#0F%! M.D,&N#/0,.! =&0/%&0,.! 5(M=8! #.4! A#*%2! M.D,&N#/0,.! =&0/%&0,.! 5AM=8! 5]<,4%+/%2/! QLQLHR!
",2#4#6! PQQW86! O)01)! 04%./0D0%4! /)%! gB?lg! N,4%+! /,! @%! N,2/! 230/#@+%L! (! $)*+,-%.%/01!
N#U0N3Nf+0F%+0),,4! /&%%! O#2! 1,.2/&31/%4! 320.-! ?(U<G! 5>/#N#/#F026! PQQ'8! O0/)! HQQ!
@,,/2/&#$! &%$+01#/%2L! (! A#*%20#.! 0.D%&%.1%! 4%.4&,-&#N! O#2! 1,N$3/%4! 320.-! /)%! $&,-&#N!
<&A#*%2! XLP! 5?,.b302/! 2)( *066! PQHP8L! BO,! 0.4%$%.4%./! &3.26! %#1)! ,D! D,3&! <%/&,$,+02f1,3$+%4!
<#&F,K! 1)#0.2! <,./%1#&+,! 5<=<=86! O%&%! +#3.1)%4! D,&! HQ! U! HQ'! -%.%&#/0,.26! /&%%2! O%&%!
2#N$+%4!%K%&*!HQQ!-%.%&#/0,.2L!>/#/0,.#&0/*!O#2!#22%22%4!@*!%U#N0.0.-!/)%!2/#.4#&4!4%K0#/0,.!
,D! 2$+0/! D&%b3%.10%2! #.4! @*! $+,//0.-! /)%! m+.!?!$%&! -%.%&#/0,.! 320.-! B&#1%&! KHL[! 5?#N@#3/! #.4!
a&3NN,.46!PQQ^86!#.4!/)%!D0&2/!P[QQQ!/&%%2!-%.%&#/%4!@%D,&%!2/#/0,.#&0/*!O%&%!4021#&4%4!#2!
nn@3&.f0.oL!B)%!#.#+*2%2!O%&%!&3.!/)&%%!/0N%26!#++!,D!O)01)!&%23+/%4!0.!04%./01#+!/,$,+,-0%2L!
:,&! 1,N@0.%4! #.#+*202! 5HW>! #.4! [LW>! -%.%286! (F#0F%! M.D,&N#/0,.! =&0/%&0,.! 5(M=8! #.4! A#*%2!
M.D,&N#/0,.! =&0/%&0,.! 5AM=8! #-#0.! 04%./0D0%4! /)%! gB?lg! N,4%+! /,! @%! N,2/! 230/#@+%L! (!
$)*+,-%.%/01! N#U0N3Nf+0F%+0),,4! /&%%! O#2! 1,.2/&31/%4! 320.-! ?#U<G6! O0/)! HQQ! @,,/2/&#$!
&%$+01#/%6!O)0+%!#!A#*%20#.!0.D%&%.1%!4%.4&,-&#N!O#2!1,N$3/%4!320.-!/)%!$&,-&#N!<&A#*%2!
XLPL! BO,! 0.4%$%.4%./! &3.26! %#1)! ,D! D,3&! <=<=! O%&%! +#3.1)%4! D,&! ^! U! HQ'! -%.%&#/0,.26! /&%%2!
O%&%!2#N$+%4!%K%&*!HQQ!-%.%&#/0,.2L!>/#/0,.#&0/*!O#2!#22%22%4!320.-!B&#1%&!KHL[!5?#N@#3/!i!
a&3NN,.46! PQQ^86! #.4! /)%! D0&2/! H^[QQ! /&%%2! -%.%&#/%4! O%&%! 4021#&4%4! #2! @3&.f0.L! B)%!
#.#+*2%2!O%&%!&3.!/)&%%!/0N%26!#++!,D!O)01)!&%23+/%4!0.!04%./01#+!/,$,+,-0%2L!
!
!1.&0*)$13(*3*0"5$5(
M.40K043#+2! $%&/#0.0.-! /,! /)%! 7$%&13+#&004#! ,&4%&! O%&%! #.#+*9%4! D,&! 0./&#2$%10D01! #.4!
0./%&2$%10D01! p0N3&#! Pf$#&#N%/%&2! 5pP"8! 402/#.1%2! @%/O%%.! )#$+,/*$%26! @*! 320.-! <Eg([! @*!
320.-!MB>H!#.4!MB>PL!!
!
!
!
"#&!
!*.2+(: !
!
5-4(2$4!
aT(! 2%b3%.10.-! O#2! 2311%22D3+! D,&! #! 2N#++! .3N@%&! ,D! 0.40K043#+! $&,/,9,#6! ,&0-0.#++*! D&,N!
/)&%%! E3&,$%#.! 1,3./&0%26! M&%+#.46! ",&/3-#+! #.4! M/#+*L! :,3&! 2311%22D3+! 2%b3%.1%2! D,&! HW>! #.4!
/)&%%!D,&!MB>Hf[LW>fMB>P!O%&%!,@/#0.%4!5B#@+%!H8L!EK%.!/),3-)!/)02!N#F%2!D,&!#!1,.204%&#@+*!
2N#++! 2#N$+%6! /)%&%D,&%! O0/)! +0//+%! 2/#/02/01! &%+%K#.1%R! /)%! %3/#&40-&#4%! 7*'*991))$&5( 486(
1@2+/*2&52+$!O#2!/)%!N,2/!1,NN,.!),2/L!
!
6172-!.!/!>#N$+0.-!20/%26!),2/!2$%10%2!#.4!g%.A#.F!&%D%&%.1%2!D,&!HW>!#.4!MB>Hf[LW>fMB>P!2%b3%.1%2!,D!#++!!6(
)*+%$,+*%&'L!!
!"#$%&' !$&()#&#!
=P^[[!
=P^[[!
=XPJW!
=XHQW!
A!
=!
(!c=HfA!
"P!
*+("%),-!
.+/,'/$&()&/'
=+#&%!M2+#.46!M&%+#.4!
7*'*991))$&5(486(1@2+/*2&52+$(
=+#&%!M2+#.46!!M&%+#.4!
7*'*991))$&5(486(1@2+/*2&52+$!
c30./#!4,!=,.4%6!",&/3-#+! 7*'*991))$&5(486(1@2+/*2&52+$!
<,4%.#6!M/#+*!
A$0325$&'(486()*+%$,+*%&'!
!
B)%!/O,!HW>!0.D%&%.1%!/&%%2!5:0-2L!P!#.4!X8!2),O!1,.202/%./!&%23+/2j!#++!D,3&!!6()*+%$,+*%&'!#&%!
1+32/%&%4!/,-%/)%&6!2%$#&#/%!D&,N!#.*!,/)%&!-%.32L!7.!#!2+0-)/+*!O04%&!&#.-%6!/)%*!N#F%!D,&!#!
1,.202/%./!1+32/%&!/,-%/)%&!O0/)!#++!#K#0+#@+%!B.2+4&0*+$*!#.4!C*'.*3200*!5,.+*!,.%!2%b3%.1%!
%U02/2! D,&! /)%! +#//%&8! #.4! 2,N%! &%$&%2%./#/0K%2! ,D! /)%! D.$5)"0$5! -%.32L! <#U0N3N! +0F%+0),,4!
0.D%&%.1%!/&%%!$,0./!/,!2,N%!0.4%D0.0/0,.!,D!/)%!E1+)$4200*(#.4!F11)/*'3$&'!-%.%&#6!O)0+%!/)%!
A#*%20#.!#.#+*202!1+%#&+*!2/#/%2!/)#/!D.$5)"0$5!02!.,/!N,.,$)*+%/01L!!!
:0-3&%2!J!#.4![!$&%2%./!A#*%20#.!#.4!<#U0N3N!+0F%+0),,4!0.D%&%.1%!/&%%2!D,&!#++!"%&0/&01)042!
O0/)!#K#0+#@+%!2%b3%.1%2!D,&!@,/)!HW>!#.4![LW>L!B)%2%!#.#+*2%2!O%&%!$%&D,&N%4!O0/)!@#202!,.!
@,/)! 10/%4! 2%b3%.1%26! /,-%/)%&L! A,/)! /&%%2! 2),O! 1,)%&%./! &%23+/2L! B)%! /)&%%! !6( )*+%$,+*%&'!
0.40K043#+2!1+32/%&!/,-%/)%&6!#2!4,!#++!,/)%&!-%.%&#6!O0/)!.,!43@0,32!20/3#/0,.26!%U1%$/0,.!N#4%!
D,&!D.$5)"0$5L!7.!/)%!<#U0N3N!+0F%+0),,4!/&%%6!D6(/23)54/20$!/#F%2!#.!0./%&0,&!$,20/0,.!O)0+%!D6(
4/+"52'"%$5!02!@#2#+R!,.!/)%!A#*%20#.!/&%%6!/)%!/O,!2$%10%2!#&%!2%$#&#/%46!%#1)!#$$%#&0.-!0.!,.%!
N#],&!1+#4%L!
!
!
"#'!
!
-(./"01,232)$4(5)&%"(13(!"#$%$&'()*+%$,+*%&' !
(
!
+*,(%-!8!9!<#U0N3N!+0F%+0),,4!/&%%!,D!HW>!-%.%!D,&!#++!#K#0+#@+%!"%&0/&01)0#LL!A,,/2/&#$!K#+3%2!1,N$3/%4!#D/%&!HQQ!
&%$+01#/%26!G2)+*/"'23*(*'2+$4*3$5!#.4!G+$4/1%$3*('&)*@$0$5!O%&%!32%4!#2!,3/-&,3$L!!B)%!/&%%!O#2!1,N$3/%4!,.!#!
'XJ!@$!4#/#2%/L!
!
"#(!
!*.2+(: !
!
!
+*,(%-! :! 9! A#*%20#.! 0.D%&%.1%! /&%%! ,D! HW>! -%.%6! 1,N$3/%4! ,.! HQQQQQQQ! -%.%&#/0,.2! O0/)! #! @3&.f0.! ,D! ! P[QQQ!
-%.%&#/0,.26!D,&!#++!#K#0+#@+%!"%&0/&01)0#L!G2)+*/"'23*(*'2+$4*3$5!#.4!G+$4/1%$3*('&)*@$0$5!O%&%!32%4!#2!,3/-&,3$L!!
B)%!/&%%!O#2!1,N$3/%4!,.!#.!'XJ!@$!4#/#2%/L!
"#)!
!
-(./"01,232)$4(5)&%"(13(!"#$%$&'()*+%$,+*%&' !
(
!
!
+*,(%-!;!m!<#U0N3N!+0F%+0),,4!/&%%!@#2%4!,.!HW>l[LW>6!D,&!#++!#K#0+#@+%!"%&0/&01)0#L!A,,/2/&#$!K#+3%2!1,N$3/%4!
#D/%&! HQQ! &%$+01#/%2L! G2)+*/"'23*( *'2+$4*3$5! #.4! G+$4/1%$3*( '&)*@$0$5! O%&%! 32%4! #2! ,3/-&,3$L! B)%! /&%%! O#2!
1,N$3/%4!,.!#.!WQP!@$!4#/#2%/L!
!
B)%! N,&%! K#&0#@+%! MB>H! #.4! MB>P! 2%b3%.1%2! O%&%! #+2,! ,@/#0.%4! D,&! /)&%%! !6( )*+%$,+*%&'!
2$%10N%.L! a02/#.1%! N#/&0U%2! 5B#@+%2! P! #.4! X8! O%&%! %2/0N#/%4! #N,.-! /)%! $&%K0,326! 0.1+340.-!
#+2,! ,/)%&! 7$%&13+#&004#%! 2$%10%2! O0/)! $3@+02)%4! MB>! 4#/#L! :,&! MB>H! 5B#@+%! P86! /)%! /O,! D6(
4/+"52'"%$56!/)%!/O,!M&02)!!6()*+%$,+*%&'6!#.4!,.%!C6(&'@200*+$*!O0/)(D6(,*02*!%#1)!2)#&%!/)%!
%U#1/! N#/1)0.-! )#$+,/*$%2! O0/)! 0/2! 1,.2$%10D01L! B)%! /O,! C6( &'@200*+$*! #&%! 402/#.1%4! @*! HL^Y!
#.4! /)%! /O,! D6( /23)54/20$! @*! QLIYL! MB>H! -%.%/01! 402/#.1%! @%/O%%.! /)%! /O,! M&02)! #.4! /)%!
",&/3-3%2%!!6()*+%$,+*%&'!02!,D!'L'YL!B)02!+#/%!2$%10N%.!02!/)%!N,2/!02,+#/%4!,.%!0.!/)%!%./0&%!
N#/&0U6!O0/)!402/#.1%2!/,!,/)%&!-%.%&#!&#.-0.-!D&,N!J^L[Y!/,!'WLJYL!
!
!
"#*!
!*.2+(: !
!
!
+*,(%-! <! 9! A#*%20#.! 0.D%&%.1%! /&%%! @#2%4! ,.! HW>l[LW>6! 1,N$3/%4! ,.! ^QQQQQQ! -%.%&#/0,.2! O0/)! #! @3&.f0.! ,D!!
H^[QQ! -%.%&#/0,.26! ! D,&! #++! #K#0+#@+%! "%&0/&01)0#L! G2)+*/"'23*( *'2+$4*3$5! #.4! G+$4/1%$3*( '&)*@$0$5! O%&%! 32%4! #2!
,3/-&,3$L!!B)%!/&%%!O#2!1,N$3/%4!,.!#.!WQP!@$!4#/#2%/L!
!
:,&!MB>P!#!2N#++%&!N#/&0U!5B#@+%!X8!O#2!,@/#0.%46!#2!,.+*!/)&%%!2%b3%.1%2!O%&%!#K#0+#@+%!D&,N!
/)%!+0/%&#/3&%L!_%&%6!0./&#-%.%&01!-%.%/01!402/#.1%2!&#.-%!D&,N!H[LIY!@%/O%%.!/)%!/O,!D.$5)"0$5(
2$%10%26! /,! JWY! @%/O%%.! /)%! ",&/3-3%2%! !6( )*+%$,+*%&'! #.4! D6( 4/+"52'"%$5L! B)%! /O,!
$,$3+#/0,.2!,D!!6()*+%$,+*%&'!#&%!2%$#&#/%4!@*!XLIY!MB>P!@#2%4!-%.%/01!402/#.1%L!C32/!+0F%!0.!
O0/)!MB>P6!/)%!M&02)!!6()*+%$,+*%&'!0.40K043#+2!2)#&%!/)%!2#N%!)#$+,/*$%L!
!
""#!
!
-(./"01,232)$4(5)&%"(13(!"#$%$&'()*+%$,+*%&' !
(
!
6172-! ! 8! 9! 01$%*=! &>! ,-#-$*)! '*4$1#)-4! ?@8AB! &>! "6C! .! ,-#-! >&%! $3-! DE-%)(21%**'1-! ?721)FB! 1#'! 4$1#'1%'!
-%%&%4!?72(-BG!714-'!&#!1!'1$14-$!&>!.8H!7EI!
! !
H(
P(
X(
J(
[(
'(
^(
W(
I(
HQ(
HH(
H! gS[W'HW^!
!
Q6QQQ! Q6QXW! Q6QX'! Q6Q'^! Q6Q^I! Q6Q'^! Q6Q'I! Q6HH'! Q6HH'! Q6H'I!
D.$5)"0$5(4/+"52'"%$5(
P! (:JPIWW^!
Q6QQQ!
!
Q6QXW! Q6QX'! Q6Q'^! Q6Q^I! Q6Q'^! Q6Q'I! Q6HH'! Q6HH'! Q6H'I!
D.$5)"0$5(4/+"52'"%$5(
X! gS[W'HW'!
Q6HX^! Q6HX^!
!
Q6QQI! Q6Q^Q! Q6QWX! Q6Q'I! Q6Q^P! Q6HHX! Q6HHX! Q6H[Q!
D.$5)"0$5(/23)54/20$(
J! (:JPIWWI!
Q6HP'! Q6HP'! Q6QQI!
!
Q6Q'W! Q6QWQ! Q6Q'^! Q6Q'I! Q6HQI! Q6HQI! Q6HJX!
D.$5)"0$5(/23)54/20$(
[! (:JPIWWW!
Q6XP'! Q6XP'! Q6XJW! Q6XXX!
!
Q6Q^'! Q6QQQ! Q6QHP! Q6QII! Q6QII! Q6HPH!
D.$5)"0$5(,*02*(
'! (:JPIWIX!
Q6XI[! Q6XI[! Q6JHW! Q6JQH! Q6JQH!
!
Q6Q^'! Q6QWQ! Q6HPP! Q6HPP! Q6H[H!
B.2+4&0*+$*(
'$4+1%$54&'(
^! gS[W'HWW!
Q6XPP! Q6XPP! Q6XJJ! Q6XPI! Q6QQQ! Q6JQH!
!
Q6QHP! Q6QI^! Q6QI^! Q6HH^!
C*'.*3200*(&'@200*+$*(
W! (:JPIWW[!
Q6XX'! Q6XX'! Q6X[W! Q6XJJ! Q6QH^! Q6JXP! Q6QH^!
!
Q6HQH! Q6HQH! Q6HPH!
C*'.*3200*(&'@200*+$*(
I! =P^[[!A!
Q6JIW! Q6JIW! Q6JIW! Q6J^'! Q6JH[! Q6JWW! Q6JQW! Q6JPW!
!
Q6QQQ! Q6QXQ!
HQ! =P^[[!=!
Q6JIW! Q6JIW! Q6JIW! Q6J^'! Q6JH[! Q6JWW! Q6JQW! Q6JPW! Q6QQQ!
!
Q6QXQ!
HH! =XPJW!(!c=HA!
Q6'WJ! Q6'WJ! Q6[WI! Q6['J! Q6JWJ! Q6'H'! Q6J^[! Q6JIW! Q6Q''! Q6Q''!
!
!
6172-! :! 9! 01$%*=! &>! ,-#-$*)! '*4$1#)-4! ?@8AB! &>! "6C! 8! ,-#-! >&%! $3-! DE-%)(21%**'1-! ?721)FB! 1#'! 4$1#'1%'!
-%%&%4!?72(-BG!714-'!&#!1!'1$14-$!&>!8JH!7EI!
!
!
H(
P(
X(
J(
[(
'(
H! gS[W'HW^!D.$5)"0$5(4/+"52'"%$5!
!
Q6QXH! Q6Q[Q! Q6Q'I! Q6Q'I! Q6Q^Q!
P! gS[W'HW'!D.$5)"0$5(/23)54/20$!
Q6H[I!
!
Q6QJ^! Q6Q[W! Q6Q[W! Q6Q'X!
X! gS[W'HWW!C*'.*3200*(&'@200*+$*! Q6XH[! Q6PIH!
!
Q6Q['! Q6Q['! Q6Q'W!
J! =P^[[!A!
Q6J'X! Q6XW'! Q6X[^!
!
Q6QQQ! Q6QHJ!
[! =P^[[!=!
Q6J'X! Q6XW'! Q6X[^! Q6QQQ!
!
Q6QHJ!
'! =XPJW!(!c=HA!
Q6JWQ! Q6JPH! Q6JP'! Q6QXI! Q6QXI!
!
!
K*4)(44*&#!
>311%22D3+!aT(!4#/#!O%&%!)#&4!/,!,@/#0.!D&,N!0.40K043#+!2$%10N%.2!,D!!6()*+%$,+*%&'6!43%!/,!
40DD013+/0%2!0.K,+K0.-!)#.4+0.-!,D!231)!2N#++!+0D%!D,&N26!#2!O%++!#2!/,!/)%!2N#++!#N,3./!,D!.31+%01!
#1042! $&%2%./! 0.! %#1)! 2$%10N%.L! B)%! -%.%/01! 4#/#! $&%2%./%4! )%&%! #&%! /)%! D0&2/! %K%&( ,.! !6(
)*+%$,+*%&'L!
B)%!HW>!#.#+*2%2!$+#1%!!6()*+%$,+*%&'(0.!/)%!&%1%./+*!%&%1/%4!,&4%&!7$%&13+#&004#!5S/9!2)(*0L6!
PQHQ86! 3.4%&! /)%! 7$%&13+#&004#%! D#N0+*L! >31)! 1,.1+320,.! N#F%2! 2%.2%! D&,N! #! N,&$),+,-01#+!
$,0./! ,D! K0%O6! 20.1%! /)%2%! #&%! 4%D0.%4! #2! 2%220+%! 1,+,.0#+! 10+0#/%2! O0/)! &0-04! 2/#+F2! 5C#.F,O2F06!
HIWQ86! #.4! !6( )*+%$,+*%&'! 1,+,.0%2! )#K%! @%%.! &%-02/%&%4! 5%L-L6! ;01%./%( 2)( *0L6! PQQW8L! A,/)!
A#*%20#.!#.#+*202!#.4!@,,/2/&#$!K#+3%2!0.401#/%!/)#/!D."5)"0$5!02!$,+*$)*+%/01L!(11,&40.-!/,!S/9!
2)( *0L! 5PQHQ86! /)%! D."5)"0$5! -%.32! N#*! #1/3#++*! &%$&%2%./! #! 2%/! ,D! @#2#+! 2$%10%2! &%/#0.0.-!
!
"""!
!*.2+(: !
!
#.1%2/&#+! 1)#&#1/%&2L! B)02! 02! 1,.202/%./! O0/)! ,3&! &%23+/2! D&,N! /)%! HW2l[LW2! 0.D%&%.1%2L! 73&!
HW2l[LW2! &%23+/2! 2/&,.-+*! 23$$,&/! /)%! /)&%%! %U02/0.-! D#N0+*! 1+#4%2! ,D! 7$%&13+#&004#%6!
;,&/01%++04#%!#.4!q,,/)#N.004#%L!!
M.4%$%.4%./!MB>H!#.4!MB>P!-%.%/01!402/#.1%!N#/&0U%2!2),O!1,)%&%./!&%23+/26!O0/)!K#+3%2!@%%.!
2+0-)/+*!+,O%&!D,&!/)%!MB>P6!1,N$#&#/0K%+*!/,!/)%!MB>HL!a02/#.1%2!,D!QY!@%/O%%.!0.40K043#+2!,D!
/)%!2#N%!2$%10%26!,D!HPfHXY!#N,.-2/!2$%10%2!,D!/)%!2#N%!-%.32!#.4!,D!X[f'WY!#N,.-!-%.%&#6!
N#F%! ,D! /)%2%! /O,! 2%b3%.1%2! -,,4! 1#.404#/%2! /,! 4%+0N0/! /)%! 2$%10%2! #.4! -%.32! @#&&0%&2! ,D!
"%&0/&01)42L!!
B)%! 0./&0-30.-! 1#2%! ,D! D6( ,*02*! @%%.! 2%$#&#/%4! @*! QfHL^Y! D&,N! C6( &'@200*+$*! 2/&%.-/)%.2! /)%!
$&%K0,32!2/#/%N%./2!0.!D#K,3&!,D!/)%!$,+*$)*+%/01!.#/3&%!,D!D."5)"0$56!#.4!1#++2!D,&!#!&%4%D0.0/0,.!
,D!/)%!-%.32L!!
=,.204%&0.-! /)%! )0-)%&! 402/#.1%2! &%-02/%&%4! #N,.-! -%.%&#6! 0/! #$$%#&2! &%#2,.#@+%! /,! 2#*! /)#/!
XLIY!40DD%&%.1%!D,&!MB>P!#.4!'L'Y!D,&!MB>H!@%/O%%.!/)%!",&/3-3%2%!#.4!M&02)!$,$3+#/0,.2!,D!!6(
)*+%$,+*%&'! D#++2! ,.! /)%! 0.204%! ,D! /)%! 2$%10%2! +0N0/L! B)02! O,3+4! N#F%! 2%.2%! O0/)! /)%! .,.f
K%&0D01#/0,.!,D!N,&$),+,-01#+!4021&%$#.10%2!#N,.-!/O,!E3&,$%#.!$,$3+#/0,.2!#.4!#.!(20#.!,.%!
52%%! ;01%./%( 2)( *0L6! PQQW8! #.4! O,3+4! /)32! 1,&&,@,&#/%! /)%! 232$010,.! /)#/! !6( )*+%$,+*%&'! 02! #!
1,2N,$,+0/#.!2$%10%2L!_,O%K%&6!/,!4#/%6!,.+*!/)%!+0N0/%4!#N,3./!,D!aT(!4#/#!$&%2%./%4!)%&%!
%U02/26! #.4! /)%2%! #&%! 1+%#&+*! 0.23DD010%./! /,! N#F%! 231)! #! 2/#/%N%./L! <,&%! -%.%/01! 2/34*0.-! 02!
.%%4%4!#.4!D&,N!#!23$%&0,&!.3N@%&!,D!2$%10N%.26!0.!,&4%&!/,!#113&#/%+*!4%/%&N0.%!/)%!+0N0/2!
,D!0./&#!$,$3+#/0,.!-%.%/01!K#&0#@0+0/*L!(440/0,.#++*6!4#/#!D&,N!#!O04%&!+02/!,D!,&0-0.2!O,3+4!@%!
&%b30&%4!/,!&%2$,.46!,.1%!#.4!D,&!#++6!/,!/)%!b3%2/0,.!#@,3/!O)%/)%&!,&!.,/!O%!#&%!4%#+0.-!O0/)!
#!3@0b30/,32!2$%10%2!,D!$&,/,9,#L!!!!!!
!
L)F#&M2-',-N-#$4!
B)02!O,&F!O#2!$#&/0#++*!D3.4%4!@*!/)%!",&/3-3%2%!H&3%*IJ1(.*+*(*(C$K34$*(2(*(G243101,$*!O0/)!
#! -&#./! 5>:?_kAakXIPXJkPQQ^8! /,! /)%! D0&2/! #3/),&L! B)%! &%2%#&1)! 02! #+2,! $#&/! ,D! /)%! $&,]%1/!
<,aT(!23$$,&/%4!@*!:,.4#90,.%!=#22#!40!?02$#&N0,!40!<,4%.#!5M/#+*8!#.4!/)%!S.0K%&20/*!,D!
<,4%.#!#.4!?%--0,!EN0+0#!5<,4%.#6!M/#+*8L!
!
!
""+!
!
-(./"01,232)$4(5)&%"(13(!"#$%$&'()*+%$,+*%&' !
(
!
5->-%-#)-4!!
=%2#&06! <L6! A%&/,+#.06! ?L6! ?%@%11)06! GL! i! g304%//0L! ?L! 5PQQI8! aT(! @#&1,40.-! 0.! B#&40-&#4#j! /)%!
D0&2/! 1#2%! 2/34*! ,.! A*4+1@$1)&5( '*4+14*0$#( A%&/,+#.0! #.4! ?%@%11)0! HIIX! 5E3/#&40-&#4#6!
<#1&,@0,/04#%8L!A10(D410(725!Hj'IIm^Q'L!
a#2/*1)6!_L!5HIWJ8!B)%!/#&40-&#4%!D&,N!(./#&1/01!O0/)!4%21&0$/0,.!,D!2%K%&#+!.%O!2$%10%2L!-4)*(
F110(C+*41L!8OjX^^mJX'L!!
g&0DD0/)2fC,.%26! >L6! <,U,.6! >L6! <#&2)#++6! <L6! p)#..#6! (L6! E44*6! >L?L6! A#/%N#.6! (L! 5PQQ[8L! ?D#Nj!
#..,/#/0.-!.,.f1,40.-!?T(2!0.!1,N$+%/%!-%.,N%2L!=&4(-4$%5(725!XXjHPHfHPJL!!
_#++#26!BL!EL!5HI^^8!>3&K%*!,D!/)%!/#&40-&#4%2!,D!:0.+#.4L!-33(F110(H233!.;jH^XmHWXL!
M)#&,26!gL!5HI''8!(!A#F,.*f)%-*2%-!B#&40-&#4#fD#3.#]#L!MMML!-00*))(M190!<:j'Im^WL!
C#.F,O2F06! (L! `L! 5HIWQ8L! =,.2$%1/32! ,D! #! .%O! 2*2/%N! ,D! /)%! $)*+3N! =0+0,$),&#L! G+&%"(
F1101,$4/25N1,1(O35)$)&)*(?23$3,+*%!H;jHQX!mHPHL!
C%..0.-26! "L! 5HI^'8! B)%! B#&40-&#4#! ,D! >0-.*! M2+#.46! >,3/)! 7&F.%*! M2+#.426! O0/)! #! .,/%! ,.! /)%!
?,/0D%&#L!P+(-3)*+4)(;&+L(P&00!;;jHmP[L!
<0#,6!`L6!:%.6!`L!>L6!r36!rL!_L6!q)#.-6!sL!rL!i!>)%.6!rL!:L!5PQQJ8!")*+,-%.%/01!?%+#/0,.2)0$2!,D!/)%!
>3@1+#22! "%&0/&01)0#! 57+0-,)*N%.,$),&%#6! =0+0,$),&#8! M.D%&&%4! D&,N! >N#++! >[email protected]/! &?T(!
g%.%!>%b3%.1%2L!Q(D&N*+"1)(A$4+1@$10!<.?8BjHWQmHW'L(
<#&+%*6! TL! CL6! `&0-)/6! aL! EL! 5HIIJ8! "*U0403N! /#&40-&#43N! K#.! 4%&! G#.46! #! &#&%+*! &%1,&4%4!
2*N$),&0#./!,.!O#/%&@%#&2!5B#&40-&#4#8L!R&2N2))(Q(A$4+154!:OjPXPmPXXL!
<,&-#.6! =L! ML! 5HI^'8! >/340%2! ,.! /)%! A&0/02)! /#&40-&#4%! D#3.#L! >,N%! 9,,-%,-&#$)01#+! #.4!
%1,+,-01#+!.,/%2L!Q(=*)(S$5)!.Jj'Q^m'PXL!
T,&D6! _L! i! :,022.%&6! `L! 5PQHQ8! (! T%O! :+#-2)0$! "%&0/&01)! 5=0+0,$),&#6! "%&0/&01)04#8! D&,N! /)%!
?0K%&!?)0.%6g%&N#.*j!-.14*+4/25$&'(*+3%)$!.L!2$L!Q(D&N*+"1)(A$4+1@$10!<O?:BjP[QmP'JL!
?#N@#3/6!
(L!
i!
a&3NN,.46!
(L!
CL!
5PQQ^8L!
B&#1%&!
KHLJL!
(K#0+#@+%!
#/!
)//$jkk@%#2/L@0,L%4L#1L3FkB&#1%&L!
>3.6! "L6! =+#N$6! CL! =L! i! s36! aL! 5PQHQ8! (.#+*202! ,D! /)%! 2%1,.4#&*! 2/&31/3&%! ,D! MB>! /&#.21&0$/2! 0.!
$%&0/&01)! 10+0#/%2! 5=0+0,$),&#6! 7+0-,)*N%.,$),&%#8j! MN$+01#/0,.2! D,&! 2/&31/3&#+! %K,+3/0,.!
#.4!$)*+,-%.%/01!&%1,.2/&31/0,.L!A10(!/"01,23(DL10!<PjPJPmP[HL!
>/#N#/#F026!(L!5PQQ'8L!?(U<Gf;Mf_"=j!N#U0N3N!+0F%+0),,4f@#2%4!$)*+,-%.%/01!#.#+*2%2!O0/)!
/),32#.42!,D!/#U#!#.4!N0U%4!N,4%+2L!P$1$381+'*)$456!88jP'WWmP'IQL!
B#N3&#6! pL6! "%/%&2,.6! aL6! "%/%&2,.6! TL6! >/%1)+%&6! gL6! T%06! <L! i! p3N#&6! >L! 5PQHH8! <Eg([j!
N,+%13+#&! %K,+3/0,.#&*! -%.%/012! #.#+*202! 320.-! N#U0N3N! +0F%+0),,46! %K,+3/0,.#&*!
402/#.1%6!#.4!N#U0N3N!$#&20N,.*!N%/),42L!A10(P$10(DL10!8QjP^XHmP^XIL!
!
""$!
!*.2+(: !
!
S/96! GL! ?L! "L6! >0Nt,6! BL! GL! GL6! >#D06! GL! >L! GL! i! E0&090F6! EL! 5PQHQ8! EU$#.4%4! ")*+,-%.%/01!
?%$&%2%./#/0,.! ,D! g%.%&#! 7$%&13+#&0#! #.4! E$02/*+02! >)%42! G0-)/! ,.! /)%! EK,+3/0,.! #.4!
_0-)%&fG%K%+! B#U,.,N*! ,D! "%&0/&01)! =0+0#/%2! 5=0+0,$),&#j! "%&0/&01)0#8! Q( D&N*+"1)( A$4+1@$10!
<O?<BjJH[mJPQL!
`&0-)/6! CL! =L! 5HIIH8! B)%! 20-.0D01#.1%! ,D! D,3&! U%&01! $#&#N%/%&2! 0.! /)%! %1,+,-*! ,D! /%&&%2/&0#+!
B#&40-&#4#L!Q(F110!88;j[Im^^L!
;#.!4%&!G#.46!CL!5HI'J8!(!.%O!$%&0/&01),32!10+0#/%!#2!#!2*N$),&0,./!,.!#!/#&40-&#4%L(F110(A2%2%!
:HRW[mWWL!
;01%./%6! :L6! <01)#+19*F! uL6! p#19N#&%F! uL! i! A,#K04#! <L! CL! 5PQQW8! 7@2%&K#/0,.2! ,.! !"#$%$&'(
)*+%$,+*%&'!5=0+0,$),&#86!#!$&,/,9,#.!+0K0.-!,.!E3/#&40-&#4#j!0.D%2/#/0,.6!N,&$),+,-*!#.4!
D%%40.-!@%)#K0,&L!!*+*5$)10(725!.J:RHXPXmHXXHL!
""%!
!
!
Concluding remarks Concluding remarks and future perspectives If I had to find a metaphor that would summarise the main conclusions of this thesis, it would be the famous quote by Jacques Cousteau: “People protect what they love, and they love what they understand”. We find ourselves in a time in human history where the rate at which biodiversity is being lost is without parallel. Climatic changes together with the pressure exerted on natural habitats by human activities are accelerating the pace at which living species disappear, mainly in consequence of habitat loss. This is a mater of the greatest importance and should seriously concern all of those who care about the future of life on this planet. We should not be naïve. Significant loss of biodiversity over a short period of time will most likely result in the disruption of natural balances that are guarantee of major ecological services. Since there are no reversal perspectives of the accelerated process of biodiversity loss in the near future, we should expect consequences, e.g., more common and more devastation plagues and diseases affecting both human population as well as crops. In order to address this mater, something is paramount, and that is knowledge. Until we do not fully understand a problem, we will not be able to properly act upon it. It has been shown here that there are serious gaps in our knowledge of conservational statuses of a major slide of the biodiversity cake. Tardigrades have been used to set an example for all the taxa that require studying under the discipline of Conservation Biology. It has also been demonstrated that populations of these small animals are negatively affected by habitat destruction, in a similar way to macroscopic fauna. Hopefully, this will have been the first of many more future studies alike, focusing on other groups of life that have so far never been considered under this topic. Another necessary step forward in the path for a sharper understanding of the biodiversity of the Phylum Tardigrada is the update of methods used for describing new taxa, in particular (but not only) species. If future findings make use of the integrative approach that I have made a case for, incorrect descriptions shall be created with increased rarity, and old ones may fall at a faster rate. I believe I have 116 Tardigrada: a study on integrative taxonomy, impacts on biodiversity and concerns with conservation
shown the advantages of integrative taxonomy, not only in resolving puzzling cases involving synonymous species, but also in determining the actual phylogenetic distances between morphologically distinct populations. This will help determine the true evolutionary importance of morphological characters. Making use of only traditional taxonomy, it is much harder or even impossible, at times, to establish the line that separates in-­‐species morphological variability from intra-­‐species differences. It is my strong conviction that the taxonomy of tardigrades will meet a revolution in the near future thanks to integrative taxonomy, and our perspective of the biological diversity of these animals will change significantly, with a more common adoption of this integrative perspective, particularly with a generalization of genetic analysis. On the mater of the eutardigrade colonizer Peritrichid species, considerable advances have been made, since very little knowledge existed to date on Pyxidium tardigradum. The first ever live and SEM images were obtained, its morphology looked into in detail, its feeding behaviour studied and registered,. Infestation rates were measured and a change in the classification of the animal-­‐protozoan relationship was proposed. The phylogenetic position of this species was successfully determined and the first insight into population’s genetic variability was given. Nevertheless, much is left to be done. Proper quantification of the detrimental effects imposed on eutardigrades is required. Nothing is known about the way in which the protozoan binds to eutardigrades’ cuticle; or if some eutardigrade species are more affected than others and play a more prominent role in dispersing the ciliate. Finally, only a superficial look was given to P. tardigradum’s genetic richness and the question of whether it is a true cosmopolitan or an ensemble of closely related species remain unanswered. 117