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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

5

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

Filipe Vicente – Doutoramento em Biologia

6

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.


7

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.

Filipe Vicente – Doutoramento em Biologia

8

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.


9

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.

General Introduction

10

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).


11

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,

General Introduction

12

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.


13

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.

Paper 1

Micro-‐invertebrates conservation: forgotten biodiversity.

Filipe Vicente (2010) Biodiversity Conservation 19: 3629-‐3634.

Micro-‐invertebrates conservation

17

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.

Paper 1

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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,


19

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-‐

Paper 1

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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


21

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

Paper 1

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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;


23

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.

Paper 1

24

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.

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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

31

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.

Paper 2

32

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.


33

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.

Paper 2

34

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.


35

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).

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36

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

Macrobiotus cf. macrocalix 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 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,


37

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 -‐ -‐

Macrobiotus vladimiri F1 -‐ -‐ -‐ -‐ F1

Minibiotus furcatus -‐ G1 -‐ -‐ G2 -‐

Bryodelphax parvulus -‐ H1 H1 -‐ -‐ -‐

Hypsibius pallidus -‐ -‐ -‐ -‐ -‐ -‐

Ramazzottius cf. oberhaeuseri -‐ -‐ -‐ -‐ -‐ -‐

Echiniscus quadrispinosus I1 -‐ -‐ -‐ -‐ -‐

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).

Paper 2

38

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.


39

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.

Paper 2

40

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).


41

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).

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Paper 3

Considerations on the taxonomy of the Phylum Tardigrada

Filipe Vicente & Roberto Bertolani

Zootaxa (submitted)


47

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

Paper 3

48

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, Calohypsibius, Parhexapodibius, Thulinius

5

Testechiniscus, Astatumen, Microhypsibius, Insuetifurca 4 Archechiniscus, Dipodarctus, Wingstrandarctus, Raiarctus, Rhomboarctus, Anisonyches, Oreella, Bryochoerus, Auteruseus, Hebesuncus, Halobiotus, Ramajendas, Xerobiotus, Apodibius

3

Euclavarctus, Parmursa, Chrysoarctus, Orzeliscus, Parechiniscus, Eohypsibius, Haplomacrobiotus, Mesocrista, Parascon, Eremobiotus

2

Trogloarctus, Clavarctus, Exoclavarctus, Moebjergarctus, Proclavarctus, Ligiarctus, 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

1

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


49

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

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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.,

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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.


51

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.

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

55

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

Integrative taxonomy allows the identification of synonymous species

56

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.

Paper 4

57

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,


58

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

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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 18S

Testechiniscus spitsbergensis (Scourfield, 1897)

HM193419 HM193385, EU266967-‐8

Echiniscus spiniger Richters, 1904b HM193408 HM193376 Echiniscus wendti Richters, 1903 GU329528 Echiniscus merokensis Richters, 1904a FJ435813 FJ435719 Echiniscus bigranulatus Richters, 1908 HM193406 HM193373 Echiniscus viridissimus Péterfi, 1956 HM193409 AF056024 Echiniscus trisetosus Cuénot, 1932 FJ435815 FJ435718 Echiniscus canadensis Murray, 1910 FJ435814 Echiniscus testudo (Doyère, 1840) EF620368-‐81,

EU244601 GQ849022

Echiniscus blumi Richters, 1903 EF620382, EU046090, EU046098, EU046168, EU046197-‐8, HM193407

HM193375, EU049476, EU049482, EU049486

Echiniscus jenningsi Dastych, 1984 EU266969 Echiniscus granulatus (Doyère, 1840) DQ839606 Echiniscus sp. EF620367 EF632453, EF632457,

EF632458, EU266964, EU266971, EU266974, EU266976

Bryodelphax parvulus Thulin, 1928 HM193371 Bryodelphax sp. EF632434 Cornechiniscus lobatus (Ramazzotti, 1943)

EU038077, EU038079, HM193372

Mopsechiniscus granulosus Mihelčič, 1967 HM193379 Hypechiniscus exarmatus (Murray, 1907a) HM193377 Hypechiniscus gladiator (Murray, 1905) HM193378 Parechiniscus chitonides Cuénot, 1926 HM193380 Proechiniscus hanneae (Petersen, 1951) HM193381 Pseudechiniscus islandicus (Richters, 1904c)

HM193383

Pseudechiniscus facettalis Petersen, 1951 FJ435720, HM193382 Pseudechiniscus sp. EU266965 Milnesium tardigradum Doyère, 1840 GQ925696


60

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).

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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).


62

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

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(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).


64

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

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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).


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Table 3 – Measurements of specimens attributable to E. oihonnae and E. multispinosus (values in µm).

Structures Mean Standard deviation

Min Max Number of 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.

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 1 C3039 Et.01 E. oihonnae 2 C3040 V03 E. oihonnae 0.034 3 C3041 V02 E. oihonnae 0.000 0.034 4 C3042 V07 E. oihonnae 0.012 0.040 0.012 5 C3250 V01 E. oihonnae 0.007 0.041 0.007 0.014 6 C3250 V04 E. oihonnae 0.000 0.034 0.000 0.012 0.007 7 C3250 V08 E. oihonnae 0.019 0.040 0.019 0.024 0.024 0.019 8 C3250 V11 E. multispinosus 0.018 0.038 0.018 0.022 0.022 0.018 0.000 9 C2257 V03 T. spitsbergensis 0.197 0.195 0.197 0.202 0.196 0.197 0.199 0.196 10 HM193419 T. spitsbergensis 0.192 0.189 0.192 0.194 0.191 0.192 0.196 0.195 0.006

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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 D

1 E. oihonnae-‐multispinosus 0.019 2 T. spitsbergensis 0.195 0.006 3 E. spiniger 0.196 0.197 NP 4 E. wendti 0.208 0.221 0.218 NP 5 E. merokensis 0.213 0.206 0.222 0.229 NP 6 E. blumi-‐canadensis 0.199 0.191 0.191 0.200 0.194 0.134 7 E. bigranulatus 0.201 0.222 0.190 0.192 0.215 0.198 NP 8 E. viridissimus 0.180 0.208 0.182 0.163 0.249 0.189 0.189 NP 9 E. trisetosus 0.196 0.217 0.185 0.191 0.180 0.088 0.190 0.190 NP 10 E. testudo 0.200 0.187 0.177 0.210 0.182 0.194 0.192 0.188 0.200 0.007 11 Echiniscus n. sp. 0.208 0.192 0.184 0.189 0.208 0.184 0.182 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.


68

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.

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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.


70

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.

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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.


72

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

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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.


74

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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 on Pyxidium tardigradum

81

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.

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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.


83

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

width w are known: 2

2234

⎟⎠

⎞⎜⎝

⎛⋅⋅=wlv π . We also estimated volumes of eighty three random fixed

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, 21χ =0.04).

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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.


85

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).

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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.


87

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

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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)


89

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.

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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)


91

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)

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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)


93

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

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94

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


95

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.

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96

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).


97

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

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Dastych, H. (1984) The tardigrade from Antarctic with description of several new species. Acta

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Concluding remarks

116

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


117

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.

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