Speeding up the Snail's Pace Bird

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Please be advised that this information was generated on 2021-10-07 and may be subject to change. SPEEDING UP THE SNAIL’S PACE Bird-mediated dispersal of aquatic organisms

Casper H.A. van Leeuwen

Speeding up the snail’s pace

Bird-mediated dispersal of aquatic organisms The work in this thesis was conducted at the Netherlands Institute of Ecology (NIOO-KNAW) and Radboud University Nijmegen, cooperating within the Centre for Wetland Ecology.

This thesis should be cited as: Van Leeuwen, C.H.A. (2012) Speeding up the snail’s pace: bird-mediated dispersal of aquatic organisms. PhD thesis, Radboud University Nijmegen, Nijmegen, The Netherlands

ISBN: 978-90-6464-566-2

Printed by Ponsen & Looijen, Ede, The Netherlands Speeding up the snail’s pace

Bird-mediated dispersal of aquatic organisms

PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op het gezag van de rector magnificus prof. mr. S.C.J.J. Kortmann, volgens besluit van het College van Decanen in het openbaar te verdedigen op woensdag 27 juni 2012 om 13.00 uur precies

door

Casper Hendrik Abram van Leeuwen

geboren op 18 september 1983 te Odijk Promotoren: Prof. dr. Jan van Groenendael Prof. dr. Marcel Klaassen (Universiteit Utrecht)

Copromotor: Dr. Gerard van der Velde

Manuscriptcommissie: Prof. dr. Hans de Kroon Dr. Gerhard Cadée (Koninklijk NIOZ) Prof. dr. Edmund Gittenberger (Universiteit Leiden) Prof. dr. Andy Green (Estación Biológica de Doñana, CSIC, Spain) Prof. dr. Luc de Meester (KU Leuven, Belgium) Contents

1. General introduction 7

2. Gut travellers: internal dispersal of aquatic organisms by waterfowl 17

3. Experimental quantification of long distance dispersal potential of 49 aquatic snails in the gut of migratory birds

4. Vector activity and propagule size affect dispersal potential by vertebrates 67

5. Prerequisites for flying snails: external transport potential of aquatic 85 snails by waterbirds

6. How did this snail get here? Multiple dispersal vectors revealed for an 101 aquatic invasive species

7. A snail on four continents: bird-mediated dispersal of a parasite vector? 121

8. Synthesis 141

Summary 161

Nederlandse samenvatting 165

Dankwoord 169

Affiliations of co-authors 173

Publication list 174

Curriculum vitae 175

Chapter 1

General introduction

Casper H.A. van Leeuwen

7 Chapter 1

Dispersal Movement of species in the landscape is essential for the long term survival of their populations. It enables species to colonize new suitable habitat and escape potential deteriorating conditions in their present habitat, which is crucial for their prolonged existence in a continuously dynamic environment (Cain et al., 2000; Holt, 2003; Lest- er et al., 2007). Biological dispersal, defined as the directional movement away from a source location to establish or reproduce, occurs in nearly all species and has important consequences at the individual, population and community level (Clobert et al., 2001; Bullock et al., 2002; Nathan, 2006). It occurs at all spatial scales, ranging from vertical movements of larvae in the water column (Sundelöf & Jonsson, 2011) to pollen drifting in wind over many kilometres in rainforests (Ndiade-Bourobou et al., 2010). Dispersal thereby influences most ecological and evolutionary processes (Dieckmann et al., 1999). However, for many species it is still unknown how and in which directions they disperse (Clobert et al., 2001). Dispersal allows individuals to find other locations where there may be more nutrients and less diseases and predators (McKinnon et al., 2010; Altizer et al., 2011), and can dilute negative effects of inbreeding or loss of genetic variation in populations (Hamilton & May, 1977; Fayard et al., 2009). Nowadays, in an attempt to cope with global change processes, a species’ ability to escape from rapidly deteriorating environmental conditions or exploit new suitable areas may be of even greater relevance. Dispersal can thereby positively affect biodiversity if it allows species to e.g. follow changing climatic boundaries, maintain populations in increasingly fragmented landscapes or avoid genetic drift in small populations (Kokko & Lopez- Sepulcre, 2006; Pearson, 2006). On the other hand, biodiversity can be negatively affected by increased dispersal rates if species with very good dispersal abilities expand their home ranges drastically and thereby outcompete other species after invasion (Phillips et al., 2006; Van der Velde et al., 2010).

Vector-mediated dispersal Despite the ecological significance of dispersal for many species, the heterogeneity of the earth’s landscape may restrict their movement. Whereas it is often relatively easy to travel within a certain habitat type (e.g. a forest, ocean or lake), it becomes more challenging when this involves crossing a different type of terrain over a longer distance, i.e. an ecological barrier (Cain et al., 2000). Nevertheless, even remote islands in the ocean and isolated ponds in the desert often harbour a high biodiversity (Schabetsberger et al., 2009; Jocque et al., 2010). Many species are able to cross ecological barriers to suitable patches using their own propulsion, i.e. active dispersers (Jenkins et al., 2007, see also Fig. 1.1). Others, so-called passive dispersers, require transport by vectors (Zickovich & Bohonak, 2007). Organisms may be blown across land or sea by the wind (anemochory, e.g. Soons & Ozinga, 2005), can be carried by water (hydrochory, e.g. Van de Meutter et al., 2006), or may be transported by more mobile animals (zoochory, e.g. Enders & Vander Wall, 2011; Pollux, 2011) including humans (e.g. Wichmann et al., 2009). Although passive dispersal can involve transport of whole organisms, it frequently involves transport of dispersal units called “propagules”. These are here considered all (parts of) organisms that

8 General introduction can disperse and regenerate, regardless of their dispersal mode or metabolic state (e.g. plant seeds, fruits, algae spores, cladoceran ephippia, bryozoan statoblasts, pieces of plants that can regenerate). Islands provide ideal model systems to investigate vector-mediated dispersal of propagules (MacArthur & Wilson, 1967), because island biodiversity is largely determined by which species are able to disperse across another habitat type from (distant) source populations (Gillespie et al., 2008). Freshwater habitats, often referred to as “islands in a sea of land” (essay of 1844 of C. Darwin, in F. Darwin 1909), are particularly suitable to study dispersal of aquatic organisms over land. The fact that many wetlands harbour a high biodiversity (Green et al., 2002b; Junk et al., 2006) seems contrasting to their often high degree of isolation, and can usually not entirely be explained by wind- and water-mediated dispersal. This paradox already fascinated Darwin over 150 years ago (Darwin, 1859). It made him propose waterbirds as potential dispersal vectors for aquatic organisms. Waterbirds travel fast, directed and in large numbers between ecologically similar habitats (Figuerola & Green, 2002; Green et al., 2002a; Nathan et al., 2008). Terres- trial birds are already known to be effective dispersers of plant seeds and fruits that survive digestion after ingestion (internal transport or endozoochory, reviewed by Traveset, 1998). Seeds and fruits may also be transported on feet or between feathers of birds (external transport or ectozoochory, reviewed by Sorensen, 1986). How- ever, in comparison to what is known in terrestrial ecosystems, knowledge on the potential of waterbirds to disperse aquatic organisms is still limited (reviewed by Figuerola & Green, 2002; Green & Figuerola, 2005).

Species distributions

Capacity to Capacity to disperse survive in environment

Active dispersal Passive dispersal by vectors (own propulsion)

Animals Wind Water

Birds Other animals, e.g. Chapter 6 mammals, fish, insects at small scale

Internal External Chapter 7 dispersal dispersal at large scale

Chapter 2,3,4 Chapter 5 Molecular genetic Mechanistic approach approach

Figure 1.1: Conceptual diagram of the topics addressed in this thesis. Species distributions are determined by their capacity to disperse through as well as survive in their environment. Dispersal can be either passive by vectors such as animals, wind or water, or active by own propulsion. In this thesis the mechanistic approach is used to investigate internal and external dispersal by birds, whereas the molecular genetic approach addresses dispersal on small scales by multiple vectors and dispersal on a large scale by birds.

9 Chapter 1

This is surprising, given the presumably even higher relevance of dispersal between discrete, isolated aquatic systems than within more continuous terrestrial habitats. The aim of this thesis was therefore to assess the importance of waterbirds for the dispersal of aquatic organisms (Fig. 1.1).

Waterbirds as vectors for small aquatic species The idea of waterbirds as dispersal vectors originates from Darwin (1859), followed by Kew (1893) and Ridley (1930). After the idea was mentioned, scientists started to report observations of small species adhering to waterbirds that were caught in the field for other purposes (Adams, 1905; McAtee, 1914). These field observations were extended by a series of experiments on waterbird-mediated dispersal by Proctor and Malone in the 1960’s (e.g. Proctor et al., 1967; Proctor, 1968). The reports remained anecdotal for a long time, until the topic received renewed interest during the last two decades (Fig. 1.2). This new interest was probably initiated by new questions on species conservation and the rapid spread of invasive species (Kokko & Lopez- Sepulcre, 2006), and encouraged by the increasing availability of new research techniques such as analyses of genetic variability. The most recent studies today indicate that many intact propagules of aquatic plants and invertebrates can be found in droppings of waterbirds (e.g. Figuerola et al., 2003; Frisch et al., 2007). Many of these species found in droppings can also survive digestion when they are fed experimentally to birds in captivity (reviewed by Charalambidou & Santamaría, 2002). Nevertheless, even now a large proportion of all studies is still anecdotal, especially with respect to external transport (Fig. 1.2). Experiments on internal transport have been three times as frequent as those on external transport (Fig. 1.2). The literature that is currently available leaves many questions on waterbird- mediated dispersal unexplored, of which we address part in this thesis. To further specify which knowledge is still lacking on waterbird-mediated dispersal, Chapter 2 first of all investigates the taxonomic diversity of aquatic species dispersed by waterbirds. Therefore, data is extracted from all peer-reviewed literature on endozoochorous dispersal by waterfowl to date. Using a meta-analysis approach, it is shown which aquatic species are currently known to be dispersed by waterbirds. Quantitative analyses on the data specify how different bird species may contribute to dispersal of different propagules, and how much birds may quantitatively contribute to dispersal of aquatic species. The data exposes patterns in characteristics of propagules as well as their vectors, which are subsequently investigated in the following chapters of this thesis. Chapters 3, 4 and 5 thereby use a mechanistic approach, while Chapters 6 and 7 use a molecular genetic approach (Fig. 1.1). The large variety of aquatic organisms with potential for bird-mediated dispersal identified in Chapter 2 first of all raised the question to what extent aquatic organisms may need specific adaptations for endozoochorous transport. Could it be that propagules are suitable for endozoochory without specific adaptations? This question is addressed mechanistically by investigating the taxonomic extent of endozoochory in Chapter 3, by studying the dispersal potential of a number of aquatic

10 General introduction

Field observations: 19 na l r

Experiments: 33 e t n I Anecdotal: 9

Field observations:14 na l r e

Experiments: 12 t x Anecdotal: 22 E

Distribution patterns: 23

1850 1875 1900 1925 1950 1975 2000

Year ! Figure 1.2: Scientific publications on waterbird-mediated dispersal of aquatic organisms. Internal (triangles) and external (squares) transport have both been investigated by catching birds in the field (Field observations), experimentally (Experiments) and have been reported after accidental observation (Anecdotal). Publications inferring dispersal from species distributions or genetic variability between populations are indicated by open circles (Distribution patterns). The numbers on the y-axis indicate the total number of publications to date in the different categories. The frequency distribution of all studies is shown on top of the figure. snail species (Gastropoda). Aquatic snails are well adapted to survive in a variety of environmental circumstances, but are not known to be adapted for, or even capable of, endozoochory. Bird-mediated dispersal could thus be an explanation for the widespread distributions and invasive behaviour of many aquatic snails. The survival and retrieval of four species of aquatic snails was investigated in experimental setups with mallards (Anas platyrhynchos), and the successful gut passage of one of these species is discussed in an evolutionary context. What does their survival imply for snail distributions, which characteristics allow them to survive passage through a bird’s digestive system and what does the survival of aquatic snails learn us about endozoochory? Whereas experimental assessment of retention times and survival of various propagules such as described in Chapter 2 is valuable, a common problem of this

11 Chapter 1 type of experiments is that the potential dispersal distance of propagules (i.e. the dispersal kernel, the function that describes the probability of dispersal to different distances) is calculated from retrieval patterns of resting animals. Since vectors in natural situations will be actively moving during effective transport rather than resting, we need to know how physical activity of vector animals might affect dispersal kernels and survival of propagules. InChapter 4, digestive characteristics are compared between swimming, wading (i.e. resting in water) and isolated (i.e. resting in a dry cage) mallards. By feeding differently sized plastic markers, the effect of propagule size on retention time was included in the experiment, and by feeding aquatic snails the effect of activity on digestive intensity was investigated. Chapter 4 discusses the importance of propagule size for dispersal, and how the lack of vector activity in many previous experiments may have affected inferred propagule release patterns. Whereas the above chapters addressed only endozoochory, snails may also be transported by adhering to birds on the outside, i.e. by ectozoochory. Many anecdotal observations exist of snails carried by birds, but there has been little experimental verification of these observations. In Chapter 5, this is addressed mechanistically by monitoring snail attachment under varying experimental circumstances and determining their potential to stay adhered. Whether aquatic snails have the prerequisites for successful transport by birds was assessed by monitoring snail adhesion behaviour and survival of desiccation during potential transport for a selection of common species from The Netherlands. The results are discussed with respect to the ectozoochorous dispersal potential of aquatic snails. The above chapters indicated that internal and external transport of multiple organisms is mechanistically possible. However, can these processes also be important in natural situations in the field? Since transport of individuals often leads to transport of genes, genetic analyses are a suitable tool to measure actual dispersal in the field. InChapter 6, genetic analyses were used to investigate dispersal of Physa acuta, an invasive freshwater snail that occurs abundantly in a group of isolated, temporary wetlands in Southern Spain. Because its dispersal vectors to and from these remote wetlands were unknown, population genetic structure was inferred from microsatellites and used to identify potential dispersal vectors. Special focus was on the potential role of waterbirds as dispersal vectors. Dispersal was investigated on a much larger scale in Chapter 7, by analysing the world-wide distribution and phylogenetic position of a specific aquatic snail, Galba truncatula, which is an intermediate host of trematode parasites such as the Liver fluke (Fasciola hepatica). Migration routes of waterbirds were related to the current distribution of this snail, and the worldwide populations were compared based on ribosomal DNA (internal transcribed spacer 1). The potential role of waterbirds for dispersal of aquatic snails between continents is addressed. In Chapter 8, a synthesis of all the previous chapters is presented, after which the impact of bird-mediated dispersal on aquatic systems is discussed.

12 General introduction

References Adams, L.E. (1905) A plover with Anodonta cygnea attached to its foot. Journal of Conchology, 11, 175 Altizer, S., Bartel, R. & Han, B.A. (2011) Animal migration and infectious disease risk. Science, 331, 296-302 Bullock, J.M., Kenward, R.E. & Hails, R.S. (2002) Dispersal ecology. Blackwell Science. Cain, M.L., Milligan, B.G. & Strand, A.E. (2000) Long-distance seed dispersal in plant populations. American Journal of Botany, 87, 1217-1227 Charalambidou, I. & Santamaría, L. (2002) Waterbirds as endozoochorous dispersers of aquatic organisms: a review of experimental evidence. Acta Oecologica-International Journal of Ecology, 23, 165-176 Clobert, J., Danchin, E., Dhondt, A.A. & Nichols, J.D. (2001) Dispersal. Oxford University Press, Oxford. Darwin, C. (1859) On the origin of species by means of natural selection, or the preservation of fa- voured races in the struggle for life. John Murray, London. Darwin, F. (1909) The foundations of The origin of species. Two essays written in 1842 and 1844. University Press, Cambridge. Dieckmann, U., O’Hara, B. & Weisser, W. (1999) The evolutionary ecology of dispersal. Trends in Ecology & Evolution, 14, 88-90 Enders, M.S. & Vander Wall, S.B. (2011) Black bears Ursus americanus are effective seed dispersers, with a little help from their friends.Oikos , 121, 589-596 Fayard, J., Klein, E.K. & Lefèvre, F. (2009) Long distance dispersal and the fate of a gene from the colonization front. Journal of Evolutionary Biology, 22, 2171-2182 Figuerola, J. & Green, A.J. (2002) Dispersal of aquatic organisms by waterbirds: a review of past research and priorities for future studies. Freshwater Biology, 47, 483-494 Figuerola, J., Green, A.J. & Santamaría, L. (2003) Passive internal transport of aquatic organisms by waterfowl in Donana, south-west Spain. Global Ecology and Biogeography, 12, 427-436 Frisch, D., Green, A.J. & Figuerola, J. (2007) High dispersal capacity of a broad spectrum of aquatic invertebrates via waterbirds. Aquatic Sciences, 69, 568-574 Gillespie, R.G., Claridge, E.M. & Roderick, G.K. (2008) Biodiversity dynamics in isolated island communities: interaction between natural and human-mediated processes. Molecular Ecology, 17, 45-57 Green, A.J., Figuerola, J. & Sanchez, M.I. (2002a) Implications of waterbird ecology for the dispersal of aquatic organisms. Acta Oecologica-International Journal of Ecology, 23, 177-189 Green, A.J., Hamzaoui, M.E., El Agbani, M.A. & Franchimont, J. (2002b) The conservation status of Moroccan wetlands with particular reference to waterbirds and to changes since 1978. Biological Conservation, 104, 71-82 Green, A.J. & Figuerola, J. (2005) Recent advances in the study of long-distance dispersal of aquatic invertebrates via birds. Diversity and Distributions, 11, 149-156 Hamilton, W.D. & May, R.M. (1977) Dispersal in stable habitats. Nature, 269, 578-581 Holt, R.D. (2003) On the evolutionary ecology of species’ ranges. Evolutionary Ecology Research, 5, 159-178 Jenkins, D.G., Brescacin, C.R., Duxbury, C.V., Elliott, J.A., Evans, J.A., Grablow, K.R., Hille- gass, M., Lyon, B.N., Metzger, G.A., Olandese, M.L., Pepe, D., Silvers, G.A., Suresch, H.N., Thompson, T.N., Trexler, C.M., Williams, G.E., Williams, N.C. & Williams, S.E. (2007) Does size matter for dispersal distance?Global Ecology and Biogeography, 16, 415-425

13 Chapter 1

Jocque, M., Vanschoenwinkel, B. & Brendonck, L. (2010) Freshwater rock pools: a review of habitat characteristics, faunal diversity and conservation value. Freshwater Biology, 55, 1587- 1602 Junk, W., Brown, M., Campbell, I., Finlayson, M., Gopal, B., Ramberg, L. & Warner, B. (2006) The comparative biodiversity of seven globally important wetlands: a synthesis. Aquatic Sciences - Research Across Boundaries, 68, 400-414 Kew, H. (1893) The dispersal of shells: An inquiry into the means of dispersal possessed by fresh-water and land Mollusca. Kegan Paul, Trench, Trubner & Co., London, England. 291 pp Kokko, H. & Lopez-Sepulcre, A. (2006) From individual dispersal to species ranges: perspectives for a changing world. Science, 313, 789-791 Lester, S.E., Ruttenberg, B.I., Gaines, S.D. & Kinlan, B.P. (2007) The relationship between dispersal ability and geographic range size. Ecology Letters, 10, 745-758 MacArthur, R.H. & Wilson, E.O. (1967) The theory of island biogeography. Monographs in Popula- tion Biology. Princeton University Press. McAtee (1914) Birds transporting food supplies. Auk, 31, 404-405 McKinnon, L., Smith, P.A., Nol, E., Martin, J.L., Doyle, F.I., Abraham, K.F., Gilchrist, H.G., Morrison, R.I.G. & Bêty, J. (2010) Lower predation risk for migratory birds at high latitudes. Science, 327, 326-327 Nathan, R. (2006) Long-distance dispersal of plants. Science, 313, 786-788 Nathan, R., Schurr, F.M., Spiegel, O., Steinitz, O., Trakhtenbrot, A. & Tsoar, A. (2008) Mecha- nisms of long-distance seed dispersal. Trends in Ecology & Evolution, 23, 638-647 Ndiade-Bourobou, D., Hardy, O.J., Favreau, B., Moussavou, H., Nzengue, E., Mignot, A. & Bouvet, J.M. (2010) Long-distance seed and pollen dispersal inferred from spatial genetic structure in the very low–density rainforest tree, Baillonella toxisperma Pierre, in Central Africa. Molecular Ecology, 19, 4949-4962 Pearson, R.G. (2006) Climate change and the migration capacity of species. Trends in Ecology & Evolution, 21, 111-113 Phillips, B.L., Brown, G.P., Webb, J.K. & Shine, R. (2006) Invasion and the evolution of speed in toads. Nature, 439, 803-803 Pollux, B.J.A. (2011) The experimental study of seed dispersal by fish (ichthyochory). Fresh- water Biology, 56, 197-212 Proctor, V.W., Malone, C.R. & DeVlaming, V. (1967) Dispersal of aquatic organisms - viability of disseminules recovered from intestinal tract of captive killdeer. Ecology, 48, 672-676 Proctor, V.W. (1968) Long-distance dispersal of seeds by retention in digestive tract of birds. Science, 160, 321-& Ridley, H.N. (1930) The distribution of plants throughout the world. Reeve & Company, Ashford, United Kingdom. Schabetsberger, R., Drozdowski, G., Rott, E., Lenzenweger, R., Fersabek, C.D., Fiers, F., Traun- spurger, W., Reiff, N., Stoch, F., Kotov, A.A., Martens, K., Schatz, H. & Kaiser, R. (2009) Los- ing the bounty? Investigating species richness in isolated freshwater ecosystems of Oceania. Pacific Science, 63, 153-179 Soons, M.B. & Ozinga, W.A. (2005) How important is long-distance seed dispersal for the regional survival of plant species? Diversity and Distributions, 11, 165-172 Sorensen, A.E. (1986) Seed dispersal by adhesion. Annual Review of Ecology and Systematics, 17, 443-463 Sundelöf, A. & Jonsson, P.R. (2011) Larval dispersal and vertical migration behaviour – a

14 General introduction

simulation study for short dispersal times. Marine Ecology, online early Traveset, A. (1998) Effect of seed passage through vertebrate frugivores’ guts on germination: a review. Perspectives in Plant Ecology Evolution and Systematics, 1, 151-190 Van de Meutter, F., Stoks, R. & De Meester, L. (2006) Lotic dispersal of lentic macroinverte- brates. Ecography, 29, 223-230 Van der Velde, G., Rajagopal, S. & Bij de Vaate, A. (2010) The Zebra mussel in Europe. W. Back- huys, Leiden/ Margraf Publishers, Weikersheim. Wichmann, M.C., Alexander, M.J., Soons, M.B., Galsworthy, S., Dunne, L., Gould, R., Fairfax, C., Niggemann, M., Hails, R.S. & Bullock, J.M. (2009) Human-mediated dispersal of seeds over long distances. Proceedings of the Royal Society B-Biological Sciences, 276, 523-532 Zickovich, J.M. & Bohonak, A.J. (2007) Dispersal ability and genetic structure in aquatic invertebrates: a comparative study in southern California streams and reservoirs. Freshwater Biology, 52, 1982-1996

15 16 Chapter 2

Gut travellers: internal dispersal of aquatic organisms by waterfowl

Casper H.A. van Leeuwen, Gerard van der Velde, Jan M. van Groenendael & Marcel Klaassen

Journal of Biogeography (under revision)

17 Chapter 2

Abstract High biodiversity of less mobile organisms at isolated locations suggests passive dispersal is a regularly occurring process. However, to what extent remains unclear. We aimed to determine the contribution of birds as potential vectors transporting plant seeds and macro-invertebrates. Birds are renowned vectors for terrestrial plants, but less acknowledged for their role in more dispersal-dependent aquatic systems. We therefore performed a meta-analysis on bird-mediated endozoochorous dispersal of aquatic species. We analysed data from 81 peer-reviewed publications on endozoochorous dispersal of aquatic plant seeds and macro-invertebrates by waterbirds. In total, 38% of 1649 waterbird droppings collected in the field contained one or more intact propagules, with macro-invertebrates found almost as frequent as plant seeds. Positive droppings contained on average 3.3 intact propagules, of which one-third viable. In 728 trials in 17 published feeding experiments, on average 24% of the ingested propagules were retrieved intact, with ~6.5% both viable and intact. As many as 17 species of Anatidae and Rallidae were found involved in dispersing at least 39 species of macro-invertebrates and 97 species of plant seeds from a wide taxonomic range. Smaller propagules seemed less affected by digestion than larger ones. We provide a first quantitative model that can be used for any system of interest to estimate bird-mediated propagule dispersal between wetlands. It indicates an average bird can potentially disperse five viable propagules after flying more than 100 km, and one additional propagule after flying 300km. Birds have the potential to transport a wide variety of aquatic plants and animals over several hundreds of kilometres. High survival of propagules might be explained by propagule adaptations or by the fact that birds maximize energy absorption over time rather than assimilation efficiency. Our meta-analysis suggests that waterbirds might contribute significantly to wetland biodiversity around the world, but also identified many limitations of our current knowledge for which we outline avenues for future research.

Key words: Anatidae, aquatic propagules, digestive physiology, long-distance dispersal, macro-invertebrates, plant seeds, Rallidae

18 Gut travellers: internal dispersal of aquatic organisms by waterfowl

Introduction The presence of organisms at remote and isolated locations in the landscape has fascinated scientists for long (Darwin, 1859; Ridley, 1930), and this interest is ongoing (Gittenberger et al., 2006). Species distributions often range across deserts and include remote oceanic islands (Schabetsberger et al., 2009; Jocque et al., 2010), suggesting high species mobility in the landscape (Lester et al., 2007). However, for many species it is still unknown how and how often they can reach remote habitat (Cain et al., 2000). An increased understanding of potential dispersal vectors and dispersal frequency is essential for understanding (meta)-population functioning and community dynamics (Puth & Post, 2005), and forms the basis for understanding the movement of invasive species and species threatened by habitat fragmentation or global change (Kokko & Lopez-Sepulcre, 2006). To study dispersal and meta-population dynamics, islands provide excellent model systems because of their discrete character (MacArthur & Wilson, 1967; Gil- lespie et al., 2008). Wetlands, which can be considered “islands in a sea of land” (Dar- win, 1909), are particularly suitable to study dispersal of aquatic organisms. Many wetlands are isolated from other aquatic areas but still harbour a high biodiversity of aquatic organisms, and are often even colonized by species with low mobility. This apparent paradox already fascinated Darwin over 150 years ago (Darwin, 1859), and made him hypothesize dispersal of aquatic organisms by waterbirds over land. Waterbirds might be suitable transport vectors of aquatic organisms because of their frequent, directed movements between ecologically similar habitats (Figuerola & Green, 2002; Green et al., 2002; Bohonak & Jenkins, 2003; Nathan et al., 2008), notably in comparison to other potential vectors, such as randomly dispersing wind (Jenkins & Underwood, 1998; Soons, 2006), unidirectional water flows limited to river or flood occurrence (Jacquemyn et al., 2010; Pollux, 2011), irregular anthropogenic activities (Wichmann et al., 2009; Waterkeyn et al., 2010; Kappes & Haase, 2011) or slower, less abundant non-avian animals (Bilton et al., 2001; Vanschoenwinkel et al., 2008). Nowadays, birds are identified as important dispersers of many terrestrial plant seeds (for review see Traveset, 1998), but our knowledge on their potential role in the dispersal of aquatic organisms, including animals, is still relatively limited. Anatidae (ducks, geese and swans) and Rallidae (coots, rails, gallinules and crakes) have been found to carry small propagules, such as algae spores (Schlichting, 1960; Kristiansen, 1996) and viruses (e.g. Winker & Gibson, 2010) between aquatic habitats. More recently, also evidence for transport of larger propagules such as aquatic plant seeds and aquatic macro-invertebrates has accumulated (Figuerola & Green, 2002). Increasingly more large propagules are being found to either survive digestion (endozoochory) or stay attached externally (ectozoochory) to flying waterbirds. We here review the currently existing publications on endozoochorous transport of aquatic organisms by waterbirds. We include both the relatively well studied dispersal of plant seeds (e.g. Charalambidou & Santamaría, 2002) and the still less acknowledged transport of freshwater macro-invertebrates. Our review complements earlier reviews addressing bird-mediated dispersal of aquatic invertebrates (Bilton

19 Chapter 2 et al., 2001; Figuerola & Green, 2002; Malmqvist, 2002; Okamura & Freeland, 2002; Bohonak & Jenkins, 2003; Green & Figuerola, 2005). By analysing the currently available field and experimental data, we now provide a first (quantitative) meta-analysis of bird-mediated dispersal. We focus on both the vector and propagule species, thereby restricting ourselves to endozoochorous dispersal because of the even more limited data available on ectozoochory. We provide a taxonomic overview of which aquatic organisms have been suggested capable of internal dispersal by waterbirds, and consider physiological traits of the birds that can potentially explain their suitability for passive dispersal. Bird species are ranked according to their suitability for carrying propagules internally, which reflects their potential impact on community composition of aquatic organisms. Our meta-analysis provides a first quantitative estimate of the number of viably dispersed propagules of plants and macro-invertebrates. Collectively, this will increase our understanding on how birds might provide connections between fragmented or changing habitats (Brooker et al., 2007) or transport invasive species (Puth & Post, 2005; Suarez & Tsutsui, 2008).

Methods

Data collection Publications were collected by searching the online databases of e.g. ISI, PubMed, Scopus and Google scholar. Most publications from before ~1980 were retrieved from the Zoological Record and library databases of Dutch universities as well as the literature archive of NCB Naturalis, Leiden, The Netherlands. Initial search words included “dispersal”, “waterbirds”, “water fowl”, “endozoochory”, “internal transport”, and combinations of these, which was combined with the use of cited references. We reviewed a total of 81 peer-reviewed papers referring to bird mediated dispersal in freshwater habitats to date (listed in Appendix Table 2.S1). The analysis was restricted to endozoochorous dispersal of freshwater macro-invertebrates (>0.5mm) and aquatic and semi-aquatic macrophyte propagules (mainly angiosperm seeds, but including liverwort spores and Characeae oogonia) found in or on the shores of freshwater habitats. Brackish and saline species were excluded, except for the particularly well studied, salt-tolerant Ruppia maritima. Although birds are also known as important dispersal vectors for e.g. terrestrial plants and algae, this has previously been treated elsewhere (algae: Kristiansen, 1996; terrestrial plants: Traveset, 1998). Related review publications of potential interest to the read- er as an additional source are listed at the end of Appendix Table 2.S1. We focused on Anatidae and Rallidae as vectors, and their capacity to disperse propagules by ingestion, transport and release in droppings. Dispersal by mammals, reptiles and fishes was excluded, as were publications on the external transport of invertebrates and seeds by birds. We made a distinction between bird species as predominantly omnivores and predominantly herbivores following Bruinzeel et al. (1997), and depicted this separation in Fig. 2.1a/b and Table 2.3. We consider this division as representing predominant diet, assuming some herbivores may feed opportunistically on invertebrates from time to time.

20 Gut travellers: internal dispersal of aquatic organisms by waterfowl

Meta-analysis data In all 81 publications we found i) 9 anecdotal publications in which bird-mediated dispersal was merely indicated as possible dispersal mechanism or evidence consisted of a single observation; ii) 23 publications on distribution patterns and genetic analyses: dispersal by birds was inferred or suggested based on species distribution patterns or genetic variability analyses;iii ) 30 publications describing endozoochory experiments: waterbirds were fed a known amount of propagules and retrieval was monitored; iv) 16 published field observations: publications that present results of dropping collections in the field;v ) 18 previously published reviews of relevance: reviews related to dispersal by water birds potentially of interest to readers interested in transport of aquatic propagules by birds. Three additional publications contained data for both category iii and iv. All used publications are indicated in Appendix Table 2.S1. Data for the meta-analysis was extracted from publications in categories iii and iv, Seventeen publications from category iii carried out propagule feeding experiments in a comparable way. Fifteen of the studies also tested the viability of the retrieved propagules. The setup of studies varied to some extent (e.g. the food availability to the birds during the experiments, time of the year studies were conducted, bird species used), which we either included explicitly in the statistical models or considered in the discussion. From these studies, we extracted the number of birds per species that was fed propagules; the number of propagules fed to experimental birds; the percentages of propagules retrieved at 4h, 8h, 12h and in total during the experiment and the percentage of these retrieved propagules that was viable. Nine publications in category iv collected fresh droppings of Anatidae or Ralli- dae and presented faeces contents in a comparable way and were used in the analysis (indicated in Appendix Table 2.S1). The total number of droppings examined in these studies was 1649, from 17 different bird species. From these publications we extracted the number of droppings examined; the number of droppings containing at least one propagule; the number of propagules per dropping; and the percentage of propagules that was viable for each bird species that was investigated. Publica- tions investigating lower gut contents rather than dropping contents were included in the taxonomic identification of dispersed species, but were not included in the quantitative analyses whereas the number of propagules per dropping remained unknown.

Statistical analyses The influence of physiological and anatomical differences between duck and Ralli- dae species on their ability as dispersal vectors was tested using two generalized linear models and functions in R (R-Development-Core-Team, 2011). In the first model, the square root transformed average number of propagules per dropping followed a Poisson distribution and was modelled using a quasi-Poisson error function to correct for non-integer values, with log link function using “glm” of package “stats”. In the second model, the viability of propagules was analysed with a binomial error distribution with log link function, using function “lm”.

21 Chapter 2

Table 2.1: Overview of macro-invertebrates suggested capable of transport by birds. The dispersal of 4 phyla, 7 classes, 24 families and at least 39 species is described in 44 publications. The “+” indicates groups of organisms for which the exact number of species being dispersed is unknown, yet manifold. Dispersal vectors are divided over dabbling ducks, diving ducks and Rallidae, with specific species codes corresponding to the first three letters of the specific Latin name. If no dispersal vectors are given, the literature referred to “waterbirds” and evidence is anecdotal or inferred from distributions and flyways of waterbirds in general.

# of propagule # of Dabbling Diving Phylum Class Families species papers ducks ducks Rallidae Bryozoa Phylactolaemata Cristatellidae 2 6 ACU CLY ANG Fredericellidae 1 1 PLA Pectinatellidae 1 1 PLA CLY CRE FER ATR Plumatellidae 2 5 PLA STR RUF POR Mollusca Gastropoda Hydrobiidae 2 5 PLA Lithoglyphidae 1 1 Lymnaeidae 1 1 PLA Physidae 2 2 PLA Valvatidae 1 1 Arthropoda Branchiopoda Artemiidae 1 4 PLA Branchinectidae 1 1 Cercopagididae 1 1 ACU CLY Chydoridae 2 2 CRE PLA ACU CAR CLY CRE Daphniidae 6 11 PEN PLA ATR CLY CRE Moinidae 1 3 PLA Sididae 2 2 PLA Streptocephalidae 1 2 PLA Thamnocephalidae 2 2 SUP ATR Malacostraca Atyidae 2 2 Gammaridae 1 1 AFF Triopsidae 1 2 PLA Insecta Chironomidae 1 1 ANG Corixidae 2 2 ACU CLY FER ATR PEN PLA ANG STR Crysomelidae 1 1 Ostracoda + 5 CLY CRE ATR PLA Maxillopoda + 1 PLA (Copepoda) Nematoda + 3 ANG ATR

22 Gut travellers: internal dispersal of aquatic organisms by waterfowl

Table 2.2: Overview of wetland macrophytes suggested capable of transport by birds: internal dispersal by birds has been found in 4 orders of Monocots and 10 orders of Eudicots, one species of Nymphaeales and one species of Marchantiophyta. In total the dispersal of 97 wetland plant species from 26 plant families have been described in 34 publications. Dispersal vectors are divided over dabbling ducks, diving ducks and Rallidae, with specific species codes corresponding to the first three letters of their specific Latin name. If no dispersal vectors are given, the literature referred to “waterbirds” and evidence is anecdotal or inferred from distributions and flyways of waterbirds in general.

# of propagule # of Dabbling Diving Rallidae Division/Clade Order Families species papers ducks ducks Angiospermae/ Alismatales Alismataceae 2 3 CRE PLA Monocotyledoneae Araceae 1 1 ATR Najadaceae 1 1 PLA

Potamogetonaceae 7 11 CLY CRE ANG ATR

PEN PLA

Ruppiaceae 1 4 ACU CLY ANG ATR

CRE PEN FER

PLA STR RUF

Asparagales Iridaceae 1 1 PLA

Commelinales Pontederiaceae 2 2 CRE Poales Cyperaceae 31 14 ACU CAR ANG ATR CLY CRE COL POR PLA SUP RUF Juncaceae 3 4 CRE PLA Poaceae 7 7 CRE PLA Typhaceae 6 4 CRE PLA ATR SUP Angiospermae/ Apiales Apiaceae 2 1 PLA Eudicotyledoneae Brassicales Brassicaceae 1 1 PLA Caryophyllales Amaranthaceae. 3 3 ACY CLY ANG ATR CRE PEN FER PLA STR Droseraceae 1 1

Polygonaceae 8 7 CRE PLA

Portulaceae 1 1 Ericales Myrsinaceae 1 1 PLA Fabales Fabaceae 1 1 Leguminosae 1 1 CRE Lamiales Lamiaceae 2 1 PLA Myrtales Lythraceae 1 1 PLA

Onagraceae 3 2 CRE PLA

Ranunculales Ranunculaceae 1 2 CRE PLA ANG Rosales Rosaceae 2 2 CRE PLA Saxifragales Haloragaceae 2 3 CRE SUP ATR Angiospermae/ Nymphaeales Nymphaeaceae 1 1 PLA Nymphaeales Marchantiophyta Sphaerocarpales Riellaceae 1 1 PLA Charophyta Characeae 3 7 ACU CRE ANG ATR PLA

23 Chapter 2

Macro-‐invertebrate propagules in droppings Plant seeds in droppings (a) (b) Anas clypeata n= 71 Marmarone?a angusFrostris n= 22 Anas acuta n= 64 Anas clypeata n= 109 Tadorna tadorna n= 3 Tadorna tadorna n= 7 Fulica atra n= 283 Ne?a ruﬁna n= 10 Anas penelope n= 16 Anas crecca n= 201 Anas acuta n= 95 Anas platyrhynchos n= 209 Anas platyrhynchos n= 341

Aythya ferina n= 22 Anas strepera n= 38 Anas gracilis n= 30 Anas penelope n= 44 Marmarone9a angus>rostris n= 12 Anas gracilis n= 30 Ne9a ruﬁna n= 11 Fulica atra n= 346 Anas strepera n= 31 Anas crecca n= 218 Porphyrio porphyrio n= 17 Aythya ferina n= 33 Gallinula chloropus n= 5

0 20 40 60 80 100 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 % with at least one propagule % viable % with at least one propagule % viable

Figure 2.1a/b: Percentage of droppings with at least one intact macro-invertebrate propagule (a) or seed (b) and the percentage of these propagules that was viable. On the vertical axes bird species are ranked according to decreasing quantitative dispersal capacity, calculated from both the prevalence and viability of propagules in their droppings. n denotes the number of droppings collected. Underlined species are considered predominantly herbivores.

24 Gut travellers: internal dispersal of aquatic organisms by waterfowl

0 20 40 60 80 100 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 % with at least one propagule % viable % with at least one propagule % viable

25 Chapter 2

The number of droppings examined in the field was included as weighting factor in both models, because data based on more droppings collected (i.e. number of propagules per dropping calculated over more droppings) was considered to be more accurate than counts based on fewer droppings. Body mass, gizzard mass and gut length of the birds were extracted from the avian literature and included as centred covariates. Since these covariates were unique per bird species, bird species could not be included in the models as an additional factor. Because only a limited number or even unique bird species were tested per study, including study as random factor (using “glmer” from package “lme4”) inhibited the detection of effects of covariates; observed differences were thus caused by analysing between rather than within studies. For this reason, results should be treated with caution, keeping in mind that this is a meta-analysis of different studies using different methodologies that cannot all statistically be unravelled (yet). Interactions and the fixed factors feeding guild and “subfamily” (i.e. diving duck, dabbling ducks or Rallidae) were not included because they resulted in overdetermination of the models. This data is presented informatively in Tables 2.1, 2.2 and 2.3.

Results

Propagules collected from droppings in the field Dispersal by waterbirds is recurrently investigated by analysing freshly collected droppings from the field for intact and viable propagules. Our database indicated plants to be present in 45.4% and invertebrates in 32.3% of the samples. Aquatic macro-invertebrates were found in droppings almost as often as plant seeds (W = 1055, p = 0.06; n = 1332 and 1024 droppings investigated for content of plant and invertebrate propagules, respectively). Of all positive droppings, on average 3.32 (+ 0.70 SE) intact propagules were found per dropping (plants: 2.85 + 0.70SE, invertebrates: 3.75 + 1.16SE, W = 783, p = 0.89). Across the 53% (1257) of all examined droppings for which propagule viability was tested, 35.8% of the plant propagules and 30.3% of the macro-invertebrates was viable (which was not significantly different between plants and invertebrates, W = 159, p = 0.17). On average, ~12% of all droppings contained either one viable plant seed or one viable macro-invertebrate propagule (Fig. 2.1a/b).

Taxonomy of dispersed aquatic propagules To date, at least 39 species of macro-invertebrates, divided over 24 families from 7 classes in 4 phyla, have been suggested to be dispersed by waterbirds, based on data from 44 different publications (Table 2.1). Bryozoa, several species of water fleas (Daphnia sp.) and some Insecta (Corixidae) are among the best documented passively transported species. Findings of Mollusca are mainly based on anecdotal observations with limited experimental evidence. Ostracods, nematods and copepods are probably transported in large numbers but not always assigned to species level, thus they are included in Table 2.1 as “multiple species”. Species from 26 angiosperm families have been found capable of bird-mediated dispersal by endozoochory, divided over four orders of monocots and 10 orders of

26 Gut travellers: internal dispersal of aquatic organisms by waterfowl eudicots. In total the dispersal of 97 wetland macrophyte species has been described in 33 publications (Table 2.2). The salt-tolerant Ruppia maritima was found capable of dispersal by all vector species for which this was tested (Figuerola et al., 2002). The number of species per family that were identified to date as capable of dispersing by birds, correlates to the number of publications that tested this (r = 0.75). We made a survey of vectors involved in dispersing the different propagules, and indicated their division over “subfamilies” (Tables 2.1 and 2.2).

Propagule survival in feeding experiments Up to now, seventeen publications included endozoochorous dispersal experiments from which data could be extracted, in all of which either seeds or aquatic macro-invertebrates were fed in a total of 668 trials with dabbling ducks, 38 trials with diving ducks and 22 trials with Rallidae (total 728 of which 387 with mallards, Anas platyrhynchos). Smaller propagules were significantly retrieved in larger quantities (Fig. 2.2), although note that this relation had an R2 value of 0.06 only. The regression was mostly based on experiments in which plant seeds were fed (95% of the data), but the available macro-invertebrate data was in line with the plant seed data (Fig. 2.2). 100 80 60 40 ●

y = −1.1x + 21.9 20

Percentage of propagules retrieved after feeding of propagules retrieved Percentage ● ●

●

0 ●

0 2 4 6 8 10 12 14

Propagule size in mm

Figure 2.2: The total percentage of ingested propagules retrieved intact over the duration 2 of experiments was lower for larger propagules (lin regr: F1,125 = 5.86, p < 0.02, R = 0.06). Effect size of propagule size was -1.1 + 0.45 SE as indicated in the equation in the graph. Open symbols denote plant seeds, and closed symbols macro-invertebrate propagules. Fifty- three per cent of the experimental birds were mallards (triangles), circles are other duck and Rallidae species. The regression was also significant for mallards only, as well as for all other species excluding mallards.

27 Chapter 2

Six published studies, reporting the results of 281 feeding trials, reported passage rates at four hour intervals allowing direct comparison of propagule passage rates. Retrieval was typically finished after 12 hours: on average 68.1% (+ 19.7 SD) of all retrieved propagules were excreted within 4 hours, 85.0% (+ 14.7 SD) within 8 hours and 92.3% (+ 10.8 SD) within 12 hours. Across all studies, on average 23.5% of the ingested propagules were retrieved intact, of which 27.9% were retrieved viable. This indicates that experimentally, on average ~6.5% of the initially ingested propagules was retrieved both intact and viable.

Taxonomic patterns in vector species Diving ducks (Aythyinae), dabbling ducks (Anatinae) and Rallidae (predominantly Fulica atra) did not differ in their quantitative dispersal capacity based on the limited dropping collection data currently available. The (sub)families had on average 3.4 (+ 5.0 SD, n = 79 droppings), 3.6 (+ 7.4, n = 1028) and 2.1 (+ 3.4, n= 450) propagules per dropping, respectively. Qualitatively in terms of dispersed species, dabbling ducks have been found to disperse more invertebrate and plant species (Table 2.3), but not if corrected for the larger number of droppings investigated for dabbling ducks to date (diving ducks: ~4 droppings investigated per newly identified species, dabbling ducks: ~10 droppings per species, Rallidae: ~20 droppings per species). Not all species dispersed by diving ducks and Rallidae are also dispersed by dabbling ducks, as indicated by the overlap in Table 2.3. Geese were mainly involved in the dispersal of terrestrial seeds (Willson et al., 1997; Chang et al., 2005; Bruun et al., 2008) and thus not included. Viability of propagules was similar between (sub)families based on the currently available data, i.e. 48% of the propagules was viable for diving ducks (tested for n=45 droppings), 34% for dabbling ducks (n=772) and 34% for Rallidae (n=379). The viability of propagules (Fig. 2.1a/b) was on average 32% for herbivores (n = 453) and 34% for omnivores (n =743), with respectively 2.0 + 3.4SD and 3.9 + 7.2SD propagules per dropping. However, note that species considered as herbivores also dispersed invertebrates (Table 2.3).

Digestive physiology and anatomy Birds with a higher body mass and smaller gizzard mass excreted more propagules 2 per dropping (GLM: F3,48 = 14.4, R = 0.47 for the entire model; body mass: p < 0.01; effect size = 0.19 propagules per dropping for an increase of 100 g body mass per bird; gizzard mass p < 0.04, effect size = -0.005 propagules per dropping with a 1 g increase of gizzard mass; gut length had no effect). In the model testing for propagule viability there was a small positive effect of bird body mass on viability of the excreted propagules after removing insignificant effects of gizzard mass and gut 2 length (log regr: F1, 64 = 6.05, R = 0.09 for the entire model; body mass: p < 0.02, 1.4% more chance of retrieving a viable propagule if body mass increases 100 grams).

Quantitative dispersal estimate The collected data allowed us to calculate a first quantitative estimate of the average

28 Gut travellers: internal dispersal of aquatic organisms by waterfowl

Table 2.3: Number of plant and invertebrate species known to be dispersed by different waterbird vectors grouped by (sub)family, including overlap between (sub)families.

vector species (sub)family # invertebrate species # plant species Unspecified "waterbirds" 17 2

Anas acuta Dabbling 5 6 Anas carolinensis Dabbling 1 3 Anas clypeata Dabbling 9 4 Anas crecca Dabbling 6 26 Anas penelope Dabbling 3 3 Anas platyrhynchos Dabbling 19 61 Anas strepera Dabbling 4 2 Anas superciliosa Dabbling 1 4 Dabbling ducks total variety 24 80

Aythya affinis Diving 1 0 Aythya collaris Diving 0 3 Aythya ferina Diving 4 2 Marmaronetta angustirostris Diving 5 8 Netta rufina Diving 2 3 Diving ducks total variety 8 11

Fulica atra Rallidae 9 13 Porphyrio porphyrio Rallidae 1 4 Rallidae total variety 9 15

Overlap dabbling and diving 4 10 Overlap Rallidae and diving 5 5 Overlap Rallidae and dabbling 7 10 number of propagules dispersed by waterbirds over distance. We combined the average number of propagules per dropping, their viability, bird dropping rates, the flight speed of waterfowl, and retention times of ingested propagules. Thirty-eight per cent of the droppings were found positive, with an average of 3.32 propagules per positive dropping. Dropping rates ranged between 6.5 and 9.7 droppings per hour for herbivorous vector species (Bruinzeel et al., 1997; Hahn et al., 2008). For car- nivores this is generally lower, i.e. 2.4 to 3.1 droppings per hour (Hahn et al., 2007). We modelled dropping rates from the full range of 2.4 to 9.7 droppings per hour, considering it to vary between these values due to differences between species, bird feeding guild, and factors like the diet of an individual bird at a certain moment. Propagule release decreased exponentially over time based on the six feeding experiment studies that reported detailed propagule release over time intervals of 4 hours

29 Chapter 2

up to now (Charalambidou et al., 2003a; Charalambidou et al., 2003b; Charalambidou et al., 2003c; Pollux et al., 2005; Wongsriphuek et al., 2008; Figuerola et al., 2010). We fitted an exponentially declining function to the data retrieved from these six publications (for equation see Fig. 2.3). This exponential decline (68% after 4h, 85% after 8h, and 92% after 12h) was further supported by an experiment monitoring nightin- gales (Luscinia megarhynchos) flying in a wind tunnel, of which dropping rates were measured continuously and decreased exponentially to 25% of its initial rate after 8 hours (M. Klaassen, A. Kvist, Å. Lindström, unpublished data). Viability of aquatic propagules has also been shown to decrease exponentially with increasing retention time (Charalambidou et al., 2003c; Charalambidou et al., 2005; Pollux et al., 2005), with an average regression coefficient of 0.0688+ 0.0243 (limited to n=4, but consistent over studies). Viability of propagules collected in the field was on average 33.6%. Since 92% of the propagules would have been excreted in the first 12 hours according to feeding experiments, we fitted an exponential declining function with a regression coefficient of 0.0688 and an average of 33.6% over the first 12 hours as the declining viability curve (for equation see Fig. 2.3). We deliberately did not include possible positive effects of gut passage, such as enhanced germination (Santamaría et al., 2002), because this is a variable phenomenon with both intra- and interspecific differences still hardly understood (Traveset, 1998; Espinar et al., 2004; Traveset et al., 2008; Brochet et al., 2010a). The average flight speed of waterfowl is ~75km h-1, but is known to vary with flight conditions and species from as low as 42km h-1 up to 116km h-1 (Welham, 1994; Bruderer & Boldt, 2001; Clausen et al., 2002; Miller et al., 2005). Using this flight speed data and our model depicted in figure 2.3, we can now calculate quantitative dispersal for any hypothetical bird. For example, an average bird flying at 75 km h-1 will produce 6.1 droppings per hour, and would thus excrete 3 viable propagules during the first hour of flying (visualized in Fig. 2.3). These will be excreted between the point of ingestion and the location of the birds’ destination, at a maximum possible distance of 75km from the origin. Assuming the bird continues its flight without foraging or resting, dropping rate and propagule viability will continue to decrease exponentially over time, but the bird will still release cumulatively 4.1 propagules over the next 3 hours (i.e. 2.0 + 1.2 + 0.9 = 4.1 propagules). After 4 hours (if spent in flight at a maximum of 4*75km = 300 kilometres from the propagules’ origin), it will continue to release propagules at the hourly rates indicated in Fig. 2.3. Each bird departing after a foraging baud and flying at average speed should thus transport at least one propagule over the first 300 kilometres. Similarly, we can estimate propagule transport over distance for any bird species, if we know its behaviour over time including flight speed and direction.

Discussion Our results indicate that waterbirds can transport a variety of aquatic organisms by endozoochory, with more than twice as many plant (97) than macro-invertebrate species (39) being implicated in this dispersal process (Tables 2.1 and 2.2). These numbers can easily be much larger and we hope that our overview stimulates future work on underrepresented groups of organisms for which bird-mediated dispersal

30 Gut travellers: internal dispersal of aquatic organisms by waterfowl is suspected, as well as vector species of which relatively little is known to date. The correlation between the number of identified dispersed species and the number of publications investigating these same species suggested future work on more organisms will also reveal dispersal of more species. In this respect investigating by screening dispersal of macro-invertebrates in a systematic way is of special interest, because these appear to be equally abundant in droppings as plant seeds. As a first step towards assessing the quantitative importance of dispersal by waterbirds, a model is presented with which viable propagule release can be calculated (Fig. 2.3). Although dispersal itself does not directly inform on the potential of species to establish in a new situation because unfavourable conditions can limit species establishment (Jenkins & Buikema, 1998; De Meester et al., 2002), a first step is to know the amount of potential propagules that might be introduced. The model predicted that true long distance dispersal of propagules (>1000 km) only occurs at a very low frequency, and will be restricted to fast flying birds during long-distance migratory movements in spring and autumn. Propagule viability rapidly decreases with time in the digestive system, and dropping rate will likewise decrease without new input of food. The dispersal potential seems most significant for propagules remaining in birds for only several hours.

5 Viable High dropping rate propagules released 4 per hour

Low dropping rate 0 0 1 2 3 4 5 6 7 8 Hours after ingestion

Figure 2.3: The potential number of viable propagules excreted by waterbirds per hour based on feeding experiment data extracted from the literature. Dropping release over time was calculated by [Dropping release = e 2.465 - T * 0.196, with T representing time in hours since ingestion of a propagule]. Viability was found decreasing exponentially over time according to [Percentage of propagules viable = e 3.938 - T * 0.0688 / 100, with T representing time in hours since ingestion]. The number of viable propagules dispersed at time T was then calculated by [Dropping release * Percentage of propagules viable]. The upper and lower dashed lines represent bird species with high and low dropping rates, respectively, compared to average dropping rates (solid line).

31 Chapter 2

Within this time period, birds on migration might cover several hundreds of kilometres. Birds moving at a more local scale during the breeding season or making flights between feeding and roosting areas are estimated to frequently connect habitats tens of kilometres apart. Although the model only provides a first estimate and true dispersal will depend on more factors than included at this stage, it offers indicative propagule transport of birds of interest with known foraging and flight behaviour. For instance, the potential connectivity of two wetlands on a migratory route might be calculated based on the distance between these wetlands and the number of birds that yearly visit both sites during migration. In case there is information on the size of bird colonies on an island that are known to feed on the mainland, transport of a propagule species of interest to the island might likewise be estimated. However, the model provides an estimate rather than an exact value. Whether or not model calculations are correct for specific field situations would still require testing specific systems.

High quantitative dispersal explained The high frequency with which intact propagules can be found in bird droppings suggests an underlying mechanism. First of all, propagule survival might be due to characteristics of the propagules. Adaptations for gut passage have to date been found in some plant seeds, of which germination increased after gut passage (e.g. Santamaría et al., 2002). However, for many other surviving plant seeds and macro-invertebrates, no obvious adaptations have yet been identified. Most dispersed species seem merely adapted to survival in stochastic environments, which adaptations also provide suitability for dispersal by birds. It would be of interest to use our overview of dispersed taxa for a phylogenetic analysis of the dispersed species and their characteristics in the future. If we can identify species characteristics or potential adaptations in propagules related to bird-mediated dispersal, we can specifically explore the dispersal potential of species with similar characteristics, or even start to understand potential underlying evolutionary processes of bird-mediated dispersal. A second potential explanation for the survival of (non-adapted) propagules may be found in a digestive trade-off in the vector animals. Although survival of propagules seems inefficient for foraging vectors because they effectively excrete undigested food, foraging birds will achieve maximum net energy and nutrient gain over time at lower than maximal assimilation efficiency (defined as the energy absorbed as a proportion of energy ingested (Castro et al., 1989)). An increase in assimilation efficiency increases retention time (e.g. Prop & Vulink, 1992) and therewith decreases total food intake over time if food is abundant. In natural situations, the assimilation efficiency of a range of food types is therefore ~75% in most birds (Castro et al., 1989; Karasov & Levey, 1990). Assimilation efficiency can become even lower if for instance the food intake rate increases (Kersten & Visser, 1996; Clauss et al., 2007; Van Gils et al., 2008) or the bird’s digestive system is atrophied in prepa- ration for migration (e.g. McWilliams & Karasov, 2001). Often, part of all ingested food items will remain undigested, which provides a window of opportunity for

32 Gut travellers: internal dispersal of aquatic organisms by waterfowl propagules to be excreted intact. How digestive flexibility varies between bird species, within and between seasons, and is dependent on activities such as flying and swimming, diet shifts, and changes in amount and timing of food ingestion, are interesting future directions of (experimental) research that will increase our understanding of propagule dispersal.

Differences between birds as vectors The fact that various bird species are involved in the dispersal of different propagules (Tables 2.1, 2.2, and 2.3) can first of all be attributed to differences in investigation effort. In both experiments and field collections, most studies focused on dabbling ducks (and notably mallards). There is a bias of investigated vector species (Table 2.3), and the number of investigated droppings per bird species (Fig. 2.1a/b). This should give future researchers a handle to concentrate their studies on the least investigated species. Diving ducks are of special interest, because despite the relatively small amount of collected droppings and performed experiments, the number of identified species per investigated dropping was highest. Both diving ducks and Rallidae are known as dispersal vectors for some propagules not (yet) known to be dispersed by dabbling ducks, in particular several macro-invertebrate species. True variation between dispersal vectors, to date only sporadically investigated (but see Charalambidou et al., 2003c), can probably be attributed to a combination of differences in diet and digestive physiology (e.g. Figuerola et al., 2003; Green et al., 2008). The diet and thereby qualitative dispersal of all species varies with season and food availability, because most of the investigated species are opportunistic feeders. Seasonal and dietary effects on the dispersal of propagules have been included in some studies (see Figuerola et al., 2003), but did not provide enough data in our meta-analysis for a more elaborate investigation and requires more attention in future studies. Potential effects of bird physiology were addressed statistically. Despite the limited available data, we found quantitative dispersal slightly increasing with increasing body mass and decreasing with increasing gizzard mass, although these factors together only explained 47% of the observed variability. Birds with high body mass were also found to excrete a higher percentage of all excreted propagules viably. The causes for this remain speculative. Although our calculations suggest potential higher importance of larger bird species for dispersal, larger birds typically have a lower dropping rate (Hahn et al., 2008) and smaller population sizes, thus other factors might reduce the number of propagules excreted over time by birds with higher body masses. The available data to date should best be expanded experimentally, to enable investigating differences between bird species in similar circumstances (e.g. Charalambidou et al., 2003c). Preferably their digestive systems should be allowed to adjust to the diet of interest (Charalambidou et al., 2005), and effects of fasting and timing of eating should be included (e.g. Figuerola & Green, 2005). We found no differences in quantitative dispersal capacity between bird species classified as predominantly herbivorous or omnivorous (feeding guild is indicated in Table 2.3 and Fig. 2.1a/b). Both guilds dispersed macro-invertebrates and plant

33 Chapter 2 seeds. Since assimilation efficiency of animal matter is usually higher than that of plant matter (Swanson & Bartonek, 1970; Castro et al., 1989), it is plausible that birds classified as predominant herbivores deliberately forage on macro-invertebrates as opportunistic feeders. In addition, the abundance of macro-invertebrates on or around macrophytes or floating between accumulated seeds may promote accidental ingestion (Janzen, 1984). Consumed intentionally or not, both omnivorous and herbivorous birds are likely dispersal vectors for aquatic macro-invertebrates.

Propagule variation The dispersal success of a propagule depends on vector characteristics, but is also influenced by the characteristics of the propagule itself. Propagule size has been put forward as an important adaptive trait for terrestrial (e.g. Traveset et al., 2001) and aquatic propagules (e.g. DeVlaming & Proctor, 1968; Mueller & Van der Valk, 2002; Soons et al., 2008; Wongsriphuek et al., 2008). However, investigations into the effect of size on survival and retrieval of aquatic propagules have yielded contrasting results in past studies (Figuerola et al., 2010) and hardly included macro-invertebrates. Our meta-analysis indicated that smaller propagules were retrieved in higher total percentages than larger propagules, confirming previous similar findings on plant seeds in mallards (Soons et al., 2008) across studies. However, the low statistical power of this relation and lack of invertebrate studies indicates more future work is needed. The available data suggested a propagule of 1 mm to have almost twice as much chance of being endozoochorously dispersed after ingestion than a propagule of 10 mm (Fig. 2.2). In future work aimed at substantiating this estimate, one of the challenges is to isolate the effect of propagule size from associated variation on morphological characteristics. This can be resolved by using indigestible markers in feeding experiments (e.g. Charalambidou et al., 2005; Figuerola & Green, 2005), although these also have limitations. They can be retained as grit or invoke a different response of the digestive system on indigestible objects. Studies comparing effects of propagule size should preferably feed a mix of differently sized propagules to avoid different loading volumes of the digestive system when feeding equal numbers of differently size propagules. The amount of propagules fed should be a trade-off between finding a low percentage of surviving organisms and overfeeding unrealistic large amounts of propagules resulting in regurgitation. Indicative, successful previous studies fed on average 100 - 500 propagules with an average size of 3.5 mm per trial to birds the size of a mallard. Smaller propagules are not only excreted in larger amounts, but they have also been shown to pass the digestive system slower (Figuerola et al., 2010) and are thus retained longer. Since longer retention implies more inflicted damage (e.g. DeVlam- ing & Proctor, 1968; Afik & Karasov, 1995; Bauchinger et al., 2009), a future question arises whether smaller propagules escape digestion more easily or are more resistant to digestion. In conclusion, smaller propagules will have both highest survival and farthest dispersal by long retention, and will likely be produced and ingested in higher numbers in natural situations (Bruun & Poschlod, 2006; Brochet et al., 2010b). Conclusions and implications

34 Gut travellers: internal dispersal of aquatic organisms by waterfowl

We have shown that waterbirds have the potential to disperse a large variety of aquatic organisms in quantitatively substantial numbers, but we also showed that our knowledge on this subject is still limited. Our meta-analysis indicates large variation between studies and many opportunities for additional research. Today, dispersal is of considerable interest because of the spread of invasive species and continuous fragmentation of habitats, (climatic) changes of habitats that require species to move as the spatial arrangement of suitable habitat also moves in the landscape. Furthermore, the relevance of increasing our knowledge on this subject is indicated by the worldwide geographical range of the studies involved: we included information from European migratory stopover sites and their bird and propagule species (Figuerola et al., 2003; Pollux et al., 2005; Soons et al., 2008; Brochet et al., 2009), North American birds and propagules (DeVlaming & Proctor, 1968; Mueller & Van der Valk, 2002; Wongsriphuek et al., 2008) and these organisms in Australia (Green et al., 2008; Raulings et al., 2011). This suggests bird-mediated dispersal occurs on a global scale and in many different wetland systems. The impact of bird-mediated dispersal on specific aquatic systems can thus be expected to be dependent on the local abundance of waterbirds. In areas with high waterbird densities such as stopover sites on migratory routes and wetlands frequently used as roosting sites, bird-mediated transport might be especially important. The limited knowledge on dispersal of aquatic organisms by other vectors such as wind, humans and other animals prevents an estimation of the relative importance of birds for the biodiversity of wetlands. We therefore provided a first estimation of the transport potential by birds, so this can now be compared to the transport potential of other vectors in specific systems. If waterbirds indeed play a considerable role in transport of other organisms, this important contribution to the biodiversity of wetlands should be recognized. Both the habitat and the birds that act as vectors maintaining this biodiversity should be protected.

Acknowledgements We would like to thank Andy J. Green, David G. Jenkins and one anonymous reviewer for useful comments on a previous version of this manuscript.

References

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40 Gut travellers: internal dispersal of aquatic organisms by waterfowl

Appendix Chapter 2

Table 2.S1: Publications used for the meta-analysis. Grouped as either i) anecdotal publications: bird-mediated dispersal was merely indicated as possible dispersal mechanism or evidence consisted of a single observation; ii) distribution patterns and genetic analyses: dispersal by birds was inferred or suggested based on species distribution patterns or genetic variability analyses; iii) endozoochory experiments: waterbirds were fed a known amount of propagules and retrieval was monitored; iv) field observations: publications that present results of dropping collections in the field; v) previously published reviews of relevance: reviews related to dispersal by water birds potentially of interest to readers interested in transport of aquatic propagules by birds. The addressed taxa are indicated with either Plantae, Animalia or a mix of plants and animals addressed in the same publication. In the column “use in analyses”, “field data” indicates publications used in the quantitative meta-analysis of dropping contents; “no field data: gut contents” indicates the part of the field collection publications that examined gut contents rather than dropping contents, these studies were excluded from the quantitative dropping calculations; “experimental data” indicates the publications used as endozoochory experiments; “data reported dissimilar” indicates publications that were dissimilar of setup or that reported data in an incomparable way and were thus excluded from the analyses. All publications were included in the tables of taxonomic species dispersed (Tables 2.1 and 2.2).

Author - Year – Journal Taxa addressed Use in analyses

Anecdotal publications Bondesen & Kaiser 1949 Oikos Animalia Cejka 1994 Biologia Animalia Cotton 1960 South Australian Ornithologist Animalia Darwin 1859 Animalia Frömming 1956 Duncker & Humblot, Berlin Animalia Glöer & Georgiev 2011 Journal of Conchology Animalia Guppy 1897 Prococeedings of the Royal Physiological Plantae Society of Edinburgh Löffler 1963 Vogelwart Animalia Luther 1963 Acta Vertebrata Mixed

Distribution pattern and genetic analyses Crease et al. 1997 Heredity Animalia Dillon & Wethington 1995 Systematic Biology Animalia Elansary et al. 2010 Aquatic Botany Plantae Figuerola et al. 2005 American Naturalist Plantae Freeland et al. 2000 Journal of Evolutionary Biology Animalia Hatton-Ellis et al. 2002 Archiv für Hydrobiologie Animalia Hawes 2009 Antarctic Science Animalia Hubendick 1950 Zoologiska bidrag fran Uppsala Animalia Ketmaier et al. 2008 Aquatic Sciences Animalia Mader et al. 1998 Aquatic Botany Plantae Mende et al. 2010 European Journal of Entomology Plantae Miller et al. 2006 Freshwater Biology Animalia Okamura & Freeland 2002 Dispersal Ecology Animalia Page & Hughes 2007 Limnology and Oceanography Animalia Page et al. 2007 Molecular Phylogenetics and Evolution Animalia Saunders et al. 1993 Journal of Crustacean Biology Animalia Taylor et al. 1998 Evolution Animalia Von Oheimb et al. 2011 PLoS One Animalia Weider & Hobaek 1997 Heredity Animalia Weider et al. 1996 Molecular Ecology Animalia Wesselingh et al. 1999 Geologie en Mijnbouw Animalia Wilmer et al. 2008 Molecular Ecology Animalia Worthington-Wilmer & Wilcox 2007 Conservation Genetics Animalia

41 Chapter 2

Author - Year – Journal Taxa addressed Use in analyses

Endozoochory experiments Agami & Waisel 1986 Oecologia Plantae experimental data Brochet et al. 2010 Aquatic Botany Plantae experimental data Brown 1933 Transactions of the American Microscopical Animalia experimental data Society Camenisch & Cook 1996 Aquatic Botany Plantae experimental data Charalambidou et al. 2003 Archiv für Hydrobiologie Animalia experimental data Charalambidou et al. 2003 Diversity and Distributions Animalia experimental data Charalambidou et al. 2003 Functional Ecology Plantae experimental data Charalambidou et al. 2005 Functional Ecology Animalia experimental data Devlaming & Proctor 1968 American Journal of Botany Plantae results reported dissimilar Figuerola & Green 2005 Revue d' Écologie-La terre et la Plantae experimental data Vie Figuerola et al. 2010 Naturwissenschaften Plantae experimental data Grandy 1972 Auk Animalia dissimilar experiment Horne 1966 Transactions of the American Microscopical Animalia in vitro experiment Society Imahori 1954 Kanazawa University, Japan. Plantae dissimilar experiment Johnson & Carlton 1996 Ecology Animalia dissimilar experiment Low 1937 State University, Utah Plantae dissimilar experiment Malone 1965 Texas Technological College Animalia dissimilar experiment Pascal 1891 Journal de Conch Animalia dissimilar experiment Pollux et al. 2005 Freshwater Biology Plantae experimental data Proctor & Malone 1965 Ecology Mixed dissimilar experiment Proctor 1961 Bryologist Plantae results reported dissimilar Proctor 1962 Ecology Plantae results reported dissimilar Proctor 1964 Ecology Mixed results reported dissimilar Proctor et al. 1967 Ecology Plantae results reported dissimilar Proctor 1968 Science Plantae results reported dissimilar Santamaría et al. 2002 Archiv für Hydrobiologie Plantae experimental data Soons et al. 2008 Journal of Ecology Plantae experimental data Smits et al. 1989 Aquatic Botany Plantae experimental data Wongsriphuek et al. 2008 Wetlands Plantae experimental data Thompson & Sparks 1977 American Midland Naturalist Animalia experimental data

Field observations and experiments Mueller & Van Der Valk 2002 Wetlands Plantae experimental data field data Powers et al. 1978 Journal of Wildlife Management Plantae experimental data no field data: gut contents Bell 2000 Phd-thesis Plantae experimental data field data reported dissimilar

Field observations Brochet et al. 2009 Ecography Plantae no field data: gut contents Brochet et al. 2010 Freshwater Biology Plantae no field data: gut contents Brochet et al. 2010 Hydrobiologia Animalia no field data: gut contents Bruun et al. 2008 Oecologia Plantae field data Charalambidou & Santamaría 2005 Wetlands Animalia field data Devlaming & Proctor 1968 American Journal of Botany Plantae no field data: gut contents Figuerola et al. 2002 Journal of Ecology Plantae field data Figuerola et al. 2003 Global Ecology and Biogeography Mixed field data Figuerola et al. 2004 Canadian Journal of Zoology Animalia field data Frisch et al. 2007 Aquatic Sciences Mixed field data

42 Gut travellers: internal dispersal of aquatic organisms by waterfowl

Green & Sanchez 2003 Bird Study Mixed results reported dissimilar Green & Selva 2000 Revue d' Écologie-La Terre et la Vie Animalia field data Green et al. 2008 Freshwater Biology Mixed field data Guppy 1894 Science-gossip Plantae no field data: gut contents Guppy 1906 Vol. 2. Plant Dispersal. Macmillan Co, UK. Plantae no field data: gut contents Raulings et al. 2011 Freshwater Biology Plantae no field data: gut contents

Previously published reviews of relevance Allen 2007 Oecologia Zooplankton Barrat-Segretain 1996 Plant Ecology Plantae Bilton et al. 2001 Annual Review of Ecology and Animalia Systematics Bohonak & Jenkins 2003 Ecology Letters Animalia Brochet et al. 2009 Ecography Plantae Cain et al. 2000 American Journal of Botany Plantae Charalambidou & Santamaría 2002 Acta Oecologica- Endozoochory International Journal of Ecology experiments Clausen et al. 2002 Acta Oecologica-International Journal Plantae of Ecology Figuerola & Green 2002 Freshwater Biology Animalia and plants Gittenberger 2012 Journal of Biogeography Animalia Green et al. 2002 Acta Oecologica-International Journal of Animalia and plants - Ecology endozoochory Green & Figuerola 2005 Diversity and Distributions Animalia Kew 1893 Kegan Paul, Trench, Trubner & Co., London, Mollusca England. 291 pp Kristiansen 1996 Hydrobiologia Algae Malmqvist 2002 Freshwater Biology Animalia in river systems Okamura & Hattonellis 1995 Experientia Bryozoa Rees 1965 Journal of Molluscan Studies Mollusca Swanson 1984 Journal of Wildlife Management Amphipoda

References for Appendix Chapter 2

Agami, M. & Waisel, Y. (1986) The role of mallard ducks (Anas platyrhynchos) in distribution and germination of seeds of the submerged hydrophyte Najas marina L. Oecologia, 68, 473- 475. Allen, M.R. (2007) Measuring and modeling dispersal of adult zooplankton. Oecologia, 153, 135-143. Barrat-Segretain, M.H. (1996) Strategies of reproduction, dispersion, and competition in river plants: A review. Plant Ecology, 123, 13-37. Bell, D. (2000) The ecology of coexisting Eleocharis species. PhD-thesis Bilton, D.T., Freeland, J.R. & Okamura, B. (2001) Dispersal in freshwater invertebrates. Annual Review of Ecology and Systematics, 32, 159-181. Bohonak, A.J. & Jenkins, D.G. (2003) Ecological and evolutionary significance of dispersal by freshwater invertebrates. Ecology Letters, 6, 783-796. Bondesen, P. & Kaiser, E.W. (1949) Hydrobia (Potamopyrgus) jenkinsi Smith in Denmark illustrated by its ecology. Oikos, 1, 252-281. Brochet, A.-L., Guillemain, M., Gauthier-Clerc, M., Fritz, H. & Green, A.J. (2010) Endozoo- chory of Mediterranean aquatic plant seeds by teal after a period of desiccation: Deter- minants of seed survival and influence of retention time on germinability and viability. Aquatic Botany, 93, 99-106. Brochet, A.L., Guillemain, M., Fritz, H., Gauthier-Clerc, M. & Green, A.J. (2009) The role of migratory ducks in the long-distance dispersal of native plants and the spread of exotic plants in Europe. Ecography, 32, 919-928. Brochet, A.L., Gauthier-Clerc, M., Guillemain, M., Fritz, H., Waterkeyn, A., Baltanas, A. &

43 Chapter 2

Green, A.J. (2010) Field evidence of dispersal of branchiopods, ostracods and bryozoans by teal (Anas crecca) in the Camargue (southern France). Hydrobiologia, 637, 255-261. Brochet, A.L., Guillemain, M., Fritz, H., Gauthier-Clerc, M. & Green, A.J. (2010) Plant dispersal by teal (Anas crecca) in the Camargue: duck guts are more important than their feet. Fresh- water Biology, 55, 1262-1273. Brown, C. (1933) A limnological study of certain fresh-water Polyzoa with special reference to their statoblasts. Transactions of the American Microscopical Society, 52, 271-316. Bruun, H.H., Lundgren, R. & Philipp, M. (2008) Enhancement of local species richness in tundra by seed dispersal through guts of muskox and barnacle goose. Oecologia, 155, 101-110. Cain, M.L., Milligan, B.G. & Strand, A.E. (2000) Long-distance seed dispersal in plant populations. American Journal of Botany, 87, 1217-1227. Camenisch, M. & Cook, C.D.K. (1996) Wiesneria triandra (Dalzell) Micheli (Alismataceae): a rare and unusual south Indian endemic. Aquatic Botany, 55, 115-131. Cejka, T. (1994) 1st record of the New-Zealand mollusk Potamopyrgus antipodarum (Gray, 1843), (Gastropoda, Hydrobiidae) from the Slovak section of the Dunaj river. Biologia, 49, 657-658. Charalambidou, I. & Santamaría, L. (2002) Waterbirds as endozoochorous dispersers of aquatic organisms: a review of experimental evidence. Acta Oecologica-International Journal of Ecology, 23, 165-176. Charalambidou, I., Ketelaars, H.A.M. & Santamaría, L. (2003) Endozoochory by ducks: influence of developmental stage of Bythotrephes diapause eggs on dispersal probability. Diver- sity and Distributions, 9, 367-374. Charalambidou, I., Santamaría, L. & Figuerola, J. (2003) How far can the freshwater bryozoan Cristatella mucedo disperse in duck guts? Archiv für Hydrobiologie, 157, 547-554. Charalambidou, I., Santamaría, L. & Langevoord, O. (2003) Effect of ingestion by five avian dispersers on the retention time, retrieval and germination of Ruppia maritima seeds. Func- tional Ecology, 17, 747-753. Charalambidou, I. & Santamaría, L. (2005) Field evidence for the potential of waterbirds as dispersers of aquatic organisms. Wetlands, 25, 252-258. Charalambidou, I., Santamaría, L., Jansen, C. & Nolet, B.A. (2005) Digestive plasticity in mallard ducks modulates dispersal probabilities of aquatic plants and crustaceans. Functional Ecology, 19, 513-519. Clausen, P., Nolet, B.A., Fox, A.D. & Klaassen, M. (2002) Long-distance endozoochorous dispersal of submerged macrophyte seeds by migratory waterbirds in northern Europe - a critical review of possibilities and limitations. Acta Oecologica-International Journal of Ecology, 23, 191-203. Cotton, B.C. (1960) Mollusca eaten by birds.South Australian Ornithologist, 23, 44-47. Crease, T.J., Lee, S.K., Yu, S.L., Spitze, K., Lehman, N. & Lynch, M. (1997) Allozyme and mtD- NA variation in populations of the Daphnia pulex complex from both sides of the Rocky Mountains. Heredity, 79, 242-251. Darwin, C. (1859) On the origin of species by means of natural selection, or the preservation of fa- voured races in the struggle for life. John Murray, London. Devlaming, V. & Proctor, V.W. (1968) Dispersal of aquatic organisms - viability of seeds revo- vered from droppings of captive killdeer and mallard ducks. American Journal of Botany, 55, 20-26. Dillon, R.T. & Wethington, A.R. (1995) The biogeography of sea islands - clues from the pop-

44 Gut travellers: internal dispersal of aquatic organisms by waterfowl

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45 Chapter 2

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46 Gut travellers: internal dispersal of aquatic organisms by waterfowl

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Zhang, H.C. & Wilke, T. (2011) Freshwater biogeography and limnological evolution of the Tibetan Plateau - insights from a plateau-wide distributed Gastropod taxon (Radix spp.). PLoS One, 6, e26307. Weider, L.J., Hobaek, A., Crease, T.J. & Stibor, H. (1996) Molecular characterization of clonal population structure and biogeography of arctic apomictic Daphnia from Greenland and Iceland. Molecular Ecology, 5, 107-118. Weider, L.J. & Hobaek, A. (1997) Postglacial dispersal, glacial refugia, and clonal structure in Russian/Siberian populations of the Arctic Daphnia pulex complex. Heredity, 78, 363-372. Wesselingh, F.P., Cadée, G.C. & Renema, W. (1999) Flying high: on the airborne dispersal of aquatic organisms as illustrated by the distribution histories of the gastropod genera Tryo- nia and Planorbarius. Geologie en Mijnbouw, 78, 165-174. Wilmer, J.W., Elkin, C., Wilcox, C., Murray, L., Niejalke, D. & Possingham, H. (2008) The influence of multiple dispersal mechanisms and landscape structure on population clustering and connectivity in fragmented artesian spring snail populations. Molecular Ecology, 17, 3733-3751. Wongsriphuek, C., Dugger, B.D. & Bartuszevige, A.M. (2008) Dispersal of wetland plant seeds by mallards: Influence of gut passage on recovery, retention, and germination. Wet- lands, 28, 290-299. Worthington-Wilmer, J. & Wilcox, C. (2007) Fine scale patterns of migration and gene flow in the endangered mound spring snail, Fonscochlea accepta (Mollusca:Hydrobiidae) in arid Australia. Conservation Genetics, 8, 617-628.

48 Chapter 3

Experimental quantification of long distance dispersal potential of aquatic snails in the gut of migratory birds

Casper H.A. van Leeuwen, Gerard van der Velde, Bart van Lith & Marcel Klaassen

PLoS ONE 7:3 e32292

49 Chapter 3

Abstract Many plant seeds and invertebrates can survive passage through the digestive system of birds, which may lead to long distance dispersal (endozoochory) in case of prolonged retention by moving vectors. Endozoochorous dispersal by waterbirds has nowadays been documented for many aquatic plant seeds, algae and dormant life stages of aquatic invertebrates. Anecdotal information indicates that endozoochory is also possible for fully functional, active aquatic organisms, a phenomenon that we here address experimentally using aquatic snails. We fed four species of aquatic snails to mallards (Anas platyrhynchos), and monitored snail retrieval and survival over time. One of the snail species tested was found to survive passage through the digestive tract of mallards as fully functional adults. Hydrobia (Peringia) ulvae survived up to five hours in the digestive tract. This suggests a maximum potential transport distance of up to 300 km may be possible if these snails are taken by flying birds, although the actual dispersal distance greatly depends on additional factors such as the behaviour of the vectors. We put forward that more organisms that acquired traits for survival in stochastic environments such as wetlands, but not specifically adapted for endozoochory, may be sufficiently equipped to successfully pass a bird’s digestive system. This may be explained by a digestive trade-off in birds, which maximize their net energy intake rate rather than digestive efficiency, since higher efficiency comes with the cost of prolonged retention times and hence reduces food intake. The resulting lower digestive efficiency allows species like aquatic snails, and potentially other fully functional organisms without obvious dispersal adaptations, to be transported internally. Adopting this view, endozoochorous dispersal may be more common than up to now thought.

Key words: aquatic invertebrates, digestive intensity, endozoochory, Gastropoda, propagules, retention time

50 Long distance dispersal potential of aquatic snails in the gut of migratory birds

Introduction Widespread geographical ranges and fast colonization by aquatic organisms have fascinated biologists for a long time (Darwin, 1859; Kew, 1893; Ridley, 1930). How can isolated wetlands, with varying water quality and short life spans, harbour a high biodiversity? A plausible explanation is long distance dispersal, whereby remote, new or only temporarily suitable wetlands are (repeatedly) colonized from a larger pool of biodiversity (e.g. Levins, 1969; Okamura & Freeland, 2002). However, this requires aquatic species to either disperse actively across land over long distances, or be suitable for passive transport by vectors such as wind (anemochory) (e.g. Brendonck & Riddoch, 1999; Caceres & Soluk, 2002), water (hydrochory) (e.g. Nilsson et al., 2010) or other animals (zoochory: e.g. Vanschoenwinkel et al., 2008; Cousens et al., 2010; Pollux, 2011). Waterbirds function as passive dispersal vectors for smaller organisms and are considered especially suitable because of their high abundances and directed flights between ecologically comparable habitats (Bilton et al., 2001; Figuerola & Green, 2002a; Green & Figuerola, 2005). Birds have been caught while carrying seeds, algae and aquatic invertebrates between their feathers, on their bill or on their feet (ectozoochory) (e.g. Kristiansen, 1996; Figuerola & Green, 2002b; Brochet et al., 2010b; Raulings et al., 2011). Additionally, birds have been found to carry viable aquatic organisms in their digestive system (endozoochory), which probably occurs at even higher frequency than external transport (Brochet et al., 2010b). This indicates that not only parasites can survive in the digestive systems of animals, but also some free-living aquatic organisms (e.g. Atkinson, 1980; Kristiansen, 1996; Figuerola et al., 2004; Charalambidou & Santamaría, 2005; Green et al., 2005; Frisch & Green, 2007; Brochet et al., 2010a). However, our taxonomic knowledge on which species are capable of surviving passage through the digestive system of waterbirds is still limited. Most of the propagules recovered from droppings have so far been plant seeds or cryptobiotic life stages of aquatic invertebrates (Charalambidou et al., 2003a; Charalambidou et al., 2003b; Soons et al., 2008). However, also some aquatic organisms have been retrieved from droppings while they were in fully functional, non- cryptobiotic life stages (Cadée, 1988; Green & Sanchez, 2006; Frisch et al., 2007; Green et al., 2008; Anders et al., 2009; Cadée, 2011). For these fully functional organisms we still lack knowledge on their actual dispersal potential, on which we focus here. Al- though their presence in droppings indicates that they can survive passage through the digestive tract, it still remains unknown whether or not they were retained in the digestive system long enough for dispersal over a significant distance. They might have been excreted shortly after ingestion, and thus their survival might contribute little to actual dispersal. For many of the cryptobiotic life stages found in droppings, their potential for long-distance dispersal has been assessed by combining field observations with experimental assessment of retention times and survival rates of the various propagules (Charalambidou & Santamaría, 2002). To date, we are aware of only one study addressing this for fully functional organisms, i.e. adult ostracods have been shown to survive long retentions in the digestive system of killdeer (Charadrius vociferus) (Proctor et al., 1967).

51 Chapter 3

We experimentally investigated the role of endozoochory for fully functional aquatic organisms when passing the digestive tract of waterbirds to assess its significance for long distance dispersal. We investigated this by determining the ability of aquatic snails (Gastropoda) to pass the digestive tract of mallards (Anas platyrhynchos). One snail species, Hydrobia (Peringa) ulvae, has previously been found to survive gut passage of shelducks (Tadorna tadorna) (e.g. Cadée, 1988). However, whether or not these snails were retained long enough for effective long distance dispersal remains unknown (Wesselingh et al., 1999). Endozoochory could be a plausible explanation for the widespread distributions and fast colonization of many aquatic snail species, and there are many invasive aquatic snails, such as Physella (Haitia) acuta (e.g. Albrecht et al., 2009), Bithynia tentaculata (e.g. Mills et al., 1993) and Potamopyrgus antipodarum (e.g. Stadler et al., 2005) with unknown dispersal vectors. Since snails may be important vectors for parasites such as the trematodes Fasciola sp. (liver fluke) and Microphallus sp. that can harm waterbirds and cattle (Morley, 2008), it is also of applied relevance to know the dispersal capabilities of aquatic snails. We performed two complementary experiments in which we tested both the survival potential and the associated retention times of four aquatic snail species after ingestion by mallards. The two experiments differed in emphasis. Experiment 1 tested the survival and potential dispersal distances of the four species, while experiment 2 concentrated on the two snail species with the highest potential of survival, for which faeces were sampled at a higher frequency and the number of mallards and snails was increased. To mimic accidental ingestion of invertebrates by herbivorous waterbirds, we added the simultaneous ingestion of macrophytes and snails to experiment 2. We hypothesize that aquatic snails can be retained long enough in the digestive system of mallards to be successfully dispersed over long distances.

Materials and methods

Snails Four snail species were chosen for the experiments, each with a widespread distribution throughout the Netherlands suggesting good dispersal capacities. With respect to their chance of surviving passage through the digestive tract we note that Pota- mopyrgus antipodarum is known as a successful invasive species (e.g. Stadler et al., 2005) and has a wide tolerance to environmental conditions (Gittenberger et al., 2004; Alonso & Castro-Diez, 2008). Bithynia leachii was chosen as native species. Hydrobia (Peringia) ulvae is a marine species related to P. antipodarum, and known to survive digestion of shelducks (e.g. Cadée, 1988; Cadée, 2011). All these three prosobranch species posses an operculum, which is a calcareous or horny lid that can close the shell aperture. The fourth species was Bathyomphalus contortus, a common planorbid species that was included because of its different shell morphology (flat) compared to the other species. This pulmonate species does not have an operculum. Appendix Table 3.S1 provides the sampling locations, shells sizes and further morphological information on the species. All snails involved in the two experiments were collected a maximum of two days prior to their use in an experiment. They were kept in aquaria that we filled

52 Long distance dispersal potential of aquatic snails in the gut of migratory birds with water collected at their sampling locations, at a constant temperature of 15 °C. Before each experiment a random subset of each species was measured for length and width to the nearest 0.1 mm with calipers. Thereby shell size was defined as the maximum measurable size of the shell (shell height in the case of the prosobranchs, and shell diameter in the case of the planorbid species) (Gittenberger et al., 2004). In the morning of each experimental day, the snails were taken from the aquaria and portions of 50 individuals were surrounded by a 1 to 2 mm layer of dough (i.e. moisturized grinded wheat seeds) to create pill-shaped “pellets” that facilitated feeding. We previously assessed 100% survival of snails in pellets over a period of 4 hrs (n=50 per pellet, tested 2 pellets for each species), all snails thus entered the mallards in good condition as if they were swallowed simultaneously with a minor amount of grinded seeds.

Experiments The procedure during both experiments was to take the mallards from their outdoor aviary at 0800 hours on each experimental day. They were weighed and fed 100 to 300 snails depending on the treatment, and subsequently kept in individual hardboard cages (LWH: 0.54×0.46×0.48m) for 24 hours. The birds had continuous access to water but not to food, resembling flying conditions as much as possible. The front of each cage was made of 12mm mesh wire and the cages were placed side by side, so that the birds could see their surroundings but not each other. The floor was constructed of the same mesh wire, which allowed us to collect faeces in a removable tray without disturbing the birds. The removable trays were filled with filtered water from the snail species’ sampling location to dissolve faeces immediately after excretion. At regular intervals (depending on the experiment, see above) the content of the removable trays was sieved using a 0.5 mm mesh. Viability of snails was checked immediately upon retrieval by looking for movement or retraction reactions after touch under a microscope. If in doubt, survival was subsequently checked every four hours up to 48 hours after excretion, until ascribed to the categories “alive” or “dead”. Viability was monitored for three months after retrieval by keeping the viable snails in aquaria at 15 °C. Shells without viable snails showing no visible damage, and of which length and width could be measured, were defined as ‘intact shells’. Broken shells and parts of shells were defined as ‘damaged shells’. Experiment 1 was conducted between 5 and 27 September 2007. All four snail species were fed once to each of six male and six female mallards in a random block design. Due to low availability of Bathyomphalus contortus and Bithynia leachii we fed 100 individuals of these species to each of the 12 mallards, whereas for P. antipodarum and H. ulvae we fed 200 individuals per mallard, maximizing the effect of detecting potential survival. Faeces were sampled at 1, 2, 4, 8, 12 and 24 hours after feeding. Mallards were allowed one week of recovery in between experimental days. Experiment 2 was conducted between 6 and 20 August 2008. Seven male and seven female mallards were used, of which four individuals had also been used in experiment 1. Each mallard was fed 300 H. ulvae, 300 P. antipodarum, or a mixture of

53 Chapter 3

150 P. antipodarum and 1.0 gram fresh weight Elodea nuttallii. Feeding was done in a random block design over three weeks, again allowing mallards a one-week recovery between experiments. Faeces were sampled every hour for the first 12 hours, and once after 24 hours.

Mallards Mallards were chosen because they represent common omnivorous, migratory waterbirds with a widespread distribution (e.g. Cramp & Simmons, 1977). Both freshwater and marine aquatic snails are part of their regular diet (e.g. Swanson et al., 1985; Gruenhagen & Fredrickson, 1990; Baldwin & Lovvorn, 1994; Rodrigues et al., 2002). In addition, mallards are opportunistic feeders with their diet composition greatly determined by availability of the potential food items in their habitat (Combs & Fredrickson, 1996). They thus potentially ingest large amounts of similar propagules. For instance, up to 1200 snails of P. antipodarum were found per mallard shot in Ireland (Whilde, 1977). Mallards behave well in captivity and can be used with minimal stress during experiments. They are therefore suitable and frequently chosen for dispersal studies (Charalambidou et al., 2005; Soons et al., 2008). All experimental mallards were of Dutch origin, captive bred and originally obtained from a waterfowl breeder (P. Kooy and Sons, ‘t Zand, The Netherlands). They had been housed in the outdoor aviary of the Netherlands Institute of Ecology in Heteren, The Netherlands, for at least 2 years prior to the experiments. They were kept on a stable diet of commercial pellets (Anseres 3®, Kasper Faunafood, Waalwi- jk, the Netherlands) and seed-based mixed grains (HAVENS Voeders ®, Maashees, Cary, NC, USA). One week prior to each experiment, the mallards were subjected to the experimental protocol to habituate them to the procedures and reduce stress. Male mallards (ranging from 1008 to 1288 g, with mean 1130 + 16 SD) were on average heavier than females (870 to 1155 g, mean 1001 + 22 SD, t = 5.7, df = 12, p <0.001).

Statistical analyses Because no intact shells were retrieved from B. contortus, and only 1.0% retrieval of shells from B. leachii, we concentrated statistical analyses on retrieval of intact H. ulvae and P. antipodarum. Where appropriate, data from both experiments were combined by calculating the retrieved snails per 4, 8 and 12 hours after ingestion. For each trial and sampling interval, the probability that H. ulvae and P. antipodarum were retrieved intact was used as the binomial dependent variable in a generalized mixed model with binomial error distribution and logit link function (Appendix Ta- ble 3.S3). Fixed factors included in the best model were snail species, mallard gender and whether or not macrophytes were fed together with the snails. Individual mallard was included as random factor nested in fixed factor gender. Retention time, number of propagules fed and mallard body mass at the start of each experimental day were taken as covariates. Covariates were centered to allow interpretation of the estimates at mean values. After model selection based on AIC criteria (see Appendix Table 3.S3), interactions left in the model were “macrophytes and retention time” and “mallard body mass and retention time”.

54 Long distance dispersal potential of aquatic snails in the gut of migratory birds

The effect of retention time on the size of excreted intact snails was tested using a general linear model with the normally distributed length of excreted snails depending on retention time as factor and gender and individual mallards as random factors, using only the more detailed data from experiment 2. Whether the average length of excreted snails was different from that of ingested snails was tested using an ANOVA with post-hoc Tukey’s HSD. All calculations were performed in R for statistics (R-Development-Core-Team, 2011).

Results Retrieval of viable snails and intact or damaged shells differed between snail species and changed with retention time (Appendix Tables 3.S2 and 3.S3, Fig. 3.1). Viable snails were retrieved up to five hours after feeding, but only for H. ulvae (Fig. 3.1A). Most viable snails of H. ulvae were retrieved in the first four hours after ingestion,

0.5 0.4 A Bathyomphalus contortus 0.3 Bithynia leachii Hydrobia (Peringia) ulvae 0.2 Potamopyrgus antipodarum 0.1

Viable snails (%) Viable 0.0

5 4 B 3 2 1 Intact shells (%) 0

5 4 C 3 2 1

Damaged shells (%) 0 0−4 hrs 4−8 hrs 8−12 hrs

Figure 3.1: Percentage of ingested snails retrieved viable (A), intact (B) or damaged (C) as a function of retention time. Data for the two experiments combined.

55 Chapter 3 and most intact shells of all snail species together between four and eight hours after ingestion (235 versus 366, respectively, Fig 3.1B). Only 99 shells were retrieved between eight and 12 hours, and 26 shells between 12 and 24 hours after feeding (Fig. 3.1B). Birds with higher body mass excreted less intact snails, and this relation became more pronounced with increasing retention time (indicated by the negative interaction coefficient in a generalized mixed model, Appendix Table 3.S3). Viable snails stayed alive for at least three months after retrieval. The interaction between retention time and mallard body mass was the most important predictor in the model indicated by the highest standardized coefficient (Appendix Table 3.S3). Based on the effect size of the interaction coefficient, but given large confidence intervals, an increase of body mass by 100 grams (our experimental birds ranged between 870 and 1288 g) would decrease the chance a bird excretes an intact snail by 3.9% at 4 hours after ingestion. The effect became more pronounced at longer retention times, with an increase of 100 g leading to a 22% and 37% reduced chance of intact snail retrieval at 8 and 12 hours after ingestion, respectively. Body mass was a more important predictor than mallard gender as indicated by the higher standardized coefficient in the model. Addition of macrophytes to the feeding of the snails changed the release pattern of intact shells over time (indicated by the significant interaction with retention time in Appendix Table 3.S3, and visualized in more detail in Fig. 3.2).

2.5 P. antipodarum P. antipodarum + M H. ulvae

2.0 ed (%) v ie 1.5

1.0 Intact shells retr

0.5

0.0 12345678910111224

Retention time (in hours)

Figure 3.2: Percentage of ingested snails retrieved as intact shells (mean + SE) as a function of snail species and retention time. ”P. antipodarum + M” is feeding including macrophytes. Data from Experiment 2 exclusively.

56 Long distance dispersal potential of aquatic snails in the gut of migratory birds

The average size of excreted shells was smaller than that of ingested snails (both in terms of length and width) for all species (except for B. contortus where we did not retrieve any intact shells, Fig. 3.3). The size of retrieved intact shells did not differ with retention time (GLM, t = -1.21, df = 346, p = 0.22).

6 4 r = 0.84 Size ingested Size excreted 3 2 5 Width (mm) 1 p<0.001 0 01234567 4 Length (mm) p<0.01 p<0.001

e (in mm) 3 Snail siz

660 279 870 435 120 0 120 12 0 Hydrobia (Peringia) ulvae Potamopyrgus antipodarum Bathyomphalus contortus Bithynia leachii

Figure 3.3: Average shell size of snails ingested and excreted for the four different species. Average shell size of excreted snails was smaller than the average shell size of ingested snails for all three species of which snails were retrieved (post-hoc Tukey’s HSD, p-values indicated in the graph). Error bars indicate 95% CI of the mean and samples sizes are indicated at the bottom of the bars. Results for shell width were identical due to a strong correlation between shell length and width for all species (r = 0.84, p< 0.001).

Discussion Our experiments showed that the aquatic snail Hydrobia ulvae is capable of surviving up to five hours in the digestive system of waterfowl. This indicates that not only cryptobiotic life stages, algae and plant seeds may use endozoochorous transport by surviving several hours in the digestive system of birds, but also fully functional, active life stages of invertebrates have endozoochorous dispersal potential. H. ulvae has previously also been retrieved alive from shelduck droppings (Ca- dée, 1988; Anders et al., 2009; Cadée, 2011). Our results now show that these snails

57 Chapter 3 may have originated from another location then where they were retrieved in droppings. Whereas many waterbirds can maintain sustainable flight speeds of up to 70km h-1 (Welham, 1994; Bruderer & Boldt, 2001), a bird in straight flight might cover a distance of over 300 kilometres in five hours. Although the majority of snails that are potentially ingested before departure will be excreted within the first tens of kilometres (Bohonak & Jenkins, 2003), there is potential for birds to disperse snails over distances not easily achieved by the snails themselves. The actual dispersal distance and frequency will depend on more additional factors than could be included in our experiments, such as the timing and place of ingestion, behavior and activity of the birds during digestion, domestic or wild origin of the experimental birds, and bird species. Nevertheless, our results indicate there is potential for small operculated aquatic snails to survive prolonged digestion, which is an important requirement for successful dispersal. Since travelling birds will generally not forage during actual moving we only provided water during the experiments. Continuous foraging during digestion has been shown to reduce digestive intensity (Clauss et al., 2007), while fasting more likely improves digestive intensity by recirculation of food in the digestive system (Clench & Mathias, 1995). We choose to remove the food during the experiments, to estimate snail survival during the trials as conservatively as possible. To minimize stress of the mallards in the experiments we habituated them in a test trial the weeks before the start of each experiment. We rolled the snails in a thin layer of bread dough (grinded wheat seeds with some water) to minimize handling stress during feeding and allowing exact timing of ingestion. Starving the mallards before offering snails to make them forage voluntarily would not allow feeding of a known quantity of snails at a known time, which was required for estimating survival as well as retention times. We expected minimal influence of feeding by pellets, since each pellet contained only a minor amount of flour compared to the amount of snails. This resembles ingesting a minor amount of seeds simultaneous with the snails, which we also expect to occur in natural situations. This technique has frequently been applied successfully in previous studies on endozoochory (e.g. Charalambidou et al., 2003b; Charalambidou et al., 2005; Soons et al., 2008; Figuerola et al., 2010).

Why do snails survive? One potential explanation for survival of aquatic organisms is that transported organisms have evolved special adaptations for endozoochorous dispersal. This has been shown in many terrestrial seeds and fruits (e.g. Traveset, 1998; Traveset et al., 2008). For aquatic seeds, these adaptations have only been suggested more recently (Soons et al., 2008), and for aquatic invertebrates detected only sporadically (Okamu- ra & Freeland, 2002). While many resting stages of aquatic invertebrates are known to be adapted to survive temporarily unfavorable environmental conditions, to date it is unclear whether they are especially adapted for internal dispersal by birds. Alternatively, such as in the case of aquatic snails, characteristics that make them suitable for internal dispersal may be attributed to adaptations likely acquired to survive normal environmental conditions. Both marine and freshwater habitats can

58 Long distance dispersal potential of aquatic snails in the gut of migratory birds be very dynamic, with fluctuating water levels, oxygen and nutrient concentrations and temperatures, requiring adaptations for survival. This requires comparable traits to the ones needed for endozoochory. Prosobranchs snails have a strong shell and operculum (a calcareous or horny lid). These characteristics probably evolved to protect them from predation and desiccation (e.g. Gibson, 1970), but at the same time protect them from crushing forces in the gizzard and from digestive enzymes and juices entering the shell. Pulmonate snails lack these characteristics, and during our experiments, indeed no remnants of the pulmonate snail B. contortus were retrieved. The relatively weak shells of this species likely dissolved completely during digestion, while for the three operculated snails remains of their shells were retrieved. Malone (1965a) experimented with two other pulmonate snail species with relatively weak shells and without operculum, Physa anatina and Helisoma trivolvis. He also did not retrieve intact shells after feeding to birds. A strong shell thus facilitates both protection and survival during endozoochory. Additionally, the small size of many snail species may have several advantages in wetlands, such as survival during low food conditions, fast generation times in a stochastic environment or survival in small moist crevices. However, it is also an important trait of propagules for dispersal (e.g. DeVlaming & Proctor, 1968; Traveset, 1998; Soons et al., 2008), and also led to increased probability of retrieval in our experiments (Fig. 3.3). The above mentioned examples illustrate that characteristics needed for successful dispersal by endozoochory are similar to those required for survival in stochastic environments such as wetlands. This explanation for the survival of snails can be combined with an explanation involving the physiology of the birds as vectors. Generally, the efficiency with which birds digest their food is ~75%, and can even be as low as 50% (Castro et al., 1989; Prop & Vulink, 1992). An increase in digestive intensity will come with the cost of an increase in retention time (e.g. Prop & Vulink, 1992; Afik & Karasov, 1995). Hence, this may reduce food intake over time, at least in situations with high food availability. A maximum long-term average energy intake rate may thus be achieved at a less than 100% digestive intensity. This trade-off can provide a window of opportunity for all kinds of organisms to pass the digestive system undigested, even though they are not specially adapted for endozoochory.

Significance of dispersal The small percentage of the ingested snails that survived digestion raises the question of its significance for snail populations. However, due to the low energy content of one snail compared to the energy requirements of one water bird (Evans et al., 1979; Zwarts & Blomert, 1992), waterbirds can ingest large amounts of snails. Even low survival frequencies can lead to many individuals surviving. Many duck species are opportunistic feeders that ingest much of the same food source once abundantly available (Cramp & Simmons, 1977). Shelducks have been estimated to ingest up to 33 000 H. ulvae per day (Cadée, 1988; Anders et al., 2009), and up to 1200 individuals of P. antipodarum have been collected per mallard in Ireland (Whilde, 1977). Illustrative, the shelduck droppings in which H. ulvae snails were retrieved in

59 Chapter 3 the field contained multiple viable snails per dropping (Cadée, 2011). However, what is the significance of endozoochorous dispersal for snails such as H. ulvae? Aquatic snails may also be transported by other vectors (reviewed by Bilton et al., 2001), or externally by adhering to the outside of waterbirds (although evidence for “ectozoochory” by snails is still very limited (e.g. Boag (1986), see also Malone (1965a) and Wesselingh et al. (1999). Furthermore, H. ulvae is a marine snail that produces free swimming pelagic larvae, can float attached to the water surface (Newell, 1962) and has the capability to raft on drifting wood or plants. In contrast to freshwater snails, populations seem less dependent on long distance dispersal. Nevertheless, given the numerous waterbirds that forage on aquatic snails, even low frequency endozoochory may provide a constant dispersal mechanism connecting (marine) populations, and may connect different populations than other vectors. Marine populations have been shown to be (genetically) separated by ecological barriers in the sea (e.g. Hohenlohe, 2004), thus long-distance dispersal may enable range expansions along coastlines with more and less suitable sections, strong out- going currents of rivers, or connect populations of coastlines separated by land or open water. The fact that P. antipodarum, the freshwater snail closest related to H. ulvae, did not survive digestion indicates freshwater snail endozoochory might be less plausible than that of H. ulvae. Despite that P. antipodarum is such an effective invasive species (Stadler et al., 2005; Alonso & Castro-Diez, 2008) for which dispersal by waterbirds between freshwater habitats may be even more relevant than for H. ulvae, it did not survive digestion of mallards in the quantities we offered. Based on our experiments, the success of P. antipodarum as an invasive species at this point cannot be attributed to birds as dispersal vectors, but is more likely caused by its other characteristics (Gittenberger et al., 2004). Nevertheless, very recently, a terrestrial snail species was found to survive digestion of passerine birds (Wada et al., 2012). This indicates that more snail species than H. ulvae may be capable of endozoochory, including other fully functional aquatic organisms.

Vectors Mallards in this experiment represent omnivorous, common waterbirds with a highly variable diet including aquatic snails (e.g. Campbell, 1947; Gruenhagen & Fredrickson, 1990; Baldwin & Lovvorn, 1994; Rodrigues et al., 2002). Extrapolating our results to a field situation with other vector species has to be done conservatively. Although different species of Anas have shown similar capacity to disperse aquatic propagules (Charalambidou et al., 2003c; Pollux et al., 2005; Raulings et al., 2011), marked interspecific differences have also been found (e.g. Atkinson, 1980; Charalambidou & Santamaría, 2002; Figuerola et al., 2003). Nevertheless, besides surviving mallard digestion, H. ulvae is also capable of surviving shelduck digestion (Cadée, 2011). Specific experiments will be necessary to assess the dispersal potential of each vector-propagule pair separately, but the survival of snails in both shelducks and mallards strengthens the idea that potentially more (duck) species are capable of passing viable aquatic snails through their digestive systems.

60 Long distance dispersal potential of aquatic snails in the gut of migratory birds

More intact snail shells were retrieved from smaller mallards (Appendix Table 3.S3), which may be due to smaller gut or gizzard sizes in smaller birds (e.g. Figuerola et al., 2002; Figuerola et al., 2004). Given that gut length and gizzard size are generally correlated to body mass (e.g. Kehoe et al., 1988), we expect smaller individuals to have shorter retention times leading to retrieval of more intact propagules. The effect of body mass became more pronounced with increasing retention time, although the large confidence intervals should be kept in mind. This suggests smaller mallards may not only marginally excrete more intact propagules, they may also continue to do so at longer distance from the location of ingestion.

The effect of adding macrophytes Digestive intensity is known to vary with the quality and type of food ingested (Kar- asov & Levey, 1990). Therefore we also tested snail survival and retention times when snails were ingested simultaneously with macrophytes. This resembles the field situation of accidental ingestion of invertebrates by herbivores, or a mixed diet by omnivores. Retrieval of intact snail shells was accelerated due to the addition of macrophytes to the diet (Fig. 3.2 and Appendix Table 3.S3). This is in accordance with previous observations where retention times of brine shrimp eggs (Artemia salina) decreased when ingested with macrophytes (Malone, 1965b). A general decrease of viability with retention time observed for propagules (Charalambidou et al., 2003c; Charalambidou et al., 2005; Pollux et al., 2005) suggests that aquatic snails ingested with macrophytes will have increased survival chances, but shorter dispersal distances. Herbivorous birds that ingest invertebrates accidentally and omnivores that forage on invertebrates with macrophytes, may thus contribute to dispersal of invertebrates in natural situations. How this depends on macrophyte species, bird species and other parameters remain interesting avenues for future research.

Conclusions We have shown that the aquatic snail H. ulvae can survive long enough in the digestive tract of birds to potentially be dispersed over significant distances. We suggest this is possible with the adaptations this snail already acquired for surviving unfavorable circumstances in their natural habitat. We put forward that a digestive trade-off in birds makes endozoochory possible for propagules without special adaptations for endozoochory. The fact that besides cryptobiotic life stages of invertebrates, algae and plant seeds also aquatic snails, as fully functional free-living aquatic organisms, can successfully be dispersed in the digestive system of birds suggests endozoochory is a more common mode of transport than currently realized.

Acknowledgements We thank Naomi Huig for assistance during the experiments, Wim Kuijper for help with snail sampling and John Endler, Andy Green, Gerhard C. Cadée and one anonymous reviewer for valuable comments on previous versions of the manuscript. This is publication 5209 of the Netherlands Institute of Ecology (NIOO-KNAW).

61 Chapter 3

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zoan Cristatella mucedo disperse in duck guts? Archiv für Hydrobiologie, 157, 547-554 Charalambidou, I., Santamaría, L. & Langevoord, O. (2003c) Effect of ingestion by five avian dispersers on the retention time, retrieval and germination of Ruppia maritima seeds. Func- tional Ecology, 17, 747-753 Charalambidou, I. & Santamaría, L. (2005) Field evidence for the potential of waterbirds as dispersers of aquatic organisms. Wetlands, 25, 252-258 Charalambidou, I., Santamaría, L., Jansen, C. & Nolet, B.A. (2005) Digestive plasticity in mallard ducks modulates dispersal probabilities of aquatic plants and crustaceans. Functional Ecology, 19, 513-519 Clauss, M., Streich, W.J., Schwarm, A., Ortmann, S. & Hummel, J. (2007) The relationship of food intake and ingesta passage predicts feeding ecology in two different megaherbivore groups. Oikos, 116, 209-216 Clench, M.H. & Mathias, J.R. (1995) Motility responses to fasting in the gastrointestinal-tract of 3 avian species. Condor, 97, 1041-1047 Combs, D.L. & Fredrickson, L.H. (1996) Foods used by male Mallards wintering in Southeast- ern Missouri. Journal of Wildlife Management, 60, 603-610 Cousens, R.D., Hill, J., French, K. & Bishop, I.D. (2010) Towards better prediction of seed dispersal by animals. Functional Ecology, 24, 1163-1170 Cramp, S. & Simmons, K.E.L. (1977) The birds of the Western Palearctic: Volume 1. Oxford Uni- versity Press, Oxford, Darwin, C. (1859) On the origin of species by means of natural selection, or the preservation of fa- voured races in the struggle for life. John Murray, London, DeVlaming, V. & Proctor, V.W. (1968) Dispersal of aquatic organisms - viability of seeds revo- vered from droppings of captive killdeer and mallard ducks. American Journal of Botany, 55, 20-26 Evans, P.R., Herdson, D.M., Knights, P.J. & Pienkowski, M.W. (1979) Short-term effects of reclamation of part of Seal Sands, Teesmouth, on wintering waders and Shelduck. Oecologia, 41, 183-206 Figuerola, J. & Green, A.J. (2002a) Dispersal of aquatic organisms by waterbirds: a review of past research and priorities for future studies. Freshwater Biology, 47, 483-494 Figuerola, J. & Green, A.J. (2002b) How frequent is external transport of seeds and invertebrate eggs by waterbirds? A study in Donana, SW Spain. Archiv für Hydrobiologie, 155, 557-565 Figuerola, J., Green, A.J. & Santamaría, L. (2002) Comparative dispersal effectiveness of wig- eongrass seeds by waterfowl wintering in south-west Spain: quantitative and qualitative aspects. Journal of Ecology, 90, 989-1001 Figuerola, J., Green, A.J. & Santamaría, L. (2003) Passive internal transport of aquatic organisms by waterfowl in Donana, south-west Spain. Global Ecology and Biogeography, 12, 427-436 Figuerola, J., Green, A.J., Black, K. & Okamura, B. (2004) Influence of gut morphology on passive transport of freshwater bryozoans by waterfowl in Donana (southwestern Spain). Canadian Journal of Zoology, 82, 835-840 Figuerola, J., Charalambidou, I., Santamaría, L. & Green, A.J. (2010) Internal dispersal of seeds by waterfowl: effect of seed size on gut passage time and germination patterns. Naturwis- senschaften, 97, 555-565 Frisch, D. & Green, A.J. (2007) Copepods come in first: rapid colonization of new temporary ponds. Fundamental and Applied Limnology, 168, 289-297 Frisch, D., Green, A.J. & Figuerola, J. (2007) High dispersal capacity of a broad spectrum of

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64 Long distance dispersal potential of aquatic snails in the gut of migratory birds

Okamura, B. & Freeland, J.R. (2002) Gene flow and the evolutionary ecology of passively dispersing aquatic invertebrates. In. Dispersal Ecology (ed. by J.M. Bullock, R.E. Kenward and R.S. Hails), pp. 194-216. Blackwell Publishing, Oxford. Pollux, B.J.A., Santamaría, L. & Ouborg, N.J. (2005) Differences in endozoochorous dispersal between aquatic plant species, with reference to plant population persistence in rivers. Freshwater Biology, 50, 232-242 Pollux, B.J.A. (2011) The experimental study of seed dispersal by fish (ichthyochory). Fresh- water Biology, 56, 197-212 Proctor, V.W., Malone, C.R. & DeVlaming, V. (1967) Dispersal of aquatic organisms - viability of disseminules recovered from intestinal tract of captive killdeer. Ecology, 48, 672-676 Prop, J. & Vulink, T. (1992) Digestion by barnacle geese in the annual cycle - the interplay between retention time and food quality. Functional Ecology, 6, 180-189 R-Development-Core-Team (2011) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http:// www.R-project.org. Raulings, E., Morris, K.A.Y., Thompson, R. & Nally, R.M. (2011) Do birds of a feather disperse plants together? Freshwater Biology, 56, 1390-1402 Ridley, H.N. (1930) The distribution of plants throughout the world. Reeve & Company, Ashford, United Kingdom, Rodrigues, D., Figueiredo, M. & Fabiao, A. (2002) Mallard (Anas platyrhynchos) summer diet in central Portugal rice-fields. Game & Wildlife Science, 10, 55-62 Soons, M.B., Van der Vlugt, C., Van Lith, B., Heil, G.W. & Klaassen, M. (2008) Small seed size increases the potential for dispersal of wetland plants by ducks. Journal of Ecology, 96, 619- 627 Stadler, T., Frye, M., Neiman, M. & Lively, C.M. (2005) Mitochondrial haplotypes and the New Zealand origin of clonal European Potamopyrgus, an invasive aquatic snail. Molecular Ecology, 14, 2465-2473 Swanson, G.A., Meyer, M.I. & Adomaitis, V.A. (1985) Foods consumed by breeding mallards on wetlands of south-central North Dakota. Journal of Wildlife Management, 49, 197-203 Traveset, A. (1998) Effect of seed passage through vertebrate frugivores’ guts on germination: a review. Perspectives in Plant Ecology Evolution and Systematics, 1, 151-190 Traveset, A., Rodriguez-Perez, J. & Pias, B. (2008) Seed trait changes in dispersers’ guts and consequences for germination and seedling growth. Ecology, 89, 95-106 Vanschoenwinkel, B., Gielen, S., Seaman, M. & Brendonck, L. (2008) Any way the wind blows - frequent wind dispersal drives species sorting in ephemeral aquatic communities. Oikos, 117, 125-134 Wada, S., Kawakami, K. & Chiba, S. (2012) Snails can survive passage through a bird’s digestive system. Journal of Biogeography, 39, 69-73 Welham, C.V.J. (1994) Flight speeds of migrating birds - a test of maximum range speed predictions from 3 aerodynamic equations. Behavioral Ecology, 5, 1-8 Wesselingh, F.P., Cadée, G.C. & Renema, W. (1999) Flying high: on the airborne dispersal of aquatic organisms as illustrated by the distribution histories of the gastropod genera Tryo- nia and Planorbarius. Geologie en Mijnbouw, 78, 165-174 Whilde, A. (1977) The autumn and winter food of Mallard, Anas p. platyrhynchos (L.) and some other Irish wildfowl. The Irish Naturalists’ Journal, 19, 18-21 Zwarts, L. & Blomert, A.M. (1992) Why knot Calidris canutus take medium-sized Macoma balthica when 6 prey species are available. Marine Ecology-Progress Series, 83, 113-128

65 Chapter 3

Appendix Chapter 3

Table 3.S1: Snail species used in the experiments.

Respiratory Sampling location Size in mm Snail species Family; Order Operculum Notes system (The Netherlands) (Average + SD) strong, Vechten (ditch) Bithyniidae; L 2.9 + 0.9 horn- Bithynia leachii gill yes 52°03’33 N Prosobranchia W 2.1 + 0.6 shaped 05°10’14 E shell Paesens (coast) horn- Hydrobia Hydrobiidae; L 4.0 + 0.8 gill yes 53°24’19 N shaped, (Peringia) ulvae Prosobranchia W 2.1 + 0.4 06°05’14 E marine Ooijpolder (lake) horn- Potamopyrgus Hydrobiidae; L 3.4 + 0.9 gill yes 51°51’12 N shaped, antipodarum Prosobranchia W 1.8 + 0.4 05°53’18 E invasive Loosdrecht (ditch) flat, Bathyomphalus Planorbidae; L 3.3 + 0.3 lungs No 52°09’42 N round contortus Pulmonata W 1.7 + 0.2 05°01’52 E shaped

Table 3.S2: The number of intact, damaged and viable snails retrieved after 24 hours for the two experiments combined. Species Snails ingested Damaged shells Intact shells Viable snails (average + 95%CI) (average + 95%CI) (average + 95%CI) Hydrobia (Peringia) ulvae 12×200 + 14×300 = 6600 11.4 (+7.0): 4.33% 11.2 (+8.5): 4.23% 0.8 (+1.2): 0.32% Potamopyrgus antipodarum 12×200 + 14×300 = 6600 4.8 (+3.6): 1.88% 13.9 (+10.7): 5.47% - Potamopyrgus antipodarum 14×150 = 2100 0.8 (+0.5): 0.52% 5.3 (+3.7): 3.52% - + macrophytes Bithynia leachii 12×100 = 1200 - 1.0 (+1.3): 1.0% - Bathyomphalus contortus 12×100 = 1200 - - -

Table 3.S3: Results of the generalized mixed model for the probability of retrieval of ntact shells. Significant and marginally significant factors are in bold. Repeatability for random factor mallard was 16.5%. Standardized coefficients were calculated to indicate the relative contribution of the different factors (including binomial factors) to the model. Initial AIC of the full model including “experiment” and all second order interactions was 278.1. After model selection and removal of insignificant interactions and insignificant “experiment”, AIC was reduced to 260.8 with a delta AIC of 2.7 to the next best model.

St. coefficients SE coef Z-value Pr(>|z|) (Intercept) - 0.22 0.42 0.87 0.39 Snail species - 0.61 0.36 - 1.73 0.08 Mallard gender - 0.053 0.60 - 0.088 0.93 Macrophytes - 0.70 0.68 - 1.28 0.20 Retention time - 0.53 0.055 - 0.37 0.71 Number of snails fed - 1.04 0.0042 - 1.92 0.055 Mallard body mass - 1.56 0.0036 - 2.54 < 0.05 Retention time : Mallard body mass - 2.10 0.00062 - 3.00 < 0.01 Retention time : Macrophytes - 1.56 0.13 - 2.25 < 0.05

66 Chapter 4

Vector activity and propagule size affect dispersal potential by vertebrates

Casper H. A. van Leeuwen, Marthe L. Tollenaar & Marcel Klaassen

Oecologia (in press)

67 Chapter 4

Abstract Many small organisms in various life stages can be transported in the digestive system of larger vertebrates, a process known as endozoochory. Potential dispersal distances of these “propagules” are generally calculated after monitoring retrieval in experiments with resting vector animals. We argue that vectors in natural situations will be actively moving during effective transport rather than resting. We here test for the first time how physical activity of a vector animal might affect its dispersal efficiency. We compared digestive characteristics between swimming, wading (i.e. resting in water) and isolation (i.e. resting in a cage) mallards (Anas platyrhynchos). We fed plastic markers and aquatic gastropods, and monitored retrieval and survival of these propagules in the droppings over 24 h. Over a period of 5 h of swimming, mallards excreted 1.5 times more markers than when wading and 2.3 times more markers than isolation birds, the pattern being reversed over the subsequent period of monitoring where all birds were resting. Retention times of markers were shortened for approximately 1 h for swimming, and 0.5 h for wading birds. Shorter retention times imply higher survival of propagules at increased vector activity. However, digestive intensity measured directly by retrieval of snail shells was not a straightforward function of level of activity. Increased marker size had a negative effect on discharge rate. Our experiment indicates that previous estimates of propagule dispersal distances based on resting animals are overestimated, while propagule survival seems underestimated. These findings have implications for the dispersal of invasive species, meta-population structures and long distance colonization events.

Key words: digestion, endozoochory, metabolic rate, physiology, retention time

68 Vector activity and propagule size affect dispersal potential by vertebrates

Introduction Many small organisms can be transported alive in the digestive system of more mobile vertebrates, i.e. by endozoochory. The potential importance and generality of this process was recognized a long time ago (Darwin, 1859; Ridley, 1930). Mam- mals, such as bears, foxes and musk ox, forage on seeds and fruits, and defecate surviving seeds after travelling tens of kilometres across the landscape (e.g. Bruun et al., 2008; Koike et al., 2011). Many migratory animals, such as wildebeest, reindeer, fish, turtles, and numerous species of birds, can potentially transport seeds and invertebrates in various life stages (hereafter referred to as “propagules”) over even hundreds of kilometres (Liu et al., 2004; Anne Bråthen et al., 2007; Traveset et al., 2008; Brochet et al., 2009; Pollux, 2011; Raulings et al., 2011). This dispersal potential of propagules is often assessed experimentally. Captive animals, ranging from waterbirds to monkeys to foxes, are fed a known quantity of specific propagules, and kept in cages while feces are examined for retrieval of viable organisms (Varela & Bucher, 2006; Spiegel & Nathan, 2007; Brochet et al., 2010; Figuerola et al., 2010; Tsuji et al., 2010). This way, the survival of gut passage and the timing of retrieval is assessed and used to estimate expected and maximum dispersal distances. However, vector organisms will have to move to other locations to enable dispersal. None of the experiments to date have addressed the potential effects of the movement of vectors on their digestive performance and the resulting dispersal kernels (i.e. the function that describes the probability of dispersal to different distances). Movement, and high levels of activity in general, likely require reallocation of blood flow from the digestive system to muscle tissues and other organs supporting the activity (Brouns & Beckers, 1993). Hence, retention times and propagule survival might be different for actively moving (and dispersing) vectors than for inactive animals in cages. Previous experiments have conducted in which smaller propagules are retrieved in higher numbers and are retained longer in the digestive system (e.g. DeVlaming & Proctor, 1968; Soons et al., 2008; Figuerola et al., 2010). However, not all studies found similar effects of seed size (Varela & Bucher, 2006; Wongsriphuek et al., 2008; Brochet et al., 2010). Most experiments compared passage of propagules that not only differ in size but inevitably also in other characteristics such as resistance to digestion and shape. Knowledge on how propagule size per se affects dispersal distance and how this might interact with the level of activity of the vector is still limited. We here present the first experiment in which propagule dispersal is investigated in active animals. In the experiment, we also investigated the effect of propagule size. We compared digestive intensity and propagule retention times between mallards (Anas platyrhynchos) swimming in a flume tank and inactive controls. Swim- ming was expected to increase the metabolic rate of the birds, and hence affect their digestion. We used retrieval of aquatic snails [Hydrobia (Peringia) ulvae], previously found to survive digestion by ducks (Anders et al., 2009; Cadée, 2011; Van Leeuwen et al., 2012), to measure changes in digestive intensity. The effect of propagule size was included by feeding plastic markers as surrogate propagules that differ in size, but are otherwise identical.

69 Chapter 4

Materials and methods

Training and the flume tank Sixteen adult mallards (Anas platyrhynchos) were trained twice a week to swim in water flowing at 0.7 m/s in an outdoor setup, starting May 2009 for 8 weeks. In late June, training was continued in an indoor flume tank until 12 mallards swam voluntarily and continuously for 5 h at a velocity of 1.11 m/s. The flume was oval shaped, made of PVC and filled with tap water. Two silent outboard engines on 12V batteries positioned at the start of either long side of the oval each produced a 214 N thrust, creating a near-laminar flow of 1.11 m/s (Appendix Fig. 4.S1). At the end of each long side, two rectangular cages (LWH: 0.72×0.46×0.10m) kept the mallards in the flume tank. This allowed four mallards to be in the flume tank simultaneously. In the cages closest to the engines, a horizontal 12 mm mesh wire layer was placed 0.02 m below the water surface. Therefore, mallards in these cages could not swim but instead sat on the mesh wire in the same situation and water current as the swimming birds. Behind each set of two cages, droppings were retrieved in sieves with 1.5mm mesh (the two mallards at the same side of the tank were fed different propagules during each experiment, allowing collection from two individuals in the same sieve). Control birds were individually housed in isolation cages constructed of 12 mm thick wood (LWH: 0.54x0.46×0.48m). The floor and part of the front of each cage was made of 12mm mesh wire, and the cages were placed side by side. This allowed the birds to see their surroundings but not their conspecifics, and allowed us to collect their feces in a removable tray without disturbing the birds. Average air and water temperatures during the experiment were 23 and 18°C, respectively.

The experiment Propagule retrieval was compared between mallards subjected to three different treatments: isolation, swimming and wading; (1) two isolation birds were kept in the isolation cages for 24 h, (2) two swimming birds were swimming continuously for 5 h at 1.11 m/s in the flume tank, after which they were also transported to isolation cages and kept there for an additional 19 hours and (3) two wading birds sat on the mesh wire in the water in a flume tank for 5 hrs, and were thereafter transported to isolation cages. After 24 h all birds returned to the outdoor aviary. The total experiment took place over 18 days (11-28 July 2009) with 12 birds and three treatments in a random block design. Each experimental day, six birds performed trials simultaneously, and two consecutive experimental days were followed by one resting day. Each of the 12 individuals was therefore used once every 3 days in an experiment. We fed two propagule types; therefore each bird was involved in each treatment twice (12 birds, three treatments, two propagule types, totalling 72 trials in 12 experimental days with 6 rest days). During the experiment, all birds had access to freshwater ad libitum but not to food, to resemble the situation of travelling. At the start of each experiment, all mallards were weighed and fed either of the two propagule types: round plastic markers (150 Polyoxymethylene balls, POM ko- gels; DIT Holland, Hilvarenbeek, 50×2 mm, 50×3 mm, 50×4 mm diameter) or live aquatic snails (300 Hydrobia (P.) ulvae, L: 4.3 mm + 0.5, W: 2.0 mm + 0.2, n = 100,

70 Vector activity and propagule size affect dispersal potential by vertebrates mean + SD, randomly selected and measured to the nearest 0.1 mm with callipers). Aquatic snails are part of the regular diet of mallards (Swanson et al., 1985; Gruen- hagen & Fredrickson, 1990; Baldwin & Lovvorn, 1994; Rodrigues et al., 2002), and Hydrobia (P.) ulvae is a common marine snail with an operculum, of which a small percentage can survive passage through duck guts (Anders et al., 2009; Cadée, 2011; Van Leeuwen et al., 2012). This species also occurs in brackish environments, and is not affected by short-term exposure to freshwater (Fenchel, 1975). Designated propagules were divided into portions and surrounded by a 1 to 2 mm layer of dough (i.e. moisturized ground wheat seeds) that created six pill- shaped “pellets” to facilitate feeding. A known quantity of propagules could be fed within minutes to each mallard, while minimizing handling stress and allowing exact determination of the time between ingestion and retrieval of propagules. The pellets did not affect the snails, as 100% of snails in control pellets (n = 50 per pellet, two pellets tested) survived at least 4 h. Droppings were collected hourly until 12 h after ingestion, and once after 24 h. Retrieved plastic markers were sorted by size and counted, while snails were categorized into intact shells, fragments of shells, or viable snails. Intact shells were shells that did not show visible damage, and were measured for length and width with callipers. Broken shells and shell parts were counted as fragments. Viability of all intact shells was checked immediately by returning the snails to seawater and looking for movement or retraction reactions after touch under the microscope. In case of uncertainty, survival was checked every 4 h up to 48 h after excretion.

Statistical analyses The number of markers retrieved per trial and sampling interval followed a (over- dispersed) Poisson distribution (based on normality of the residuals of the model) and was analyzed using repeated measures generalized mixed-effects models with Poisson error distribution, log- link function and random slope (Table 4.1). Treat- ment (swim, wade or isolation) was set as the fixed factor, with swimming as reference level to compare to both isolation and wading ducks. Retention time over the first 12 h (1-12, log-transformed, included linearly and squared), marker size (2, 3 and 4 mm) and mallard body mass at the start of each experimental day were set as covariates after centering (Raudenbush & Bryk, 2002). Interactions initially included in the model were treatment:marker size, treatment:retention time, treatment:retention time2, marker size:retention time, and marker size:retention time2. Initial model AIC was 2915, which lowered to 2912 by removing insignificant treatment:marker size. Further removal of interactions lowered the AIC by <2, so these models were considered equivalent and no further interactions were removed. Additive overdispersion was modelled by adding an extra random factor according to Nagakawa and Schielzeth (2010), i.e. overdispersion is absorbed by this added term, consisting of a random variable with a random level for each observation. To correct for possible differences between mallards in intercept, individual mallard was taken as random factor. Retention time was included as random slope for this random factor, to account for the possibility that individual mallards could differ

71 Chapter 4 not only in mean number of markers excreted (which is indicated by the random intercept) but also in pattern of excretion over time (indicated by the random slope of each mallard) (Schielzeth & Forstmeier, 2009). Model output was consistent when calculated with or without combinations of covariates as random slopes. Because the linear component of retention time was considered the most relevant covariate involved in significant interactions in the final model, we present output with only retention time included as random slope for random factor mallard. The number of intact snails or markers retrieved during different phases of the experiments all followed Poisson distributions. Differences of retrieval between treatments were therefore compared in repeated measures generalized mixed-effects models with Poisson error distribution and log-link function. As dependent variables, we used either the total number of snails retrieved intact during the 5 h active phase, or the total retrieved during the subsequent 6-24 h inactive interval. For the markers, we analysed their total number retrieved over 24h only, since their retrieval over the first 12 h was already addressed in the GLM including the more detailed retention time analysis enabled by the more frequent retrieval of markers than snails. In the three similar models, treatment was included as fixed factor (swimming as intercept) and centered mallard body mass as covariate. Individual mallard was taken as random factor. A potential size difference between ingested and excreted snails was tested using a Student’s t test. All calculations were performed using package lme4 in R for statistics (R-Development-Core-Team, 2011).

Results

Timing of marker retrieval During the first 5 h of the experiment, in which theswimming birds were active, marker excretion in this group was increased compared to the isolation and wading birds. Swimming birds excreted 2.3 times more markers than isolation, and 1.5 times more than wading birds (Fig. 4.1a).

St. coef z-value Pr(>|z|) Table 4.1: Results of the generalized mixed model for the chance of (Intercept) 0.58 11.4 <0.001 retrieval of markers including Treatment wade (contrast swim) - 0.098 - 1.4 0.17 retention time. Swimming birds were set as reference level of Treatment isolation (contrast swim) - 0.099 - 1.4 0.17 treatment. Significant effects are Retention time - 0.57 - 5.0 <0.001 in bold. Standardized coefficients 2 indicate the relative contribution of Retention time - 1.2 - 7.7 <0.001 the different factors to the model Marker size - 0.26 - 4.4 <0.001 (Gelman, 2008). The standard deviations for the random slopes Mallard body mass 0.022 0.51 0.61 of retention times were 0.18, i.e. Treatment wade : retention time 0.25 1.8 0.07 95% of the retention time slopes Treatment isolation: retention time 0.43 3.0 <0.01 varied between -0.92 and -0.21 (Schielzeth & Forstmeier, 2009). Treatment wade : retention time2 0.34 1.5 0.14

The repeatability for the intercept Treatment isolation: retention time2 0.34 1.5 0.14 of random factor mallard was 3.4%, and additive overdispersion 0.50. Marker size : retention time 0.15 1.3 0.20 Marker size : retention time2 0.41 2.2 <0.05

72 Vector activity and propagule size affect dispersal potential by vertebrates

a. Active phase (0−5h) b. Inactive phase (6−12h) 6 4.0%

4mm 3.0%

4 ) − 1 ) − 1

3mm 3 4mm 2.0% 4mm Number of markers retrieved (h retrieved Number of markers Percentage of markers retrieved (h retrieved of markers Percentage 2 4mm 3mm 3mm 4mm

1.0% 3mm 2mm 3mm 4mm 1 3mm 2mm 2mm 2mm 2mm 2mm

0 0% Isolation Wade Swim Isolation Wade Swim Treatment Figure 4.1: The mean number of 2, 3, and 4 mm markers retrieved per hour (left y-axes) and the percentage retrieved per hour (right y-axes) from mallard ducks in isolation, wading, or swimming treatment during the a) active phase (i.e. the first 5 hours of the experiment where wading and swimming birds were in the flume tank) and b) the inactive phase (6-12 hours after the active phase), n = 12 individuals per treatment group. The significant interaction between wade and swim treatments as found by the GLM in Table 4.1 is visible.

After 5 h, when all birds were placed in the isolation cages, the swimming birds con- trastingly excreted less than the isolation and wading birds. Swimming birds excreted 2.2 times fewer markers than birds in isolation, and 1.4 times fewer markers than wading birds between 6 and 12 h after ingestion (Fig. 4.1b). This caused both linear and non-linear effects of retention time (Table 4.1). The curvilinear component of retention time described the retention time curve most clearly as indicated by its highest standardized coefficient (Table 4.1, the parabolic curve visualized in Ap- pendix Fig. 4.S2). Treatment affected only the linear component of retention time,

73 Chapter 4 dominated by the differences during the initial 5 h of the experiment rather than the overall retrieval over 12 h or 24 h. This interaction (representing pattern of retrieval) was significantly different between swimming and isolation birds (Table 4.1), with wading birds intermediate but not significantly different from swimming birds (although p = 0.07, visualised in more detail in Appendix Fig. 4.S2). Average retention times of markers during the first 12 h were 5 h 20 min for swimming, 5 h 55 min for wading and 6 h 30 min for isolation. Total marker retrieval over 24 h was on average (+SD) still slightly higher for swimming birds (77.5 + 27.0), compared to wading (68.9 + 24.2) and isolation (70.5 + 24.8). Swimming birds only differed significantly from wading birds, and marginally from isolation birds (GLM effect size swim-wade = -0.11, z = -2.3, p <0.05; GLM effect size swim-isolation = -0.09, z = -1.84, p = 0.07. This implies that birds that had been swimming retained the fewest markers longer than 24 h.

100

60 al of intact shells (%) v i e e ret r 40

ulati v Cu m

Swimming Wading Isolation 20

0 2 4 6 8 10 12 Retention time (h)

Figure 4.2: Cumulative percent of intact snail shells retrieved from mallards swimming, wading or in isolation after 1 to 12 hours propagule retention.

74 Vector activity and propagule size affect dispersal potential by vertebrates

Digestive intensity during exercise The number of snails viably retrieved was too low to compare between treatments, but viable snails were retrieved from all three treatment groups (Appendix Table 4.S1). The last viable snail was retrieved after 7 h. The number of excreted intact shells, representing digestive intensity, was fewer for wading birds than for swimming birds over the active period (5 h) (average (+SD) for wading 1.6 + 3.6, for swimming 4.8 + 12.2, GLM effect size = -1.0, z = -3.7, p <0.001). However,isolation birds did not differ from swimming birds (isolation 3.8 + 5.6, GLM effect size = - 0.34, z = -1.6, p = 0.10. After removal of all birds from the flume tank after 5 h there was no longer an effect of treatment in the 6-24 h interval (average +SD on the number of excreted intact shells), although the analysis indicates a trend towards less excretion by swimming birds, intermediate excretion for wading and most excretion for isolation birds (swimming 1.0 + 3.2, wading 1.75 + 5.2, isolation 1.9 + 4.9, wading effect size 0.47, z = 1.2, p = 0.22, isolation effect size = 0.65, z = 1.8, p = 0.07). This pattern is supported by a relatively fast retrieval pattern of intact shells fromswimming birds, intermediate for wading and relatively slowest retrieval from isolation birds (Fig. 4.2). The total number of intact shells recovered after 12 h was 70 for swimming, 40 for wading, and 68 for control birds in isolation. No intact shells were retrieved after more than 12 h.

Propagule size Markers of 2 mm were retrieved 1.6 times more than 3mm markers and 2.6 times more than 4 mm markers during the 24 h of the experiment, which was consistent over treatments (Fig. 4.1, Table 4.1, significant marker size but no interaction with treatment). The non-linear retrieval patterns of markers varied with marker size (see interaction, Table 4.1), but the cumulative release pattern of differently sized markers was similar over time (Appendix Fig. 4.S3). Consistent with the effect of plastic marker size, the average length of excreted snails (3.9mm + 0.04 SE, n = 178) was smaller than the average length ingested (4.3mm + 0.06 SE, n = 100, t = 5.98, p < 0.001).

Discussion

Activity affects propagule retention Physical activity of mallards was found to modulate retention of ingested propagules in their digestive system. By inducing swimming in a flume tank, propagule excretion increased in comparison to inactive birds in dry cages or in the water. The metabolic rate of swimming mallards likely increased to more than four times that of birds resting in water (wading) (Prange & Schmidt-Nielsen, 1970). The higher thermal conductivity of the wading birds in water might have increased their metabolic rate by as much as 25-30% compared to isolation birds (Prange & Schmidt-Nielsen, 1970; Richman & Lovvorn, 2011). We therefore suggest that the increased excretion of plastic markers is associated with an increase of metabolic rate. This corresponds to the intermediate number of markers excreted by wading birds and the fact that marker excretion of active animals was only higher during the actual active phase of the experiment, and again reduced after the activity stopped. Digestive rate is likely a flexible and rapidly adjustable process influenced by the metabolic rate of vectors.

75 Chapter 4

Wading birds were included as a treatment because factors such as the ability to see conspecifics or the increase of thermal heat loss in water might have caused differences between isolation and swimming birds. The isolation birds allow comparison to the general situation of birds in previous endozoochory experiments, since most experiments with waterbirds to date have monitored retention times of inactive birds in such small and dry cages (Appendix Table 4.S2). Indeed, the wading birds with an increased thermal conductivity to water of only 18°C, excreted more makers than isolation birds. Such cage-effects by themselves can therefore already change experimental results. Comparing swimming and wading birds to assess the effect of activity alone resulted in a 50% increase of propagule excretion during activity. Propagule retention times in experiments with inactive animals are therefore probably overestimated. That swimming birds also excreted the most propagules during the whole experiment over 24 h, although they were only active for 5 h of this time, indicates the potential for extreme long-distance transport may be lower than thus far inferred from experimental data in inactive birds. On the other hand, the experiments show that plastic markers were retained longer than 24 h in all treatments, suggesting a greater potential for long distance dispersal than previously anticipated in the literature for those propagules potentially able to survive such long retention.

Digestive intensity Propagule survival is known to decrease exponentially with increasing retention time (Charalambidou et al., 2003c; Charalambidou et al., 2005; Pollux et al., 2005). Hence, the shorter retention of propagules we found in active animals should result in higher viability, assuming digestive intensity is not influenced by activity. To compare digestive efficiencies between treatments directly, we included aquatic snails, Hydrobia (P.) ulvae, as propagules in our experiments. H. ulvae is an operculated snail that can close its shell and survive digestion by waterbirds (Anders et al., 2009; Cadée, 2011; Van Leeuwen et al., 2012). Because of low retrieval of viable snails, we used retrieval of intact snail shells as a proxy for digestive intensity. As expected, the retrieval of snail shells in all treatments decreased with longer retention in birds (Appendix Table 4.S1). We expected that activity would increase the blood flow to the birds’ lungs and muscles involved in physical activity, which would reduce the potential to allocate blood to the digestive system (Brouns & Beckers, 1993). This could reduce digestive intensity. However, we found no clear evidence for this conjecture. Although swimming birds were less efficient in digesting snails thanwading birds, the isolation birds were also less efficient than wading birds. The effect of activity on digestive intensity thereby remains inconclusive. Nevertheless, the shorter retention times of intact shells (Fig. 4.2) as well as markers (Fig. 4.1, Table 4.1) provides a mechanism that increases propagule survival. Shorter retention times for more active birds, without indications for increased digestive intensity, will result in higher survival of propagules but dispersal over shorter distances.

76 Vector activity and propagule size affect dispersal potential by vertebrates

Effect of propagule size on dispersal distance and retention Propagule size is considered an important trait determining dispersal success. Smaller propagules are often retrieved in higher numbers and after longer retention in experiments (e.g. Traveset, 1998; Soons et al., 2008; Figuerola et al., 2010). How- ever, not all studies obtain similar results (Wongsriphuek et al., 2008; Brochet et al., 2010), and the propagules in most experiments did not only differ in size, but inevitably also in other aspects such as shape and structure (e.g. Mazer & Wheelwright, 1993). In contrast, our experiment with indigestible markers that only differ in size indicates that larger propagules actually have potential for longer retention times than smaller propagules. This is similar to previous marker studies with other bird species (Grajal & Parra, 1995; Figuerola & Green, 2005). While larger organic propagules (that can be digested) are mostly retrieved at short retention times only, larger indigestible markers are on average excreted after longer retention. The indigestible markers were even occasionally retrieved from birds involved in a subsequent experimental treatment 3 days after initial ingestion (distinguishable by their yellow colour). Since not all plastic markers were excreted within the 24 h of the experiments (see Appendix Fig. 4.S3), they were likely slowly released over the days following the feeding, perhaps after being retained as grit (Mateo et al., 2000). This supports the original suggestion that larger propagules are more likely to become trapped in the gizzard or other parts of the digestive system, and stay there for prolonged periods of time (DeVlaming & Proctor, 1968). Because most large organic propagules are increasingly damaged during extremely long retention (up to days), they are mostly retrieved intact in experiments only after relatively short retention times. Our indestructible plastic markers indicate a potential for extreme long distance dispersal by larger propagules (>2 mm) in case of sufficient resistance to digestion. Overall, smaller propagules (<2mm) will have shorter exposure to digestive damage by passing the digestive system faster, and therefore have a higher success rate for dispersal. This is indeed what we observed for the aquatic snails. Smaller propagules will be quantitatively more dispersed but at shorter distances. Larger propagules might be transported over extremely long distances, but only if they can survive such long retention. Given the generally small size of propagules retrieved in waterbird droppings (e.g. Charalambidou & Santamaría, 2005; Frisch et al., 2007), large propagules with this potential may be scarce.

Implications Our experiment involved waterbirds, chosen because of their suggested high importance as passive dispersal vectors (Bilton et al., 2001; Figuerola & Green, 2002; Green & Figuerola, 2005). In previous studies with waterbirds that assessed retention times of propagules experimentally, inactive animals had cage sizes varying between 3.0x3.0m (LxW) and as small as 0.20x0.20x0.30m (LxWxH), in which a duck can hardly move (Appendix Table 4.S2). Most frequently used cages measured 0.60x0.50x0.50m, thus for comparison our isolation birds were kept in cages of similar size. However, the problem of artificially reduced activity in endozoochorous ex-

77 Chapter 4 periments goes beyond experiments with waterbirds. Retention times and dispersal distances of frugivorous terrestrial birds have also been inferred from birds held in small cages or even cotton bags (e.g. Spiegel & Nathan, 2007; Lehouck et al., 2011). Feeding experiments with fish are performed in small tanks (e.g. Pollux et al., 2006; Anderson et al., 2009), and seed retention by mammals such as foxes, monkeys and elephants are inferred from animals retained in cages ranging from one to a several square metres (e.g. Graae et al., 2004; Varela & Bucher, 2006; Campos-Arceiz et al., 2008; Tsuji et al., 2010). Although all mentioned experiments make important contri- butions to our knowledge on dispersal, their estimated dispersal distances should be refined by correcting for potential activity of vectors. Thereby it should be borne in mind that different modes of transport may affect digestion differently. While swimming presumably increased the metabolic rate of the ducks fourfold, flying by the mallards would likely affect metabolic rate and digestive processes differently. Whether or not an even higher increase of metabolic rate (e.g. by flying) will further accelerate digestion or may instead reduce propagule excretion requires further research.

Conclusions The fact that active animals have shorter retention times and higher propagule survival rates implies that past estimates of long distance dispersal potential using captive vertebrates may have overestimated dispersal distances, while underestimating propagule survival. These findings are of importance when constructing dispersal kernels to estimate dispersal distances of invasive species, assessing the capability of individuals to disperse across fragmented habitats, or estimating the colonization potential of rare species. Cage characteristics and circumstances (in water or on land) affect experimental outcomes. Experimentally monitoring propagule survival and retention times in flying birds, swimming fish, and moving mammals therefore provides interesting avenues for future research, but will require creative solutions for the practical issues involved in experiments with moving animals.

Acknowledgements We thank Bart van Lith, Naomi Huig, Ralf Kurvers, Ab and Gillis Wijlhuizen for help during the experiment, and Jillis Kinkel for drawing Figure 4.S1. We thank David J. Marcogliese, Andy J. Green and one anonymous reviewer for comments on an earlier version of this manuscript. This experiment has been carried out under license number CL09.01 of the KNAW animal experiments committee. This is publication 5210 of the Netherlands Institute of Ecology (NIOO-KNAW).

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fishes in Amazonian floodplain habitats. Oecologia, 161, 279-290 Anne Bråthen, K., González, V.T., Iversen, M., Killengreen, S., Ravolainen, V.T., Ims, R.A. & Yoccoz, N.G. (2007) Endozoochory varies with ecological scale and context. Ecography, 30, 308-320 Baldwin, J.R. & Lovvorn, J.R. (1994) Habitats and tidal accessibility of the marine foods of dabbling ducks and brant in Boundary Bay, British Columbia. Marine Biology, 120, 627-638 Bell, D. (2000) The ecology of coexisting Eleocharis species. PhD-thesis Bilton, D.T., Freeland, J.R. & Okamura, B. (2001) Dispersal in freshwater invertebrates. Annual Review of Ecology and Systematics, 32, 159-181 Brochet, A.-L., Guillemain, M., Gauthier-Clerc, M., Fritz, H. & Green, A.J. (2010) Endozoo- chory of Mediterranean aquatic plant seeds by teal after a period of desiccation: Deter- minants of seed survival and influence of retention time on germinability and viability. Aquatic Botany, 93, 99-106 Brochet, A.L., Guillemain, M., Fritz, H., Gauthier-Clerc, M. & Green, A.J. (2009) The role of migratory ducks in the long-distance dispersal of native plants and the spread of exotic plants in Europe. Ecography, 32, 919-928 Brouns, F. & Beckers, E. (1993) Is the gut an athletic organ - digestion, absorption and exercise. Sports Medicine, 15, 242-257 Bruun, H.H., Lundgren, R. & Philipp, M. (2008) Enhancement of local species richness in tundra by seed dispersal through guts of muskox and barnacle goose. Oecologia, 155, 101-110 Cadée, G.C. (2011) Hydrobia as “Jonah in the whale”: shell repair after passing through the digestive tract of shelducks alive. Palaios, 26, 245-249 Campos-Arceiz, A., Larrinaga, A.R., Weerasinghe, U.R., Takatsuki, S., Pastorini, J., Leimgru- ber, P., Fernando, P. & Santamaría, L. (2008) Behavior rather than diet mediates seasonal differences in seed dispersal by asian elephants.Ecology , 89, 2684-2691 Charalambidou, I., Ketelaars, H.A.M. & Santamaría, L. (2003a) Endozoochory by ducks: influence of developmental stage of Bythotrephes diapause eggs on dispersal probability. Diver- sity and Distributions, 9, 367-374 Charalambidou, I., Santamaría, L. & Figuerola, J. (2003b) How far can the freshwater bryozoan Cristatella mucedo disperse in duck guts? Archiv für Hydrobiologie, 157, 547-554 Charalambidou, I., Santamaría, L. & Langevoord, O. (2003c) Effect of ingestion by five avian dispersers on the retention time, retrieval and germination of Ruppia maritima seeds. Func- tional Ecology, 17, 747-753 Charalambidou, I. & Santamaría, L. (2005) Field evidence for the potential of waterbirds as dispersers of aquatic organisms. Wetlands, 25, 252-258 Charalambidou, I., Santamaría, L., Jansen, C. & Nolet, B.A. (2005) Digestive plasticity in mallard ducks modulates dispersal probabilities of aquatic plants and crustaceans. Functional Ecology, 19, 513-519 Darwin, C. (1859) On the origin of species by means of natural selection, or the preservation of fa- voured races in the struggle for life. John Murray, London. DeVlaming, V. & Proctor, V.W. (1968) Dispersal of aquatic organisms - viability of seeds revo- vered from droppings of captive killdeer and mallard ducks. American Journal of Botany, 55, 20-26 Fenchel, T. (1975) Factors determining the distribution patterns of mud snails (Hydrobiidae). Oecologia, 20, 1-17 Figuerola, J. & Green, A.J. (2002) Dispersal of aquatic organisms by waterbirds: a review of

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past research and priorities for future studies. Freshwater Biology, 47, 483-494 Figuerola, J. & Green, A.J. (2005) Effects of premigratory fasting on the potential for long distance dispersal of seeds by waterfowl: An experiment with marbled teal. Revue d’ Écologie- La terre et la Vie, 60, 283-287 Figuerola, J., Charalambidou, I., Santamaría, L. & Green, A.J. (2010) Internal dispersal of seeds by waterfowl: effect of seed size on gut passage time and germination patterns. Naturwis- senschaften, 97, 555-565 Frisch, D., Green, A.J. & Figuerola, J. (2007) High dispersal capacity of a broad spectrum of aquatic invertebrates via waterbirds. Aquatic Sciences, 69, 568-574 Gelman, A. (2008) Scaling regression inputs by dividing by two standard deviations. Statistics in Medicine, 27, 2865-2873 Graae, B.J., Pagh, S. & Bruun, H.H. (2004) An experimental evaluation of the arctic fox (Alopex lagopus) as a seed disperser. Arctic Antarctic and Alpine Research, 36, 468-473 Grajal, A. & Parra, O. (1995) Passage rates of digesta markers in the gut of the hoatzin, a foli- vorous bird with foregut fermentation. The Condor, 97, 675-683 Green, A.J. & Figuerola, J. (2005) Recent advances in the study of long-distance dispersal of aquatic invertebrates via birds. Diversity and Distributions, 11, 149-156 Gruenhagen, N.M. & Fredrickson, L.H. (1990) Food use by migratory female mallards in Northwest Missouri. Journal of Wildlife Management, 54, 622-626 Koike, S., Masaki, T., Nemoto, Y., Kozakai, C., Yamazaki, K., Kasai, S., Nakajima, A. & Kaji, K. (2011) Estimate of the seed shadow created by the Asiatic black bear Ursus thibetanus and its characteristics as a seed disperser in Japanese cool-temperate forest. Oikos, 120, 280-290 Lehouck, V.L.V., Spanhove, T. & Lens, L. (2011) Avian fruit ingestion differentially facilitates seed germination of four fleshy-fruited plant species of an Afrotropical forest.Plant Ecology and Evolution, 144, 96-100 -Liu, H., Plat, S.G. & Borg, C.K. (2004) Seed dispersal by the Florida box turtle (Terrapene caro lina bauri) in pine rockland forests of the lower Florida Keys, United States. Oecologia, 138, 539-546 Mateo, R., Guitart, R. & Green, A.J. (2000) Determinants of lead shot, rice, and grit ingestion in ducks and coots. Journal of Wildlife Management, 64, 939-947 Mazer, S.J. & Wheelwright, N.T. (1993) Fruit size and shape - allometry at different taxonomic levels in bird-dispersed plants. Evolutionary Ecology, 7, 556-575 Mueller, M.H. & Van der Valk, A.G. (2002) The potential role of ducks in wetland seed dispersal. Wetlands, 22, 170-178 Nakagawa, S. & Schielzeth, H. (2010) Repeatability for Gaussian and non-Gaussian data: a practical guide for biologists. Biological Review, 85, 935-956 Pollux, B.J.A., Santamaría, L. & Ouborg, N.J. (2005) Differences in endozoochorous dispersal between aquatic plant species, with reference to plant population persistence in rivers. Freshwater Biology, 50, 232-242 Pollux, B.J.A., De Jong, M., Steegh, A., Ouborg, N.J., Van Groenendael, J.M. & Klaassen, M. (2006) The effect of seed morphology on the potential dispersal of aquatic macrophytes by the common carp (Cyprinus carpio). Freshwater Biology, 51, 2063-2071 Pollux, B.J.A. (2011) The experimental study of seed dispersal by fish (ichthyochory). Fresh- water Biology, 56, 197-212 Powers, K.D., Noble, R.E. & Chabreck, R.H. (1978) Seed distribution by waterfowl in South- western Louisiana. Journal of Wildlife Management, 42, 598-605

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Prange, H.D. & Schmidt-Nielsen, K. (1970) The metabolic cost of swimming in ducks. Journal of Experimental Biology, 53, 763-777 R-Development-Core-Team (2011) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http:// www.R-project.org. Raudenbush, S.W. & Bryk, A.S. (2002) Hierarchical linear models: Applications and data analysis methods., 2nd edn. Sage, Newbury Park, CA. Raulings, E., Morris, K.A.Y., Thompson, R. & Nally, R.M. (2011) Do birds of a feather disperse plants together? Freshwater Biology, 56, 1390-1402 Richman, S.E. & Lovvorn, J.R. (2011) Effects of air and water temperatures on resting metabo- lism of auklets and other diving birds. Physiological and Biochemical Zoology, 84, 316-332 Ridley, H.N. (1930) The distribution of plants throughout the world. . Reeve & Company, Ashford, United Kingdom. Rodrigues, D., Figueiredo, M. & Fabiao, A. (2002) Mallard (Anas platyrhynchos) summer diet in central Portugal rice-fields. Game & Wildlife Science, 10, 55-62 Santamaría, L., Charalambidou, I., Figuerola, J. & Green, A.J. (2002) Effect of passage through duck gut on germination of fennel pondweed seeds. Archiv für Hydrobiologie, 156, 11-22 Schielzeth, H. & Forstmeier, W. (2009) Conclusions beyond support: overconfident estimates in mixed models. Behavioral Ecology, 20, 416-420 Smits, A.J.M., Van Ruremonde, R. & Van der Velde, G. (1989) Seed dispersal of three nym- phaeid macrophytes. Aquatic Botany, 35, 167-180 Soons, M.B., Van der Vlugt, C., Van Lith, B., Heil, G.W. & Klaassen, M. (2008) Small seed size increases the potential for dispersal of wetland plants by ducks. Journal of Ecology, 96, 619- 627 Spiegel, O. & Nathan, R. (2007) Incorporating dispersal distance into the disperser effectiveness framework: frugivorous birds provide complementary dispersal to plants in a patchy environment. Ecology Letters, 10, 718-728 Swanson, G.A., Meyer, M.I. & Adomaitis, V.A. (1985) Foods consumed by breeding mallards on wetlands of south-central North Dakota. Journal of Wildlife Management, 49, 197-203 Traveset, A. (1998) Effect of seed passage through vertebrate frugivores’ guts on germination: a review. Perspectives in Plant Ecology Evolution and Systematics, 1, 151-190 Traveset, A., Rodriguez-Perez, J. & Pias, B. (2008) Seed trait changes in dispersers’ guts and consequences for germination and seedling growth. Ecology, 89, 95-106 Tsuji, Y., Morimoto, M. & Matsubayashi, K. (2010) Effects of the physical characteristics of seeds on gastrointestinal passage time in captive Japanese macaques. Journal of Zoology, 280, 171-176 Van Leeuwen, C.H.A., Van Der Velde, G., Van Lith, B. & Klaassen, M. (2012) Experimental quantification of long distance dispersal potential of aquatic snails in the gut of migratory birds. PLoS ONE, 7, e32292 Varela, O. & Bucher, E.H. (2006) Passage time, viability, and germination of seeds ingested by foxes. Journal of Arid Environments, 67, 566-578 Wongsriphuek, C., Dugger, B.D. & Bartuszevige, A.M. (2008) Dispersal of wetland plant seeds by mallards: Influence of gut passage on recovery, retention, and germination. Wet- lands, 28, 290-299

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Appendix Chapter 4

Table 4.S1: Sum of propagules ingested per treatment and retrieved from all 12 mallards per treatment.

Markers Hydrobia (Peringia) ulvae 2mm 3mm 4mm Shells intact Fragments Living snails Ingested 600 600 600 3600 Active (0-5h) Retrieved Isolation 122 82 54 45 (1.3%) 26 (0.7%) 8 Wading 186 137 76 19 (0.5%) 65 (1.8%) 5 Swimming 280 201 108 58 (1.6%) 88 (2.4%) 1 0-12h Retrieved Isolation 354 267 143 68 (1.8%) 110 (3.1%) 9 Wading 337 246 134 40 (1.1%) 115 (3.2%) 5 Swimming 382 280 157 70 (1.9%) 134 (3.7%) 1 12-24h Retrieved Isolation 23 31 28 0 (0%) 0 (0%) 0 Wading 31 49 30 0 (0%) 0 (0%) 0 Swimming 32 46 33 0 (0%) 0 (0%) 0

Table 4.S2: Cage sizes for the sixteen most recent endozoochorous experiments with waterbirds. In all publications birds were individually kept and the most cages had floors of mesh wire (ranging from 9 to 12-mm). Publications are sorted starting with the largest cage size.

Cage size, LxW(xH) Publication 3.00x3.00m (Figuerola & Green, 2005) 1.70x1.70 m (Charalambidou et al., 2003c) 2.00x0.80x0.60m or (Smits et al., 1989) 1.0x0.79x0.58m (with small water basin) 0.50x1.50m (Bell, 2000) 0.60×0.50×0.50m (Santamaría et al., 2002; Charalambidou et al., 2003a; Charalambidou et al., 2003b; Charalambidou et al., 2005; Pollux et al., 2005; Soons et al., 2008; Brochet et al., 2010; Figuerola et al., 2010) 0.35x0.75x0.60 (Powers et al., 1978) 0.20x0.20x0.30m (Wongsriphuek et al., 2008) not mentioned (Agami & Waisel, 1986) “pens” (Mueller & Van der Valk, 2002)

82 Vector activity and propagule size affect dispersal potential by vertebrates

Figure 4.S1: The indoor flume tank, with 1. Sieves for collecting droppings, 2.Swimming ducks, ! 3. Wading ducks sitting on mesh wire beneath the water surface preventing the mallards from swimming, 4. Electric outboard engines. The total construction was 4.80 m by 2.05 m, with tank width of 0.41 m and water depth of 0.37 m; the arrows indicate the direction of the current.

0.40

0.35 Isolation 0.30 Wading Swimming 0.25 ed (%) v 0.20 ie

0.15 ers retr k

Mar 0.10

0.05

0.00 123456789101112

Retention time (in hours)

Figure 4.S2: Retrieval of markers over time for the different treatments, average over all sizes (mean + SE).

83 Chapter 4

100

2 mm 3 mm 4 mm

80 ers (%)

k 60

al of ma r v i e

e ret r 40

ulati v Cu m

0 2 4 6 8 10 12 14 16 18 20 22 24

Retention time (h) Figure 4.S3: Cumulative retrieval of different sizes markers over time, averaged for all treatments and plotted as percentage of total ingested.

84 Chapter 5

Prerequisites for flying snails: external transport potential of aquatic snails by waterbirds

Casper H. A. van Leeuwen & Gerard van der Velde

Freshwater Science (in press)

85 Chapter 5

Abstract The widespread distributions of aquatic species often contrast with their limited ability to disperse by their own propulsion among wetlands isolated by land. Stud- ies of the potential role of water birds as dispersal vectors have been focused mainly on internal transport (endozoochory). However, many anecdotal observations that small species adhere to flying birds also exist (ectozoochory). We addressed the hypothesis that ectozoochory may contribute to the widespread distributions of aquatic snails (Gastropoda) in several experiments. We tested the likelihood that snails would attach to mallards (Anas platyrhynchos) leaving macrophyte vegetation with high densities of 3 snail species. All species tested (Gyraulus albus, Anisus vortex, and Radix balthica) readily attached to the mallards’ bodies. The rate of attachment was proportional to snail density, and the birds’ feathers contained most snails. How- ever, ⅔ of the snails detached when mallards subsequently walked for 3 m. Snails of 12 species attached within minutes to any surroundings available when floating in the water, a result indicating that active crawling onto birds may facilitate dispersal. Snails we attached deliberately to duck bills with mud could remain attached for up to 8 h. We measured desiccation tolerance of 13 common aquatic snail species. Al- most all snail species survived 48 h of desiccation at 10 to 20°C. The ability to retain water did not differ between species with an operculum and species that form a mucus layer (epiphragm) in their shell openings. Our experiments indicate that aquatic snails possess a range of prerequisites for successful bird-mediated dispersal, but the capacity of snails (and other propagules) to remain attached during flight and successfully colonize new habitats upon arrival must still be assessed.

Key words: Gastropoda, ectozoochory, desiccation, dispersal, adhesion.

86 External transport potential of aquatic snails by waterbirds

Introduction Biodiversity in isolated aquatic wetlands or on remote oceanic islands often includes species with limited ability to locomote (Page et al., 2007; Schabetsberger et al., 2009). Dispersal of these often small species by vectors, such as wind (anemochory), water (hydrochory), or larger, more mobile animals (zoochory) may explain their widespread distributions (e.g. Couvreur et al., 2005; Hogan & Phillips, 2011). For many species that are restricted to aquatic habitats, this passive dispersal is essential for their persistence in wetland metapopulations that can cover multiple “islands in a sea of land” (Darwin, 1909). Several potential vectors could help disperse aquatic species, and water birds were suggested as particularly suitable dispersal vectors long ago (Darwin, 1859). Birds move quickly, are generally abundant, and migrate long distances between similar habitats. Observations of birds carrying smaller organisms have accumulated steadily since Darwin’s time, and the number of publications on this subject has increased (Figuerola & Green, 2002a; Green & Figuerola, 2005). Terrestrial and water birds can transport plants seeds, algae, and invertebrates internally if these organisms can survive passage through their digestive system (endozoochory) or externally if species adhere to their exterior during flight (ectozoochory) (reviewed by e.g. Maguire, 1963; Sorensen, 1986; Kristiansen, 1996; Traveset, 1998; Bilton et al., 2001; Figuerola & Green, 2002a; Green & Figuerola, 2005). Brochet et al. (2010) regarded external transport of aquatic plants by adhesion as less important for dispersal than internal transport because they retrieved greater diversity and abundance of aquatic propagules from feces and lower guts than attached to birds. However, most knowledge of ectozoochory is still anecdotal (e.g. Mcatee, 1914; Cockerell, 1921; Bondesen & Kaiser, 1949; Roscoe, 1955; Cotton, 1960; Daborn, 1976). Only some field observations (e.g. Viviansmith & Stiles, 1994; Figuer- ola & Green, 2002b; Frisch et al., 2007; Brochet et al., 2010; Raulings et al., 2011) and targeted experiments (e.g. Davies et al., 1982; Boag, 1986; Barrat-Segretain, 1996; Johnson & Carlton, 1996) have been published. Ectozoochory may be possible for aquatic snails (Gastropoda). Numerous anecdotal reports exist that snails adhere to birds (see review by Rees, 1965), but only few experiments have been done to strengthen these observations (e.g. Darwin, 1859; Boag, 1986). Active dispersal by snails is limited to only few km/y (Kappes & Haase, 2011), but dispersal by water birds may be an explanation for the generally rapid colonization of new suitable habitat by aquatic snails, their widespread distributions (Hubendick, 1951), and the existence of multiple rapidly spreading invasive aquatic snail species (e.g. Dillon et al., 2002; Alonso & Castro-Diez, 2008). The need to understand snail dispersal mechanisms is urgent because they are important dispersal vectors for human and livestock parasites (Brown, 1978; Morley, 2008). We investigated the potential for ectozoochory in aquatic snails with 4 complementary experiments. First, we tested whether aquatic snails could attach to water birds and persist in this attachment. Second, we investigated whether snails stayed attached in drying mud on water birds. Third, we addressed the potential of snails to crawl onto birds by active movement. Last, we assessed the desiccation tolerance

87 Chapter 5 of 13 common aquatic snail species with various shell sizes and under various temperatures because desiccation tolerance strongly affects survival of aquatic species during external transport (Barrat-Segretain, 1996; Figuerola & Green, 2002a).

Methods

Transport experiment I We tested the ability of snails to adhere to mallards (Anas platyrhynchos) in our bird facilities in Heteren, The Netherlands, in July 2009. We chose mallards because they are common, interact frequently with snails (e.g. Gruenhagen & Fredrickson, 1990; Baldwin & Lovvorn, 1994), and adjust easily to captivity and experimental setups (Charalambidou et al., 2005; Soons et al., 2008). We ran a total of 48 trials with 12 different male mallards over a period of 4 d. We ran trials in the morning, early afternoon, and late afternoon. Each time, we took 4 mallards from the outdoor aviary and introduced them individually into 1 of 4 cages (0.9 × 0.7 × 1.2 m [l × w × h]). Cages were constructed of 10-mm-thick wood with 0.1-m-deep metal removable trays as bases. One hour before each trial, we filled the trays to a depth of 0.05 m with water containing aquatic snails associated with a mixture of macrophytes dominated by Elodea sp. and Lemna sp. We collected this vegetation from a ditch at Driemond, The Netherlands (lat 52°17′36′′N, long 05°01′13′′E), <1 d before each trial, and held it overnight in aquaria under ambient temperature conditions. The snail species in this vegetation were Gyraulus albus, Anisus vortex, and Radix balthica (mean ± SD densities = 13.0 ± 6.5, 6.9 ± 3.7, and 4.6 ± 5.7 snails/10 g plant material, respectively; n = 15). We defined snail size as the maximum measurable shell dimension (shell height for cone-shaped R. balthica and shell diameter for planorbid species; (Gittenberger et al., 2004). Snail size was 4.1 ± 0.8 mm for G. albus, 3.6 ± 0.4 mm for A. vortex, and 4.5 ± 0.6 mm for R. balthica (n = 10 snails/species), and size was normally distributed. We created 4 snail densities by adding 1.0, 2.0, 3.0, or 4.0 kg of the vegetation/ snail mixture to the water in the trays. This procedure yielded linearly increasing snail densities of, on average, 1500, 3000, 4500, and 6000 snails/m2 in a ratio of 3:4:8 (R. balthica:A. vortex:G. albus). Over the course of the experiment, we placed each mallard in a cage with each snail density once in a random block design. For each trial, we kept mallards individually in the cages with vegetation for 60 min. Subsequently, we allowed them to exit the cage through a polyvinyl chloride (PVC) tunnel (0.41 m wide, 0.41 m high, 3.0 m long) covered by mesh wire. The tunnels connected the cages with vegetation to identical, but clean, cages where we examined the birds for adhering snails. We checked the tunnels for detached snails after each trial. Before and after each trial, we inspected all mallards for snails with methods similar to those regularly used in field investigations (see e.g. Viviansmith & Stiles, 1994; Figuerola & Green, 2002b; Brochet et al., 2010). First, we visually inspected the feet and bills of the mallards and rinsed their feet rinsed in clean tap water. Second, we brushed each bird with a soft shoe brush above an empty tray and checked for the presence of snails between its feathers. We sieved the water used to rinse the

88 External transport potential of aquatic snails by waterbirds feet and to wash the birds separately through a 1-mm-mesh sieve. We distinguished between those snails that remained attached to the birds (feathers, feet, or bill), those that detached while the bird walked through the tunnel (tunnel), and those that we detached during inspection of the bird for presence of snails in the final tray (tray).

Transport experiment II We ran a 2nd transport experiment with Potamopyrgus antipodarum collected from the Ooijpolder, The Netherlands (lat 51°51′12′′N, long 05°53′18′′E). Mean snail size was 3.6 ± 0.7 mm (SD, n = 10). We mimicked snail ectozoochory that might occur after a mallard foraging bout in mud to test how long snails could potentially remain attached to birds after initial attachment. We used P. antipodarum as the focal species because of its relatively neutral characteristics for attachment by mud. It does not have a flat shell (like Planorbidae) or extensive mucous secretion (like R. balthica) that might facilitate attachment. We ran 25 trials, starting in the mornings) over 4 d with 8 different male mallards. In each trial, we deliberately attached 10 snails to the bill of each mallard with a ~2- to 5-mm layer of mud. After attachment, we placed the mallards in clean cages (described above) without access to food or water. We checked the bills for detachment of snails at 30-min intervals over 8 h or until all 10 snails were detached from all birds during a trial.

Adhesion experiments In May 2008 and May 2009, we tested the readiness with which 11 freshwater and 1 marine snail species would attach to objects in their environment. We collected snails from their natural habitat in The Netherlands (for species and location details see Ta- ble 5.1; note that P. planorbis is in the table but was included only in the desiccation experiment described below). We held snails in aquaria filled with water from their sampling location at 15°C for no longer than 2 days before testing. For each species, we removed 100 snails from their aquarium and placed them in a 0.2 × 0.2-m plastic tray with 0.01 m of their natural water at 20°C for 5 min. At t = 0, we made sure all snails were detached, and then counted attached snails at 1-min intervals for 10 min.

Desiccation experiments We monitored mass loss caused by evaporation of water and survival of 12 freshwater and 1 marine snail species in May 2008 and May 2009. We collected snails from their natural habitat in The Netherlands (see Table 5.1 for species and location details). For each species, we divided 75 snails evenly among 3 desiccation treatments (ambient temperatures = 10, 15, or 20°C). We measured shell length and width as previously described, and weighed snails with a Sartorius Microbalance ME5 (reso- lution d = 0.001 mg; Sartorius AG, Goettingen, Germany) after removing outside moisture from their shells by rolling them in filter paper. Immediately after weigh- ing them, we placed the snails individually in 10 × 10 × 10-mm cubicles in a 0.1 × 0.1 × 0.01-m tray covered with 1-mm mesh to prevent the snails from crawling out. We placed the trays over water in temperature-controlled aquaria to control humidity and held them at the appropriate temperature for 48 h. We monitored the air

89 Chapter 5 temperature in the trays with temperature loggers (Tinytagg Talk 2, TK-4014-MED, Gemini Data Loggers [UK] Ltd., Chichester, United Kingdom) (mean ± SD temperatures over 48 h = 10.2 ± 0.5, 14.7 ± 0.6, and 20.0 ± 0.6). In all cases, relative humidity of the air was between 80 and 85%. We measured snail mass after desiccation for 48 h and then resubmerged them in their natural water (taken from the sampling location) at 20°C. We assessed survival by monitoring movement over the next 7 d, and when in doubt, we checked by monitoring foot-retraction reactions after touch under a microscope.

Statistical analysis For transport experiment I, we used linear regression to describe the relationship between the number of transported snails and snail density for all species together because of the limited available data (function lm in R; R Development Core Team, Vienna, Austria). For transport experiment II, we used the lm function to describe the relationship between the (log[x]-transformed) number of snails that remained attached to bills for ≥0.5 h and over time. We tested the effects of snail size and possession of an operculum and desiccation temperature on survival with a generalized linear model (GLM) with binomial error distribution and logit link function (package lmer in R). We used viability of snails after 48 h as a binomial dependent variable and included snail size and desiccation temperature as covariates after centering (Raudenbush & Bryk, 2002).

Table 5.1: Snail species per family, the year in which they were collected, their sampling location, presence of an operculum, the size range of tested individuals, and the shape of the shell.

Family Species Year Latitude Longitude Location Operculum Size Shape

Planorbidae Planorbis planorbis 2009 52°8′39′′N 5°1′36′′E Breukelen ditch No 7–14 Flat

Planorbis carinatus 2008 52°12′40′′N 5°2′18′′E Loenen small No 8–12 Flat

pond

Gyraulus albus 2008 52°17′36′′N 5°1′13′′ E Driemond ditch No 2–3 Flat

Bathyomphalus contortus 2008 52°9′42′′N 5°1′52′′E Loosdrecht ditch No 2–4 Flat

Anisus vortex 2009 52°17′36′′N 5°1′13′′E Driemond ditch No 4–8 Flat

Lymnaeidae Stagnicola palustris 2009 53°8′22′′N 6°1’40′′E Nijega ditch No 9–19 Cone

Radix balthica 2008 51°5′19′′N 5°28′48′′E Meeuwen ditch No 8–15 Cone

Lymnaea stagnalis 2008 52°12′40′′N 5°2′18′′E Loenen small No 5–13 Cone

pond

Valvatidae Valvata piscinalis 2009 51°56′31′′N 5°46′37′′E Driel ditch Yes 2–5 Cone

Hydrobiidae Potamopyrgus 2008 51°51′12′′N 5°53′18′′E Ooijpolder lake Yes 2–4 Cone

antipodarum

Hydrobia ulvae 2008 53°24′17′′N 6°4′59′′E Paesens coast Yes 3–5 Cone

Bithyniidae Bithynia tentaculata 2009 52°17′36′′N 5°1′13′′E Driemond ditch Yes 5–11 Cone

Bithynia leachii 2008 52°3′33′′N 5°10′14′′E Vechten ditch Yes 2–5 Cone

90 External transport potential of aquatic snails by waterbirds

We included operculum as a fixed factor, and year and snail species as random factors because we tested during 2 subsequent seasons, and the snail species had different size ranges (Table 5.1). We nested snail species in the fixed factor, operculum. We analyzed the effects of desiccation and shell size with separate linear models for each species. The % mass loss over 48 h was the dependent variable and snail length and desiccation temperature were centered covariates.

Results

Transport experiments In transport experiment I, all 3 snail species present in the vegetation were transported by mallards. Snails were transported out of the cages in 34 of 48 trials (71%). A linear increase in snail density resulted in a linear increase in total number of snails transported (linear regression calculated for all species pooled, R2 = 0.94, p < 0.01). More A.vortex and G. albus were transported in total (found in the tray, in the tunnel, and on birds) than R. balthica (39 transported [0.33% of snails in the tray], 36 [0.23%], and 4 [0.04%]), respectively; Fig. 5.1). Of 2.5 A the total number of snails 2.0 transported, 65% detached either in the tunnel or in the 1.5 ● tray, whereas 35% was still 1.0 ● attached to the bird upon ● examination. The feathers 0.5 ● of the mallards contained 0.0 almost 5× more snails than the bill and feet together (23 2.5 B c(400, 800, 1200, 1600) vs 5 for all trials). 2.0

1.5 ● 1.0 ● ● 0.5 ● 0.0

2.5 C c(800, 1600, 2400, 3200) 2.0 Figure 5.1: Mean (± 95% 1.5 CI, n = 12) number of snails 1.0 transported from vegetation/ trial as a function of snail of snails transportedMean number per trial (+/− 95% CI) 0.5 density for Anisus vortex (A), ● ● 0.0 ● ● Gyraulus albus (B), and Radix balthica (C). 0 600 1200 1800 2400 3000 3600 2 Snailc(300, density 600, 900, (no./m 1200) )

91 Chapter 5

Table 5.2: The number of snails that attached to the tray during 10 min. The number is indicated in bold if no additional snails attached until termination of the experiment.

Time (min) Species 1 2 3 4 5 6 7 8 9 10 Radix balthica 100 Bithynia leachii 98 100 Lymnaea stagnalis 45 95 100 Hydrobia ulvae 90 93 98 99 100 Potamopyrgus antipodarum 96 97 98 98 100 Stagnicola palustris 66 82 83 88 93 100 Bithynia tentaculata 60 89 93 94 94 94 94 95 96 98 Bathyomphalus contortus 90 95 96 97 Gyraulus albus 93 95 95 96 96 97 Valvata piscinalis 41 66 75 86 90 94 96 Anisus vortex 5 11 23 34 46 56 66 76 84 89 Planorbis carinatus 27 39 43 44 49 50 50 50 51 Mean 68 80 84 86 89 91 92 93 93 94

10.00

y = 1.97 − 0.25x R 2 = 0.80

1.00

0.10 Log(Snails attached)

0.01

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

Time (h)

Figure 5.2: Mean (±1 SD) number of snails embedded in mud that remained attached to the bills of mallards (Anas platyrhynchos) over time. The number of snails decreased more than exponentially until the first check at t = 0.5 h, after which it followed an exponential decreasing function over the rest of time as indicated by the equation. Note log scale on y-axis.

92 External transport potential of aquatic snails by waterbirds

In transport experiment II, 74% of P. antipodarum individuals that were deliberately attached to the bills of the mallards with mud had detached before the first check after 30 min. The percentage of snails that detached thereafter declined exponentially over time (Fig. 5.2). Thirty-one snails (12.4%) remained attached in the dry mud for 3 h, and 3 (1.2%) remained attached for 8 h.

Desiccation and adhesion experiments In the adhesion experiment, >50% of individuals in 8 of 12 species adhered to their direct surroundings in <1 min (Table 5.2). In 10 of the 12 species (all except Anisus vortex and Planorbis carinatus), >90% of all individuals adhered in <10 min. In the desiccation experiment, over all snail species and all 3 temperature treatments, >50% of the 25 individuals in each temperature treatment survived. The only exceptions were that no P. antipodarum individuals survived the 20°C treatment and 48% of Anisus vortex survived this treatment. Mass loss and number of surviving individuals varied among species and shell sizes (Fig. 5.3, Table 5.3). Snail survival was negatively affected by the increase of temperature in the treatments (GLM, z = –8.9, p < 0.001, effect size = 1.7% less chance to survive if temperature increases by 1°C, based on all snail sizes and species pooled). Average mass loss (± SD) of snails was 7.0 ± 7.7% at 10°C, 11.6 ± 10.3% at 15°C, and 20.2 ± 14.2% at 20°C, calculated over all species (details per species in Table 5.3).

Table 5.3: The effect of shell length and desiccation temperature on its % mass loss over 48 h of desiccation. Coefficients (Coef) indicate the associated change in % mass loss given a change of 1 mm in shell length or 1°C in temperature. Full-model R2 indicates the quality of model fit. Mean (±1 SE) % mass loss and survival are indicated per species for the 3 temperatures. Bold indicates significant p-values.

Shell length Temperature % survival % mass loss

Species p Coef p Coef R2 10 15 20 10 15 20

Planorbis planorbis 0.04 –0.7 <0.001 0.9 0.62 100 100 92 5.5 ± 0.5 7.8 ± 0.4 15.0 ± 0.9

Planorbis carinatus 0.47 0.3 0.11 0.1 0.04 100 100 100 3.6 ± 0.5 4.4 ± 0.3 4.5 ± 0.3

Gyraulus albus 0.16 –8.2 <0.001 1.4 0.18 100 68 56 12.3 ± 2.2 25.0 ± 2.2 26.3 ±3.4

Bathyomphalus contortus <0.001 –11.0 <0.001 2.5 0.74 100 92 72 6.3 ±0.9 19.5 ± 1.4 30.0 ± 2.4

Anisus vortex 0.85 0.1 <0.001 1.6 0.54 68 60 48 2.3 ± 0.8 12.1 ± 1.4 18.0 ± 1.2

Stagnicola palustris 0.40 –0.2 <0.001 1.2 0.59 76 80 52 6.2 ± 0.8 8.0 ± 0.6 17.9 ± 0.8

Radix balthica <0.001 –1.6 <0.001 1.9 0.72 100 100 100 6.1 ±1.0 10.8 ± 1.0 25.1 ± 1.2

Lymnaea stagnalis 0.04 –1.0 <0.001 1.4 0.40 100 96 84 10.5 ± 1.1 10.2 ± 1.1 23.4 ± 1.5

Valvata piscinalis <0.01 –3.0 <0.001 1.2 0.42 84 88 52 8.3 ± 0.6 8.3 ± 0.8 20.6 ± 1.9

Potamopyrgus antipodarum 0.01 –5.0 <0.001 3.3 0.72 92 68 0 18.8 ± 2.2 27.9 ± 1.4 50.0 ± 0.9

Hyrobia ulvae 0.21 –1.3 <0.001 0.7 0.39 100 100 100 2.5 ± 0.6 2.0 ± 0.4 10.1 ± 0.9

Bithynia tentaculata <0.01 –2.6 0.06 0.5 0.19 100 100 72 3.1 ± 2.2 3.5 ± 0.7 10.1 ± 2.7

Bithynia leachii <0.01 –4.4 0.04 0.7 0.13 100 60 60 5.1 ± 1.1 11.7 ± 3.4 11.3 ± 2.2

93 Chapter 5

% weight loss over 48 h

0 10 20 30 40 50 60

Bithynia leachii Operculated Bithyniidae Bithynia tentaculata

Hydrobia ulvae Hydrobiidae Potamopyrgus antipodarum

Valvatidae Valvata piscinalis

Lymnaea stagnalis

Lymnaeidae Radix balthica

Stagnicola palustris No operculum Anisus vortex

Bathyomphalus contortus

Planorbidae Gyraulus albus

Planorbis carinatus

Planorbis planorbis

Maximum % weight loss life animals

Figure 5.3: Box-and-whisker plots for % mass loss of different species of snails during aerial exposure over 48h at 15°C. Species are ordered by families. Presence of an operculum is indicated for each species. Lines in boxes show medians, box ends show 25th and 75th percentiles, whiskers show 95% confidence intervals, and solid squares indicate the observed maximum % mass loss of live specimens, and thereby, the upper range of % mass loss during the experiments.

The maximum mass loss before mortality differed among species (Fig. 5.3). Mass loss did not difer between species with and without opercula (GLM, z = 0.017, p = 0.98). Larger snails had a higher probability of surviving (GLM, z = 2.7, p < 0.01, effect size = 1.4% more chance to survive if shell size is 1 mm larger) and lost a smaller percentage of their initial mass, calculated over all species (details per species in Table 5.3).

94 External transport potential of aquatic snails by waterbirds

Discussion Waterbirds leaving macrophyte vegetation with snails carried a small percentage of these snails on their feathers, feet, and bill. The number of snails attached to their bodies increased proportionally with snail density, and snails adhering in drying mud could remain attached for several hours. Individuals belonging to many snail species actively attached to their available surroundings in minutes, and individuals in all snail species survived prolonged aerial exposure. Thus, initial attachment of snails to living waterbirds, subsequent adherence, and survival of desiccation during transport are unlikely to be limiting factors for ectozoochory of aquatic snails.

Initial attachment At a density of 1000 snails/m2, up to 10 snails attached per bird, but the number of attaching snails varied with snail species (Fig. 1A–C). Densities of snails intheir natural environment can easily exceed several thousand/m2 (Heitkamp & Zemella, 1988; Gittenberger et al., 2004; Anders et al., 2009; Cadée, 2011), so water birds that leave the water by walking directly from vegetation may initially carry multiple snails. Snails probably attach both passively and actively. Passive adhesion of snails on birds may be facilitated by the low mass of snails relative to the large contact surface of their shells. Many aquatic snails float at the water surface by adhering to the water surface film (e.g. Bimler, 1976). Planorbidae have a flat shell shape, and their large surface-to-volume ratio facilitates flotation. This shell shape may assist passive adhesion to birds in a similar way. Our sample sizes were low, but the 2 Planorbidae species (flat shell shape) were carried more frequently than R. balthica (cone-shaped shell) (Fig. 1A–C). In addition, snails attached rapidly to the surface of the tray, which was at that moment their only available surface (Table 5.2). This active behavior supports observations that snails crawl actively onto birds’ feet and floating feathers (Darwin, 1859; Boag, 1986) and may lead to dispersal. Many egg-laying species lay their egg capsules on substrates (Gittenberger et al., 2004) like the feet of birds or produce sticky mucous layers (Darwin, 1859; Boag, 1986; Smith, 2002; Gittenberger et al., 2004), that may facilitate attachment. Both passive and active attachment of snails may facilitate their initial attachment.

Prolonged adhesion After initial attachment, many snails detached rapidly from the mallards in both transport experiments (Fig. 5.2). This result suggests that ectozoochorous dispersal will usually result in only short-distance dispersal and confirms the results of previous experiments in which snails adhered for only 15 min to duck feathers during simulated flight (Boag, 1986). Snails attached by drying mud probably have the greatest potential for long-distance dispersal. Most snails that remained attached during the first 30 min (during which the mud dried) detached quickly, but some snails stayed attached for up to 8 h. The mallards in the experiment did not actively clean the snails from their bills, although birds could move freely in their cages. Therefore, the snails that remained attached for hours were released after birds sub-

95 Chapter 5 merged their bills, analogous to arrival in another aquatic habitat (Malone, 1965). Facilitation of ectozoochory by mud on waterbirds has been suggested for plant seeds (Barrat-Segretain, 1996 and references therein), and both seeds and aquatic invertebrates have been retrieved from mud transported by boars (Sus scrofa) (Vansch- oenwinkel et al., 2008). Autonomous attachment might result in short (<1 h) attachment (Boag, 1986), but longer adhesion may be possible with mud as an adhesive. This idea is supported by a previous observation of Figuerola and Green (2002b) who noted that birds in a muddy habitat carried more propagules than those in a sandy habitat.

Propagule survival The high survival rate of all snail species tested during the desiccation experiments suggests that aerial exposure does not prohibit snail dispersal. Many aquatic snails live in habitats that dry occasionally, such as temporary freshwater ponds or tidal areas, and are adapted to desiccation (e.g. Wiggins et al., 1980). Shells can be perme- able to water (Van Aardt & Steytler, 2007), but most water loss occurs through the shell aperture and from the surface of the foot (Storey, 1972). Prosobranchs reduce such losses by closing their aperture with an operculum (Gibson, 1970) and pulmonate species by producing a mucous layer (epiphragm) (Storey, 1972; Eckblad, 1973; Jokinen, 1977). Both adaptations allow survival in extreme conditions, such as drought or freezing in winter, when most snail species go into dormancy (aestivation; (Storey, 1972; Jokinen, 1977; Frentrop, 1998a, b). Our 48-h desiccation experiments indicate that water loss does occur during short-term desiccation, but that the desiccation is mostly nonlethal (Fig. 5.3, Table 5.3). Water loss and survival did not differ between operculated snails and snails with an epiphragm. However, smaller snails lost a larger percentage of their body mass and had lower survival than large snails, a common pattern for small aquatic species (Ricciardi et al., 1995; Paukstis et al., 1999; Facon et al., 2004). Thus, small propagules are generally more successfully transported and are found more often between feathers than large snails (Sorensen, 1986; Brochet et al., 2010), but larger snails may have higher survival. Together, our results indicate that snails of intermediate size (3–5 mm) might be most suitable for transport because this size class attached readily during transport experiments and survived desiccation. Mass loss of snails in our desiccation experiments varied with temperature (Table 5.3), and varied with humidity in experiments done by (Heitkamp & Zemella, 1988). Interesting in this respect is that Winterbourn (1970) found that P. antipodarum could survive for up to 30 h in dry situations, whereas survival for >30 days was possible in damp situations. Since migratory birds also face dehydration risks during migratory flight (Gerson & Guglielmo, 2011) and, therefore, may opt for flight routes and conditions that minimize water loss (Klaassen, 2004), also potential stowaways could profit.

Conclusions Aquatic snails can attach to living mallards passively or actively, remain attached in

96 External transport potential of aquatic snails by waterbirds drying mud for several hours, and survive long periods of aerial exposure. There- fore, ectozoochorous dispersal by waterbirds might be a plausible explanation for the wide distributions of many snail species. Whether long distance dispersal of aquatic snails truly occurs in natural situations and whether or not snails that arrive successfully in another habitat may also become established, remain challenges for future research. However, our study shows that many aquatic snail species have the necessarily prerequisites for successful dispersal.

Acknowledgements We thank Karen Vreugdenhil, Bart van Lith, Naomi Huig, André van den Beld, Re- inhoud de Blok, Cindy van der Leije, Bart Raven, Anita Stargardt, and Falkje van Wirdum for help during the experiments and Marij Orbons for help with species identification. We thank Marcel Klaassen for valuable comments during the experiments and on the manuscript. This research has been carried out under license number CL08.06 of the KNAW animal ethics committee, and is publication 5269 of the Netherlands Institute of Ecology.

References

Alonso, A. & Castro-Diez, P. (2008) What explains the invading success of the aquatic mud snail Potamopyrgus antipodarum (Hydrobiidae, Mollusca)? Hydrobiologia, 614, 107-116 Anders, N.R., Churchyard, T. & Hiddink, J.G. (2009) Predation of the shelduck Tadorna tadorna on the mud snail Hydrobia ulvae. Aquatic Ecology, 43, 1193-1199 Baldwin, J.R. & Lovvorn, J.R. (1994) Habitats and tidal accessibility of the marine foods of dabbling ducks and brant in Boundary Bay, British Columbia. Marine Biology, 120, 627-638 Barrat-Segretain, M.H. (1996) Strategies of reproduction, dispersion, and competition in river plants: A review. Plant Ecology, 123, 13-37 Bilton, D.T., Freeland, J.R. & Okamura, B. (2001) Dispersal in freshwater invertebrates. Annual Review of Ecology and Systematics, 32, 159-181 Bimler, A. (1976) Effects of overcrowding on floating behaviour in the mud whelk Cominella glandiformis (Mollusca: Gastropoda). New Zealand Journal of Marine and Freshwater Research, 10, 715-720 Boag, D.A. (1986) Dispersal in pond snails - potential role of waterfowl. Canadian Journal of Zoology-Revue Canadienne De Zoologie, 64, 904-909 Bondesen, P. & Kaiser, E.W. (1949) Hydrobia (Potamopyrgus) jenkinsi Smith in Denmark illustrated by its ecology. Oikos, 1, 252-281 Brochet, A.L., Guillemain, M., Fritz, H., Gauthier-Clerc, M. & Green, A.J. (2010) Plant dispersal by teal (Anas crecca) in the Camargue: Duck guts are more important than their feet. Fresh- water Biology, 55, 1262-1273 Brown, D. (1978) Pulmonate molluscs as intermediate hosts for digenetic trematodes. Pulmo- nates (ed. by V. Fretter and J. Peake), pp. 287-333. Academic Press, London. Cadée, G.C. (2011) Hydrobia as “Jonah in the whale”: Shell repair after passing through the digestive tract of shelducks alive. Palaios, 26, 245-249 Charalambidou, I., Santamaría, L., Jansen, C. & Nolet, B.A. (2005) Digestive plasticity in mallard ducks modulates dispersal probabilities of aquatic plants and crustaceans. Functional Ecology, 19, 513-519

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Cockerell, T. (1921) The dispersal of snails by birds. Nature, 108, 496-497 Cotton, B.C. (1960) Mollusca eaten by birds.South Australian Ornithologist, 23, 44-47 Couvreur, M., Cosyns, E., Hermy, M. & Hoffmann, M. (2005) Complementarity of epi- and endozoochory of plant seeds by free ranging donkeys. Ecography, 28, 37-48 Daborn, G.R. (1976) Colonisation of isolated aquatic habitats. Canadian Field-Naturalist, 90, 56–57 Darwin, C. (1859) On the origin of species by means of natural selection, or the preservation of fa- voured races in the struggle for life. John Murray, London. Darwin, F. (1909) The foundations of the origin of species. Two essays written in 1842 and 1844. University Press, Cambridge. Davies, R., Linton, L. & Wrona, F. (1982) Passive dispersal of four species of freshwater leech- es (Hirudinoidea) by ducks. Freshwater invertebrate biology, 1, 40-44 Dillon, R.T., Wethington, A.R., Rhett, J.M. & Smith, T.P. (2002) Populations of the european freshwater pulmonate Physa acuta are not reproductively isolated from american Physa heterostropha or Physa integra. Invertebrate Biology, 121, 226-234 Eckblad, J. (1973) Population studies of three aquatic gastropods in an intermittent backwater. Hydrobiologia, 41, 199-219 Facon, B., Machline, E., Pointier, J.P. & David, P. (2004) Variation in desiccation tolerance in freshwater snails and its consequences for invasion ability. Biological Invasions, 6, 283-293 Figuerola, J. & Green, A.J. (2002a) Dispersal of aquatic organisms by waterbirds: A review of past research and priorities for future studies. Freshwater Biology, 47, 483-494 Figuerola, J. & Green, A.J. (2002b) How frequent is external transport of seeds and invertebrate eggs by waterbirds? A study in Donana, SW Spain. Archiv für Hydrobiologie, 155, 557-565 Frentrop, J.G.J. (1998a) Zoetwaterslakken en droogte (i). Natura, 95, 67-69 Frentrop, J.G.J. (1998b) Zoetwaterslakken en droogte (ii). Natura, 95, 104-105 Frisch, D., Green, A.J. & Figuerola, J. (2007) High dispersal capacity of a broad spectrum of aquatic invertebrates via waterbirds. Aquatic Sciences, 69, 568-574 Gerson, A.R. & Guglielmo, C.G. (2011) Flight at low ambient humidity increases protein ca- tabolism in migratory birds. Science, 333, 1434-1436 Gibson, J.S. (1970) Function of operculum of Thais lapillus (l.) in resisting desiccation and predation. Journal of Animal Ecology, 39, 159-168 Gitenberger, E., Janssen, A.W., Kuijper, W.J., Kuiper, J.G., Meijer, T., Van der Velde, G. & De Vries, J.N. (2004) De Nederlandse Zoetwatermollusken. Recente en fossiele weekdieren uit zoet en brak water, 2nd edn. Nationaal Natuurhistorisch Museum Naturalis, European Invertebrate Survey-Nederland, KNNV Uitgeverij Utrecht. Green, A.J. & Figuerola, J. (2005) Recent advances in the study of long-distance dispersal of aquatic invertebrates via birds. Diversity and Distributions, 11, 149-156 Gruenhagen, N.M. & Fredrickson, L.H. (1990) Food use by migratory female mallards in Northwest Missouri. Journal of Wildlife Management, 54, 622-626 Heitkamp, U. & Zemella, B. (1988) Untersuchungen zur phänologie und autökologie von An- isus leucostomus (Millet, 1813) (Gastropoda, Planorbidae). Archiv für Hydrobiologie, Suppl. 79, 227-266 Hogan, J.P. & Phillips, C.J.C. (2011) Transmission of weed seed by livestock: A review. Animal Production Science, 51, 391-398 Hubendick, B. (1951) Recent Lymnaeidae. Their variation, morphology, taxonomy, nomen- clature and distribution. Kungliga Svenska Vetenskapsakademiens Handlingar, 4, 1-223, 5 plts

98 External transport potential of aquatic snails by waterbirds

Johnson, L.E. & Carlton, J.T. (1996) Post-establishment spread in large-scale invasions: Disper- sal mechanisms of the zebra mussel Dreissena polymorpha. Ecology, 77, 1686-1690 Jokinen, E.H. (1977) The aestivation pattern of a population of Lymnaea elodes (say) (Gastrop- oda: Lymnaeidae). The American Midland Naturalist, 100, 43-53 Kappes, H. & Haase, P. (2011) Slow, but steady: Dispersal of freshwater molluscs. Aquatic Sci- ences - Research Across Boundaries, 74, 1-14 Klaassen, M. (2004) May dehydration risk govern long-distance migratory behaviour? Journal of Avian Biology, 35, 4-6 Kristiansen, J. (1996) Dispersal of freshwater algae — a review. Hydrobiologia, 336, 151-157 Maguire, B. (1963) Passive dispersal of small aquatic organisms and their colonization of isolated bodies of water. Ecological Monographs, 33, 161-185 Malone, C.R. (1965) Killdeer (Charadrius vociferus Linnaeus) as a means of dispersal for aquatic gastropods. Ecology, 46, 551-552 Mcatee, W.L. (1914) Birds transporting food supplies. Auk, 31, 404-405 Morley, N. (2008) The role of the invasive snail Potamopyrgus antipodarum in the transmission of trematode parasites in europe and its implications for ecotoxicological studies. Aquatic Sciences, 70, 107-114 Page, T.J., Von Rintelen, K. & Hughes, J.M. (2007) An island in the stream: Australia’s place in the cosmopolitan world of Indo-west pacific freshwater shrimp (Decapoda:Atyidae:Caridina). Molecular Phylogenetics and Evolution, 43, 645-659 Paukstis, G.L., Tucker, J.K., Bronikowski, A.M. & Janzen, F.J. (1999) Survivorship of aerially- exposed zebra mussels (Dreissena polymorpha) under laboratory conditions. Journal of Fresh- water Ecology, 14, 511-517 Raudenbush, S.W. & Bryk, A.S. (2002) Hierarchical linear models: Applications and data analysis methods., 2nd edn. Sage, Newbury Park, CA. Raulings, E., Morris, K.a.Y., Thompson, R. & Nally, R.M. (2011) Do birds of a feather disperse plants together? Freshwater Biology, 56, 1390-1402 Rees, W.J. (1965) The aerial dispersal of mollusca. Journal of Molluscan Studies, 36, 269-282 Ricciardi, A., Serrouya, R. & Whoriskey, F.G. (1995) Aerial exposure tolerance of zebra and quagga mussels (Bivalvia, Dreissenidae) - implications for overland dispersal. Canadian Journal of Fisheries and Aquatic Sciences, 52, 470-477 Roscoe, E. (1955) Aquatic snails found attached to feathers of white-glossy ibis. The Wilson Bulletin, 67, 66 Schabetsberger, R., Drozdowski, G., Rott, E., Lenzenweger, R., Fersabek, C.D., Fiers, F., Traun- spurger, W., Reiff, N., Stoch, F., Kotov, A.A., Martens, K., Schatz, H. & Kaiser, R. (2009) Los- ing the bounty? Investigating species richness in isolated freshwater ecosystems of oceania. Pacific Science, 63, 153-179 Smith, A.M. (2002) The structure and function of adhesive gels from invertebrates. Integrative and Comparative Biology, 42, 1164-1171 Soons, M.B., Van Der Vlugt, C., Van Lith, B., Heil, G.W. & Klaassen, M. (2008) Small seed size increases the potential for dispersal of wetland plants by ducks. Journal of Ecology, 96, 619- 627 Sorensen, A.E. (1986) Seed dispersal by adhesion. Annual Review of Ecology and Systematics, 17, 443-463 Storey, R. (1972) Dormancy in Lymnaea peregra (müller) during periods of dryness. Journal of Conchology, 27, 377-386

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Traveset, A. (1998) Effect of seed passage through vertebrate frugivores’ guts on germination: A review. Perspectives in Plant Ecology Evolution and Systematics, 1, 151-190 Van Aardt, W.J. & Steytler, S.S.J. (2007) Shell permeability and desiccation physiology of the freshwater snail Bulinus (Bulinus) tropicus (Krauss). Malacologia, 49, 339-349 Vanschoenwinkel, B., Waterkeyn, A., Vandecaetsbeek, T., Pineau, O., Grillas, P. & Brendonck, L. (2008) Dispersal of freshwater invertebrates by large terrestrial mammals: A case study with wild boar (Sus scrofa) in mediterranean wetlands. Freshwater Biology, 53, 2264-2273 Viviansmith, G. & Stiles, E.W. (1994) Dispersal of salt-marsh seeds on the feet and feathers of waterfowl. Wetlands, 14, 316-319 Wiggins, G.B., Mackay, R.J. & Smith, I.M. (1980) Evolutionary and ecological strategies of animals in annual temporary ponds. Archiv für Hydrobiologie/Suppl. 58, 1, 97-206 Winterbourn, M.J. (1970) The new zealand species of Potamopyrgus (Gastropoda: Hydrobii- dae). Malacologia, 10, 283-321

100 Chapter 6

How did this snail get here? Multiple dispersal vectors revealed for an aquatic invasive species

Casper H.A. van Leeuwen, Naomi Huig, Gerard van der Velde, Theo van Alen, Cornelis A.M. Wagemaker, Craig D.H. Sherman, Marcel Klaassen & Jordi Figuerola

Freshwater Biology (under revision)

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Abstract How species can reach and persist in isolated habitats remains an open question in many cases, especially for rapidly spreading invasive species. One of the most puz- zling habitats in this respect are temporary freshwater ponds, which can be remote and may dry out annually, but may still harbour high biodiversity. Populations of aquatic organisms in these areas largely depend on recurrent colonization or extreme survival capacities of individuals, which provides an ideal system to investigate dispersal and connectivity. Here we test the hypothesis that the wide distributions and invasive potential of aquatic snails is due to their ability to exploit multiple dispersal vectors in different landscapes. We therefore explored the population structure of Physa acuta (recent synonyms: Haitia acuta, Physella acuta, Pulmonata: Gastropoda), an invasive aquatic snail originating from North America, established in temporary ponds in Doñana National Park, Southern Spain. In this area, landscape configuration limits the relative importance of hydrological, waterbird- and mammal-mediated connectivity of aquatic populations. Population demography and genetic analyses using six microsatellite loci of 271 snails in 21 sites indicated that i) geographically and hydrologically isolated snail populations in the park were genetically similar to a large snail population in rice fields more than 30 kilometres away ii) these isolated ponds showed an isolation- by-distance pattern, however, this pattern broke down for those ponds with high visitation rates by large mammals such as cattle, red and fallow deer and wild boars iii) snail populations were panmictic in flooded and hydrologically connected rice fields. These results support the ideas that aquatic snails disperse readily by water connections in the flooded rice fields, can be carried by waterbirds flying between the rice fields and the park, and may disperse between ponds within the park by attaching to large mammals.The potential for aquatic snails such as Physa acuta to exploit multiple dispersal vectors may contribute to their wide distribution on various continents and their success as invasive species. We suggest that the interaction between multiple dispersal vectors, their relation to specific habitats and consequences at different geographical scales should be considered when attempting to control invasive freshwater species as well as protecting endangered species.

Key words: Physa acuta, large mammals, birds, flooding, microsatellite, temporary ponds

102 Multiple dispersal vectors revealed for an aquatic invasive species

Introduction Many freshwater species lack the ability to actively disperse across the landscape using their own propulsion, despite the importance of dispersal for organisms living in such a discrete and, in many cases, temporary habitat type (Hoffsten, 2004; Van de Meutter et al., 2007). Vector assisted transport of aquatic species, i.e. dispersal whereby organisms or their propagules are carried by more mobile vectors, is therefore an important aspect determining the biodiversity of wetlands and riverine systems (Bilton et al., 2001; Malmqvist, 2002). Many aquatic species live in meta-community structures that significantly rely on transport by vectors such as water, wind, or animals for their maintenance (reviewed by Bilton et al., 2001). Water may carry aquatic plants and invertebrates across great distances during flooding and in river systems (hydrochory, e.g. Malmqvist, 2002; Frisch & Threlkeld, 2005). Wind may be a suitable vector (anemochory) for propagules that can be lifted into the air (Soons & Ozinga, 2005; Soons, 2006; Vanschoenwinkel et al., 2008a), and dispersal by animals (zoochory) is increasingly discovered for a growing number of taxa (e.g. Figuerola et al., 2003; Frisch et al., 2007). However, zoochory requires individuals to survive digestion by vertebrates or stay adhered to animals long enough for transport. Dispersal by water requires hydrological connectivity of habitats, and only certain propagules may be lifted into the air. Species that are only able to exploit one of these potential dispersal vectors, will thus still be limited in where they can go. Nevertheless, even propagules that are specifically adapted for transport by one of the above mentioned vectors, can still exploit other vectors (Nathan et al., 2008). Polychory, i.e. transport by multiple dispersal vectors, allows transport across more landscape types and over a larger scale. For (aquatic) plant seeds, the ability to be carried by multiple vectors has been shown to increase their dispersal success (Oz- inga et al., 2004). For aquatic invertebrates, however, the capacity to use multiple dispersal vectors has been less explored. Mostly only singular dispersal vectors have been identified in a single system (Vanschoenwinkel et al., 2008b; Wilmer et al., 2008). Since dispersal of aquatic invertebrates has received little scientific consideration until recently (Figuerola & Green, 2002; Green & Figuerola, 2005), the importance of the various potential dispersal vectors for aquatic invertebrates across diverse landscape types is largely unknown (Vanschoenwinkel et al., 2011). Enhanced dispersal may assist freshwater invertebrates to disperse in fragmented or endangered habitats, but can also facilitate dispersal of invasive species (e.g. Wilson et al., 1999; Shurin & Havel, 2002). The initial introduction of invasive aquatic species into a new geographic area is often linked to human activities, e.g. through ballast water of ships, commerce or intentional introduction for fishing, or the aquarium trade (e.g. Briski et al., 2011; Clarke Murray et al., 2011). After introduction using one vector, invasive species may then use a variety of vectors to disperse and expand their geographical range (Carlton, 1993; Van der Velde et al., 2006; Van der Velde et al., 2010). One species for which the ability to use multiple dispersal vectors might explain its recent success as invasive species, is Physa (Costatella) acuta (recent synonyms:

103 Chapter 6

Haitia acuta, Physella acuta) (Gastropoda; Pulmonata; Physidae). Other synonyms are Physa heterostropha and Physa integra, as previous work has indicated that these all belong to a single widespread species (Dillon et al., 2002). This species is capable of living in a broad range of freshwater habitats and shows tolerance to a wide range of environmental conditions (e.g. Kefford & Nugegoda, 2005; Turner & Montgomery, 2009). Originating from North America and introduced by the aquarium trade, it can now be found on almost all continents and has been called the most cosmopolitan snail (Dillon et al., 2002; Wethington & Lydeard, 2007). P. acuta is known as a rapid (re)colonizer of freshwater systems with changing environmental conditions (Chly- eh et al., 2006) and can efficiently disperse via water connections (Van de Meutter et al., 2006). It is carried in plant material on boats between lakes (Albrecht et al., 2009), and can potentially disperse to isolated waters by waterbirds (e.g. McAtee, 1914; Roscoe, 1955; Malone, 1965a). P. acuta is a hermaphroditic pulmonate snail capable of self fertilization (Bousset et al., 2004). Adult snails generally survive for one season in which they lay one clutch of 18-50 eggs (Gittenberger et al., 2004). Both adult snails and eggs are covered in sticky mucus, frequently used to attach eggs to water plants or other substrate (Gittenberger et al., 2004). Here we investigate the dispersal capabilities of this invasive freshwater snail in relation to landscape structure. We studied the genetic structure of P. acuta in a system of geographically and hydrologically isolated ponds in Doñana National Park (SW Spain) in relation to the occurrence of different potential vectors: water, birds and large mammals. Population genetic structure has been successfully used to unravel dispersal vectors in other aquatic invertebrate species (e.g. Wilson et al., 1999; Chlyeh et al., 2006; Zickovich & Bohonak, 2007; Wilmer et al., 2008), and if used correctly assesses effective dispersal rather than dispersal potential (Bohonak & Jenkins, 2003; Marko & Hart, 2011). We used six highly polymorphic microsatellite markers to assess the genetic differentiation and population structure ofP. acuta populations in isolated temporary ponds within the national park, and compared this to a more permanent population in rice fields ~30 km away. By investigating the dispersal of P. acuta in Doñana National Park, we aim to understand the abundant occurrence of invertebrates in this system (e.g. Florencio et al., 2011) as well as the ability of (invasive) aquatic invertebrates to disperse passively on large and small scales by multiple vectors in general.

Materials and Methods

Study area Doñana National Park is a protected wetland area in SW Spain (6°W, 37° N), bordered by the Guadalquivir River in the east and the Atlantic Ocean in the south and west (Fig. 6.1). The national park comprises marshlands, shallow streams, sand dunes, and shrubland. In the park summers are hot and dry, whereas winters are cool and wet (see Serrano et al., 2006 for detailed description). The sandy area of the park is geologically young having formed only ~6000 years ago. It contains more than 3000 small ponds, of which ~200 are man-made to provide cattle with drinking water during dry periods (zaccalones). In this study, we refer to this area as “the

104 Multiple dispersal vectors revealed for an aquatic invasive species park”. A detailed description of the ponds characteristics can be found in Gómez- Rodríguez (2009) and Florencio et al. (2011). The sampled ponds varied in size, depth and elevation (indicative average size: 50 m2; average depth: ~1 m) but were all hydrologically isolated. About half of the sampled ponds dry completely during summer. Occasionally, during years of heavy rainfall, the park may partially flood, caus- ing lower lying ponds to become hydrologically connected. Cows (Bos primigenius), fallow deer (Dama dama), red deer (Cervus alaphus), horses (Equus ferus) and wild boars (Sus scrofa) are abundant in the park. About 30 kilometres east of the park rice fields cover an area of about 360 km2 on former marshlands that were reclaimed during the 20th century. The water level in the rice fields is regulated and the fields are actively flooded every year at the end

! Figure 6.1: Location of the studied area: Doñana National Park and the rice fields. Between the park and the rice fields are brackish fish ponds “Veta la Palma”, bordered by the Guadalquivir river. Closed circles denote ponds where P. acuta were present but not sampled, closed squares denote locations of sampling. At open circles, ponds contained water but no snails were found. Crosses indicate dry ponds at time of sampling. The inset denotes the location of Doñana National Park in Europe, and light shaded area indicates protected areas.

105 Chapter 6 of May with water pumped from the nearby Guadalquivir River. The fields remain flooded until the end of November or December, but this period may expand until April in rainy winters (Toral et al., 2011). We refer to this area as “the rice fields”.

Field sampling Physa acuta samples were collected between 10 and 20 November 2009, before the start of the autumn-winter rainfalls. In the park, 69 out of 83 visited ponds contained water at the time of sampling, of which 40 (48%) contained Physa acuta (Fig. 6.1). Six concrete drinking trays specially made for cattle also contained P. acuta in high densities, and were mainly present in the north of the park. In the rice fields, all 25 visited locations contained P. acuta in high densities (>50 individuals m-2, Fig. 6.1). Standard latitude/longitude coordinates for each location were recorded using a handheld GPS unit. After conversion to WGS1984 coordinates, pairwise geographic distances between sites were calculated using R for statistics (R-Development-Core- Team, 2011). We sampled snails from 29 ponds in the park and 9 locations in the rice fields (indicated in Fig 6.1), collecting between 5 and 20 snails per location (n = 271). The collected snails were stored in separate containers per site in a CTAB-DMSO solution (1.5% cetyltrimethylammonium bromide 20% dimethyl sulfoxide in deionised water) immediately after sampling. Unfortunately, logistical constraints and sampling impact on small snail populations lowered the number of snails sampled per pond. Since estimating allele frequencies based on only 5 individuals per pond was unrealistic, we pooled samples from ponds geographically close together and of similar altitude to increase sample sizes. Pooled samples were in Hardy-Weinberg equilibrium suggesting unlimited gene flow between pools or homogenous colonization from the same source population, allowing comparative analysis of pooled samples. This resulted in 16 locations (hereafter referred to as sites) in the park and 5 in the rice fields (average 13 snails per site). The average geographical distance between the sites was 7.3 KM (+ 5.2 SD) while the average distance between ponds within a site was 1.6 KM (+ 1.4 SD). The average altitude of the ponds in the park was 10.0 m (+ 8.7 SD), ranging from 1.4 to 28.6 m above sea level; the average difference in altitude within sites was 1.7 m (1.7+SD). A potential category of dispersal vectors in the park were large mammals, vis- iting ponds for drinking water. To investigate their potential involvement in the transport of snails, we used the frequency of mammal densities in Doñana National Park from Soriguer et al. (2003). These authors estimated the density of deer, sheep, cows and horses by counting dropping density in 422 transects covering 139 km in the park. Dropping density data ranged from 0 to over a 1000 droppings per hectare per mammal species, which we categorized based on the median droppings densities. Sites in areas where over 500 droppings per hectare were found for one or more of the mammal species were considered to be frequently visited by large mammals. Sites with less than 500 droppings for all mammals were considered only occasionally visited. This resulted in 8 sites in the park with low (<300) and 8 sites in the park with high (>800) mammal densities, probably because the large mammals tended to

106 Multiple dispersal vectors revealed for an aquatic invasive species prefer similar locations or aggregate together. Average altitude of more frequently visited ponds (4.7 m + 3.2 SD) was lower than that of ponds not visited (16.7 + 8.7, Students t-test, p<0.001).

Genetic analyses Six microsatellite markers were used to assess levels of genetic diversity and genetic differentiation within and between sites and habitats (Table 6.1).

Table 6.1: Microsatellite markers used. Number of alleles (N) is the number of alleles detected in all 271 individuals. Repeated motive and size range of alleles (in base pairs) are given. Accession No is the number on GenBank.

Locus N Repeated motive Size range Publication Accession No.

Pac1 3 (TG)22 100-118 (Sourrouille et al., 2003) AF532037

Pac2 5 (TATC)15 157-181 (Sourrouille et al., 2003) AF532038

Pac4 5 (TATC)24(TGTC)9(TG)4 209-257 (Sourrouille et al., 2003) AF532039

Pac5 4 (YGTC)20 241-265 (Sourrouille et al., 2003) AF532040

Pac7 10 (GT)7(ATGT)21(CTGT)22(ATGT)3 302-432 (Sourrouille et al., 2003) AF532041

27 5 (TG)28 151 (Monsutti & Perrin, 1999) AF108764

DNA was isolated using a Maxwell ® 16 (Promega, Madison, WI) with the accompa- nying Maxwell Tissue DNA purification kit. DNA was checked for quality by running 5 µl DNA on a 0.7% agarose gel, and quantitatively by using a Qubit fluorom- eter (Invitrogen, Carlsbad, CA). DNA concentrations ranged from 50 to 300 ng/ml, and were sufficient for subsequent PCR. PCR’s were performed separately for each locus to be able to control product concentrations on the gel, but products from two loci with non-overlapping alleles were combined for genotyping analysis. PCR’s were performed in volumes of 25 µl with a Biometra T Gradient Personal Thermal Cycler (Westburg, The Netherlands), containing the following components: 1 μl of template DNA, 2.0 μl Purified Bovine Serum Albumen (10mg/ml, New England Bio- labs, Inc), 1 μl of 5pmol/ul forward primer (Biolegio, Nijmegen, The Netherlands), 1 μl of 5 pmol/μl IR800 dye (LI-COR Biosciences, Lincoln, NE) labelled reverse primer (Biolegio, Nijmegen, The Netherlands), 2.5 μl reaction buffer (Bioline, Luckenwalde, Germany), 0.2 μl Taq polymerase (BioTAQ 5 u/μl), 1.0 μl dNTP’s (5.0 mM) and 1.0 μl 50 mM MgCl. Cycling conditions were a hot start at 95°C for 2 minutes, followed by 35 cycles of denaturation at 95°C for 1.5 minutes, annealing at 60°C for 1 minute and elongation at 72°C for 1.5 minutes. After a final elongation step for 5 minutes at 72°C, samples were stored at 4°C until separation using a LICOR 4200 s2 within 24 hours. Alleles were sized by comparison to a 50-350 Sizing Standard IRD800 ladder (LI-COR Biosciences, Lincoln, NE).

Statistical analyses Prior to all analyses, data were checked for the presence of null alleles, scoring errors due to stuttering and large allele dropout with Microchecker (2.2.3, Van Oosterhout et al., 2004). There was no evidence for scoring errors due to stuttering or evidence

107 Chapter 6 for large allele dropouts. Null-alleles were only present in one locus (pac7), for which observed heterozygosity was lower than expected. Since the frequency of null-alleles in pac7 was on average only 7%, which is considered low enough to have little impact on FST estimates (Oddou-Muratorio et al., 2009), we present results including pac7. Pair-wise linkage disequilibrium was tested to determine whether each locus as- sorted independently from each other locus using Arlequin 3.5 (Excoffier & Lischer, 2010), using sequential Bonferroni correction (Rice, 1989). Deviations from Hardy- Weinberg equilibrium were tested for all sites and deviations expressed as fixation index, F (Wright, 1978), where positive and negative values represent deficits or excess of heterozygotes respectively. Chi-square tests were used to determine whether the observed number of heterozygotes was significantly different from those expected under Hardy-Weinberg equilibrium using the genetics program Arlequin 3.5 (Ex- coffier & Lischer, 2010). The levels of population subdivision were quantified using hierarchical analysis of standardized genetic variance (F) statistics (Wright, 1969).

We used FST to denote the total variation among all sites, FHT for the variation among habitats (park versus rice fields) and FSH for the variation among sites within each habitat. These parameters were calculated using the formulations of Weir and Cock- erham (1984) in TFPGA 1.3 (Miller, 1997), with 95%CI determined by bootstrapping over loci with 10 000 replicates. Statistical significance of values of F was assumed when the 95% confidence intervals of the mean did not cross zero. In order to test for relative differences in gene flow between sites, the average number of effective -1 migrants per generation was derived by calculating Nem = (FST – 1)/4. The level of allelic differentiation between sites and habitats in relation to geographic distance (i.e. isolation by distance) was tested by comparing the pairwise FST

/ (1 - FST) matrix to the pairwise matrix of linear geographical distances in kilometres. Significance was assessed using a Mantel test with 10 000 permutations (Sokal, 1979) in TFPGA 1.3 (Miller, 1997). We ran STRUCTURE 2.2.3 (Pritchard et al., 2000) for multiple selections of the dataset to perform assignment tests, on datasets including either all sites, all sites in the park or all sites in the rice fields. We ran the analysis with and without prior information on which individuals were sampled at each site. In all cases we used an admixture model with 750 000 MCMC repeated permutations after a burnin period of 500 000 permutations.

Results

Genetic variation among sites and habitats The six microsatellite loci showed high levels of polymorphism with an average of 5.1 alleles, ranging from 3 to 10 alleles per locus. The mean within-population diversity represented by the expected heterozygosity (Nei, 1973) was 0.48 + 0.30SD in both the park and the rice fields. The observed heterozygosities were also similar for park and rice fields, i.e. 0.35 + 0.23SD and 0.34 + 0.21SD, respectively. In 16 out of the 21 sites, single-locus heterozygosity departed from Hardy-Weinberg equilibrium, involving 27 of the 126 estimates. However, only 13 of these remained significant after a sequential Bonferroni correction and all represented heterozygous deficit suggest-

108 Multiple dispersal vectors revealed for an aquatic invasive species ing deviations due to mating system (i.e. inbreeding/self-fertilization) rather than a Wahlund effect due to the pooling of samples from different populations. Tests for linkage disequilibrium over a total of 315 pairwise comparisons revealed 16 inter- locus associations, however, none of these associations remained significant after application of a sequential Bonferroni correction.

Population structure based on microsatellites The hierarchical analysis of F-statistics revealed significant levels of population subdivision among all sites and habitats (Table 6.2). The mean Fst (± SE) across six loci was 0.075 ± 0.016, with approximately 32% of this variation attributed to variation among habitats (Fht = 0.024 ± 0.01). We detected no significant genetic subdivision among sites within the rice field (FsH = - 0.0047 ± 0.01) indicating substantial gene flow within this habitat. In contrast, we detected significant genetic differentiation among sites within the park, with ponds more frequently visited by large mammals having lower levels of genetic differentiation compared with less visited ponds (FsH: high mammal = 0.077 ± 0.03 vs low mammal = 0.11 ± 0.03).

Table 6.2: Population structure as indicated by hierarchical F statistics. n denotes the number of populations sampled. Structure over all populations is indicated by FST values, between rice and park populations by FHT values and for the rice fields and the park in isolation byF SH values (with for the park the distinction between only those ponds with high or only those with low dropping densities of large mammals). Standard deviations (SD) and confidence intervals (95%CI) are indicated. FIS is the within population structure and Nem the estimated number of migrants per population. Significant values are in bold.

n FST/HT/SH SD 95%CI FIS SD 95%CI Nem

All populations (FST) 21 0.075 0.016 0.096 to 0.040 0.24 0.13 0.42 to 0.026 3.1

Rice fields vs park (FHT) 2 0.024 0.010 0.039 to 0.0019 10.2

Rice fields only (FSH) 5 -0.0047 0.010 0.014 to -0.021 0.32 0.17 0.56 to -0.0019 >50

Park: high mammals (FSH) 8 0.077 0.030 0.13 to 0.027 0.19 0.14 0.37 to -0.058 3.0

Park: low mammals (FSH) 8 0.11 0.037 0.16 to 0.037 0.24 0.094 0.41 to 0.086 2.0

There was a large and significant heterozygote deficit across all sites (FIS = 0.24 ± 0.13, Table 6.2) suggesting significant levels of inbreeding including the extreme of self-fertilization. This pattern was driven primarily by significant heterozygous deficits with ponds isolated in the park and which receive few or no visits from large vertebrates. In contrast, while heterozygous deficits were detected in other ponds in the park and sites within the rice fields, these were not significantly different from zero (Table 6.2).

Isolation by distance Mantel tests revealed significant isolation-by-distance patterns between geographic distance and pairwise FST estimates when calculated over all ponds in the park (Fig. 6.2a). However, for those ponds frequently visited by large mammals (cows, horses,

109 Chapter 6 (a) 0.35

0.3

0.25 r = 0.60 0.2 ) ) ST 0.15 /(1-‐F ST F 0.1

0.05

0 0 10 20 30 40 -‐0.05 Geographic distance between ponds (in km)

(b) 0.35

0.3

0.25 r = 0.76 0.2 ) ) SH 0.15 /(1-‐F SH F 0.1 No large mammals 0.05 Large mammals

0 0 10 20 30 40

-‐0.05 Geographic distance between ponds (in km)

Figure 6.2a/b: Genetic distance based on all six loci is plotted against geographic distance between ponds in the park. (a) Data including all sites in the park indicates significant isola-

tion by distance in the park based on Mantel test correlations (r = 0.60, p = 0.004, FST /(1- FST) = 0.0074(GeoDist) + 0.016) (b) Data including all sites in the park but separated by either pairs of ponds where both ponds had high densities of large mammals (closed triangles) or where both ponds had low densities of large mammals (open diamonds). Only for the latter Mantel

test correlations indicated significant isolation by distance (r = 0.76, p = 0.001; FSH /(1- FSH) = 0.0122(GeoDist) + 0.014; indicated by the solid line).

110 Multiple dispersal vectors revealed for an aquatic invasive species wild boars and fallow and red deer), the pattern of isolation by distance broke down (Fig. 6.2b). There was no isolation by altitude in the park, when correlating altitudinal distance between ponds to genetic distance (Mantel test, r = 0.01, p = 0.54). In the rice fields, no isolation by distance was detected suggesting well mixed population with little structuring (Mantel test, r = 0.33, p = 0.15).

Assignment tests The assignment tests had limited power to identify individual dispersers, most likely due to the strong pattern of isolation by distance present in the park (which tends to give mixed memberships in multiple groups and reduces the biological meaning of the K-value (Pritchard et al., 2000)) and possibly due to small sample sizes for some sites. Clear dispersal patterns and population origins could not be determined. Al- though results should be interpreted with caution, running assignments tests for the park data only, revealed two clusters in the park (K=2) when running with prior site information and four clusters (K=4) when running without prior site information. Overall, the population farthest to the south and the population farthest to the north were most different from each other and from all core populations in the centre of the park. These two populations also had the highest average FSH value compared to the core populations. Similarly, two clusters were found in the rice fields when running the test with prior site information, but four clusters were found when running without site information. In the rice fields, all populations consisted of a mixture of these clusters in all cases. Running the assignment tests for both park and rice field data together indicated three clusters (K=3), both with and without site included as prior information.

Discussion The degree of gene flow between aquatic snail populations in Doñana National Park was found to vary with habitat type and structure. Inferred population structures could not be attributed to a single passive dispersal vector, but instead showed patterns consistent with dispersal by multiple dispersal vectors acting on different spatial scales. P. acuta is known to be capable of dispersal by water connecting ponds (Van de Meutter et al., 2006) and in irrigation systems (Chlyeh et al., 2006), and has been found to be transported in macrophytes attached to boats (Albrecht et al., 2009). Snails have been observed on the feathers of trapped waterbirds (e.g. McAtee, 1914; Roscoe, 1955), observations more recently supported by inferring bird-mediated dispersal from genetic analyses of snail populations (Wilmer et al., 2008; Von Oheimb et al., 2011) including populations of P. acuta (Dillon & Wethington, 1995). The occurrence of P. acuta in the isolated, temporary ponds of Doñana National Park was most consistent with combined dispersal by waterbirds, large mammals and water.

Dispersal potential by waterbirds Allelic richness, heterozygosity and levels of genetic differentiation in the park and the rice fields were very similar (FHT, Table 6.2), suggesting that occasional exchange of snails between (part of) the park and the rice fields could exist. Although the rice fields and the marsh formerly formed a unique habitat with clear hydrologic

111 Chapter 6 connections, these connections were broken during the second half of the 20th century. Since the establishment of these new habitats less than 70 years ago, a range of additional potential vectors are now likely to contribute to dispersal over differing spatial scales. The small ponds in the park, that fluctuate in population size and may even completely dry out during part of the season, were expected to show genetic structure associated with bottle necks or founder effects in case of no dispersal. In- stead, similarity with snails from the rice fields suggested snails either maintained stable populations over time since isolation (without effects of reduced population sizes when ponds dry), or might occasionally exchange individuals with the rice field population. Since there are no more connections by water or humans between the rice fields and the park, but many waterbirds visit both areas (Rendon et al., 2008), we propose birds are the most plausible vector for transport between these habitats on this scale. Zoochory has previously been suggested as the most plausible transport mechanism on this scale for aquatic snails found on oceanic islands and ponds in the desert (Dil- lon & Wethington, 1995; Wilmer et al., 2008). The idea of bird-mediated transport is founded upon previous records of up- land sandpipers (Bartramia longicauda) and white-faced glossy ibises (Plegadis mexi- cana) with numerous Physa snails found between their wings (McAtee, 1914; Roscoe, 1955). The sticky nature of the eggs and adult snails (Gittenberger et al., 2004) is further support for avian dispersal. In his early and only review of the importance of birds as dispersal vectors for aquatic snails, Rees (1965) already concluded birds to be important vectors for snails, and suggested that mud may be an important facili- tator for attachment of snails to birds. Since the rice fields are described as “mudflats with water” throughout a large proportion of the year (Toral et al., 2011) with very high snail densities, attachment probability may be sufficient for occasional transport. Whereas external attachment and dispersal of eggs and/or adults is the most plausible mode of transport (Malone, 1965b), egg capsules of Physa anatina might also be transported in the digestive system or the crop of killdeer (Charadrius vociferus) and mallards (Anas platyrhynchos) (Malone, 1965b). More recently, successful internal transport has even been found for whole snail individuals (Cadée, 2011; Git- tenberger, 2012; Wada et al., 2012). Dispersal by waterbirds between rice fields and the park, whether internal or external, may be sufficient to explain genetic similarity at this large spatial scale.

Potential dispersal by large mammals Large mammals such as boars, elephants (Loxodonta africana) and buffalos (Syncerus caffer caffer) have previously been found to carry invertebrates in drying mud (e.g. Vanschoenwinkel et al., 2008c; Vanschoenwinkel et al., 2011). The abundance of cattle, wild boars and fallow and red deer in the park provides a possible mechanism for snails to disperse between ponds by attaching to their legs or bodies, potentially facilitated by mud. This idea is strengthened by the observation that all drinking trays of large mammals in the park contained high snail densities. To test the potential for this mode of dispersal in our system, we divided the ponds in the park

112 Multiple dispersal vectors revealed for an aquatic invasive species in groups with either high and low mammal visitation rates. If large mammals indeed disperse snails, the more frequent exchange of snails between ponds visited by mammals would reduce genetic differentiation between these populations (Mader et al., 1998; Wada et al., 2012). Over all ponds of the park, gene flow seemed to occur at low frequency at the scale of several kilometres. When the FSH value of the park (0.11, Table 6.2) was considered in comparison to the expected heterozygosity (0.48, the maximum possible value of FSH), the populations in the park were relatively isolated (Charlesworth, 1998; Meirmans & Hedrick, 2011). Significant isolation by distance in the park (Fig. 6.2a) indicated more exchange of snails between neighbouring ponds than between ponds farther apart (e.g. a distinct pattern of isolation by distance). However, after selecting only ponds frequently visited by large mammals for the analysis, the pattern of isolation by distance broke down (Fig. 6.2b). The FSH value calculated among only these ponds was small and non-significant (0.077, Table 6.2). This observation was supported by the assignment tests that, despite their limited explanatory power, indicated relative isolation and high average FSH values of the most northern and southern populations where overall visitation rates of mammals were lowest. Our data therefore support the idea that large mammals transport snails between some ponds inside the park. The fact that the ponds more frequently visited by mammals also had an average lower altitude, does not rule out the possibility these ponds may be occasionally connected by flooding in the park. However, our sampling points lie outside the flooding area of the marsh (http://mercurio.ebd.csic.es/imgs2/), and although nearby ponds might be connected during rainy winters (see also Serrano et al., 2006), such connections are unlikely to occur at the large scale of our analyses. For these reasons we clustered ponds separated by a few hundreds of metres to increase sample sizes for analyses, in which case dispersal by flooding would only connect ponds within sites. A Mantel test on isolation by altitude did not find any correlation between altitude and genetic differentiation, thus favouring large mammal visitations as a predictor of the patterns on the smaller to intermediate scale between ponds inside the park. An interesting question that follows from this suggestion is whether or not the snails may actively promote dispersal on both birds and large mammals through behavioural adaptations. If success rates of dispersing individuals are sufficiently high, dispersal might be an adaptive response to high population densities or unfavourable conditions in ponds. P. acuta is known to respond to the presence of crayfish as predators in the water by crawling out (Alexander & Covich, 1991), which puts forward the question whether or not they might also crawl actively onto passive vectors (Alexander & Covich, 1991).

Dispersal potential by water Passive drifting on the water has been claimed as the most important means of transport for many aquatic invertebrates (Bilton et al., 2001). Currents and flooding events can increase gene flow (Kawata et al., 2005), and an increase in water exchange be-

113 Chapter 6 tween areas also increases exchange of invertebrates (Van de Meutter et al., 2007; Siziba et al., 2011). In our system, we attribute the higher gene flow at the small scale in the rice fields (supported by high values ofN em, Table 6.2) to their frequent flooding. Several months of the year the snails can freely move in the water throughout the rice fields, or might be moved by the incoming water (Frisch & Threlkeld, 2005; Siziba et al., 2011), in particular when they are adhered upside down to the water surface for grazing or breathing, attach to floating plant material or are crawling on bare mud (Bickel, 1965). Our analyses suggest the ability to move freely throughout the flooded rice fields caused at least 15 times more exchange of individuals between sites compared with that detected between sites in the park where hydrological isolation prevails and dispersal largely depends on alternative vectors. Assignment tests did not detect any structure in the rice fields. Although extrapolating genetic differentiation directly to dispersal rates can only be done with care (Whitlock & McCauley, 1999; Bohonak & Roderick, 2001), our relatively young populations of no more than 200 years old (the first record of the species from the Iberian Peninsula was in 1845 (Cobo et al., 2010 and literature therein) were likely already in equilibrium as indicated by the isolation by distance. For the gene flow estimates, we only compared similar populations relatively to one another, and thus can also interpret the number of migrants per generation as relative rather than absolute number of snails (Bossart & Prowell, 1998). Water connections probably contribute extensively to dispersal of aquatic snails.

Gene flow and dispersal Although snails may potentially attach to mammals or birds, or may be carried by water, this may not always result in successful dispersal. Probably a large number of snails will be dropped from their animal vectors between ponds, for example near trees where vectors clean themselves from adhering mud (Vanschoenwinkel et al., 2011). Those snails that do arrive at suitable sites, may not always establish in this new population (De Meester et al., 2002). The number of snails actually transported may therefore differ from the number that ultimately contributes to the genetic structure of the population. Our genetic data support the idea that part of the transported snails is also capable of establishing and reproducing. There were no indications for a dispersal-gene flow paradox or founder effects in the small ponds in the park, that might have prevented dispersing individuals to establish in already colonized locations (De Meester et al., 2002; Bohonak & Jenkins, 2003). We may speculatively attribute this to the relatively young populations in the park, where resources might still have been sufficient and competition low. Many ponds have still not been colonized (Fig. 6.1), which may arise from unsuitable habitat or limited opportunities for dispersal to these ponds. Direct monitoring of dispersal in this study system may reveal higher transport rates of snails that are detached from vectors before reaching suitable habitat or are unsuccessful at establishing in some ponds.

114 Multiple dispersal vectors revealed for an aquatic invasive species

Effects of dispersal on reproductive choice Sixteen of the 21 sites we investigated had single-locus heterozygosity departing from Hardy-Weinberg Equilibrium, all due to heterozygote deficits. These are most likely to result from inbreeding, including the extreme of self-fertilization, which is frequently found in Physa and pulmonates in general (Dillon & Wethington, 1995; Jarne et al., 2000). Individuals of P. acuta are known to prefer outcrossing over selfing in experimental setups, because selfing often reduces their reproductive success

(Wethington & Dillon, 1997). Therefore, we expected FIS to be greater in isolated ponds where introduction by a small number of individuals has limited the opportunity for outcrossing and promoted selfing and inbreeding of subsequent genera- tions. This is supported by significant inbreeding coefficients in those ponds that were the most isolated and that were the least visited by large mammals (Table 6.2). In contrast we detected no significant inbreeding in the rice fields or the ponds more frequently visited by large mammals, again suggesting that snails preferred outcrossing and had the opportunity to do this with introduced conspecifics. The advantages of sexual reproduction in such stochastic environments might be substantial. Although we cannot disentangle selfing from inbreeding in our research, snails seem highly suitable for isolated and temporary environments by being capable of self fertilization after initial colonization, and subsequently reproduce sexually to allow adaptations in a stochastic environment and prohibit inbreeding.

Conclusions and implications The population structure of the invasive P. acuta occurring in Doñana was found to vary between habitats and spatial scales and our genetic data is consistent with snail dispersal by multiple dispersal vectors acting at different scales. At the largest scale, waterbirds were the most plausible vector connecting the rice fields and the park, leading to only low levels of connectivity. Within the rice fields the populations were panmictic, which we attribute to the water connections that are present throughout a large part of the year, supporting extensive dispersal by water on intermediate scale. At the small to intermediate scales between the hydrologically isolated ponds in the park the connectivity was much lower than in the rice fields, but still present if large mammals were abundant. Together, this suggests the interaction of the various dispersal vectors acting at different scales facilitated the overall occurrence of P. acuta in Doñana, and especially its occurrence in the isolated, temporary ponds in the National Park. Snails in many of the isolated ponds in the park may have required dispersal by both waterbirds and large mammals to reach their final location. If P. acuta is indeed capable of dispersing by multiple passive dispersal vectors, this might explain its cosmopolitan distribution and its success as an invasive species. After initial dispersal by humans (Albrecht et al., 2009), it may be using water, waterbirds and large mammals to expand its geographical distribution. Its ability to reproduce by selfing, and subsequently sexual reproduction, may additionally facilitate both introduction into new habitat and the ability to adjust to new stochastic environments. These findings

115 Chapter 6 for P. acuta suggest that not only plant seeds, but also aquatic invertebrates such as snails may rely on multiple vectors for their dispersal, even within a single system. This suggestion has important implications for controlling protected as well as invasive freshwater species. Whereas invaders with the ability to use multiple dispersal vectors are likely the most successful and may be controlled by taking away only one vector, native species with this ability might form the most robust populations with resistance to habitat fragmentation and environmental changes (Kokko & Lopez-Sepulcre, 2006; Pearson, 2006).

Acknowledgements The authors would like to thank Begoña Arrizabalaga, Arancha Arechederra, Isidro Roman, Miguel Ángel Bravo Utrera and Rosa Rodriguez Manzano for assistance at the Doñana Scientific Reserve, and Diego Fernando López Bañez and Juan Miguel Arroyo Salas for assistance in the field. We are grateful to Ramon Soriguer for providing dropping density data, David Aragonés Borrego for calculating altitudinal data, Roos Keijzer for lab assistance and Joaquin Munoz for helpful comments on an earlier draft of this manuscript. This project was performed with all necessary permits from the national and regional authorities, and funded by the Access Pro- gramme ICTS- Doñana Scientific Reserve (ICTS-RBD) supported by European Un- ion funds through the FEDER program, and Schure-Beijerinck-Popping fonds.

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118 Multiple dispersal vectors revealed for an aquatic invasive species

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A snail on four continents: bird- mediated dispersal of a parasite vector?

Casper H.A. van Leeuwen, Gerard van der Velde, H. Wouter Wisselink, Theo van Alen, Rose Sablon & Johannes H. P. Hackstein

(submitted)

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Abstract Many aquatic snail species have a wide distribution, despite their relative limited ability to disperse long distance by own propulsion. To date still little is known on how snails may be transported over long distances and by which vectors, despite that they can be important intermediate hosts for human and livestock parasites. Through literature and genetic analyses we investigated the distribution and phylogenetic position of one particularly widespread aquatic snail, Galba truncatula, which is an important intermediate host of pathogenic trematodes. We test the hypothesis that waterbirds may be responsible for this species’ long distance dispersal. The distribution of G. truncatula was extracted from the literature and its phylogenetic position determined based on ribosomal DNA sequences from The Netherlands and Belgium, combined with sequences of other geographic locations extracted from GenBank-EMBL. The obtained data revealed that G. truncatula is documented to occur on the European, African, North- and South-American continents, on at least 14 island-groups and 14 highland areas. This suggests G. truncatula has a high mobility throughout its range of suitable habitat. The overlap between the distribution of this species and the migratory flyways of (water)birds suggests birds as the most parsimonious dispersal vector. The higher phylogenetic relatedness of European G. truncatula to American than to other European lymnaeids combined with the high endemism of the genus Galba in America, suggest birds may also have been responsible for the introduction of this species to the Eurasian and African continents in the past. G. truncatula, and potentially its associated parasites, may thus be capable of aerial dispersal over thousands of kilometres.

Key words: liver fluke, Galba truncatula, ITS-1, phylogeny, distribution

122 A snail on four continents: bird-mediated dispersal of a parasite vector?

Introduction Many freshwater snail species (Gastropoda) are widely distributed throughout the world (Hubendick, 1951). Although they depend on aquatic habitats for survival and have only limited ability to disperse by own propulsion (Kappes and Haase, 2011), they are known to rapidly colonize isolated water bodies and disperse to remote oceanic islands (e.g. Mas-Coma et al., 2001; Griffiths and Florens, 2006). Pond snails of the family Lymnaeidae are particularly widespread (Hubendick, 1951). The fact that many species in this family are intermediate hosts of trematode parasites, with considerable medical and veterinary impact (Bargues et al. 2001; Bargues and Mas-Coma, 2005; Cichy et al., 2011), increases the relevance of understanding their dispersal. However, to date little is known on how freshwater snails cross land and oceans over long distances. Widespread distributions of other aquatic species can often be explained by passive dispersal by wind (anemochory), water (hydrochory) or mobile animals such as large mammals and birds (zoochory) (Kirchner et al., 1997; Caceres and Soluk, 2002; Figuerola and Green, 2002; Van de Meutter et al., 2006). In this respect, waterbirds are particularly important dispersal vectors, because they travel fast in large numbers between ecologically similar habitats and migrate over long distances across mountain ranges and oceans (Figuerola and Green, 2002; Green et al., 2002). Their potential was first noted by Darwin (1859), who conducted experiments whereby snails were exchanged between aquaria on the feet of ducks. Since these first documented experiments, further support for dispersal of snails by waterbirds has accumulated (Gittenberger, 2012), for both internal (Cadée, 2011; Van Leeuwen et al., 2012; Wada et al., 2012) and external (Malone 1965; Boag, 1986) transport. Genetic analyses have indicated possible transport of marine snails across land (Miura et al., 2011) and land snails to islands (Gittenbergeret al., 2006) by birds. However, whether bird-mediated dispersal can explain wide distributions of freshwater snails by facilitating transport over very long distances (e.g. across and between continents) has still been little researched. Galba truncatula (Müller, 1774) is a widespread pond snail (Hubendick, 1951; Bargues et al., 2001). Its fossil record in Europe starts at the lower Pleistocene, i.e. ~2.6 million years ago, and it was commonly found throughout the Pleistocene and Holocene (Ellis, 1969; Gittenberger et al., 2004) during various climatic circumstances. It is a resistant snail that easily withstands desiccation and low temperatures (Hodasi, 1976; Chapuis and Ferdy, 2012) and has an amphibious lifestyle, which are all prerequisites that make it a good candidate species for dispersal by waterbirds. Upon arrival in a new habitat, it may reproduce by self fertilization (Trouve et al., 2003), which may further increase its chances of successful colonization after dispersal. G. truncatula is a temperate-subtropical species that is commonly found throughout such regions, but lacks in real tropical environments. In the tropics, it occurs only in highlands as this is the single suitable habitat. Galba truncatula is also known as Galba trunculata, Galba truncatuliana, Lymnaea truncatula, Limnaeus truncatulus, Fossaria truncatula, Simpsonia truncatula and Bucci- num truncatulum (Zilch, 1959; Burch, 1982; Glöer, 2002). As can be concluded from

123 Chapter 7 these many alternative names, the species status of G. truncatula has been a matter of debate. The shell shapes of snails are highly phenotypically plastic, which often results in similar shaped shells for different species. This complicates investigating the phylogenetic relations among pulmonate snails (Samadi et al., 2000). Many species can only be separated by comparing the morphological characteristics of the reproductive system obtained by anatomical dissection. Therefore, existing phylogenetic relations of the Gastropoda are increasingly updated due to the application of molecular genetic techniques (e.g. Remigio and Blair, 1997a). Also the position of G. truncatula continues to be reassessed (Bargues et al., 2011a). Here we investigate the distribution and the phylogenetic position of G. truncatula in relation to its potential dispersal by birds. We present a literature overview of the entire known distribution of G. truncatula to date, and relate this to migratory flyways of waterbirds. We reassessed the phylogenetic relations of the Lymnaeidae based on a section of ribosomal DNA, whereby we put special focus on the similarities of the various samples from different geographic locations. This combination of geography and phylogeny can be used to unravel how pond snails may historically have colonized the world and carried their associated parasites, and how G. truncatula may thereby have relied on long distance dispersal.

Methods

Distribution information of Galba truncatula We searched the literature for information on the present day geographic distribution of G. truncatula, using the searching terms “Lymnaea truncatula”, “Galba truncatula” and “truncatula”. The resulting information was subsequently mapped using package “maptools” in R for statistics (R-Development-Core-Team, 2011).

Bird movement information Information on migratory movements of waterbirds was extracted from the Water- bird Population Estimates (Wetlands-International 2007) by comparing species differences in breeding and wintering distributions. Species of the following orders were included in the analysis: Anatidae (ducks, geese and swans), Charadriidae (plovers, waders), Gruidae (cranes), Laridae (gulls), Ardeidae (herons), Sternidae (terns) and Gaviidae (divers, loons).

Internal transcribed spacer 1 sequences Internal transcribed spacer 1 (ITS-1) is a ‘non-coding’ region of the nuclear ribosomal DNA spliced from the ribosomal rRNA transcript, frequently used to unravel phylogenetic relations of species (Remigio and Blair, 1997b; Schilthuizen et al., 1999; DeJong et al., 2001; Stunzenas et al., 2004; Bargues et al., 2006a; Bargues et al., 2006b). Ribosomal DNA consists sequentially of a coding 18S region, followed by a non- coding internal transcribed spacer (ITS-1), coding 5.8S, non-coding ITS2 and coding 28S region. Whereas the coding regions of the ribosomal DNA between related species are generally highly uniform, mutations in the non-coding regions such as ITS-1 have no apparent fitness consequences and therefore contain valuable infor-

124 A snail on four continents: bird-mediated dispersal of a parasite vector? mation on phylogenetic relations between species. The ITS sequences of ribosomal DNA are therefore very useful for classification of species as well as comparing the genetic sequences within species over their geographic distribution (Remigio and Blair, 1997b; Bargues et al., 2001; Puslednik et al., 2009; Bargues et al., 2011a).

Molecular techniques - DNA extraction In 1996 and 1997, snails of seven common pond snail species were collected for sequence analysis from the Netherlands and Belgium (see Table 7.1 for locations) and stored in CTAB-DMSO solution (1.5% cetyltrimethylammonium bromide 20% dimethyl sulfoxide in deionised water). For each species DNA was extracted by ho- mogenisation of one whole snail using liquid nitrogen and hot CTAB-buffer (1.4M NaCl, 0.2% CTAB, 0.1M Tris, 0.02 M EDTA, 0.2% beta-mercapto-ethanol) (Doyle and Doyle, 1987). DNA was isolated using standard procedures (SDS and proteinase K treatment, phenol extraction and ethanol precipitation). Isolated DNA was checked visually by running 2 μl on a 1% agarose gel and concentration was determined using a spectrophotometer.

Table 7.1: Information on the snails collected in The Netherlands and Belgium for which ITS-1 sequences were determined.

Species Location GPS Length Base frequencies (%) (bp) A T C G Galba truncatula Beek, 50°56’ N 498 20.3 21.7 29.7 28.3 Netherlands 05°48’ E Lymnaea stagnalis Nijmegen, 51°50’ N 530 19.8 22.6 30.4 27.2 Netherlands 05°51’ E Omphiscola glabra Lichtaart, 51°13’ N 534 20.2 22.9 28.8 28.1 Belgium 04°55’ E Radix auricularia Nijmegen, 51°50’ N 567 20.3 25.5 28.7 25.5 Netherlands 05°51’ E Stagnicola corvus Nijmegen, 51°50’ N 556 20.1 21.6 29.9 28.4 Netherlands 05°51’ E Stagnicola fuscus Appelsvoorde, 51°07’ N 526 20.2 21.1 30.4 28.3 Belgium 04°09’ E Stagnicola palustris Slijk-Ewijk, 51°53’ N 529 19.3 21.2 30.6 28.9 Netherlands 05°47’ E

Molecular techniques - DNA sequence amplification The internal-transcribed-spacer 1 (ITS-1) of the ribosomal DNA was amplified using Goldstar Taq Polymerase (Eurogentec Belgium) and Goldstar reaction buffer. The universal eukaryotic primers “Fruit fly”, 5’-CAC ACC GCC CGC TAC TAC CGA TTG-3’, and “Silkworm”, 5’-GTG CGT TCG AAA TGT CGA TGT TCA A-3’ were used to anneal to the last section of 18S and start of 5.8S sections of the ribosomal DNA, respectively (Hillis and Dixon, 1991). PCR’s were performed in volumes of 25 µl with a Biometra T Gradient Personal Thermal Cycler (Westburg, The Nether-

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lands), with 15.5 μl H2O, 2.5 μl reaction buffer and concentrations of 0.25 mM MgCl2, 0.2 mM dNTP’s, 10 U/l Taq polymerase (BioTAQ), 0.8 mM of both primers. 1.0 μl of template DNA was added after diluting either 10, 100, 1000 or 10 000 times depending on the concentration. Cycling conditions were: 4 minutes hot start at 95°C, followed by 33 amplification cycles (melting for 60 s at 95°C, primer annealing for 30 s at 60°C and elongation for 60 s at 72°C). After a final elongation step for 10 minutes at 72°C, PCR-products were stored at -20°C.

Molecular techniques - DNA sequencing PCR products were cloned in the pCR2.1 vector (TA cloning kit, Invitrogen, Carls- bad, CA, USA) and sequences containing complete ITS-1-inserts were sequenced in both directions using ABI PRISM 310 Automated sequencer (Perkin Elmer, MA, USA). All obtained sequences were sequenced from both the 18S and 5.8S sides. For Radix auricularia sequencing was only performed with the 5.8S primer.

Table 7.2: Isolated populations of G. truncatula present on islands and highlands, for which long distance dispersal has been necessary. For islands, distance to the mainland or nearly larger islands is indicated in brackets.

Location Latitude Longitude Type Publication Iceland 64 °N 18 °W Island (450km) (Glöer 2002) Faroer Islands 62 °N 6 °W Island (300km) (Hubendick 1951) Shetland islands, Scotland 60 °N 1 °W Island (80km) (Kerney 1999) Isles of Lewis, Scotland 57 °N 7 °W Island (25km) (Kerney 1999) Ostrov Beringa, Russia 55 °N 166 °E Island (180km) (Hubendick 1951) Island of Man, England 54 °N 4 °W Island (30km) (Kerney 1999) Corsica, France 42 °N 9 °E Island (50km) (Hubendick 1951; Mas-Coma et al. 2001) Sardinia, Italy 40 °N 9 °E Island (50km) (Hubendick 1951) Balearctic Islands 39 °N 3 °E Island (80km) (Beckmann 2007) Azores, Portugal 38 °N 27 °W Island (Hubendick 1951; Backhuys (1500km) 1975; Mas-Coma et al. 2001) Crete, Greece 35 °N 24 °E Island (70km) (Hubendick 1951) Madeira 32 °N 17 °W Island (400km) (Backhuys 1975) Canary Islands 27 °N 15 °W Island (100km) (Backhuys 1975) Réunion (Mascarene islands) 21 °S 56 °E Island (650km) (Griffiths and Florens 2006) Alps, Austria 47 °N 13 °E highland areas (Sturm 2007) Central Massif, France 45 °N 2 °E highland areas (Vignoles et al. 2002; Vignoles et al. 2003) Alps, France 45 °N 6 °e highland areas (Vignoles et al. 2002; Vignoles et al. 2003) Himalaya, India 34 °N 76 °E highland areas (Hubendick 1951; Subba Rao 1989; Ramakrishna and Dey 2007) Ifrane, Morocco 33 °N 5 °W highland areas (Goumghar et al. 1997)

Sana'a, Jemen 14 °N 44 °E highland areas (Brown 1980) Highlands, Ethiopia 11 °N 41 °E highland areas (Brown 1980) Cordillera de Mérida Mountains, 8 °N 70 °W highland areas (Correa et al. 2011) Venezuela Central highlands, Kenya 1 °N 38 °E highland areas (Brown 1980) Blukwa and Kisenyi, Congo 1 °N 30 °E highland areas (Brown 1980) Altiplano, Peru 7 °S 78 °W highland areas (Vignoles et al. 2002; Vignoles et al. 2003) Northern Bolivian Altiplano, 16 °S 68 °W highland areas (Jabbour-Zahab et al. 1997; Bolivia Bargues et al. 2006b) Andes, Argentina 33 °S 69 °W highland areas (Bargues et al. 2006b) Andes, Chile 39 °S 73 °W highland areas (Artigas et al. 2011) Tunesia, oasis of Tozeur 33 °N 8 °E Oasis (Diawara et al. 2003) 126 A snail on four continents: bird-mediated dispersal of a parasite vector?

Molecular techniques - DNA sequence comparisons To complement the obtained ITS sequences from The Netherlands and Belgium, 39 ITS-1 sequences of 17 lymnaeid species collected around the globe were extracted from GenBank-EMBL. For species names we used the names used in GenBank and the respective publications. The accession numbers of the sequences are indicated in the phylogenetic tree (Fig. 7.2) and were obtained from the following publications: (Remigio and Blair, 1997b; Mas-Coma et al., 2001; Gutiérrez et al., 2003; Bargues et al., 2006a; Bargues et al., 2007; Correa et al., 2010; Artigas et al., 2011; Bargues et al., 2011a; Bargues et al., 2011c; Correa et al., 2011).

Molecular techniques - Sequence alignment All newly obtained ITS-1 sequences and GenBank extracted ITS-1 sequences were aligned automatically using Clustal-W in Mega 5 (Thompson et al., 1994). The alignment was manually checked. Phylogenetic analysis was performed using Maximum Likelihood and Neighbour-Joining computed with the number of substitutions according to the Tamora-Nei model method and complete deletion of missing data. As outgroup we used Biomphalaria pfeifferi from GenBank (AY030361), following many previous phylogenetic studies of Lymnaeidae (e.g. Bargues et al., 2006a; Artigas et al., 2011; Bargues et al., 2011a).

Results Our literature research on the distribution of G. truncatula indicates that this species is found on four different continents, including 14 highland areas, and at least 14 islands (Fig. 7.1a, Table 7.2). It occupies almost the entire Eurasian continent, several areas on the African continent and southern parts of Alaska. It has recently been discovered in the highland areas of South-America. Throughout this distribution, ribosomal DNA sequences thus far determined are highly similar between individuals from different origin (Fig. 7.2).

Phylogenetic results The phylogenetic analysis demonstrates the importance of geographic isolation in the evolution of ponds snails (Fig. 7.2). The Radix species and the stagnicoline Lym- naeidae showing the deepest split are sister groups. Within the stagnicoline Lym- naeidae branch, a separate branch is present for the European stagnicoline species (Stagnicola sp.). The sister branch to this group is dominated by American species. Within this last group, a distinct group is formed by the genus Galba (=Fossaria). Within this American Galba branch, also the European samples of Galba truncatula and the recently discovered L. schirazensis are present. These two species occurring in Europe cluster more closely to American pond snail species than to other pond snail species in Europe.

Bird migration patterns Migratory waterbirds travel along a number of flyways across and within continents, covering large parts of the world, including the entire distribution of G. truncatula. The movement of the seven major waterbird families potentially related to the distribution of G. truncatula is visualized in Fig. 7.1b.

127 Chapter 7 ● 150 100 ● 50 ● ● ● ● ● ● 0 ● ● ● ● ● ● ● ● −50 −100

−150

50 0 (a) −50 Figure 7.1: (a) Global distribution of G. truncatula. G. truncatula is considered present throughout the countries represented in grey and on locations denoted by the open circles. Filled circles indicate the occurrence of G. truncatula on islands. Filled triangles indicate highland locations of occurrence. Literature sources: (Baker, 1911; Hubendick, 1949; Hubendick, 1951; Adam, 1960; Ellis, 1969; Rajagopal, 1970; Sampaio Xavier et al., 1973; Backhuys, 1975; Brown, 1980; Subba Rao, 1989; Økland, 1990; Goumghar et al., 1997; Jabbour-Zahab et al., 1997; Glöer and Meier-Brook, 1998; Kerney, 1999; Mas-Coma et al., 2001; Glöer, 2002 and references therein; Vignoles et al., 2002; de Kock et al., 2003; Diawara et al., 2003; Vignoles et al., 2003; Mekroud et

128 A snail on four continents: bird-mediated dispersal of a parasite vector? A otal 1.1 5.3 29.1 23.7 10.3 26.9 T

0.6 0.8 Gaviidae

150

1.0 1.1 Sternidae

0.1 2.6 0.1 Ardeidae 100

2.3 1.0 0.4 1.1 F Laridae

6 4.1 2.2 Gruidae E 50 2.6 0.1 9.9 0.3 7.7 12.4 Charadriidae D 0 0.4 0.4 4.2 0.1 13.1 24.7 C Anatidae

F E C A B D 50 100 B 150

50 0

(b) 50 al., 2004; Moghaddam et al., 2004; Belfaiza et al., 2005; Bargues et al., 2006b; Griffiths and Florens, 2006; Ashrafi et al., 2007; Beckmann, 2007; Ramakrishna and Dey, 2007; Sturm, 2007; Florijancic et al., 2008; Artigas et al., 2011; Bargues et al., 2011a; Correa et al., 2011; Perez-Quintero, 2011; Relf et al., 2011). (b) Schematic migratory routes of birds of potential importance to the dispersal of G. truncatula, the width of the arrows being proportional to the number of birds involved in the various flyways. Flyway labels A thru F correspond with the number of birds per family (in millions of birds per annum) using this flyway as depicted in the table inset.

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Figure 7.2: Phylogenetic relations of pond snails based on ITS1 sequences of both newly analysed Dutch and Belgium snails (indicated in bold) and sequences downloaded from GenBank-EMBL. Branch lengths are proportional to the number of substitutions according to the Tamora-Nei model method. Results of Maximum Parsimony and Neighbour-Joining methods were identical. The Neighbour-Joining tree is depicted with 1000x bootstrap support values of above 70% indicated.

130 A snail on four continents: bird-mediated dispersal of a parasite vector?

Discussion Galba truncatula was found to be widespread and genetically similar throughout its distribution, both suggesting a high mobility. This mobility can potentially be attributed to bird-mediated dispersal, as the distribution of G. truncatula matches the area covered by the major waterbird flyways (Fig. 7.1a/b). The phylogenetic clustering of the European populations of G. truncatula with the American Galba clade rather than other European pond snails (Fig. 7.2), suggests that this species might originate from the American continent and may also in the past have relied on waterbirds for the colonization of Eurasia. Dispersal by waterbirds is increasingly quoted as an explanation for the widespread distributions of a variety of aquatic species (Figuerola and Green, 2002; Green and Figuerola, 2005). For aquatic snails, potentially accompanied by parasites, sug- gestive indications consist of notes of snails attached to caught birds (e.g. McAtee, 1914) as well as observations of snails surviving the passage through the digestive tract of birds (Cadée, 2011; Van Leeuwen et al., 2012). Genetic analyses indicate birds may transport marine snails and land snails over long distances (Gittenberger et al., 2006; Miura et al., 2011), and historical constructions suggest this may have been important in the past (Wesselingh et al., 1999). Furthermore, the overlapping distributions of snails and migratory flyways of birds have previously fostered speculations on bird-mediated dispersal of snails (Baker, 1945; Hubendick, 1951). Bird-mediated dispersal between Europe and America has been suggested for L. stagnalis (Remigio, 2002), similar to what we are suggesting here for G. truncatula. Although no publications exist on the dispersal of G. truncatula by birds, its distribution and phylogenetic position now also suggest this to be an important process for this species and its associated parasites. The high tolerances of G. truncatula to both low temperatures and desiccation (Hodasi, 1976; Chapuis and Ferdy, 2012) might enhance its potential to be dispersed by birds. In addition, G. truncatula is often found in shallow water layers, in mud at the littoral borders of lakes, on wet rocks, in marshes or in wet grassland outside the water, where they tolerate drought by dormancy and are active when water returns (Moukrim and Rondelaud, 1992). This behaviour may facilitate interactions with a large variety of bird species. Its ability to reproduce by self fertilization can enhance establishment after dispersal of only few individuals (Frömming, 1956; Trouve et al., 2003), making G. truncatula a snail with many prerequisites for successful dispersal by waterbirds.

Phylogenetic relations - an American origin? Our phylogenetic analysis including the North American and European stagnicoline snails demonstrates that the European G. truncatula in our study clustered with the American species (Fig. 7.2). This is consistent with previous studies (Bargues et al., 2006a; Artigas et al., 2011; Bargues et al., 2011a; Bargues et al., 2011c; Correa et al., 2011). Since the highest diversity of Galba species is found in North America, with initially up to 40 (sub)species in the genus Galba (Fossaria) (extremely reduced to 3 species by Hubendick (1951), the Galba clade most likely originates from America. Our phylogenetic tree (Fig. 7.2) shows 8 species in the genus Galba, including G.

131 Chapter 7 truncatula occurring in Alaska (Baker 1911; Burch 1982). Only two Galba species, including G. truncatula, can be found in Europe (Glöer, 2002; Bargues et al., 2011a) and three, including G. truncatula, occur in the Himalaya region (Ramakrishna and Dey, 2007). Based on base pairs lengths, Radix and Galba are considered the oldest taxa, with the European Stagnicola being more recent (Bargues et al., 2001). Only from the start of the Pleistocene onwards, fossil evidence of G. truncatula has been present in Eu- rope (Ellis, 1969; Bailey et al., 2003). Since there is no evidence for the existence of a land connection between North America and Europe after the completion of the break up of the Laurasian continent at the start of the Eocene (already 55 million years ago), this species must have dispersed from the American continents to Eura- sia after the formation of the Atlantic Ocean. The occasional land connections over the Bering Strait during ice ages may have served as a corridor, if large mammals transported snails from Alaska to the European continent. However, bird-assisted dispersal could also very well explain this pattern.

Distribution analysis The entire distribution range of G. truncatula overlaps with the major flyways of the most important families of waterbirds (Fig. 7.1a/b). G. truncatula’s presence in Eastern-Africa may be explained by the East-African migratory flyway, which is used by large numbers of small waders as well as the much larger cranes. The millions of ducks, geese and swans that migrate annually throughout Europe may be responsible for G. truncatula’s spread over this continent, while many ducks, geese and swans, but also cranes and waders connect Alaska to the highlands of the Andes in South-America (Laredo, 1996). Annually, over 5 million birds fly between West- ern Europe and Iceland, where G. truncatula also occurs (Fig. 7.1b). Although the number of birds flying to Alaska from the Eurasian continent is smaller, still over a million birds cross the Bering Strait twice each year. The occurrence of G. truncatula on many islands and highlands further strengthens the idea that this species is not dispersal-limited by marine boundaries which can be explained by bird-assisted dispersal (Table 7.2). The revealed genetic similarities between G. truncatula individuals throughout its distribution further suggest that this species is highly mobile and has a high self fertilization rate. Besides potential bird-assisted exchange of G. truncatula between North America and South America (Jabbour-Zahab et al., 1997) there is also high similarity of ITS-1 sequence from Europe and Northern-Africa, indicating connectivity between these continents. High similarity of snail species from different continents has also recently been revealed in another snail family, i.e. the Physidae (e.g. Dillon et al., 2002), suggesting aquatic snails in general (and their associated parasites) may have higher levels of connectivity than thus far thought.

ITS-1 as marker The ITS-1 region of the ribosomal DNA is frequently chosen and highly suitable as region to test species relations and paraphyly (Schilthuizen et al., 1999; Bargues

132 A snail on four continents: bird-mediated dispersal of a parasite vector? and Mas-Coma, 2005; Artigas et al., 2011; Bargues et al., 2011b; Bargues et al., 2011c). However, it has also been criticized because it has relatively low independence of nucleotide positions (i.e. homoplasy) (Álvarez and Wendel, 2003). This effect can be reduced by including more markers than solely ITS-1 (Feliner and Rosselló, 2007). Therefore, we compared the results of our phylogenetic analysis to other previously published pond snail phylogenies that used other marker combinations (Bargues et al., 2006a; Artigas et al., 2011; Bargues et al., 2011a; Bargues et al., 2011c; Correa et al., 2011). The main structure of our phylogenetic tree, i.e. Radix and Galba clades within the Lymnaeidae and G. truncatula present in the American rather than Euro- pean clade, was consistent with these previously published trees. This supports the idea that ITS-1 is a suitable marker for this phylogenetic analysis, and that we can be confident of its outcome that now includes our own European samples and the geographic information of the various species. ITS-1 seems sufficiently capable to inform on the distribution and mobility of G. truncatula.

Human-mediated dispersal Waterbirds are a plausible vector explaining the wide distribution and genetic similarity of G. truncatula, however, other vectors may also influence its distribution. Humans and their livestock are often put forward as vectors for G. truncatula and its parasites. Its distribution on the Azores has been suggested to be due to the presence of humans (Backhuys, 1975), and its presence in South-America has been linked to areas with livestock (e.g. Mas-Coma et al., 2001; Mas-Coma et al., 2009). Active transport of livestock by humans might facilitate long-distance dispersal of these snails and humans connect continents by boating traffic. However, while man may be responsible for the dispersal of G. truncatula in recent times, human-mediated dispersal has only become a possibility during the last few hundred years. The introduction of G. truncatula from the American to the Eurasian continent during the Pleistocene is therefore most likely attributed to birds, which in this case will also nowadays still contribute to dispersal.

Conclusions In summary, G. truncatula is a widespread snail that occurs on four continents. Throughout its range of suitable habitat, it can be found on remote islands and highlands, whereby its presence is associated with the migratory flyways of migratory birds. During the Pleistocene, this species likely dispersed from the American to the Eurasian continent by either large mammals that crossed the Bering Strait, or by migratory birds. The high genetic similarity throughout its present distribution further supports the view of a highly mobile species. The high mobility of this species also suggests a high mobility for its associated parasites.

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Casper H.A. van Leeuwen

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Waterbirds as dispersal vectors in aquatic systems Dispersal is an essential process in shaping species distributions and therefore global biodiversity (Clobert et al., 2001; Bullock et al., 2002). Whereas biodiversity at any given location will depend on a large number of factors, such as ecological differences between species competing for resources (niche theory, e.g. Tilman, 1985, 1994), random processes (neutral theory, Hubbell, 2001) and extinctions and recolonizations (metapopulation theory, Levins, 1969), an important regulator of biodiversity is the selection of species that is able to reach this location from elsewhere (Dieckmann et al., 1999). Since species movement is often restricted by the heterogeneity of the landscape, i.e. if suitable habitat is separated by other types of terrain, the ability or inability to disperse across such barriers can strongly influence global biodiversity. For fresh water species, land is a major barrier that restricts their dispersal over larger scales. Even so, dispersal is especially important for species inhabiting these systems. Freshwater habitat is continuously challenging species by fluctuating water levels, changing nutrient concentrations and salinity, and extreme abiotic differences over seasons (Wiggins et al., 1980; Jocque et al., 2010; Verdonschot et al., 2011). To en- sure their long term persistence, species have therefore evolved adaptations to cope with such fluctuations. Or, as an alternative strategy, may continuously disperse between temporary wetlands. Many species in temporary habitats are renowned to continuously (re)colonize new areas by dispersal, and thereby maintain viable populations over a larger area, i.e. forming meta-populations (Levins, 1969). Most aquatic species can therefore either actively walk or fly over land (e.g. Zickovich & Bohonak, 2007; Lowe, 2009), or use passive dispersal vectors such as wind, water or animals for transport between aquatic habitats. The discrete distribution of freshwater wetlands and clear separation by land make them ideal systems to investigate unresolved aspects of dispersal (MacArthur & Wilson, 1967; Gillespie et al., 2008). The fact that most of today’s wetland biodiversity is threatened by habitat loss (e.g. Green et al., 2002b), fragmentation or changing conditions due to climate change and nutrient loading (Hirt et al., 2005), makes it even more relevant to study dispersal in these systems. This thesis therefore investigated the dispersal of aquatic plants and invertebrates in freshwater systems, with special focus on passive dispersal of aquatic snails by waterbirds. This follows the first suggestion of Darwin (1859) that waterbirds might act as dispersal agents for smaller species to remote locations, as well as more recent revelations on this subject (Figuerola & Green, 2002; Green et al., 2002a; Green & Figuerola, 2005). Endozoochory is the internal transport of propagules by animal vectors. To sum- marize our knowledge on endozoochorous bird-mediated dispersal and gaps therein, we first reviewed all current literature on this subject through a meta-analysis (Chapter 2). It appeared that an average migrating bird disperses five viable propagules during its first 100 km of flight, and one additional propagule during the subsequent 200 km. A wide variety of Anatidae and Rallidae species appeared to be involved in dispersing a wide range of macro-invertebrates and plant seeds, supporting the view that many, if not most waterbirds may indeed significantly

142 Synthesis contribute to the connectivity of wetlands. We explained how such a wide taxonomic range of species can escape digestion by the fact that birds maximize their energy intake over time, rather than their digestive efficiency per se. A resulting sub-maximum digestive efficiency provides an opportunity for propagules to viably pass the digestive system. This idea was further explored and supported by experiments with aquatic snails (Gastropoda) (Chapter 3). These indicated that not only tough plant seeds and resting stages of invertebrates, but also even fully-functional adult aquatic snails can survive a passage of up to five hours through a bird’s digestive tract. This potentially leads to dispersal distances greatly exceeding the snail’s own, active dispersal distance (Kappes & Haase, 2011). Bird-mediated dispersal might be a process explaining the widespread distributions and invasive behaviour of many aquatic snails (Chapter 7, Dillon et al., 2002). These observations further strengthen our idea that endozoochory is a taxonomically widespread phenomenon. Although specific adaptations are present in species that heavily rely on endozoochory as a process, the incomplete digestion by animal vectors may also allow species without specific adaptations to be dispersed by endozoochory. To reliably estimate the potential dispersal distances of snails and other propagules, digestive characteristics of actively moving vectors should be known. How- ever, until now, these were unavailable. Neither for birds nor mammals, in aquatic or terrestrial systems, such data was found. Our experiments with swimming mallards now indicate that active animals release propagules earlier than resting animals, and that therefore dispersal distances in actively travelling animals will be shorter than thus far thought. As a consequence, survival of propagules may be higher than generally estimated (Chapter 4). Additionally, the faster retrieval of small propagules during our experiments implied a negative relation between propagule size and survival, and that smaller propagules will only achieve shorter dispersal distances. This finding matches most earlier observations in experimental studies with resting birds (reviewed in Chapter 2), as well as the generally small propagule size retrieved from droppings in field studies (e.g. Charalambidou & Santamaría, 2005; Frisch et al., 2007). Whereas our experiments and meta-analysis show that aquatic snails and many other species have the potential to be dispersed by endozoochory, there are also many anecdotal observations of birds and other animals transporting aquatic species externally. This is generally called ectozoochory, or epizoochory in the case of seed dispersal. Although often neglected and harder to investigate than endozoochory, the four complementary experiments in Chapter 5 illustrate that aquatic snails indeed have many of the necessary prerequisites for successful transport by external adhesion to birds. Ectozoochory could be another important mechanism explaining the wide distributions of many aquatic snails. However, whether this potential of snails that was revealed in experiments for both endo- and ectozoochory truly affects populations in field situations cannot be assessed mechanistically. We addressed this aspect of dispersal by genetic analyses. These analyses supported our view that waterbirds, combined with other vectors, indeed dispersed

143 Chapter 8 the invasive snail Physa acuta (Gastropoda, Pulmonata, Physidae). In Doñana Na- tional Park in Southern Spain, genetic variability in snail populations indicated a high similarity throughout rice fields that flooded during part of the year. Snails thus dispersed readily by water connections in this large area. A high similarity between these rice fields and multiple ephemeral ponds in Doñana National Park tens of kilometres away, supported the idea that waterbirds transport snails when flying between the rice fields and these ponds. The ephemeral ponds within the park were likely connected by large mammals that used the ponds for drinking water, because ponds with higher mammal densities showed higher genetic similarities. These results emphasize that waterbirds may disperse snails, but also that they interact with other dispersal vectors while doing so. Several dispersal vectors each provide part of the dispersal kernel and allow species to cross certain boundaries. The wide distribution of P. acuta on various continents and its success as invasive species may be attributed to its ability to use multiple vectors. Such interactions between multiple dispersal vectors and their relation to specific habitats should therefore be considered when attempting to control invasive freshwater species as well as protecting endangered species. How birds can contribute to genetic variability over a larger scale can be shown by the distribution of Galba truncatula (Gastropoda, Pulmonata, Lymnaeidae), an important vector for human and livestock parasites. This aquatic snail is, just like Physa acuta, widespread. It occurs on the Eurasian, African, North- and South-American continents (Chapter 7). The fact that it is also found on at least 14 islands and 14 highland areas, suggests it has very good dispersal abilities. Similarity between its distribution and the major migratory flyways of (water)birds implies birds may also be an important dispersal vector for G. truncatula. In Chapter 7 we found support for this idea. The phylogenetic relation of species of Lymnaeidae can mostly be related to the geographic distribution of species, but the European populations of G. truncatula seem to be more related to American than to other European stagnicoline pond snail species. This suggests that this snail has been transported in the past from the American to the Eurasian continent. Further supported by its wide distribution and the ease with which it colonizes remote locations over sea and land, bird-mediated dispersal seems the most parsimonious explanation for this species’ wide distribution. In summary, many aquatic plants and invertebrates can be dispersed by waterbirds internally (Chapter 2) and a series of aquatic snail species have at least the necessary prerequisites for external transport (Chapter 5). These modes of dispersal not only affect the occurrence of species on a local scale (Chapter 6), but also their worldwide distribution (Chapter 7). This is important knowledge when dealing with e.g. invasive species or important parasite vectors such as Galba truncatula (Chapter 7). However, despite that we now showed that bird-mediated dispersal is possible, and that this can affect certain species in certain landscapes, the true relevance of bird- mediated dispersal will only become clear if we compare its efficiency to other possible ways for aquatic species to disperse.

144 Synthesis

The relative importance of waterbirds as connectors While many small aquatic species have the potential to be dispersed by waterbirds, many less mobile aquatic species can also use other vectors, such as wind, water or other animals to disperse between aquatic habitats (e.g. Van de Meutter et al., 2006; Vanschoenwinkel et al., 2008b; Pollux, 2011). Wind may blow zooplankton, insect larvae, algae, resting eggs of various aquatic invertebrates and many aquatic plant seeds over long distances (Kristiansen, 1996; Cohen & Shurin, 2003; Soons, 2006; Vanschoenwinkel et al., 2008b). Both in terrestrial and aquatic systems, adaptations for dispersal by wind (anemochory) are common (e.g. Nathan et al., 2002; Minami & Azuma, 2003; Tackenberg, 2003; Soons, 2006). Dispersal by water currents is seen as the most prominent form of dispersal in rivers and streams, for both plants (Leyer, 2006) and aquatic invertebrates (Bilton et al., 2001). In these habitats, high water conditions may additionally carry propagules to higher ground and elevated land in the floodplains (Andersson et al., 2000; Boedeltje et al., 2003). During periods of low flow velocity, wind can move floating propagules in a multitude of directions to compensate for generally unidirectional flow in downstream direction (Boedeltje et al., 2004; Soomers et al., 2010; Soomers et al., 2011). Many plant seeds therefore have adaptations, such as air-filled structures to increase buoyancy or a seed coat that increases water repellence (Ridley, 1930). Dispersal of invertebrates in rivers may be active in the upstream direction (Shurin & Havel, 2002; Kano, 2009; Kappes & Haase, 2011) or passive by water flow (Van de Meutter et al., 2007; Siziba et al., 2011; Van Riel et al., 2011). Besides waterbirds, also mammals, fishes, reptiles, amphibians and even insects carry small propagules (Bilton et al., 2001). Data in Chapter 6 supported the idea that large mammals carry aquatic snails by ectozoochory between ponds, something also observed for plant seeds and other small invertebrates (Vanschoenwinkel et al., 2008c; Vanschoenwinkel et al., 2011). Additionally, many propagules that are known to survive digestion in birds have been shown to survive digestion in fish (Brown, 2007; Pollux, 2007; Pollux, 2011). Reptiles carry nematodes and ostracods (Lopez et al., 1999), and flying insects carry small larvae of water mites (Bohonak, 1999). Many animals can disperse small freshwater clams (Sphaeriidae), which often have their valves slightly open during resting, but will rapidly close their valves upon touch of a bird feather, insect leg or fish fin potentially facilitating dispersal (Gittenberger et al., 2004). Differences in the magnitude of these potential dispersal vectors determine the actual relevance of bird-mediated dispersal (e.g. Schupp, 1993; Pollux, 2007; Vansch- oenwinkel et al., 2008b). Even extensive dispersal by waterbirds over long distances may be of limited relevance to wetland biodiversity, in case other vectors are responsible for dispersal over longer distances and in larger numbers. When evaluating the relative importance of dispersal vectors, it should be kept in mind that an efficient dispersal vector picks up, transports, and releases propagules in another suitable habitat without negatively affecting their survival (Pollux, 2007; Gillespie et al., 2012). Dispersal can therefore be compared between vectors based on (1) the probability that propagules are being picked up, (2) the quantity of propagules that can

145 Chapter 8 be carried simultaneously, (3) the distance that propagules travel, (4) the directionality of the movement towards suitable habitat (Wenny, 2001; Gillespie et al., 2012) and (5) the survival of propagules given the potential damage incurred by a vector. Although an arduous task given the many factors and many unknowns involved, below we attempt to qualitatively compare bird-mediated dispersal to dispersal by wind, water and other animals (excluding humans that are addressed separately afterwards). At the same time, the importance of endozoochory and ectozoochory by birds is compared. Table 8.1 provides a generalized overview of the comparison as described in the next five sections.

1. Probability to be picked up The probability that aquatic propagules are picked up by waterbirds is mainly determined by the abundance of birds relative to the amount of propagules available. Birds can ingest large numbers of propagules (e.g. Whilde, 1977) and large parts of the vegetation (Hidding et al., 2010) before other potential vectors come into play. The probability that a propagule adheres to a bird to allow external transport increases proportionally to the number of floating propagules in the water. As a result, only few of the floating propagules will adhere to birds, as shown in the experiment with snails in Chapter 5. Thus many more aquatic propagules are likely to find their fate inside rather than on the exterior of a waterbird. Besides waterbirds, also other animals will forage on aquatic seeds and invertebrates. The relative abundance of birds compared to other animal vectors thus de- termines their relative importance. However, besides that birds are often abundant, they also have a relatively high energy need (e.g. 100-fold higher in birds than fish of similar mass, Klaassen & Nolet, 2008). They will have a disproportionally high intake of propagules compared to most other animal vectors. On the other hand, with respect to external transport, the fur of mammals is likely more effective a substrate for adhesion of propagules than the sheet-like feathers of waterbirds (Pauliuk et al., 2011). The probability for dispersal by wind is highest for seeds of emergent wetland plants, which release their seeds above the water. Many of these seeds will have the opportunity to be carried away from wetlands by wind (Soons, 2006) before being eaten by birds or other animals. Most aquatic invertebrate and plant propagules released on or in the water can only be lifted by wind after all water has been drained or evaporated from a pond (Vanschoenwinkel et al., 2008a; Vanschoenwinkel et al., 2008b). Only very small algae may be blown from waves (Kristiansen, 1996). For submerged macrophytes and aquatic invertebrates, wind is considered more important for dispersal from temporary ponds than from permanent waters. Water will transport all propagules not dispersed by wind or animals, but often also prior to or after wind and animals have contributed to their dispersal. With respect to the probability of dispersal, water is therefore considered the most liable vector.

2. Quantity Any number of propagules can be transported simultaneously by wind and water.

146 Synthesis

Quantitative transport by birds, however, is determined by the number of birds and their behaviour. An average bird will excrete about five viable propagules during the first two hours after food ingestion, followed by only a few more propagules later on (Chapter 2). During spring and autumn, when millions of waterbirds migrate long distances all over the globe (Wetlands-International, 2007), these five propagules per bird, per foraging baud followed by displacement, may together add up to billions of propagules dispersed by birds. Large numbers of waterbirds migrate annually covering large distances, but engage even more often in regular short-distance flights between feeding and roosting sites. Following the lower probability to be picked up by ectozoochory than by endozoochory (as pointed out above in section 1), external transport seems also quantitatively less important than internal transport. Other animals with fur may carry higher numbers of propagules attached to their outside, but likely carry fewer propagules internally than birds. Aquatic insects may be even more abundant than waterbirds and can therefore also carry many propagules, but they are also much smaller and have no structures for attaching such as feathers or hairs. The number of propagules carried per animal will therefore be very limited and will only involve very small sessile animals and algae. Regarding the quantity of propagules transported, dispersal by wind and water will probably be of greater importance than both endo- and ectozoochorous dispersal by waterbirds. Only during periods of high bird abundances such as the migratory season, quantitative importance of endozoochorous dispersal by birds may approach the magnitude of dispersal by wind and water.

3. Distance Dispersal distance estimates for waterbird-mediated dispersal based on experiments exceed several hundreds of kilometres, although propagule viability decreases with increasing distance (e.g. Chapter 2, Charalambidou et al., 2003b; Soons et al., 2008). The genetic analysis for Physa acuta in Chapter 6 supports dispersal distances in excess of 30 km, as the distance between rice fields and ephemeral ponds in Doñana National Park was at least 30 kilometres. The dispersal of Galba truncatula from the American to the European continent indicates that even ecological barriers of thousands of kilometres have been crossed with the likely assistance of birds, although over a long time and at a potentially very low rate (Chapter 7). Dispersal distances of propagules by wind seem unlimited, since airborne propagules can be carried over thousands of kilometres in hurricanes and cyclones (Gillespie et al., 2012). However, during normal wind conditions long distance transport is only possible in higher altitude winds (>100km/h), while low-altitude winds (20-30km/h) generally only result in local dispersal (Gillespie et al., 2012). The probability that propagules reach these high altitudes through thermal winds (e.g. Tackenberg, 2003) was estimated to be only 1–5% (Nathan et al., 2002). Thus, long- distance anemochory occurs for only a small selection of all airborne propagules, although this may still be a considerable amount (section 2). Potential dispersal distances by water depend on a river’s current velocity and

147 Chapter 8 the surface covered during a flood. In both cases propagules can extent their dispersal distance by staying buoyant. Many aquatic snails float by adhering to the water surface (Newell, 1962) or by attaching to floating substratum. Many seeds have morphological adaptations that increase their buoyancy (Boedeltje et al., 2003; Boedeltje et al., 2004). Seeds and vegetative parts of plants can float for several weeks up to several months (Barrat-Segretain, 1996; Barrat-Segretain et al., 1998; Barrat-Segretain et al., 1999; Pollux, 2007). Larval stages of aquatic bivalves that facilitate downstream movement last from some days to weeks (Gittenberger et al., 2004; Van der Velde et al., 2010). Floating propagules in a system with average water velocities of 0.05 to 0.5 m s-1 may be transported over 800 to 8000 km downstream (Pollux, 2007). This estimate is exceeded in the case of higher current velocities, or reduced if floating propagules are only moved by winds that blow them over stagnant water (Soomers et al., 2010). Dispersal distance may be limited by elements in rivers along the side and bends in meandering systems, trapping floating propagules (Andersson et al., 2000). Flood- ing events can carry propagules as far as the flood reaches, i.e. over thousands of kilometres (e.g. Von Oheimb et al., 2011). Dispersal distances by other animals than birds, such as wild boars (Sus scrofa), cattle and deer species, will generally be less than those covered by waterbirds. Land has many obstacles that limit these terrestrial animals to move (e.g. oceans, mountain ranges). Flying waterbirds are considered more mobile than these mammals (Sutherland et al., 2000). Fish disperse seeds and invertebrates upstream, providing continuous transport in suitable habitat (Bondesen & Kaiser, 1949; Pollux et al., 2006; Brown, 2007; Pollux, 2011). However, even substantial dispersal by fish usually does not exceed a few dozen kilometres (Pollux et al., 2006; Pollux, 2007), and propagules will only be dispersed within water systems. Flying aquatic insects may carry very small propagules between waters, although they will generally cover smaller distances than birds (except when they use thermal soaring). Thus, when it comes to dispersal distance, birds take a special place in comparison to other zoochorous dispersal vectors and water, being able to disperse propagules over long distances and across ecological barriers. Only anemochory can parallel this, although only for very small propagules.

4. Directionality Waterbirds often make directed flights between wetlands for feeding and roosting. During migration they refuel in similar wetland habitats providing favourable conditions for propagules they may have carried from distant locations. Darwin (1859) already highlighted this directionality of dispersal by waterbirds as an important factor for successful dispersal, which has since been quoted in almost all publications addressing bird-mediated dispersal (see Chapter 2 for overview). Aquatic insects have a similar directionality and connection to water bodies. Most other animals will also actively travel between areas that provide drinking water, as implied for cattle and boars in Doñana National Park in Chapter 6, and transport propagules directionally. In contrast, wind will transport propagules to a wide range of sites, of

148 Synthesis which only a limited percentage may be suitable wetlands (Cohen & Shurin, 2003). Wind dispersed propagules are therefore often produced in large numbers, and such propagules tend to loose their adaptations for dispersal by wind after establishment on oceanic islands where many propagules will be lost (Carlquist, 1966). Water transports propagules throughout aquatic habitat, which may be relatively suitable for the dispersed species, and can therefore be considered a directional vector. However, in rivers and streams, water will only provide unidirectional transport downstream (Pollux et al., 2005) and propagules can not be carried across land. Only during flooding events they may leave the aquatic habitat, although then directionality towards suitable habitat may be as low as for wind dispersed seeds. In short, directionality by waterbirds is unmatched by most other dispersal vectors.

5. Survival Whereas snails and most other aquatic propagules can readily survive desiccation during external transport by birds, internal transport requires tolerance to low pH conditions, crushing forces in the gizzard, high temperatures, lack of oxygen and the presence of digestive enzymes (Vispo & Karasov, 1997). This has a huge negative impact on endozoochorously dispersed propagules. Although many seeds (Soons et al., 2008), resting stages of invertebrates (Charalambidou et al., 2003a; Charalambi- dou et al., 2003b), and even some aquatic snails (Chapter 3) can survive such conditions, only few of all ingested propagules survive passage through the digestive tract. The number of species suitable for external transport therefore likely exceeds the number capable of internal transport, which is requiring special adaptations or is facilitated at low rates by incomplete digestion (Chapter 2 and 3). Although the viability of some seeds may even improve after passage through the digestive tract (Santamaría et al., 2002), many ingested seeds will still be completely digested. Only animal vectors that chew their food (e.g. most mammals) inflict even more damage to propagules than vectors that ingest propagules intact (e.g. birds, most herbivorous fishes). Since chewing is a conscious process that damages food until it is small enough to ingest, the duration of selecting suitable food as well as the grind- ing of the propagules may be longer than when this occurs internally by the grit in the gizzard. Although the bills of birds can also efficiently select food, after food en- ters the gizzard it can only be excreted by regurgitation or rapid further processing of indigestible material, which both result in dispersal. Wind dispersal generally only requires resistance for desiccation, as only propagules carried to higher altitudes may be exposed to low temperatures, low atmos- pheric pressures or solar radiation (Gillespie et al., 2012). Survival of propagules carried by water is expected to be the least affecting propagules.Survival of propagules is therefore a major limitation for endozoochory, and requires high tolerances of propagules compared to ectozoochory, hydrochory and anemochory.

Comparing the potential vectors in freshwater systems The comparison of transport vectors to date is not only limited by the estimates for long-distance transport by waterbirds, but also still by our knowledge on dispersal by wind and water (Nathan et al., 2002; Van de Meutter et al., 2006; Nathan et al.,

149 Chapter 8

2008). The general comparison as presented in Table 8.1 is therefore indicative of what an average propagule may experience, but further investigation is required to refine our understanding of the dispersal process in aquatic systems. The many differences between specific propagules and their potential adaptations for specific vectors make it difficult to draw general conclusions for all propagules. Addition- ally, whereas dispersal of one propagule might be enough for certain species to colonize an area, other species may require long and continuous propagule pressure into a suitable habitat before successful competition with the established community results in colonization (Jenkins & Buikema, 1998; De Meester et al., 2002). Still, some general observations may be identified when comparing vectors (as indicated in Table 8.1).

Table 8.1: Comparison of dispersal vectors based on five aspects of the dispersal phase. Suitability of the vectors for the aspects are indicated by ++ (high), + (normal), or - (low). The overall suitability is indicated as the sum of the plusses and minuses. Winged aquatic insects more closely resemble Waterbirds external than Other animals.

Vector Pick up Quantity Distance Directionality Survival Overall Water ++ ++ + + ++ ++++++++ Wind ++ ++ ++ - + ++++++ Waterbirds internal ++ + ++ ++ - ++++++ Waterbirds external - - ++ ++ + +++ Other animals + - - ++ - 0

All vectors have certain limitations for dispersal, but water seems the most effective vector. Propagules are readily picked up by water, may be transported over long distances in high quantities and are continuously in relatively suitable habitat that does not affect their survival. The only major limitation of water is that it cannot disperse propagules over land or across oceans in case of freshwater species. To travel between ponds and isolated wetlands, other vectors are required. Wind is highly effective for colonizing such isolated areas, as wind also disperses many propagules over long distances without reducing survival. However, wind is only considered effective for smaller propagules (<1 mm) (e.g. Wilkinson et al., 2012). Furthermore, a large number of all airborne propagules is lost due to the lack of directional transport. Flying propagules only have a very small chance to reach suitable habitat, which requires dispersal of large amounts of small propagules before successful colonization. Endozoochory by waterbirds severely affects propagules by digestion, resulting in survival of only few of the many ingested propagules. However, in contrast to wind, those few propagules that survive ingestion have a high chance to reach suitable habitat due to a high directionality. In contrast to water, dispersal across land is possible. Ectozoochory by birds requires fewer physical tolerances of the propagules, which increases chances of survival, and will therefore disperse a wider variety of species. Yet, the chances of propagules to be picked up and therewith the transported quantities seem much lower than for endozoochory. Other animals may be better at picking up propagules for ectozoochory, however, will contribute less

150 Synthesis to dispersal by endozoochory if they chew their food and move slower. The major advantage of waterbirds over other animal vectors is their ability to fly across major barriers in the landscape, also those limiting movement of other animal species. In summary, this comparison shows that water, wind and animals all contribute to dispersal of aquatic species, but also that they all have limitations on how far and where propagules may be transported. As a result, the most successfully transported propagules are those that use a combination of vectors.

Dispersal by multiple natural vectors, humans, and how this may have evolved through time Most propagules can be dispersed by multiple of the above mentioned vectors (Rid- ley, 1930). These different vectors can transport propagules to the same sites (parallel dispersal), but can also move them to different locations, or can pick up propagules one after another (serial dispersal). For example, birds can pick up propagules after they have been carried by water or wind. Although many propagules have adaptations aimed at one particular vector (e.g. wind), dispersal over really long distances mostly requires serial involvement of multiple vectors (e.g. wind and water) or transport by unusual vectors for which species have no apparent morphological adaptations (Higgins et al., 2003; Nathan et al., 2008). Species have long been able to adapt to the use of these multiple potential dispersal vectors. Ever since the first invertebrates such as insects crawled onto land, and the formation of the first plant seeds during the Devonian era, i.e. ~360 Mya (Gillespie et al., 1981), wind and water have moved species over the continent(s). Species likely adapted to using these vectors. During the Devonian and in particular in the Carboniferous period, winged insects (Pterygota) evolved and became an additional potential vector to which smaller species could adapt. After the final breakup of Pangaea at the end of the Triassic (~200 Mya) dispersal over longer distances was no longer only limited by land barriers and mountain ranges, but also oceans started to separate potential suitable habitat. However, by now, many other flying animals had appeared. The Pterosaurs (late Triassic-Cretaceous period) and many feathered raptors or primitive birds started to appear. Many of these potential vectors disappeared during the mass extinction of the K-T boundary (Dingus & Rowe, 1998), but the aquatic organisms that survived this already had had potential time to evolve (pre-)adaptations for endo- or ectozoochory. Around 50 million years ago, modern flying birds such as Rallidae and Anatidae started to appear (Dingus & Rowe, 1998; Elphick, 2007). Aquatic organisms could now (further) adapt to dispersal by modern birds. Birds evolved migratory flyways due to ice ages and seasonal- ity, and continued to connect the continents that drifted apart (Elphick, 2007). Wind and water had continuously dispersed species throughout all this time, but birds were now an additional vector. Only relatively recent in geological history, humans appeared. During the last thousand years, modern people became increasingly mobile and started to travel extensively on a global scale. Anthropogenic changes to the environment paved the way for the establishment of many species all around the world, and man is repeat-

151 Chapter 8 edly involved in serial dispersal with all kind of other vectors. Humans are now a major driver of initial introductions of species to novel areas over large distances, after which these introduced species use their natural modes of dispersal for further spread (e.g. Van der Velde et al., 2010). Species that were never before able to colonize certain areas by natural vectors are now suddenly dispersed over large distances and between continents. Many aquatic invertebrates and fish have intentionally been introduced for farming or sports fishing (Cambray, 2003; Cardoso et al., 2007; Mandrak & Cudmore, 2010; Schroder & de Leaniz, 2011). Species have also been accidentally introduced through ballast water of ships, attached to recreational boats transported over land, by the aquarium trade, or by shipping goods on which species hitchhiked (e.g. Madsen & Frandsen, 1989; Mills et al., 1993; Robinson, 1999; Milbrink & Timm, 2001; Bailey et al., 2003; Blakeslee et al., 2010; Waterkeyn et al., 2010; Briski et al., 2011; Clarke Murray et al., 2011). Waterbirds will nowadays often play a role in the dispersal following such initial introduction by humans. Although it has been a mechanism for dispersal of species for a long time, it may not only still be a mechanism by itself, but will additionally be a link in a chain of multiple other vectors (as shown in Chapter 6). This implies bird-mediated dispersal is important for an even larger suite of species, and this includes a role in the further spread of invasive species after initial introduction. It is therefore essential to further improve our knowledge on the relative importance of birds as vectors.

Future directions The importance of waterbirds for the short-distance dispersal (in the order of tens of kilometres) of a wide variety of aquatic organisms can be considered well-established based on this thesis and preceding work. However, with respect to long-distance bird-mediated dispersal, still much improvement of our estimates is required. For endozoochorous dispersal, the most crucial gap in our knowledge relates to the uncertainty of propagule ingestion directly prior to migration. An argument down- playing the relevance and magnitude of bird-mediated dispersal is that birds will not embark on migrations with a full digestive system (Clausen et al., 2002). Data to either support or refute that claim is largely lacking. Collecting droppings of birds just upon arrival during migration, or catching birds during active flight followed by dropping collections, may shed light on the amount and viability of propagules that have truly been dispersed over long distance. For ectozoochorous dispersal, the most crucial gap of knowledge is the uncertainty whether or not propagules are able to stay attached to flying birds, which could be addressed experimentally using birds flying in wind tunnels or homing pigeons. In addition, our estimates of potential dispersal distances by endozoochory may improve by a better understanding of avian digestion under a range of conditions. Experiments are needed to assess how the survival of propagules is affected by the amount of propagules ingested, the mixture of ingested food types (briefly incorpo- rated in Chapter 3), and the seasonal and diet related changes in the highly plastic digestive system of birds (Charalambidou et al., 2005). Although Chapter 4 already indicated that activity alters the digestion of birds that are swimming, other forms of

152 Synthesis locomotion may affect retention times and survival of propagules in different ways. As flying is more energetically demanding than swimming, this may either result in an increase of the digestive rate with higher survival of propagules or may result in longer retention of propagules because of increased blood flow to flight-supporting tissues. Again, wind tunnel experiments could greatly improve our insights and potential estimates of dispersal distances. Experimental work (e.g. Chapter 3) involving a wider variety of propagule species will further improve our understanding of which species are capable of surviving gut passage. Why are some species capable to survive digestion by birds and others not? Does this involve specific adaptations or merely traits already acquired for survival of environmental conditions? Experiments comparing propagules of closely related species with known physiological tolerances will be most valuable, because this allows identification of patterns in propagule characteristics that are important for dispersal. The recently increased capability of measuring the spatial and temporal behaviour of birds using GPS technology greatly advances our possibility to assess where and when birds forage (endozoochory), interact with floating propagules (ectozoochory), on which altitudes and how fast they fly, and where they release propagules. This data can now be integrated with physiological data of birds, such as the behaviour-specific retention times and propagule viability proposed to further expand experimentally. Including this in future quantitative predictions based on mechanistic models, such as what we started in Chapter 2, will form more realistic construction of dispersal kernels. Using genetic tools, the outcome of some of such predictive models could then be verified (Chapter 6 & 7). Thereby the challenge is to find suitable areas where human-mediated dispersal does not obscure the genetic variability caused by natural vectors.

Impact of waterbirds on aquatic systems Birds are known to contribute to many ecosystem functions and services, such as pest control, pollination and nutrient cycling (Sekercioglu, 2006; Wenny et al., 2011). They are food sources for other animals (e.g. raptors and carnivorous fish; Guillemain et al., 2007; Dessborn et al., 2011), affect aquatic invertebrate and plant communities as herbivores, omnivores and predators (e.g. Klaassen & Nolet, 2007; Hidding et al., 2010), have been identified as sources of nutrient input into aquatic systems (Hahn et al., 2007, 2008) and can transport diseases (Altizer et al., 2011). This thesis explored their potential to affect biodiversity of aquatic wetlands by dispersing aquatic species (Chapter 2-6), including their potentially associated parasites (Chapter 7). The results of this thesis strengthen Darwin’s original idea that the abundance and diversity of birds in wetlands contributes to the abundance and diversity of the species they disperse (Darwin, 1859). Almost a hundred species of aquatic plants and almost 40 species of invertebrates are currently known to be dispersed over long distances by waterbirds. This occurs either internally (Chapter 3 and 4) or externally (Chapter 5), and influences populations on a small scale by connecting wetlands (Chapter 6) up to a global scale by connecting continents (Chapter 7). Conservation of waterbirds as keystone species will therefore not only benefit

153 Chapter 8 birds themselves, but will also conserve connectivity between wetlands for many smaller species. Considering the current anthropogenic changes to the environment, whereby habitat is destroyed or fragmented but also becomes available elsewhere, bird-mediated dispersal will become even more essential in the future. While global change is predicted to move the ranges of suitable habitat for species towards the poles (Bakkenes et al., 2002; Parmesan & Yohe, 2003), only few aquatic species have sufficient active dispersal capacity to follow these range shifts (except winged aquatic insects) (e.g. Kappes & Haase, 2011). If bird-mediated dispersal can facilitate mobility of aquatic species in such a changing landscape by continuous (re)colonization of habitat, negative effects of climate change on biodiversity may be reduced (Brown & Kodric-Brown, 1977; Travis & Dytham, 2002; Kokko & Lopez-Sepulcre, 2006). On the other hand, there is also a downside of high levels of bird-mediated dispersal. Although the initial arrival of invasive species is a recent process often at the hands of humans, the ensuing enhanced dispersal by waterbirds may facilitate their further spread (e.g. Carlton, 1993; Wilson et al., 1999; Shurin & Havel, 2002; Van der Velde et al., 2006; Van der Velde et al., 2010). Understanding how this subsequent natural dispersal process will dispersal invasive species, might provide the clues to control them (e.g. Pichancourt et al., 2012). This thesis started with highlighting the importance of dispersal for the survival of species. It demonstrated that waterbirds play an important role in this dispersal, involving a wide variety of aquatic plants and invertebrates. Bird-mediated dispersal significantly contributes to species’ survival, to the biodiversity of wetlands and can help other species to cope with a changing environment. In comparison to other potential dispersal vectors in aquatic systems, waterbirds are quantitatively less important than transport by wind and water, but far more effective when it comes to transporting species directly between suitable habitats over land or ocean barriers. The fact that waterbirds importantly contribute to sustaining a wide variety of other aquatic species in wetlands around the globe, once more indicates that care should be taken to conserve these important players in aquatic ecosystems.

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160 Summary

Summary Movement of species in the landscape is essential for the long term survival of their populations. Nearly all species are therefore capable of dispersing within and between suitable habitats. However, the earth’s landscape is heterogeneous and includes many ecological barriers that restrict such movements. To cross these barriers species may travel by own propulsion, or can make use of vectors such as wind, water or other animals to disperse. Aquatic species that are bound to freshwater wetlands are often considered to live on “islands in a sea of land”, with land forming a major barrier for their dispersal. Since many small aquatic species lack the ability to disperse long distances by own propulsion, it is interesting and important to investigate vector-mediated dispersal in these systems. Inspired by the long standing suggestion of Darwin that especially waterbirds may be important dispersal vectors in aquatic systems, this thesis explores bird-mediated dispersal of aquatic organisms. Waterbirds travel fast and in large numbers between similar wetlands, and are therefore increasingly recognized as important dispersal vectors in aquatic systems. They may carry propagules of aquatic organisms either internally in their gut (endozoochory) or externally on their bill, feet and feathers (ectozoochory). The thesis starts with exploring the current knowledge on internal transport in a meta-analysis, followed by experiments on internal and external transport and genetic analyses to detect potential effects of bird-mediated dispersal on populations. The meta-analysis in Chapter 2 reviews all currently available peer-reviewed publications on waterbird endozoochory of freshwater plant seeds and macro-invertebrates. As many as 17 species of Anatidae and Rallidae are involved in dispersing at least 39 species of macro-invertebrates and 97 species of plant seeds. On average, one-third of all investigated waterbird droppings contains one or more intact propagules of an aquatic organism. One-third of these propagules is also viable and can thus potentially establish in a new habitat after transport. The literature-extracted data is used to describe a model with which the number of propagules carried by birds can be calculated between any wetlands of interest. This model indicates that the average bird disperses five viable propagules after flying more than 100 km, and one additional propagule after flying the subsequent 200 km. The wide variety of aquatic plants and macro-invertebrates that can be dispersed can be explained by the fact that birds maximize their energy intake over time, rather than their digestive efficiency itself. The associated relatively low digestive efficiency of ~70% provides an opportunity for many small species to be dispersed by endozoochory. The suggestion that many aquatic species are able to use this digestive trade-off for their dispersal is further supported by our finding that aquatic snails (Gastropo- da) can pass the guts of birds alive (Chapter 3). One of four snail species that we fed to mallards (Anas platyrhynchos), i.e. Hydrobia (Peringia) ulvae (Hydrobiidae), can survive up to five hours in the digestive tract of mallards. This resembles a maximum potential bird-assisted dispersal distance in excess of 300 kilometres and indicates that endozoochory may indeed be a widespread mode of dispersal. Although aquatic snails are adapted to survive in a variety of environmental circumstances and ex-

161 Summary treme conditions, they are not known to be specifically adapted for endozoochorous transport by birds. This further supports the view that bird-mediated dispersal may be possible for a wide variety of species, including those lacking specific adaptations for endozoochory. A common problem of experiments performed to estimate potential endozoochorous dispersal distances is that they are conducted on resting animals, whereas animal vectors in natural situations will always be actively moving during effective transport of propagules. The effect of physical activity of vectors on the release pattern and dispersal efficiency of propagules is addressed in Chapter 4.Diges- tive characteristics between mallards that are swimming, wading (i.e. resting in water) and resting in a cage were compared. Retention times of markers appear to be shortened by approximately one hour (to five hours and 20 minutes) in the swimming compared to the resting mallards, and by half an hour (to six hours) in wading birds. This implies that active birds release propagules at shorter dispersal distances than previously inferred from experiments with resting birds and mammals in both aquatic and terrestrial systems. Faster retrieval of propagules indicates shorter dispersal distances, but also higher propagule survival because viability of propagules decreases exponentially with increasing retention time. In this experiment we also vary marker size, which reveals that smaller markers were retrieved faster than larger markers. This indicates smaller propagules may be dispersed over shorter distances but have higher survival potential. Whereas our experiments and meta-analysis show that aquatic species have the potential to be dispersed internally by endozoochory, they may also be carried externally on birds. Our complementary experiments that we describe in Chapter 5 indicate that aquatic snails have many of the necessary prerequisites for successful transport by external adhesion to birds. They readily attach to birds and can remain attached to the bills of mallards in drying mud for up to eight hours. All snail species that we tested survived aerial exposure over 48 hours, which would be sufficient for dispersal over long distances. The experiments indicate that snails have ample potential for bird-mediated dispersal, and that ectozoochory can be another important mechanism explaining the wide distributions of many aquatic species. However, further assessment of the capacity of snails (and other propagules) to stay attached to birds during flight remains an interesting future challenge. Although experiments provide valuable information on the potential of snails and other propagules to be transported by waterbirds, these can not confirm that actual dispersal also occurs in field situations and has consequences at the metapopulation level. We therefore also investigate the dispersal of aquatic snails by waterbirds using molecular genetic techniques. The genetic population structure of two focal species, i.e. Physa acuta (Physidae) and Galba truncatula (Lymnaeidae), is analysed with regard to dispersal. Small scale dispersal of snails (tens of kilometres) is investigated with Physa acuta, an invasive species in Europe that originates from North America. This species is found to occur in many isolated, temporary ponds in Doñana National Park, Southern Spain. Microsatellite markers indicate the contribution of multiple dispersal vectors to the distribution of this species (Chapter 6).

162 Summary

Genetic analyses supported the idea that P. acuta exploits water connections, birds and also large mammals as vectors in this system. This highlights how multiple dispersal vectors are together important for the overall wide distribution of this species and its success as an invasive species. Whether bird-mediated dispersal also has the potential to affect species distributions on the scale of thousands of kilometres is addressed by analysing the distribution and phylogenetic position of Galba truncatula in Chapter 7. This snail is an important vector for livestock and human parasites and can be found on the Eurasian, African, North- and South-American continents. Analysing its distribution in detail reveals its presence on at least 14 islands and 14 highland areas. This distribution has many similarities with the major migratory flyways of (water)birds, suggesting birds may also be vectors for this species. The phylogenetic position of G. truncatula suggests that it has an American origin and that birds may have introduced this species into Europe after the separation of the Eurasian and American continents. Birds may have carried this snail and its associated parasites over thousands of kilometres in the past and may continue to be important for its dispersal. In conclusion, the research in the thesis indicates that many aquatic plants and invertebrates can be dispersed by waterbirds internally and a series of aquatic snail species have at least the necessary prerequisites for external transport. This dispersal may not only affect the occurrence of species on a local scale, but can also influence their worldwide distribution. Bird-mediated dispersal significantly contributes to species’ survival, to the biodiversity of wetlands and can help small aquatic organisms to cope with a changing environment. In the synthesis of this thesis, Chapter 8, this knowledge on bird-mediated dispersal is compared to dispersal by other potential vectors in aquatic systems. Waterbirds are quantitatively less important than transport by wind and water, but far more effective when it comes to transporting species between suitable habitats. Birds readily fly across major ecological barriers such as land or oceans, traveling fast and directed between similar wetlands in large numbers. The fact that they importantly contribute to sustaining a wide variety of other aquatic species in wetlands around the globe once more indicates that care should be taken to protect these important players in aquatic ecosystems.

163 164 Nederlandse samenvatting

Samenvatting Het vermogen van soorten om zich in het landschap te verplaatsen is voor hun voortbestaan op de lange termijn essentieel. Door de continue dynamiek die in de meeste habitats op aarde plaatsvindt, is het noodzakelijk dat een soort zich óf aan- past óf steeds opnieuw verplaatst binnen en naar geschikte gebieden. De meeste planten en dieren zijn hiertoe goed in staat. Veel soorten kunnen natuurlijke bar- rières overbruggen door middel van eigen voortbeweging. Indien een dergelijke ad- aptatie ontbreekt maken ze voor hun verplaatsing gebruik van vectoren als wind, water of dieren. Voor aquatische organismen, waarvan ook wel gesteld wordt dat ze op “eilanden in een zee van land” leven, is het land tussen watergebieden de belangrijkste barrière voor verdere verspreiding. Omdat veel aquatische soorten het vermogen missen om zichzelf efficiënt over land voort te bewegen, is onderzoek naar passieve verspreiding van deze soorten door vectoren tussen watergebieden interessant en belangrijk. Het onderwerp van dit proefschrift is geïnspireerd door Darwin, die 150 jaar geleden als eerste suggereerde dat watervogels mogelijk belangrijke verspreidings- vectoren voor aquatische plantenzaden en kleine organismen zijn. Watervogels vliegen snel en in grote aantallen tussen vergelijkbare watergebieden, en zouden daarbij kleinere organismen kunnen meenemen. Verspreidbare, levensvatbare delen van planten of dieren, collectief propagules geheten, kunnen, na opname in het spijsverteringsstelsel van vogels worden meegenomen (intern transport of endozo- öchorie), of gehecht aan hun snavels, poten of tussen hun veren (extern transport of ectozoöchorie). Dit proefschrift begint met een meta-analyse van intern transport, gevolgd door experimenten die intern en extern transport van met name zoetwaterslakken onder de loep nemen. Vervolgens worden genetische analyses gebruikt om de effecten van verspreiding door vogels op populatieniveau vast te stellen. De meta-analyse, beschreven in Hoofdstuk 2, geeft een overzicht van alle pub- licaties in de literatuur over intern transport van macro-evertebraten en zaden van zoetwaterplanten door watervogels. Deze analyse laat zien dat ten minste 17 soorten Anatidae (eenden, ganzen en zwanen) en Rallidae (rallen, koeten en waterhoentjes) ten minste 39 soorten macro-evertebraten en 97 soorten plantenzaden via hun spijsverteringsstelsel verspreiden. Gemiddeld bevat eenderde van alle uitwerpselen van watervogels één, maar vaak ook meerdere, intacte propagules. Eenderde van deze propagules is daadwerkelijk levensvatbaar en kan zich dus na transport mogelijk in een nieuw gebied vestigen. Door de literatuurgegevens samen te voegen hebben we een model kunnen maken waarmee berekend kan worden hoeveel propagules door vogels tussen watergebieden worden verspreidt. Dit model geeft aan dat een vogel gemiddeld wel vijf levensvatbare propagules verspreidt gedurende een vlucht van 100 kilometer, en nog één propagule gedurende de daaropvolgende 200 km. Dit kan tot grote verspreidingsafstanden leiden als vogels lange rechtlijnige vluchten maken, zoals tijdens de trek. We beschouwen dit hoge aantal propagules en het wijde taxonomische scala aan soorten die door intern transport verspreid kunnen worden als een gevolg van de verteringsinefficiëntie van vogels. Als vogels hun energieop- name in de tijd maximaliseren in plaats van de verteringsefficiëntie zelf, dan kunnen

165 Nederlandse samenvatting ze door met een lagere efficiëntie verteren van meer voedsel toch een hogere ener- gie opnamesnelheid behalen in dezelfde periode. Dit verklaart waarom de gemid- delde vogel een verteringsefficiëntie van ongeveer 70 in plaats van 100 procent heeft, en waarom het voor vele kleine organismen mogelijk is door vogels verspreid te worden in hun maag-darm kanaal. Zelfs aquatische slakken (Gastropoda) kunnen a dit mechanisme een passage door het verteringsstelsel van vogels overleven. Eén van vier soorten slakken die we experimenteel aan Wilde eenden (Anas plathyrhynchos), i.e. het Wadslakje Hy- drobia (Peringia) ulvae (Hydrobiidae) hebben gevoerd, overleeft een verblijf in het maagdarmkanaal tot wel vijf uur (Hoofdstuk 3). Dit impliceert een mogelijke maximale verspreidingsafstand van meer dan 300 kilometer. Aquatische slakken zijn aan diverse omgevingsfactoren en extreme milieucondities aangepast, maar het is niet bekend of ze ook speciale adaptaties hebben voor transport door endozoöchorie. Dit geeft nogmaals aan dat transport door vogels een wijdverspreid fenomeen is en zelfs voorkomt in soorten zonder speciale adaptaties voor endozoöchorie. Endozoöchorie-experimenten waarbij men de potentiële verspreidingsafstand van propagules wil meten, worden in het algemeen met dieren in rust uitgevoerd. Onder natuurlijke condities zijn vectoren zoals vogels echter in beweging zodat ook daadwerkelijk de propagules verspreiden. Deze fysieke activiteit zou de verter- ingssnelheid en efficiëntie kunnen beïnvloeden. In Hoofdstuk 4 hebben we daarom het effect van activiteit van vectoren (Wilde eenden) op hun verteringsefficiëntie en de mogelijke verspreidingsafstand van propagules bestudeerd. We vergeleken zwemmende eenden met drijvende eenden (rustend in het water) en met eenden die op het droge rusten (vergelijkbaar met de meeste eerdere experimenten). De retentietijden van gevoerde plastic markers waren bij zwemmende eenden een uur korter dan bij op het droge rustende eenden, en bij de drijvende eenden een half uur korter dan bij op het droge rustende eenden. Dit betekent dat de afstanden waarover propagules worden verspreid, door dieren met een hoger metabolisme waarschijnlijk korter zullen zijn dan die in het algemeen met rustende dieren in kooien zijn bepaald. Dit principe geldt voor vogels maar waarschijnlijk ook voor zoogdieren, en kan gelden voor zowel aquatische als terrestrische dieren. Een kortere retentietijd bij actieve dieren impliceert niet alleen een kortere verspreidingsafstand, maar ook een hogere overleving van propagules in het spijsverteringsstelsel omdat een kortere retentietijd ook verminderde schade aan de propagules opleverd. Toen we plastic markers van verschillende grootte voerden, vonden we de kleinere sneller terug dan de grotere. Dit geeft aan dat kleinere zaden en diertjes een hogere overlevingskans hebben dan de grotere, maar ook over een kortere maximale verspreidingsafstand zullen worden vervoerd. Vogels kunnen dus vele aquatische planten en dieren via hun maag-darmkanaal verspreiden, maar er zijn er ook veel aanwijzingen dat propagules aan de buitenkant van het lichaam van vogels meeliften. Een serie van elkaar aanvullende experimenten geeft aan dat aquatische slakken fysiologisch geschikter zijn voor ectozoöchorie dan voor endozoöchorie (Hoofdstuk 5). Slakken plakken makkelijk aan veren, poten en snavels van wilde eenden, en kunnen door middel van opdrogende modder

166 Nederlandse samenvatting tot wel 8 uur aan de snavels van eenden blijven zitten. Alle slakkensoorten die we hebben getest, overleefden gemakkelijk 48 uur zonder water, ruim voldoende voor het meeliften over langere afstanden. Tezamen geven deze experimenten aan dat niet alleen intern maar ook extern transport door vogels een verklaring kan zijn voor de vaak wereldwijde verspreiding van veel slakkensoorten. Experimenten met vliegende vogels waarbij onderzocht wordt of slakken en andere propagules ook daadwerkelijk aan vliegende vogels blijven vastzitten, zijn interessante vervolgstud- ies. Hoewel experimenten belangrijke informatie leveren over de potentie van slakken en andere propagules om door vogels getransporteerd te worden, tonen deze niet direct aan dat dit in natuurlijke situaties ook daadwerkelijk gebeurt, of dat het een proces is dat op metapopulatieniveau effect heeft. Daarom hebben we de verspreiding van aquatische slakken ook met genetische technieken onderzocht. We hebben ons daarbij geconcentreerd op twee soorten: de Puntige blaashoornslak (Phy- sa acuta, Physidae) en de Leverbotslak (Galba truncatula, Lymnaeidae). Verspreiding op kleine schaal, d.w.z. van enkele tientallen kilometers, hebben we uitgezocht met de puntige blaashoornslak, een invasieve slak van Noord-Amerikaanse oorsprong. Deze soort komt voor in vele geïsoleerde, droogvallende poeltjes in Parque Nacional de Doñana, Zuid-Spanje. Microsatellietanalyses van slakken uit deze poeltjes en uit een nabijgelegen rijstveld geven aan dat slakken in dit systeem door meerdere vectoren verspreid worden (Hoofdstuk 6). De genetische analyses ondersteunen het idee dat slakken door zowel waterconnecties, vogels als grote grazende zoogdieren verspreid worden. Meerdere dispersievectoren zijn waarschijnlijk samen verant- woordelijk voor de wijde verspreiding van deze soort over de wereld, en voor zijn succes als invasieve soort. De mogelijke verspreiding van slakken door vogels op de schaal van duizenden kilometers wordt behandeld via verspreidingsgegevens en de fylogenetische positie van de Leverbotslak (Hoofdstuk 7). De Leverbotslak is een belangrijke vector voor trematoden (parasieten die o.a. de leverbotziekte bij het vee veroorzaken) en leeft thans op zowel het Euraziatische, Afrikaanse als Noord- en Zuid-Amerikaanse continent. De verspreidingsgegevens van deze soort geven aan dat hij op ten minste 14 eilanden en in 14 hooggebergtes is aangetroffen. Het vermogen van deze slak om zulke afgelegen gebieden te koloniseren, en overeenkomsten tussen haar wereldwijde verspreiding en de migratieroutes van watervogels, ondersteunen beide het idee dat vogels vectoren zijn voor deze slakken. Een fylogenetische analyse geeft aan dat de leverbotslakken in Europa een Amerikaanse oorsprong hebben, wat suggereert dat slakken zelfs na de scheiding van de Euraziatische en Amerikaanse continenten nog tussen deze continenten zijn uitgewisseld. Mogelijk hebben vogels hierbij een rol gespeeld en zowel de slakken als hun parasieten op continentale schaal verspreid. Dit proefschrift ondersteunt het idee dat vele aquatische planten en evertebraten door watervogels verspreid kunnen worden. Dit verspreidingsmechanisme heeft zowel op kleine schaal alsook grote schaal potentiele effecten. De door vogels ver- oorzaakte verspreiding van soorten draagt daarbij zowel bij aan de overleving van

167 Nederlandse samenvatting die soorten als aan de biodiversiteit in watergebieden en kan bovendien aquatische soorten helpen veranderende milieuomstandigheden zoals klimaatverandering te overleven. In het laatste hoofdstuk van dit proefschrift (Hoofdstuk 8) plaatsen we de verspreiding door vogels in een groter perspectief door deze te vergelijken met verspreiding door andere natuurlijke vectoren in aquatische systemen. Vogels verspreiden kwantitatief minder propagules dan wind en water, maar blijken als vector veel doelgerichter te zijn dan deze alternatieve mechanismen omdat de getranspor- teerde propagules een relatief grote kans hebben in geschikte watergebieden terecht te komen. Vogels vliegen snel en in grote aantallen over land, zeeën en oceanen, die anders daadwerkelijk onoverbrugbare barrières zouden vormen als de soorten alleen afhankelijk zouden zijn van andere vectoren en verbinden daarmee vergelijkbare gebieden waarin propagules een hoge kans hebben om zich te kunnen vestigen. Het feit dat vogels daarbij bijdragen aan de biodiversiteit in aquatische gebieden geeft nogmaals aan hoe belangrijk het is deze dieren te beschermen.

168 Dankwoord

Dankwoord Hier wil ik graag het laatste stukje van mijn proefschrift wijden aan de vele leuke, enthousiaste en inspirerende mensen die ik tijdens dit project heb ontmoet. Aller- eerst Marcel Klaassen, dankzij jouw scherpe analyses, snelle reacties op mijn stuk- ken en continue enthousiasme voor het uitpluizen van biologische vraagstukken heb ik altijd met veel plezier aan mijn proefschrift gewerkt. Ik kon “in je wiel hangen” bij heftige tegenwind van tijdschriften en de positieve berichten enthousiast met je delen. Jullie besluit om naar Australië te gaan heeft mij een prachtige tijd opgeleverd, met veel gastvrijheid, ochtendritten om maar vast goed kapot aan de dag te beginnen en de inspirerende club op Deakin die mij altijd bij zal blijven. Ik hoop dan ook dat we contact houden en wens jou, Richtje en de kinderen “down under” veel verder geluk! Gerard van der Velde, ontzettend bedankt voor de vele nuttige adviezen in Ni- jmegen, de dagjes Naturalis en leerzame momenten in het veld. Jouw onuitputtelijke kennis over levende systemen, nuchtere kijk op de wetenschappelijke wereld en zijn regeltjes, en brede blik over hoe mijn kleine verhaaltje in de grote wereld past zijn continu erg waardevol geweest. Ik heb ontzettend veel van je geleerd en daarvoor hartelijk dank! Jan van Groenendael, de bezoeken aan Nijmegen waarbij mijn opzet weer even onder de loep genomen werd waren belangrijke mijlpalen tijdens mijn promotietra- ject. Je hebt me duidelijk mijn eigen weg laten volgen maar wel elke keer de prior- iteiten weer even aangescherpt en de koers weer voorzien van goede adviezen. Jan, hartelijk dank voor je stabiele rol in het soms hectische leven van een AiO. Gerhard Cadée, Edmund Gittenberger, Andy Green, Hans de Kroon and Luc de Meester, thanks a lot for your time to read all the way through my thesis! Gerhard, dank voor je enthousiasme in het veld, and Andy, thanks for the many great comments on earlier drafts of my papers. I would also like to thank the Schure-Beijer- inck-Popping fonds and the Access Programme ICTS- Doñana Scientific Reserve (ICTS-RBD) for funding part of my research. Zonder directe collega’s kun je niet, en ik wil dan ook alle collega’s op het NIOO in Nieuwersluis, in Nijmegen, in Australië en later op het nieuwe NIOO in Wagen- ingen hartelijke danken voor de mooie tijd. In een verder een beetje lukrake volgor- de wil ik toch beginnen met Bert Hidding. Bert, als kamergenoot heb ik ontzettend veel van je creativiteit geleerd. We hebben altijd veel ongein met serieuze discussies weten te combineren op congressen en daarbuiten, en vele “bakkies automaten- pleur” achterovergeslagen om R-scrippies draaiend te krijgen. Het is prachtig om je als paranimf ter ondersteuning te hebben. Bart Nolet, ondanks dat je nooit officieel bij mijn project betrokken bent geweest heb ik toch vaak in je kamer staan sparren. Je georganiseerde werkstijl en nuchtere analyses zijn nog altijd een mooi voorbeeld, en ik wil je nogmaals danken voor de kans om naar Taimyr te gaan, een prachtige er- varing! Bethany Hoye (and Andrew!) thank you for the discussions on how to make our way through the canyons of PhD writing. It was great that you were around. Anne Steenbergh, dank voor de verfrissende lunchwandelingen, lol in de feestcom- missie en altijd relativerende kijk op het leven als AiO. Daan Gerla, prachtig hoe we het zelfs ergens bovenaan de Niagara Falls of in de kroeg nog over ecologie hebben.

169 Dankwoord

Jacintha van Dijk en Sjoerd Duijns, dank voor jullie enthousiasme, aanstekelijke doorzettingsvermogen en medeleven bij rejections. Andrea Koelzsch, thanks for being an inspiring roommate! Abel Gyimesi, je was een mooi voorbeeld! Alena Gsell, Suzanne Wilkens, Liesbeth Vissers, thanks for sharing the burden of being a PhD student, and good luck on your defenses! Liesbeth Bakker, dank voor de inspirerende discussies. Ellen van Donk, bedankt voor het opnemen van mij als vreemde eend in je aquatische groep. Huub op den Kamp en Joop Ouborg, dank voor het vertrouwen dat werken aan slakjes zelfs iets op zou kunnen leveren voor moleculaire en microbiële wetenschappers. Theo van Alen en Niels Wagemaker, hartelijk dank het delen van jullie onuitputtelijke genetische laboratorium kennis en gezelligheid. John “crazy Irish” McEvoy, Palestina Guevara Fiore, Bibiana Andrea Rojas Zulu- aga, John and Lorna Endler, David and Marjo Roshier, Jutta Leyrer, Laura Kelley, BriAnne Addison, Ben and Chlöe Knott, Bill Buttemer, Kate Buchanan, Raoul Ribot, Tim Sanders and all the other people from Deakin, thanks so much for the inspi- rational talks, hospitality, dinners, surfing, biking and squashing! Craig Sherman, special thanks for teaching me how to analyse my genetic data, and “teaching me a lesson” of squash. I had a great time in Australia and that was largely because of all of you, so I wish you all the best in your scientific and non-scientific lives. From Seville I would like to thank all the helpful people from Donaña Biological Station that allowed us to work there, with special thanks for Jordi Figuerola for the inspiring cooperation and interest, Begoña Arrizabalaga and Rosa Rodriguez Manzano for all the paperwork and Diego Fernando López Bañez and Juan Miguel Arroyo Salas for the fun in the field. Wim Kuijper, hartelijk dank voor het vinden van zo’n mooie plek met Potamopyrgus. Wat moet een instituut zonder de stille krachten achter alle bergen werk, dank aan alle assistenten en zeker niet te vergeten de ondersteunende diensten zoals Dick Kroon die vele gekke apparaten tevoorschijn wist te toveren voor experimenten, dank! Naomi Huig, dank voor de gezelligheid in het lab en veld, het veldwerk in Donaña en het altijd klaarstaan om mijn gekke experimenten uit te voeren. Bart van Lith, zonder jouw had ik nooit de experimenten met de eenden kunnen doen. Dank voor het altijd meedenken en de vele handigheidjes! Peter de Vries, Thijs de Boer en Koos Swart, al direct toen ik binnenkwam als AiO werd ik ondergedompeld in de wondere wereld van het Lauwersmeer. Het ballenkoort, jaws en de pizzeria zijn elementen die een jonge AiO meteen met zijn beide pootjes in de modder zet. Dank voor het prachtige (veld)werk samen! Harry Korthals, dank voor de leerzame tijd achter de IRMS! Roos Keijzer, dank voor je hulp bij de DNA analyses. Alle studenten van de Aquatische Ecologie cursus, allemaal hartstikke bedankt voor jullie bereidwilligheid om slakjes (huh, slakjes???) te zoeken in het veld en vervolgens gekke proefjes mee te doen. Marthe Tollenaar, als masterstudent hadje meteen veel inbreng en hebben we toch samen die stroomgoot voor elkaar gebokst. Hartstikke bedankt voor je kritische momenten en inzet tijdens lange dagen in He- teren. Karen Orie-Vreugdenhil, als doorzetter heb je een prachtig begin gemaakt met de externe transport experimenten waar we op doorgeborduurd hebben om toch een mooi stuk van te kunnen publiceren, hartstikke bedankt! Erik Kleyheeg, ik geef het stokje vol vertrouwen aan je door en weet zeker dat dit veel interessants op gaat

170 Dankwoord leveren. Jim de Fouw, Roeland Bom en alles andere mede-reisgenoten naar Taimyr, dank voor de prachtige tijd in Siberië! Naast al die collega’s zijn er ook mensen die mij maar een gekke bioloog vinden, maar die zijn toch niet onbelangrijk. Dirk Volman, thanks voor de ontspannende Colberts-met-carbonara’s, het relativeren onder een bakkie koffie en je kritische de- signeroog op mijn thesis. Homme Rodenhuis en Marije Bakker, Laurens en Pauline Mondelaers, Camiel van Lenteren en Jeroen van Vliet, de fiets heeft mij dankzij jullie een heleboel frisse wind door m’n kop opgeleverd en ik hoop dat die dat nog veel langer blijft doen samen met jullie. Dit sportieve gedeelte gecombineerd met bbq’s, pasta’s, gewoon een avondje ouwehoeren en fietsvakanties is nog steeds zeer gesla- agd en een welkome ontspanning voor de altijd maar doordraaiende kransjes in het hoofd van een AiO. Ik hoop in de toekomst weer wat meer tijd voor jullie te hebben! Mattijs Volman en Pauline Sep, dank voor de pannenkoek-avondjes en gezelligheid, ik hoop dat die nog lang zo blijven doorgaan. Sven Brouns, dank voor het niet gek worden van mijn eeuwige planningsproblemen voor een weekje wintersporten of weekendje catten. Ik hoop dat het in de toekomst nog vaak gaat lukken om dit soort ongein in te plannen! Ada Kool en Joost van Gijn, Sita ter Haar en Chris Klink, en alle andere mede-studenten uit Utrecht, dank voor de gezelligheid! Familie, ooms, tantes, neven en nichten en hun “significant others”, dank voor de intresses en gezelligheid! Und alle Leute aus Tübingen, allen vielen Dank für die schöne Zeit zusammen. Janina, Alejandro, Christoph, Nicolas, Sofie und Anselm, Moni und Tom, Steffen, Caro, Cathi und Paul, und vielen anderen, vielen Dank für die viele gemütliche Abenden zusammen. Und allen Mitbewohnern der Heinlenstrasse 16, Bird, Gerd, Klaus, David, Miri, Lena und Dom: Vielen Dank, dass ich als WG-Mitglied immer willkommen war! Afsluitend, hopend dat ik niemand vergeten ben in deze laatste fase met dead- lines alom, nog een paar laatste niet onbelangrijke woorden. Andries en Jeannette, dank voor jullie enthousiasme, rustgevende boottochtjes en begrip voor zo’n gek biologenvriendje. Jan-Date, dank voor je altijd luisterend en begrijpend oor, en nog steeds heel mooi om je hier in dit lijstje te zetten! Tes, ik vind het prachtig om wederzijds paranimfen te zijn en zo mooi van elkaar te snappen hoe het wereldje werkt, en niet werkt. Je bent en blijft een mooi voorbeeld voor je kleine broertje, dank daarvoor! En dan de misschien wel twee belangrijkste elementen voor een AiO, de stabiliteit in Odijk. Pa en ma, zonder jullie eeuwige steun, nuchtere begrijpen, meedenken, inspiratie, me vrijlaten in wat ik wil doen en doorlopende vertrouwen in mij zou dit boekje er zo niet liggen. Ik zou graag meer doen dan jullie simpelweg bedanken, maar denk dat jullie wel in kunnen schatten wat er allemaal achter zit als ik zeg, “bedankt”! Marlijn, wat hebben we al veel prachtige plekken op deze aardbol gezien samen, elkaar naar gekke dingen meegesleurd en wat kijk ik er naar uit om nog veel meer met jou te beleven! Ik weet al niet beter meer dan dat je er bent ter ondersteuning en ontspanning, advies en inspiratie, maar toch wil ik er hier in dit boekje even bij stil staan hoe speciaal dat eigenlijk is. Dank voor dat je er altijd voor me bent, en vooral, voor wie je bent!

171 172 Affiliations of co-authors Theo van Alen Microbiology, Institute for Water and Wetland Research (IWWR), Radboud University Nijmegen, Nijmegen, The Netherlands

Jordi Figuerola Department of Wetland Ecology, Estación Biológica de Doñana, CSIC, Seville, Spain

Jan M. van Groenendael Aquatic Ecology and Environmental Biology, Institute for Water and Wetland Research (IWWR), Radboud University Nijmegen, Nijmegen, The Netherlands

Johannes H. P. Hackstein Microbiology, Institute for Water and Wetland Research (IWWR), Radboud University Nijmegen, Nijmegen, The Netherlands

Naomi Huig Aquatic Ecology, Netherlands Institute of Ecology, Wageningen, The Netherlands

Marcel Klaassen Centre for Integrative Ecology, Deakin University, Geelong, VIC, Australia / Animal Ecology, Netherlands Institute of Ecology, Wageningen, The Netherlands

Bart van Lith Animal Ecology, Netherlands Institute of Ecology, Wageningen, The Netherlands

Rose Sablon Royal Belgium Institute of natural Science – Invertebrates, Brussels, Belgium

Craig D. H. Sherman Centre for Integrative Ecology, Deakin University, Geelong, VIC, Australia

Marthe L. Tollenaar Landscape Ecology, University Utrecht, The Netherlands

Gerard van der Velde Animal Ecology and Ecophysiology, Institute for Water and Wetland Research (IWWR), Radboud University Nijmegen, Nijmegen, The Netherlands / Netherlands Centre for Biodiversity Naturalis, Leiden, The Netherlands

Cornelis A.M. Wagemaker Molecular Ecology, Institute for Water and Wetland Research (IWWR), Radboud University Nijmegen, Nijmegen, The Netherlands

H. Wouter Wisselink Animal Ecology and Ecophysiology, Institute for Water and Wetland Research (IWWR), Radboud University Nijmegen, Nijmegen, The Netherlands

173 Publications

Published or accepted for publication

Van Leeuwen, C.H.A., Van der Velde, G., Van Lith, B. & Klaassen, M. (2012b) Experimental quantification of long distance dispersal potential of aquatic snails in the gut of migratory birds. PLoS ONE, 7, e32292

Van Leeuwen, C.H.A., Tollenaar, M. & Klaassen, M. (2012a) Vector activity and propagule size affect dispersal potential by vertebrates. Oecologia, doi: 10.1007/ s00442-012-2293-0

Van Leeuwen, C.H.A & G. van der Velde (2012) Prerequisites for flying snails: external transport potential of aquatic snails by waterbirds. Freshwater Science (in press)

Dietz, M.W., Spaans, B., Dekinga, A., Klaassen, M., Korthals, H., van Leeuwen, C.H.A. & Piersma, T. (2010) Do red knots (Calidris canutus islandica) routinely skip Iceland during southward migration? Condor, 112, 48-5

Klaassen, R.H.G., Nolet, B.A. & Van Leeuwen, C.H.A. (2007) Prior knowledge about spatial pattern affects patch assessment rather than movement between patches in tactile-feeding mallard. Journal of Animal Ecology, 76, 20-29

Manuscripts under revision or submitted

C. H.A. van Leeuwen, G. van der Velde, J. M. van Groenendael & M. Klaassen. Gut travellers: internal dispersal of aquatic organisms by waterfowl.

C. H.A. van Leeuwen, N. Huig, G. van der Velde, T. van Alen, C. A.M. Wagemaker, C. D.H. Sherman, M. Klaassen & J. Figuerola. How did this snail get here? Multiple dispersal vectors revealed for an aquatic invasive species.

C. H.A. van Leeuwen, G. van der Velde, H. W. Wisselink, T. van Alen, R. Sablon, J. H. P. Hackstein. A snail on four continents: bird-mediated dispersal of a parasite vector?

174 Curriculum vitae Casper Hendrik Abram van Leeuwen was born on the 18th of September 1983 in Odijk, The Netherlands. His first biological experiments took place in his parent’s backyard at the age of 7, where he followed the spatial behaviour of marked land snails. After secondary school at the Montessori Lyceum Herman Jordan in Zeist in 2001 he pursued his curiosity for plants and animals by studying Biology at Utrecht University. The first internship of his masters led him to the Department of Plant-Animal Interactions at the Netherlands Institute of Ecology. Supervised by Raymond Klaas- sen he performed his first real experiment by investigating the foraging decisions of mallards in response to spatial patterns of food (J Anim Ecol: 2007, 76: 20-29). For his second internship he delved into the world of stable isotopes at the University of Groningen with Maurine Dietz, to investigate diet choice of red knots in relation to their gizzard size (The Condor: 112(1): 48-55). For his third internship he obtained a Schure-Beijerinck-Popping grant to migrate even further north, i.e. to the Yukon Delta, Alaska. He joined Sarah Jamieson for three months on the tundra to study the egg laying physiology and mate choice of dunlin and western sandpipers during the Arctic summer, followed by three months of data analysis at Simon Fraser Univer- sity, Vancouver, Canada. Upon his return to the Netherlands in 2007, he obtained his MSc degree and immediately moved on to do his PhD with the Centre for Wetland Ecology. Two of the partners within this centre formed his host institutes, i.e. the Netherlands Institute of Ecology (NIOO) and Radboud University Nijmegen. Within this project he investigated the dispersal of aquatic organisms by waterbirds with a focus on dispersal of aquatic snails. During his PhD project he supervised many MSc students, and integrated his experiments with the undergraduate course “Aquatic Ecology” to mutual benefit. Also during his PhD he was drawn abroad. With funding from Ministerio de photo Vroege Vogels Ciencia e Innovación and Estación Bi- ológica de Doñana he conducted field- work in Spain. In 2008 he joined organis- ers Bart Nolet (NIOO) and Bart Ebbinge (Alterra) on their BrentArctic expedition to Taimyr Peninsula (Russia), studying brent geese and shorebirds. After finish- ing the practical work for his PhD, he joined Marcel Klaassen at Deakin Univer- sity in Geelong (Australia) for six months to write on his thesis.

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