Sexual reproduction in chydorid cladocerans (, ) in southern Finland – implications for paleolimnology Liisa Nevalainen

Academic dissertation

To be presented, with the permission of the Faculty of Science of the University of Helsinki, for public criticism in lecture room E204 of Physicum, Kumpula, on November 14th, 2008 at 12 o’clock

Publications of the Department of Geology D16 Helsinki 2008 Ph.D. thesis No. 203 of the Department of Geology, University of Helsinki

Supervised by: Dr. Kaarina Sarmaja-Korjonen Department of Geology University of Helsinki Finland

Reviewed by: Prof. Krystyna Szeroczyńska Institute of Geological Sciences Polish Academy of Sciences Poland

Dr. Milla Rautio Department of Environmental Science University of Jyväskylä Finland

Discussed with: Dr. Marina Manca The National Research Council (CNR) Institute of Ecosystem Study Italy

Cover: morning mist on Lake Hampträsk

ISSN 1795-3499 ISBN 978-952-10-4271-3 (paperback) ISBN 978-952-10-4272-0 (PDF) http://ethesis.helsinki.fi/

Helsinki 2008 Yliopistopaino Liisa Nevalainen: Sexual reproduction in chydorid cladocerans (Anomopoda, Chydoridae) in southern Finland - implication for paleolimnology, University of Helsinki, 2008, 54 pp. + appendix, University of Helsinki, Publications of the Department of Geology D16, ISSN 1795-3499, ISBN 978-952-10-4271-3 (paperback), ISBN 978-952-10-4272-0 (PDF).

Abstract

The relationship between sexual reproduction of littoral chydorid cladocerans (Anomopoda, Chydoridae) and environmental factors in aquatic ecosystems has been rarely studied, although the sexual behavior of some planktonic cladocerans is well documented. Ecological monitoring was used to study the relationship between climate-related and non-climatic environmental factors and chydorid sexual reproduction patterns in nine environmentally different lakes that were closely situated to each other in southern Finland. Furthermore, paleolimnological ephippium analysis was used to clarify how current sexual reproduction is reflected in surface sediments of the same nine lakes. Additionally, short sediment cores from two of the lakes were studied with ephippium analysis to examine how recent climate-related and non-climatic environmental changes were reflected in chydorid sexual reproduction. Ephippium analysis uses the subfossil shells of asexual individuals to represent asexual reproduction and the shells of sexual females, i.e. , to represent sexual reproduction. The relative proportion of ephippia of all chydorid species, i.e. total chydorid ephippia (TCE) indicates the relative proportion of sexual reproduction during the open-water season. This thesis is part of the EPHIPPIUM-project which aims to develop ephippium analysis towards a quantitative climate reconstruction tool. To be able to develop a valid climate model, the influence of the environmental stressors other than climate on contemporary sexual reproduction and its reflection in sediment assemblages must be clarified so they can be eliminated from the model. During contemporary monitoring a few sexual individuals were observed during summer, apparently forced to sexual reproduction by non-climatic local environmental factors, such as crowding or invertebrate predation. Monitoring also revealed that the autumnal chydorid sexual reproduction period was consistent between the different lakes and climate-related factors appeared to act as the main inducers and regulators of autumnal sexual reproduction. However, during autumn, chydorid species and populations among the lakes exhibited a wide variation in the intensity, induction time, and length of autumnal sexual reproduction. These variations apparently act as mechanisms for local adaptations due to the genetic variability provided by sexual reproduction that enhance the ecological flexibility of chydorid species, allowing them to inhabit a wide range of environments. A large variation was also detected in the abundance of parthenogenetic and gamogenetic individuals during the open-water season among the lakes. On the basis of surface sediment samples, the general level of the TCE is ca. 3-4% in southern Finland, reflecting an average proportion of sexual reproduction in this specific climate. The variation in the TCE was much lower than could be expected on the basis of the monitoring results. This suggests that some of the variation detected by monitoring may derive from differences between sampling sites and years smoothed out in the sediment samples, providing an average of the entire lake area and several years. The TCE is always connected to various ecological interactions in lake ecosystems and therefore is always lake-specific. Hypothetically, deterioration of climate conditions can be detected in the TCE as an increase in ephippia of all chydorid species, since a shortening open-water season is reflected in the relative proportions of the two reproduction modes. Such an increase was clearly detected for the time period of the Little Ice Age in a sediment core. The paleolimnological results also indicated that TCE can suddenly increase due to ephippia of one or two species, which suggests that at least some chydorids can somehow increase the production of resting eggs under local environmental stress. Thus, some environmental factors may act as species-specific environmental stressors. The actual mechanism of the “increased” sexual reproduction seen in sediments has been unknown but the present study suggests that the mechanism is probably the increased intensity of gamogenesis, i.e. that a larger proportion of individuals in autumnal populations reproduce sexually, which results in a larger proportion of ephippia in sediments and a higher TCE. The results of this thesis demonstrate the utility of ephippium analysis as a paleoclimatological method which may also detect paleolimnological changes by identifying species-specific environmental stressors. For a quantitative TCE-based climate reconstruction model, the natural variation in the TCE of surface sediments in different climates must be clarified with more extensive studies. In addition, it is important to recognize the lakes where the TCE is not only a reflection of the length of the open-water season, but is also non-climatically forced. The results of ephippium analysis should always be interpreted in a lake-specific manner and in the context of other paleoecological proxies. Contents

List of publications Author’s contribution to the publications 1. Introduction 8 1.1. Biology of cladocerans 8 1.2. Cladoceran reproduction 12 1.3. Cladocerans in paleolimnology 15 1.4. Ephippium analysis 17 1.5. Objectives of this thesis 19 2. Material and Methods 20 2.1. Sites 20 2.2. Sampling and sample analyses 23 2.2.1. Contemporary samples 23 2.2.2. Sediment samples 24 2.3. Data analysis 25 3. Results and Discussion 25 3.1. Chydorid distribution 25 3.1.1. Species occurrence in sweep net and surface sediment samples 25 3.1.2. First findings of intact Unapertura latens sp. n. 28 3.2. Monitoring of modern sexual reproduction 29 3.2.1. Timing of gamogenesis (I) 29 3.2.2. Intensity of autumnal gamogenesis (II) 31 3.2.3. Population sizes and abundance of gamogenesis (III) 35 3.3. Sexual reproduction reflected in sediments 37 3.3.1. Chydorid ephippia in the Hampträsk core (IV) 37 3.3.2. Chydorid ephippia in surface sediments (V) 38 3.3.3. Chydorid ephippia in the Pieni Majaslampi core (V) 40 4. Conclusions 41 Acknowledgements References Appendix Publications I-V

 List of publications

This thesis is based on the following papers, which in the text are referred to by Roman numerals:

I Nevalainen, L. and Sarmaja-Korjonen, K. 2008. Timing of sexual reproduction in chydorid cladocerans (Anomopoda, Chydoridae) from nine lakes in southern Finland. Estonian Journal of Ecology 57, 21-36.

II Nevalainen, L. and Sarmaja-Korjonen, K. 2008. Intensity of autumnal gamogenesis in chydorid (, Chydoridae) communities in southern Finland, with a focus on Alonella nana (Baird). Aquatic Ecology 42, 151-163.

III Nevalainen, L. 2008. Parthenogenesis and gamogenesis in seasonal succession of chydorids (Crustacea, Chydoridae) in three low-productive lakes as observed with activity traps. Polish Journal of Ecology 56, 85-97.

IV Luoto, T.P., Nevalainen, L. and Sarmaja-Korjonen, K. 2008. Multiproxy evidence for the ‘Little Ice Age’ from Lake Hampträsk, Southern Finland. Journal of Paleolimnology 40, 1097-1113.

V Sarmaja-Korjonen, K., Nevalainen L. and Gąsiorowski, M. Chydorid ephippia in surface sediments and a short core from southern Finland – evidence of increased sexual reproduction caused by environmental forcing. Submitted to Journal of Paleolimnology.

In addition to the original papers, this thesis includes previously unpublished material analyzed by the author.

 Author’s contribution to the publications

I K. Sarmaja-Korjonen planned the study, L. Nevalainen performed the sampling and the sample and data analyses, L. Nevalainen and K. Sarmaja-Korjonen interpreted the results and prepared the manuscript

II L. Nevalainen and K. Sarmaja-Korjonen planned the study, L. Nevalainen performed the sampling and the sample and data analyses, L. Nevalainen and K. Sarmaja-Korjonen interpreted the results and prepared the manuscript

III L. Nevalainen planned the study, performed the sampling, the sample and data analyses, interpreted the results and prepared the manuscript

IV K. Sarmaja-Korjonen, T.P. Luoto and L. Nevalainen planned the study, L. Nevalainen and T.P. Luoto performed the sampling, T.P. Luoto did the chironomid and data analyses, L. Nevalainen did the Cladocera and ephippium analyses, T.P. Luoto, L. Nevalainen and K. Sarmaja-Korjonen interpreted the results and prepared the manuscript

V K. Sarmaja-Korjonen and L. Nevalainen planned the study, L. Nevalainen did the fieldwork and Cladocera and ephippium analyses, M. Gąsiorowski performed the 210Pb activity and LOI analyses, K. Sarmaja-Korjonen, L. Nevalainen and M. Gąsiorowski interpreted the results and prepared the manuscript

 1. Introduction 1.1. Biology of cladocerans

Cladocerans, commonly referred to as water fleas, are microscopic (ca. 200-1800 µm) inhabiting significantly a wide variety of different aquatic habitats. Their morphology is rather primitive (Fig. 1), with a body covered by a two-valved chitinous carapace protecting the inner, soft tissues. The four to six pairs of thoracic legs, i.e. trunk limbs, are located under the carapace. The trunk limbs move rhythmically back and forth and cause food particles to drift towards the mouth. The head is covered by a chitinous headshield and it ends in the rostrum. The two eyes in the head, the compaund eye and the ocellus eye, both sense light. The mouth is situated between the carapace and headshield and consists of mandibles and a labrum. The first antennae, antennules, mainly act as sensory organs, and the second antennae as swimming organs. The body ends with a postabdomen and two postabdominal claws. The postabdomen helps in movement and food scraping in some benthic species. Cladocerans, being crustaceans, can grow only by molting their exoskeleton during certain periods (Frey 1988a; Dodson and Frey 1991). The use of the word Cladocera to indicate a taxonomically valid group of has been under discussion for the past two decades and it has been suggested that it is an artificial grouping (e.g. Dodson and Frey 1991; Dumont 1997; Korovchinsky 1996, 2000). However, based on recent molecular evidence, cladocerans are considered to be a monophyletic group of crustaceans representing the order Cladocera (e.g. Crease and Taylor 1998). The order can be divided into four suborders: Haplopoda, , Anomopoda and . The systematics for the families varies, but usually the order is separated into 11 families, including e.g. , , , , Chydoridae, , and Holopedidae, and the families more than 80 genera in total (e.g. Flössner 1972, 2000; Røen 1995; Table 1). Europe has a long tradition of cladoceran research, beginning in the 18th century, and most chydorids (species of the family Chydoridae) were described as early as the late 19th century. At the end of the 20th century, however, it became clear that a lot of work on the of chydorids is required to clarify their classification outside Europe (reviewed by Frey 1995). Chydorids are now recognized as a monophyletic group, with four distinct subfamilies: Eurycercinae, Saycinae, Aloninae, and Chydorinae (Sacherová and Hebert 2003). The taxonomy of the family is presently undergoing great changes, and this is reflected by the state of the taxonomical research; new species and genera are being described around the world and old species are being redescribed and relocated among genera (e.g. Sinev 1997, 1998, 1999, 2001, 2004a, 2004b; Dumont and Silva-Briano 2000; Sinev and Kotov 2000, 2001; Van Damme et al. 2003; Smirnov 2007). Therefore, it is probable that the number of species in the family will grow further in the near future as this work continues. Although zooplankton has been actively investigated in Finland, studies on living chydorids are rare and thus their modern ecology, such as reproductive behavior and distribution around the country,

 DORSAL

head shield carapace

compound

eye ANTERIOR ocellus

POSTERIOR rostrum

trunk limbs 1. antenna labrum postabdomen postabdominal 2. antenna claw

VENTRAL Figure 1. An intact cladoceran, harpae of the family Chydoridae showing the main morphological features of most cladocerans. The picture is taken by the author. is relatively poorly documented. For example, Stenroos (1895, 1897, 1898) explored the occurrence of chydorid species in southern Finland and Karelia, and during the 20th century the studies of Järnefelt (1915, 1956) in southern Finland provided observations on chydorids as well. In addition, Purasjoki (1981) and Uimonen (1985) presented zoological and ecological observation on chydorids from south central Finland. The latest contemporary ecological studies on cladocerans, including chydorids, in Finland were performed by Rautio (1998, 2001), who examined zooplankton communities in the Kilpisjärvi region, northwestern Finnish Lapland. Although straight biogeographical studies of chydorids are lacking in Finland, it is probable that the species occurrence is quite similar to that of other nearby regions, e.g. Estonia, Denmark, and Norway (Mäemets 1961; Røen 1995; Walseng et al. 2006). Cladocerans form an important part of the freshwater microfauna, inhabiting all types of waters, although they are not a homogenous group based on their habitats and feeding ecology. A small minority of the taxa inhabit marine environments. Planktonic cladocerans, including the most commonly-known genus Daphnia (Daphniidae), live in the pelagic zone of lakes and are grazers (Table 1), consuming mainly planktonic algae, bacteria and detritus. Only a few planktonic species are predators (Røen 1995; Korhola and Rautio 2001). Some of the most important environmental factors affecting the diversity and occurrence of planktonic cladocerans are known to be temperature (George and Harris 1985; Stemberger et al. 1996; Gillooly and Dodson 2000), lake morphometry (Fryer 1985; Keller and Conlon 1994), solar UV-radiation (Zellmer 1998; Rautio and Korhola 2001, 2002), acidity (Nilssen and Sandøy 1990; Sarvala and Halsinaho 1990), and predation (Brooks and Dodson 1965; Dodson 1974; Kerfoot 1977; Lynch 1980). Littoral zones of lakes are mainly dominated by the families Chydoridae and Macrothricidae (Table

 Table 1. The most common genera of cladoceran families and their ecology based on Flössner (2000) and Røen (1995).

Family Genus Lifeform Habitat Leptodoridae plankton open water Sididae Sida littoral vegetation Limnosida plankton open water Diaphanosoma plankton open water Latona littoral bottom Holopedidae plankton open water Daphniidae Ceriodaphnia plankton open water / vegetation zone Scapholeberis neuston under surface film of water Daphnia plankton open water Simocephalus littoral vegetation / bottom Moinidae Moina plankton open water Bosminidae Bosmina plankton open water / vegetation zone Macrothricidae Ilyocryptus littoral bottom Lathonura littoral vegetation Acantholeberis littoral bottom Drephanothrix littoral bottom Streblocerus littoral vegetation Macrothrix littoral bottom Chydoridae Eyrycercus littoral vegetation Camptocercus littoral vegetation Acroperus littoral vegetation Alonopsis littoral vegetation / bottom Tretocephala littoral vegetation / bottom Kurzia littoral vegetation / bottom Oxyurella littoral vegetation littoral vegetation / bottom Leydigia littoral mud bottom Graptoleberis littoral vegetation Rhynchotalona littoral sand / rock bottom Monospilus littoral mud / sand bottom Alonella littoral vegetation / bottom Disparalona littoral bottom Pleuroxus littoral vegetation / bottom Chydorus littoral vegetation / bottom / open water Pseudochydorus littoral bottom Anchistropus littoral vegetation zone Polyphemidae plankton open water Podon plankton marine open water Evadne plankton open water Cercopagidae Bythotrephes plankton deep open water

1). Among chydorids some species live on littoral submerged vegetation, whereas others are found on mud, sand and rocky substrata (e.g. Goulden 1971; Whiteside et al. 1978; Duigan and Kovach 1991), and some inhabit even the profundal zone (Mäemets 1961, Røen 1995). Chydorids are an important

10 part of the littoral food web, consuming periphytic algae and detritus as grazers and being prey for larger invertebrate and vertebrate aquatic predators. It has been shown in many studies that chydorids are sensitive to many physical, chemical and ecological factors in their living environment and that the community structure of these animals varies in different environmental conditions (e.g. Whiteside 1970; de Eyto et al. 2002, 2003). Regardless of the fact that chydorids appear to be rather sensitive to some environmental conditions, the same species usually inhabit a wide range of lakes, but differ in their abundances (e.g. Mäemets 1961; Korhola 1999; Sweetman and Smol 2006). This suggests that they are ecologically rather flexible and that the major environmental requirements for many species are almost the same (Korhola and Rautio 2001). de Eyto (1999) emphasized the importance of abiotic factors in controlling chydorid communities, whereas biotic interactions would have importance only in regulating seasonal community structure. Besides the direct effects that temperature has on the metabolism of chydorids (Dodson and Frey 1991), it also indirectly affects their habitats, e.g. aquatic vegetation (Rautio 1998). Water transparency and lake morphometry also determine the extent of the littoral zone and consequently the abundance of aquatic vegetation, which are known to influence the diversity and occurrence of chydorids (Whiteside and Harmsworth 1967; Quade 1969; Whiteside 1970; Whiteside et al. 1978; Fryer 1985). In addition to temperature-related physical factors, the presence or absence of some benthic substrata, e.g. mud, sand or rock, may restrict the occurrence of chydorids (Williams 1982). In addition, chydorid communities have been shown to be different in lakes of differing nutrient status (Whiteside 1970; Hofmann 1996). Some chydorid species, e.g. Chydorus sphaericus, Alona rectangula, and Disparalona rostrata (Whiteside 1970; de Eyto et al. 2003; Røen 1995; Szeroczyńska 1998) are usually considered to be associated with meso-eutrophic and disturbed waters, whereas many other species prefer oligotrophic environments. The effect of lake water pH on chydorids has also been recognized in many studies (e.g. Nilssen and Sandøy 1986, 1990; Sandøy and Nilssen 1986; Uimonen-Simola and Tolonen 1987). The tolerance of different chydorid species to acidity has not been examined in detail, but there is general information on the pH preference of some species. For example, Alonella excisa is considered to thrive mostly in acidic lakes (Sandøy and Nilssen 1986; Krause-Dellin and Steinberg 1986), whereas Chydorus sphaericus is regarded as a generalist in relation to pH, because it thrives in very low and very high pH environments (Flössner 2000; Deneke 2000; Belyaeva and Deneke 2007). According to Nilssen and Sandøy (1990), in acidified lakes a pH range of 5.2-5.5 is thought to be critical for the survival of many aquatic animals, because aluminum content of the water increases as the pH decreases and aluminum is toxic. Althought the significance of pH on the physiology of aquatic animals is generally acknowledged, the effects of pH on chydorids usually appear to be indirect, relating to the structure of the food web or aquatic vegetation (Nilssen and Sandøy 1986, 1990). The biotic environment, in particular predation, is a very important factor in determining the occurrence and diversity of chydorids in lakes (Goulden 1971; Williams 1983; Robertson 1990). Littoral chydorids are food items for invertebrate and small vertebrate predators. Predacious cladocerans, copepods, insect larvae, fish fry and amphipods prey on these small and abundant animals andso the predators may affect their community structure considerably (Goulden 1971; Nilssen and Sandøy 1990). Predation in lakes is usually considered to be size selective, meaning that vertebrate predators use their eyesight to prey on larger zooplankton species, while invertebrate predators use their sensory organs to prey on smaller species or juveniles (Lynch 1980; de Bernardi et al. 1987). According to Goulden (1971), Williams (1983) and Robertson (1990), invertebrate predation may restrict the growth of chydorid summer populations.

11 1.2. Cladoceran reproduction

The cladoceran life cycle consists of active and dormant (diapause) stages, and cladocerans, together with only the rotifers and aphids, reproduce cyclically by alternating two reproduction modes: asexual (parthenogenesis) and sexual (gamogenesis) (Dodson and Frey 1991; Campbell et al. 1999). This type of reproductive strategy is called cyclical parthenogenesis (e.g. Larsson, 1990). Within this reproductive strategy, parthenogenesis is used as a biologically efficient mechanism for vigorous population growth and maintenance under favorable environmental conditions, as parthenogenetic eggs develop without fertilization and thus parthenogenetic individuals are genetically identical clones of their mothers (Hebert and Ward 1972; Frey 1982). In parthenogenesis, genetic recombination does not occur (Hebert and Ward 1972). Gamogenesis is the only way for cladocerans and other cyclic parthenogens to produce resistant forms (Rispe and Pierre 1998), and it aims to produce diapausal resting eggs when unfavourable environmental conditions prevail. According to Frey (1982) the functions of gamogenesis and resting eggs in cladocerans are primarily to provide a mechanism for re-establishment of populations after seasonal or aperiodic stresses, secondly to provide a means for passive dispersal and thirdly to generate new genotypes via meiosis and fertilization. Although Frey (1982) summarized clearly the main functions of the gamogenetic reproduction mode in cladocerans, the evolutionary and ecological significance of sexual reproduction is still puzzling, because the cost of sexual reproduction is higher than that of asexual reproduction; the male contribution to population growth is expressed only through females (Doncaster et al. 2000). The main advantage of sexual reproduction if compared to purely asexual reproduction is improved ability to adapt to changing environments due to to genetic variability (Maynard Smith 1968; Pękalski 2000) and the formation of diapausing stages in unfavorable environments (Rispe and Pierre 1998). Cladocerans most likely benefit not only from sexual reproduction, but from both reproduction modes: in parthenogenesis the fast population growth is advantageous and there are none of the costs of sex, and in gamogenesis the periodic sex increases the genetic variability in populations, improving their fitness (Lynch and Gabriel 1983; Hurst and Peck 1996). In spite of these advantages cyclical parthenogesis is very rare, and Lynch and Gabriel (1983) suggested that the rarity of this complex life cycle is caused by the extremely strict requirements for its maintenance. The role of gamogenesis and production of resting eggs is most prominant in northern climates, where most species cannot survive the harsh winter and must therefore enter diapause. The cladoceran resting eggs in sediment egg banks are extremely resistant to different environmental conditions, such as heat, cold, and drought, and have the ability to survive for decades or even longer (Frey 1982; Hairston 1996; Meijering 2003). In northern regions, such as in northern temperate and subarctic Finland, the active period of chydorids lasts usually through the open-water season (Mäemets 1961; Koksvik 1995). During most of the open-water season chydorids reproduce by parthenogenesis and therefore their populations consist of parthenogenetic females (Fig. 2). During the autumn, at the end of the active period, chydorids begin to reproduce by gamogenesis (Mäemets 1961; Flössner 1964; Green 1966; Shan 1969; Frey 1982; Koksvik 1995). Gamogenetic reproduction of chydorids starts with the appearance of diploid males (Figs. 2, 3), which develop from parthenogenetic eggs of parthenogenetic females. The structure of males generally resembles that of the females, with the exeption of the postabdomen, rostrum in some species and the copulatory hooks (Smirnov 1967). The males produce haploid sperm for fertilization. Parthenogenetic females usually alter their own reproductive mode somewhat later when becoming gamogenetic. Gamogenetic females (Figs. 2, 3) produce haploid eggs, which require fertilization by males. The copulation and fertilization produce one or two diploid resting eggs, which are enclosed and protected by a morphologically modified, thickened and pigmented carapace, the ephippium (Shan 1969; Vandekerkhove et al. 2004; Szeroczyńska and Sarmaja-Korjonen 2007). Because chydorids require

12 a) 200 µm b)

c) d)

Figure 2. a) Parthenogenetic female, b) male, c) gamogenetic female, and d) ephippium and resting egg of the chydorid Alonella nana. molting of their exoskeleton before they can grow, the rate of reproduction is dependent on the growth rate (Shan 1969). The resting eggs inside ephippia are preserved throughout the winter and enable re-establishment of populations during the following spring when environmental conditions once again become favorable. There are some optimum conditions for water temperature and photoperiod length in which the hatching of new parthenogenetic females occurs (cf. Vandekerkhove et al. 2005). Theoretically, a single resting egg is adequate to establish an entire population (Frey 1982). However, overwintering does not always occur in diapause as resting eggs, since active parthenogenetic chydorid individuals have been encountered during the winter under ice at least in Estonia (Mäemets 1961), central Norway (Koksvik 1995), the northern United States (Keen 1973) and even near Antarctica (Heywood 1967), suggesting that not all chydorid populations become dormant even in cold climate regions. However, this has not been examined in detail, and therefore there is no data on whether the overwintering of chydorids as active individuals is common in northern regions. In cold subarctic and arctic climates the open-water season is very short, lasting only 2-3 months, and chydorid populations must develop fast in order to produce resting eggs for the next winter. Thus, the length of the parthenogenetic period is restricted and gamogenesis begins early in the open-water season, during the summer months (Poulsen 1940; Kaiser 1959; Stross and Kangas 1969; Sarmaja- Korjonen 1999). According to Frey (1982) the strategy is to maximize the production of resting eggs, because there is no time to expand populations by parthenogenesis. In milder climatic conditions, such as in southern Finland in a northern temperate climate, the open-water season lasts considerably longer, being 6-7 months long, and thus there is time for populations to grow via parthenogenesis for many months. Consequently, gamogenesis occurs later in the autumn (Flössner 1964; Frey 1982; Koksvik 1995).

13 There is evidence of different timing of gamogenesis for different geographical Parthenogenetic egg Instar I regions. Poulsen (1940) showed that Chydorus sphaericus started gamogenesis PARTHENOGENESIS during July in eastern Greenland, and

Koksvik (1995) observed sexual chydorid Parthenogenetic female Instar II individuals from September onwards in central Norway. Farther south, Instar I Resting egg gamogenesis in chydorids is displaced Haploid egg towards winter months, as according Ephippial female to the results of Flössner (1964) from Sperm Germany, some chydorid species became GAMOGENESIS gamogenetic in late autumn-winter, Instar II Male but some remained parthenogenetic throughout the year. Green (1966) showed that in England only a few chydorid species Figure 3. Reproduction stages in chydorids, modified from Shan (1969). reproduced sexually during the year, with ephippial females and ephippia occurring mainly in December-February, and Sebestyén (1948) indicated that gamogenesis occurred in Lake Balaton, Hungary, from October to January. According to Frey (1982) and Cambell and Clark (1989) chydorid gamogenesis occurs during the springtime in the southern United States, triggered by the approaching hot summer. Yet farther south, in the subtropics and tropics, gamogenesis takes place irregularly and in an uncoordinated manner, because there is no need for large numbers of resting eggs to be produced for reestablishing populations (Frey 1982). Gamogenesis and production of resting eggs are extremely important phases of cladoceran life cycles, as discussed above. Perfect timing is crucial for gamogenesis. Initiating the process too early or too late may not lead to the optimal result, i.e. the optimal number of resting eggs, as Kleiven et al. (1992) suggested in their study on Daphnia. Usually chydorids are considered to be monocyclic, with one sexual reproduction period, but in some regions in northern Europe there are observations of a very weak summer sexual period in chydorids (Berg 1929; Järnefelt 1956; Mäemets 1961, Røen 1995), suggesting that some populations are dicyclic. Despite these few observations on dicyclism in chydorids, their reproduction is generally monocyclic with an autumnal sexual reproduction period. On the other hand, some daphniids (Daphniidae) are known to switch their reproduction mode very easily from parthenogenesis to gamogenesis even in summer, and are considered to be di- or polycyclic, having two or more sexual reproduction periods (Wesenberg-Lund 1937; Røen 1995). Frey (1982) stated that factors that induce and control gamogenesis are only partially understood and this statement pertains even today. The causes for the induction and forcing of gamogenesis in cladocerans have been investigated since the early 20th century (e.g. Banta 1925; Banta and Brown 1929). Based on these studies, the induction of gamogenesis was considered to be under environmental control. Later, in the 1960s, many studies showed that induction of gamogenesis and formation of diapausal resting eggs in Daphnia are mainly controlled by climatic cues such as water temperature and photoperiod length, but can also be controlled by some density-related factors, e.g. starvation (Stross and Hill, 1965, 1968; Stross 1969a, 1969b). Work with gamogenesis in Daphnia has continued and more recently it has been shown that many other stressors besides climate may easily and quickly interact in the induction of gamogenesis. The influences of food limitation, crowding and pressure from fish predation have been shownto interact in gamogenesis in Daphnia (Hobæk and Larsson 1990; Larsson 1991; Kleiven et al. 1992; Ślusarczyk 1995, 2001; Pijanowska and Stolpe 1996). Berner et al. (1991) showed with their study on Scapholeberis, another daphniid genus, that temperature and photoperiod controlled its gamogenesis

14 too, but other controlling factors, e.g. food depletion and high population density, were also required for sexual reproduction. Similar work on the effects of crowding and food depletion on gamogenesis has been performed on Moina (D’abramo 1980; Zadereev 2003; Zadereev and Lopatina 2007). These many studies about the cues inducing gamogenesis have mostly been done in laboratory settings. Less attention has been paid to the induction and control of chydorid gamogenesis. Shan and Frey (1968), Shan (1974) and Kubersky (1977) investigated chydorids in laboratory settings. The conclusion of these studies was that photoperiod, light intensity, and temperature interact in the induction of chydorid gamogenesis. In addition to these few laboratory examinations, Kubersky (1977), Frey (1982), and Campbell and Clark (1989) made observations on reproduction patterns of natural chydorid populations. Frey (1982) and Campbell and Clark (1989) suggested that environmental conditions associated with winter (e.g. low temperature) or summer (e.g. high temperature) cause physiological or ecological stress on chydorids in different geographical regions and induce gamogenesis. Kubersky (1977) on the other hand discussed the possibility of changes in water temperatures during the autumn partly inducing gamogenesis. Hobæk and Larsson (1990) stated in their study on sex determination in Daphnia that “environmental sex determination appears to be universal in the Cladocera, the determining environment being provided by the mother”. This phenomenon is called the maternal effect (Mousseau and Fox 1998). The role of maternal effects in the control of reproduction modes in Daphnia and Moina has recently gained some research attention in studies showing that information about environmental conditions, such as food and photoperiod, in which the mothers live and which can be relevant to the correct timing of gamogenesis, is transferred to offspring (LaMontagne and McCauley 2001; Alekseev and Lampert 2001). If there is a mismatch between the maternal and offspring environments, the offspring shift their reproduction to gamogenesis for survival (LaMontagne and McCauley 2001; Zadereev 2003). Therefore, if environmental control of gamogenesis in cladocerans is universal, as suggested by Hobæk and Larsson (1990), it is likely that the maternal effect also contributes to reproduction of chydorids, and some indication of this exists. Shan (1974) studied the effect of photoperiod length on gamogenesis of Pleuroxus populations from different geographic regions in the laboratory. The results indicated that information concerning the photoperiod environment of the mother had been transferred to the offspring. The laboratory stocks from different localities started to reproduce sexually in the laboratory with a photoperiod length that was typical for their original locality. Meijering (2003) hatched cladoceran resting eggs in the laboratory from arctic mud samples that had been frozen for 18 years. After hatching, the chydorid species Alona quadrangularis and Chydorus arcticus produced parthenogenetic eggs and resting eggs in a time period and cycle that was typical for arctic lakes, suggesting that information about the arctic environment of the mother had been transferred and retained in the frozen resting eggs for almost two decades. If environmental control works similarly in all or most cladoceran families, it is also likely that environmental stressors other than climate can also contribute to sexual reproduction of chydorids. Kiser et al. (1963) found in North America that after a toxic treatment that killed most of the zooplankton, the surviving chydorids in the littoral area underwent gamogenesis about 2 months before their normal time, suggesting that gamogenesis begins earlier under disturbed environmental conditions. However, there have been no attempts to examine the relationship between chydorid gamogenesis and non- climatic environmental stressors in more detail in laboratory or field studies.

1.3. Cladocerans in paleolimnology

After cladocerans die, their soft tissues decay and exoskeletons break into various body parts, which can be recovered from lake sediments and used as paleolimnological evidence. Frey (1958, 1960)

15 discovered that chitinous cladoceran body parts (carapaces, headshields, postabdomens, postabdominal claws, copulatory hooks, mandibles and ephippia) are preserved as subfossils and are identifiable in lake sediments (Fig. 4). The preservation varies among different bodyparts and among different families; the best preserved bodyparts are found in Bosminidae and Chydoridae. In chydorids, almost all chitinous bodyparts are preserved and can be indentified to the species level, hence increasing their potential for use in paleolimnological studies. In Bosminidae, difficulties arise in identification of the bodyparts, because there is very large-scale morphological variation and the taxonomy is far from clear at the present (e.g. Hofmann 1986, 1987; Szeroczyńska and Sarmaja-Korjonen 2007). The preservation of bodyparts of other cladoceran families is selective, or the body parts are not preserved at all (Frey 1986a; Hann 1989; Korhola and Rautio 2001). As an example, all bodyparts of common Daphnia species are not preserved similarly; claws and ephippia are usually well preserved, whereas other bodyparts, e.g. headshields, carapaces, carapace margins and tail spines, are rarely preserved; nonetheless sometimes they can be recovered under favourable environmental conditions (Frey 1991; Manca et al. 1999; Manca and Comoli 2004; Szeroczyńska and Zawisza 2005; Sarmaja-Korjonen 2007a).

a) b) c)

Figure 4. Subfossil chydorid remains: a) carapace of Alonella nana, b) headshield of Alona guttata var. tuberculata, and c) postabdomen of Alona rustica.

Current identification of subfossil remains of cladocerans has improved over that of earlier studies (Frey 1958, 1959, 1960; 1962a; Goulden and Frey 1963), which increases the usability of cladoceran remains as indicators of the past structure of aquatic ecosystems (e.g. Manca et al. 1999; Nevalainen and Sarmaja-Korjonen 2005; Szeroczyńska and Zawisza 2005; Sarmaja-Korjonen 2005, 2007a; Nevalainen 2007; Bjerring et al. 2008). Recently, Szeroczyńska and Sarmaja-Korjonen (2007) published the first identification key for subfossil cladocerans of northern and central Europe, whereas previously identification was based on contemporary faunistic guides and several published articles. Frey (1960, 1974, 1988b) noticed that subfossil chydorid assemblages in surface sediments provide a good representation of living chydorid communities and stated that identification of chydorid communities in lakes by examination of the surface sediment samples is very effective. It may be even better than traditional contemporary sampling, because chydorids have dynamic seasonal occurrences and they live in a variety of habitats. The accumulated chydorid remains mix before the final sedimentation and therefore lake sediments are considered to collect and unify the chydorid community, including those living in different habitats and over the entire active period. It has been shown that the species composition of chydorids is similar in sediment sequences taken from different parts of a single small lake basin although there is variation in percentage abundances (Sarmaja-Korjonen 2001). Subfossil Cladocera analysis has been widely used in paleolimnology to shed light on past development of aquatic environments and external changes in lake catchment and climate (reviewed in Frey 1986a; Hann 1989; Korhola and Rautio 2001; Rautio 2007). Pioneering studies were performed by e.g. Frey (1958), Goulden (1964), De Costa (1968) and Harmsworth (1968), who examined development of cladoceran fauna during the Holocene and connected the faunal changes to climate development.

16 Frey (1962b) and Hann and Karrow (1984) investigated cladoceran subfossils from earlier interglacials. In addition to their use in examining climate development, subfossil cladoceran fauna have been used to detect environmental changes in water pH (e.g. Nilssen and Sandøy 1986, 1990; Krause-Dellin and Steinberg 1986; Uimonen-Simola and Tolonen 1987), nutrients (e.g. Alhonen 1972; Hofmann 1987, 1996; Szeroczyńska 1991, 1998), water level (e.g. Alhonen 1970a; 1970b; Sarmaja-Korjonen 2001; Nevalainen et al. 2008), and food-web structure (e.g. Kerfoot 1974, 1981; Salo et al. 1989; Jeppesen et al. 2001; Manca et al. 2007). In Finland, Alhonen (1970a, 1970b) introduced and applied subfossil cladoceran research with his pioneering results on planktonic/littoral cladoceran ratio. Alhonen (1971, 1972), Donner et al. (1978) and Salomaa and Alhonen (1983) showed with their paleolimnological evidence that cladoceran fauna and its development from the last glacial in southern Finland has been mainly affected by climate development and related water-level changes, and by human activity. Since then, many studies on subfossil cladocerans have been performed in southern Finland, mainly concerning the development of lakes from the end of the last glacial up to the present, by Korhola (1990, 1992), Korhola and Tikkanen (1991), Sarmaja-Korjonen (2001, 2002), and Sarmaja-Korjonen and Alhonen (1999). Several studies on past environmental changes in northern Finland have also included cladocerans as proxies (Hyvärinen and Alhonen 1994; Sarmaja-Korjonen and Hyvärinen 1999; Korhola et al. 2000, 2005; Sarmaja-Korjonen et al. 2006, Szeroczyńska et al. 2007). Recently cladocerans have also been used in paleolimnological research as quantitative indicators of different environmental factors. Cladoceran assemblages have been found to relate significantly with e.g. temperature, lake depth, nutrients and altitude (Brodersen et al. 1998; Korhola 1999; Korhola et al. 2000; Bos and Cummings 2003; Amsinck et al. 2005; Sweetman and Smol 2006) and used as quantitative indicators to reconstruct past changes e.g. in temperatures (Duigan and Birks 2000; Lotter et al. 2000) and in water levels (Korhola et al. 2005).

1.4. Ephippium analysis

Subfossil ephippia of the planktonic cladocerans Daphnia, Bosmina and Ceriodaphnia, which are well preserved in sediments, have been used in paleolimnological research to study species abundances (Jeppesen et al. 2003) and to determine planktivorous fish abundances (Jeppesen et al. 2002; Johansson et al. 2005; Nykänen et al. 2006). In addition, Jeppesen et al. (2003) showed that the ratio of Bosmina ephippia to the sum of ephippia plus carapaces was significantly related to summer mean air temperature in a wide geographical region. They hypothesized that this ratio would, in addition to temperature, be affected by food abundance and fish predation. Moreover, cladoceran resting eggs, mainly those of the genera mentioned above, have been used to track past changes in e.g. acidification, eutrophication, heavy-metal pollution and climate (reviewed recently by Amsinck et al. 2007). Sarmaja-Korjonen (1999) found a high abundance of subfossil gamogenetic remains (headshields of ephippial females) of one chydorid species, Chydorus piger, from Lake Aitajärvi in northern Lapland. According to her results this may indicate a long gamogenetic reproduction period and relatively short parthenogenetic reproduction period under harsh climatic conditions. Sarmaja-Korjonen (1999) based her suggestion on the studies of Frey (1982) and Stross and Kangas (1969), who indicated that in northern climates, parthenogenetic generations are very few and gamogenesis starts early. Sarmaja- Korjonen (1999) concluded that subfossil gamogenetic remains of chydorids may prove to be valuable indicators of climatic conditions in lake sediment studies. Sarmaja-Korjonen (2003) subsequently showed that sexual reproduction in chydorids can be reconstructed from the subfossil record, since chydorid ephippia and carapaces preserve well and are identifiable to the species level in lake sediments. Sarmaja-Korjonen (2004) presented a method

17 called ephippium analysis, which uses the two reproduction modes of chydorids to estimate the length of the open-water season. In ephippium analysis, the shells of asexual individuals represent asexual reproduction and the shells of sexual females, i.e. ephippia, represent sexual reproduction (Fig. 5). The relative proportion (%) of chydorid ephippia of all chydorid species, i.e. the total chydorid ephippia (TCE), is calculated from the sum of parthenogenetic carapaces plus ephippia. Thus, the TCE indicates the relative proportion of sexual reproduction during the open-water season

a) b)

Figure 5. Subfossil a) carapace and b) ephippium of Alonella nana. A carapace represents a parthenogenetic female and an ephippium, a carapace modified to envelop and protect the resting egg, represents a gamogenetic female, i.e. the two reproduction modes used in ephippium analysis. The pictures are taken by K. Sarmaja-Korjonen.

The hypothesis for the use of ephippium analysis in climate reconstructions assumes that the open- water season is rather long in a relatively mild climate, and that chydorids reproduce asexually during most of the open-water season and that sexual reproduction is restricted to autumn. This results in a high proportion of shells of asexual females and a low proportion of ephippia in sediments. In a cold climate the open-water season is shorter and therefore the relative length of the asexual reproduction period is limited when compared to the sexual reproduction period. This leads to a high proportion of ephippia of all or most chydorid species in lake sediments. Recently, Kultti et al. (2006) found support for this hypothesis with their results. The surface sediment study of 73 lakes across Finland showed that the current TCE in surface sediments correlates well with current climate parameters, e.g. length of open-water season, being lowest (ca. 2-4%) in southern Finland and highest (ca. 20-30%) in northernmost Finnish Lapland at high altitudes. Sarmaja-Korjonen (2007b) also provided evidence that modern ephippium proportions are connected with climate and the length of the open-water season. In a pioneering study Sarmaja-Korjonen (2003) studied subfossil chydorid ephippia from two sediment sequences representing the Holocene in southern Finland. Both sequences gave similar results: ephippia were abundant (10-15%) during the cold early Holocene and decreased to 2-3% during the middle and late Holocene, indicating a longer open-water season. Nevalainen (2004) also found similar patterns in proportions of chydorid ephippia in Lake Arapisto, which was formed on a supra-aquatic location (i.e. it was never submerged under the Baltic waters after the retreat of the Scandinavian ice sheet) in southern Finland at the beginning of the Holocene. The TCE was very high (ca. 25%) during the earliest stage of the lake’s development, suggesting a very short open-water season. This very high TCE in Arapisto, higher than that found by Sarmaja-Korjonen (2003, 2004) in southern Finland, was probably due to its location on supra-aquatic ground in a very harsh environment near the retreating ice sheet. In Arapisto the TCE quickly decreased to ca. 10% and stayed at that level until ca. 9000 cal. yr BP, after which it decreased to ca. 2-5%, indicating a lengthened open-water season, and stayed at that

18 level until the present. Bennike et al. (2004) investigated the classic late-glacial Bølling Sø sequence in Denmark and found that the cold Younger Dryas was characterized by a high proportion (ca. 25%) of chydorid ephippia. The results of Sarmaja-Korjonen and Seppä (2007) suggested a considerable, periodic reduction in the length of the open-water season during the cold 8200 cal. yr BP event in southern Finland, e.g. at the beginning of the event the TCE increased more than threefold (from 1.4% to 5.2 %). The TCE is constructed not only from parthenogenetic carapaces and ephippial carapaces, but also includes male carapaces (which cannot be distinguished from carapaces of parthenogenetic females) and barren instar carapaces (chydorids have a “barren”, non-ephippial instar between the ephippial instars, Frey 1987) as Bennike et al. (2004) discussed. They also discussed how high the TCE can get due to the influence of non-ephippial remains, and suggested a maximum of ca. 30% ephippia. Unpublished data of K. Sarmaja-Korjonen (pers. comm.) indicate that the TCE can be even 35% in a subarctic high altitude lake. In northern lakes there may be only two or three parthenogenetic generations developed before the sexual reproduction begins in chydorids (e.g. Poulsen 1940) and the proportion of gamogenetic females during the autumn can be very high in northern lakes, nearly 40% (Nevalainen, subm.), suggesting that the proportions of ephippia accumulating in sediments may be very high. However, more contemporary ecological and paleoecological studies from a wide number of lakes in northern regions are required to clarify these ideas further. As mentioned above, Kiser et al. (1963) found early induction of chydorid gamogenesis under environmental stress following a release of toxic pesticide into the lake. A paleolimnological investigation of Sarmaja-Korjonen (2003) also indicated that chydorids may respond sensitively to some environmental stressors by increasing their gamogenesis. Sarmaja-Korjonen (2003) found evidence that some factors other than a shorter open-water season can also result in higher ephippium proportions. This became evident when ephippia of only a single species increased for a short period of time in the sediment sequence of Lake Rutikka, southern Finland, and thus was not mediated by climate. Prehistoric anthropogenic activities led first to a slight rise in the trophic state of the lake and apparently caused changes in the food web. This resulted in a stress on only a single species, Alona affinis, as suddenly its ephippia increased from 0 to 10%. When the trophic state further rose due to increased human impact, A. affinis, which formerly was the dominant chydorid, almost disappeared from the lake and no more of its ephippia were recovered. However, the actual mechanism or mechanisms that result in a higher than “normal” TCE in sediments are uncertain. Sarmaja-Korjonen (2004) proposed the term “increased gamogenesis” for this phenomenon. She suggested that one possible mechanism could be gamogenesis that begins earlier than the natural sexual period as a result of environmental stress, which would result in higher ephippium proportions in sediments. This suggestion is supported only by the results of Kiser et al. (1963), but lacks further documentation from other regions. It is known that summer gamogenesis occurs in some dicyclic chydorids in small lake basins (e.g. Berg 1929; Järnefelt 1956; Mäemets 1961; Røen 1995), but whether this is sufficient to increase the TCE remains unclear. The study of Cáceres and Tessier (2004) on Daphnia showed that the intensity of sexual reproduction, i.e. the proportion of sexual individuals in populations, varies among different lakes. If such variation occurs in chydorids too, the higher intensity of sexual reproduction could increase the TCE.

1.5. Objectives of this thesis

This thesis is part of the project EPHIPPIUM, which aims to develop ephippium analysis for quantitative climate reconstructions (temperatures), and especially for determining the length of the open-water season (i.e. the length of winter), which is difficult to detect with existing reconstruction methods

19 based on other biological proxies. The project aims to develop a climate reconstruction model based on the TCE values of surface sediments from 73 lakes in different climatic conditions in Finland. To be able to develop a valid model, the influence of other environmental stressors increasing the TCE and interferring with the model must be removed. Therefore, it is necessary to know more about chydorid reproduction and how the TCE is constructed. This thesis aims to examine by means of contemporary ecological methods how climate and other environmental stressors affect chydorid sexual reproduction patterns. It also aims to study with paleolimnological methods how these patterns are reflected in the surface sediments and the TCE. In addition, it aims to study how recent climate-related and non-climatic environmental changes are reflected in chydorid reproduction patterns detected in short sediment sequences. For these aims, nine lakes from the northern temperate climatic region of southern Finland that were in close proximity to each other (hence with similar climate) but with otherwise environmentally different conditions were chosen for the study. The lakes currently range from oligo- to eutrophy, from clear to brown water, from nearly pristine to human disturbed, and a few of them are known to have experienced major environmental changes in their recent history. This thesis is based on the following questions: 1) what are the main climatic regulators and non- climatic environmental stressors that induce and control chydorid gamogenesis, and how do they work in environmentally different lakes, 2) how do chydorids respond to non-climatic environmental stressors, 3) how are modern monitored sexual reproduction patterns reflected in recent surface sediments as the TCE and 4) how are climatic and non-climatic environmental changes in the past reflected in chydorid reproduction patterns and consequently in the TCE in sediment sequences?

2. Material and Methods 2.1. Sites

The nine study lakes are located in southern Finland, near the Helsinki district (Figs. 6, 7). The limnological variables (pH, conductivity, color, dissolved oxygen, total phosphorous and total nitrogen) of the lakes were measured during three seasons in 2005 (Table 2). The basic climate data of the study area are presented in Table 3. The duration of the open-water season in the area is about seven months, from early May to late November, depending on yearly variations in spring and autumn temperatures (Kuusisto 1986; Helminen 1987; Finnish Meteorogical Institute 2008). Lakes Hauklampi (60º18’N, 24º36’E, 76.7 m above sea level, area 2.7 ha), Iso Majaslampi (60º19N, 24º36’E, 92.7. m a.s.l., area 6.3 ha), and Pieni Majaslampi (60º19.3’N, 24º35.7’E, 97.3. m a.s.l., area about 1 ha) are located close to each other in the Nuuksio “upland” area in the municipality of Vihti. They are acidic and oligotrophic lakes, with small and nutrient-poor catchments. Patches of mire, fractured bedrock outcrops, and Pinus forests cover the catchments, the shores are inhabited by Carex spp. and Sphagnum mosses, and the aquatic macrophyte zone consists only of Nymphaea alba. The lakes were severely acidified during the 1980s as a result of acid deposition (Kauppi et al. 1990). According to Korhola and Tikkanen (1991), Pieni Majaslampi has been naturally very acidic since the early Holocene. Due to the recent acidification, the natural fish populations, consisting mainly of European perch (Perca fluviatilis), died out in the 1980s. The lakes went through a chemical recovery process during the 1990s. In 2002, perch were introduced into these lakes (35-120 per hectare), aiming to re-establish the perch populations (Kari Nyberg, pers. comm.). Iso Lehmälampi (60º20’N, 24º36’E, 91.7 m a.s.l., area about 3 ha) is also an acidic and oligotrophic upland lake. The catchment is characterized by the presence of bedrock outcrops and paludified areas. The lake also experienced severe acidification in the 1980s and a succeeding recovery process in the

20 25° 1990s. The lake became almost 70° Norway fish-free during the acidification when the pH declined to Sweden Study sites below 5.0 (Verta et al., 1990), N Arctic circle Municipality but has since had several Highway W E Russia introductions of whitefish Urban areas ea (Coregonus lavaretus), roach S Cultivation S (Rutilus rutilus), and perch Baltic Forest Water 5 km (Kari Nyberg, pers. comm.). Sphagnum mosses, Carex spp., 60° and submerged Nuphar lutea and Nymphaea alba dominate Iso Lehmälampi the littoral area and the shoreline. Kalatoin Jousjärv VIHTI Tuhkuri The rocky and paludified Hauklampi Pieni & Iso SIPOO catchment is rather small Majaslampi VANTAA Hampträsk and poor in nutrients. Lakes Hauklampi, Iso Majaslampi, Pieni Majaslampi, and Iso Lehmälampi were considered ESPOO Kangaslampi to have experienced stress to their zooplankton due to their HELSINKI acidification histories, and the recent fish introductions, and Figure 6. Location of the nine study lakes and land use in the Helsinki were therefore chosen for the district. study. Tuhkuri (60º20’N, 24º37’E, 73.7 m a.s.l.) is an oligotrophic forest lake with a slightly higher pH than the other study lakes in the Nuuksio upland. It is also larger in size than the other study lakes, at 13.7 ha. Several houses and summer houses are situated on its shores. The catchment of the lake is characterized by mixed forest (Picea, Pinus, Betula) and some bedrock outcrops. The aquatic vegetation consists of Phragmites australis, Nuphar lutea, and aquatic mosses. Sphagnum mosses cover some

Table 2. Basic limnological measurements of the nine lakes during three seasons in 2005.

21 Table 3. Basic climate data from southern parts of the shoreline and fish inhabit the lake. The Finland. The data was provided lake was chosen for this study to represent a lake by the Finnish Meteorological with a slight human impact. Institute and interpolated from the data of meteorological stations (S. Kalatoin (60º20’N, 24º37’E) is a very acidic and Kultti, pers. comm.). dystrophic small (about 1 ha) lake with paludified shores at an altitude of 89.5 m a.s.l. Sphagnum mosses and Carex spp. dominate the shores and the submerged vegetation consists of aquatic mosses and Nuphar lutea. The lake is surrounded by a small catchment with bog vegetation and several bedrock outcrops. A small, natural outlet drains to the northwest. The lake is fish-free, probably due to its very acidic conditions (Table 2), and therefore invertebrate predation predominates. Kalatoin was considered to be a nearly pristine lake in the study. Jousjärv (60º20’N, 25º11’E, 37.3 m a.s.l.) is a very small (about 0.5 ha), dystrophic lake with paludified shores, situated in the municipality of Sipoo. Sphagnum mosses and Carex spp. cover the entire shoreline. Small hills with bedrock outcrops characterize the catchment. The paludified area surrounding the lake is ditched, and therefore it is not in its natural state but nevertheless the lake is regarded here as pristine, and was therefore chosen for this study. The lake is most likely fish-free. The pH of the lake is higher that in Kalatoin.

a) b) c)

d) e) f)

g) h) i)

Figure 7. The nine study lakes and their surroundings: a) Hauklampi, b) Iso Majaslampi, c) Pieni Majaslampi, d) Iso Lehmälampi, e) Tuhkuri, f) Kalatoin, g) Jousjärv, h) Hampträsk, and i) Kangaslampi. The pictures are taken by the author.

22 Hampträsk (60º17’N, 25º15’E, 20.3 m a.s.l.) is a shallow and meso-eutrophic lake with human influence in its surroundings in the municipality of Sipoo. The surface area of the lake is about 4 ha. There are fields and houses near the lake and the paludified western shore is ditched. Theaquatic vegetation consists mainly of Phragmites australis and Nuphar lutea. During the winter of 2002 severe oxygen depletion occurred in the lake, which is probably a common phenomenon in the lake during ice-cover periods. The lake was chosen for the study due to possible changes in the food web caused by this period of oxygen depletion. The current fish status of the lake is unknown. Kangaslampi (60º13’N, 25º08’E) is a very shallow, small (about 1 ha), eutrophic, and disturbed lake of natural origin with very high human impact. The lake is located in a suburb of Helsinki near the Baltic Sea (14.6 m a.s.l.) within a densely inhabited area. The catchment is mostly built up, except for a small park. During the summer, algal blooms dominate the aquatic environment and thus oxygen depletion during the winter and summer is extensive. Some, probably unofficially transferred, fish were seen in the lake in the summer of 2005. The pond represents an extremely polluted and stressed site in this study.

2.2. Sampling and sample analyses

2.2.1. Contemporary samples

Sweep net sampling of chydorids was performed weekly during the open-water season in 2005 (early May to mid-November) in the nine study lakes (Table 4, paper I). The samples were taken with a 100- µm plankton net, which was swept back and forth over about 1 m of lakeshore after slightly disturbing the water. In each lake the chydorid samples were taken from three different locations to obtain animals from different habitats, e.g. from aquatic vegetation, soft sediment, and rock surfaces. In the field, the samples were sieved through 1000-µm mesh to extract large plant fragments, and the residue was concentrated with 100-µm mesh and stored in ethanol in small jars. The samples were placed in a coldroom for later preparation. In the laboratory the samples were heated quickly in 10% KOH, sieved through a 100-µm mesh, and mounted in glycerine jelly on preparation slides. Chydorids were examined under a light microscope at magnifications between 100 and 200 for precise identification. Chydorid individuals were counted until a minimum of 100 were encountered or four preparation slides (24 x 50 mm) were studied (paper I). Parthenogenetic chydorid females, gamogenetic females, and males were identified and marked separately. Additional subsamples from the same sweep net samples from seven lakes were prepared and studied, until a minimum of 100 individuals were encountered (Table 4, paper II). The intensity of gamogenetic individuals, i.e. the relative proportions of males and gamogenetic females, were calculated from the total number of chydorids, including the Table 4. Study lakes in the original papers I-V. parthenogenetic individuals. Quantitative sampling of chydorids was performed with activity traps at two-week intervals during the open-water season in 2006 (early May-late October) in three lakes (Table 4, paper III), following the procedure used by Whiteside and Williams (1975).

23 The activity traps were constructed as described in Örnólfsdóttir and Einarsson (2004) with slight modifications (Fig. 8). Each of the traps comprised a metal frame that contained nine semitransparent plastic bottles (250 ml). A plastic semitransparent funnel was attached to the mouth of each bottle. The funnel was 13 cm long and 7.5 cm wide at its wider end and 0.8 cm at its narrower end. The total collecting area of the nine funnels, and thus of a single trap, was 398 cm2. One trap was placed in the littoral zone of each of the three study lakes, the water depth varying between ca. 30 and 50 cm. The traps were placed at the sampling sites at 8-10 a.m. and collected at the same time the next day. In the field, chydorids were concentrated from the plastic bottles using a 100-µm mesh and stored in ethanol in small jars. In the laboratory, the samples were concentrated again with a mesh and mounted in glycerine jelly on preparation slides. All the chydorids encountered in the samples were identified as parthenogenetic females, gamogenetic females, or males under a light microscope (magnification 100-200). The numbers of trapped chydorids are expressed as individuals per square meter during one sampling day (ind. m-2 trapday-1). The identification of intact chydorids was based on Flössner (1972, 2000) and Røen (1995). Juvenile male instars of Alona affinis were identified using the description of Sinev (2000). The chydorid nomenclature follows that of Røen (1995). Chydorus sphaericus is considered as C. sphaericus sensu lato, according to Frey (1986b; see also Frey 1980; Duigan and Murray 1987).

a) b)

Figure 8. Activity traps developed and used in the research for this thesis: a) general structure, and b) in situ sampling.

2.2.2. Sediment samples

Surface sediments (the topmost 1-2 cm) from the nine study lakes were sampled with a Limnos gravity corer through ice in February and March 2005 (Table 4, paper V). A short sediment sequence (46 cm) from Hampträsk was cored through ice in late February 2005 using a Limnos gravity corer (Table 4, paper IV) and the sediment was sub-sampled at 1 cm intervals in the field. A sediment core (77 cm) from Pieni Majaslampi was sampled with a Frozen-finger corer through ice in February 2006 (V).The topmost 14 cm of frozen sediment was sliced at 0.5 cm intervals in the laboratory, and analyzed for subfossil Cladocera. In the laboratory the samples were prepared for Cladocera analyses according to methods described in Szeroczyńska and Sarmaja-Korjonen (2007). The samples were heated and stirred in 10% KOH on a hot plate for approximately 20 minutes. After heating, samples were sieved through a 44-µm mesh and mounted in glycerine jelly on preparation slides. The slides were examined under a light microscope at

24 a magnification of 100-400 until a minimum of 200 chydorid carapaces were enumerated. The relative proportion of chydorid ephippia, i.e. the total chydorid ephippia (TCE), was calculated from the sum of chydorid carapaces added to chydorid ephippia according to Sarmaja-Korjonen (2003).

2.3. Data analysis

Detrended correspondence analysis (DCA) is a unimodal ordination method that summarizes variation in ecological datasets and shows the relationships between samples in ordination diagrams. DCA was used to identify major patterns in the timing of sexual reproduction during the open-water season and applied to the data on timing of sexual reproduction among populations of the most common species, and it was also used to detect differences between the populations in nine lakes (I). In addition, DCA was applied to identify major differences in the trapped numbers of parthenogenetic and gamogenetic individuals at the sampling sites in three lakes (III), and to identify variation in cladoceran assemblages in sediment sequences from Hampträsk (IV) and Pieni Majaslampi (V). Principal component analysis (PCA) is a linear and indirect ordination technique that allows examination of the relationships between different variables (Ranta et al. 1989; ter Braak 1995). PCA was used to identify the relationships between limnological variables and intensity of gamogenesis (mean values of each species during the autumn) (II). PCA was also used to identify the relationship between limnological variables and the proportion of chydorid species, and between limnology and the proportion of ephippia of chydorid species in surface sediment samples. PCAs and DCAs were performed using the computer program CANOCO, version 4.52 (ter Braak and Smilauer 1998). Analysis of variance (ANOVA, one-way) determines the variance between group averages (F), and reports the significance level of the variation found (P). ANOVA was applied to clarify the variance in the total proportion of gamogenetic individuals between the lakes (II) and the variance in the numbers of trapped chydorid individuals and gamogenetic individuals between the lakes (III).

3. Results and Discussion 3.1. Chydorid distribution

3.1.1. Species occurrence in sweep net and surface sediment samples

The chydorid species Alonella nana, Alonella excisa, Alona guttata var. tuberculata, and Chydorus sphaericus s.l. occurred in all the lakes and in samples of both types (Table 5). The number of chydorid species between the sample types did not differ substantially, but in some lakes, e.g. in Kangaslampi and Tuhkuri more species were caught with net sampling than via sediment sampling and in some lakes, e.g. in Iso Majaslampi and Kalatoin, more species were identified from the sediment samples (Table 6). The number of species was highest in sweep net samples of Tuhkuri and lowest in the sediment sample of Kangaslampi (Table 6). Subfossil chydorid assemblages in surface sediments from the nine lakes (Fig. 9) seem to represent rather well the living chydorid communities in the same lakes (I). All of the most abundant species in subfossil assemblages occurred continuously during the entire open-water season of the lakes. However, there were some striking differences between the two sample types, the most pronounced difference being the occurrence of Alona affinis in lakes Hauklampi, Iso Majaslampi and Pieni Majaslampi. During the monitoring season, the species was encountered only during very limited periods and at low numbers in the lakes (I). When observing the surface sediment record, A. affinis was present

25 Table 5. Occurrence of chydorid species in the contemporary sweep net (water = w) and surface sediment samples (sediment = s) in the nine lakes. The species codes used in the PCA ordination diagrams (Figs. 10 and 15) are underlined.

Hauklampi Iso Majaslampi Pieni Majaslampi Iso Lehmälampi Tuhkuri Kalatoin Jousjärv Hampträsk Kangaslampi Alo nella nan a ws ws ws ws ws ws ws ws ws Alo nella exc isa ws ws ws ws ws ws ws ws ws Alo na guttata var. tub erculata ws ws ws ws ws ws ws ws ws Acr operus har pae ws ws ws ws ws ws ws ws w Gra ptoleberis tes tudinaria w w w ws ws ws ws ws ws Chy dorus sph aericus s.l. ws ws ws ws ws ws ws ws ws Alo na aff inis ws ws ws ws ws ws ws ws s Alo na gut tata ws ws ws w ws ws w ws ws Alo na rus tica ws ws ws ws ws s Alo nopsis elo ngata ws ws ws ws w Ple uroxus tru ncatus w ws ws ws w w Ple uroxus lae vis w ws ws w Ple uroxus tri gonellus w ws Alo na int ermedia s s ws w s w w Eur ycercus lam ellatus ws s ws ws ws w Dis paralona ros trata w w ws w Cam ptocercus rec tirostris s s ws ws s ws ws Alo na rec tangula w ws ws Alo nella exi gua w ws s s Alo na gua drangularis ws s ws s Chydorus latus w s Rhy ncothalona fal cata s ws s Chy dorus pig er s s s ws Psudochydorus globosus w Anchistropus emarginatus w Alona costata w Oxyurella tenuicaudis w Kurzia latissima s Una pertura lat ens w ws w s s in the assemblages with 10-20 % abundances (Fig. 9). Since Frey (1960) suggested that subfossil chydorid communities from surface sediment samples should represent well the living communities, it is possible that A. affinis populations very recently became depressed in these three lakes, which were dominated by invertebrate predation before fish introductions in 2002 (Kari Nyberg, Petri Nummi and Veli-Matti Väänänen, pers. comm.). A. affinis is a large chydorid and, since the predation in lake ecosystems is size selective (Lynch 1980, de Bernardi et al. 1987), it is possible that this large species became an important part of the food supply of perch populations. Many down-core and surface sediment data set studies in Europe and in the New World have provided information on the relationship between chydorid assemblages and limnological factors in their living environment (Whiteside 1970; Brodersen et al. 1998; Lotter et al. 1998; Korhola 1999; Manca and Armiraglio 2002; Bos and Cummings Table 6. Number of chydorid species in the sweep 2003; Sarmaja-Korjonen et al. 2003; Amsinck net and surface sediment samples. The et al. 2005; Sweetman and Smol 2006; Manca lake codes used in the PCA ordination et al. 2007). In the present study, PCA showed diagrams (Figs 10 and 15) are shown in that Chydorus sphaericus, Alona rectangula and brackets. Graptoleberis testudinaria, Disparalona rostrata and Pleuroxus trigonellus were associated with higher nutrient status, pH, and brown waters, while in contrast Acroperus harpae, Alonopsis elongata, Alona affinis, Chydorus piger, Alonella excisa and Alona rustica were linked to high oxygen concentrations (Fig. 10). These patterns fit well with general observations of chydorid occurrences in different environments in Europe,

26 ta la l. cu is ia s. tr os nar us uber ir us is a a udi at ar ic e r. t ct ll at er a re at st a a tr pa v r g te ul di sa ua me os ar ta ge us on is ig la ta angul is spha tica ta pi rc el ang ta me a r nana exci in s h is ex us er on ff us ru us ut us ce leber ect rc ut uadr nt l lla lla to la ra a a a r a g to a r ce a g a g a i a ne ydor rope ydor mp ap ry one o on on on onops onel on u on on on sp Lake Al Al Al Ch Ac Al Al Ch Ca Al Gr Al Al E Al Al Al Di

Hauklampi

Iso Majaslampi Pieni Majaslampi

Iso Lehmälampi Tuhkuri Kalatoin

Jousjärv Hampträsk Kangaslampi

20 40 60 20 40 60 20 40 60 20 40 60 20 20 10 10 10 10 10 10 10 10 10 10 10 10 % of total chydorids Anal. L.N. 2005 Figure 9. Relative proportions of chydorid species in the surface sediments of the nine study lakes. as presented by e.g. Brodersen et al. (1998), Amsinck et al. (2005), and Manca et al. (2007). The most common species in the present study, Alonella nana, was frequent in all types of lakes, although the results showed its occurrence to relate to high nutrient status (Figs. 9, 10). In other studies (e.g. Whiteside 1970; Brodersen et al. 1998; Amsinck et al. 2005) it has been documented to be associated with oligotrophic lakes in Europe. It is generally known that A. nana prefers oligo-dystrophic sites (e.g. Duigan 1992; Røen 1995), although its wide tolerance of environmental conditions has also been documented (Mäemets 1961, paper II). The current results support the idea that it has wide ecological flexibility. Subfossil chydorid communities give

1.0 a picture of the annual species abundances Alo qua HT Alo int (cf. Frey 1988b), whereas net sampling Dis ros Ple tri can be slightly inaccurate for determining TUH Alo nan TN Rhy fal community structure, depending on the Chy pig pH Alo Eur lam TP Cam rec sampling resolution and selected sampling aff ILL Chy sph sites, since some species have very 2 Gra Oxy IML Alo rec tes restricted active periods and habitats. Manca axis Acr har Cond Alo elo Alo gut Ple tru Alo exi et al. (2007) concluded that a combined PCA Col PML KL paleo-neolimnological approach provides JJ important perspectives when studying Alo rus Alo tub lake ecosystems. According to the results Una lat Ple lae presented here it also appears that paleo- HL and neoecological perspectives are valuable KAL Alo exc in offering a different time perspective to -1.0 the dynamics of chydorid populations and -1.0 PCA axis 1 1.0 therefore should be utilized collectively Figure 10. PCA ordination for the most abundant whenever possible to clarify the complexity chydorid species in the surface sediments of aquatic ecosystems. of the nine lakes and limnological variables oxygen (Oxy), color (Col), conductivity (Cond), pH, and total nitrogen (TN) and phosphorous (TP). The species codes are underlined in Table 5 and the lake codes are in brackets in Table 6.

27 3.1.2. First findings of intactUnapertura latens sp. n.

Sarmaja-Korjonen et al. (2000) described subfossil remains (carapace, headshield and postabdomen) of an unknown chydorid species from Finland, and suggested the tentative name Unapertura latens. Recently, Szeroczyńska and Sarmaja-Korjonen (2007) presented more comprehensive pictures of subfossil remains of this species, including an ephippium. Szeroczynsńska and Sarmaja-Korjonen (2007) stated that “the species is widespread at least in Finland, but usually occurs in low numbers”. In the present study, subfossil remains of U. latens were found in three of the nine lakes (Table 5), suggesting that it is a rather common part of the chydorid fauna in southern Finland. Subsequently, Sweetman and Sarmaja-Korjonen (subm.) presented remains identical to U. latens from Canada, suggesting a holarctic distribution of this species (if it is the same species on both continents). In addition to Finland and Canada, subfossil remains of U. latens have been documented from the Swiss Alps (Bigler et al. 2006). During the ecological monitoring of the nine lakes in 2005, a few intact U. latens specimens were recovered from three of the lakes (Fig. 11, Table 5). In addition, a few cast ephippia with resting eggs were found (Fig. 12). No males were detected. These are the first findings of this chydorid species as intact specimens. The specimens were recovered from the oligotrophic and low pH lakes Hauklampi, Iso Majaslampi and Iso Lehmälampi (Table 2). During the monitoring of the three lakes with activity traps, a single parthenogenetic female of U. latens (the holotype) was trapped in Kalatoin (Appendix I). Based on these findings, it appears thatU. latens thrives in acidic clear and brown water lakes. All these lakes support Sphagnum-covered shorelines and littoral zones of submerged vegetation. Therefore it is possible that the species is associated with mossy and vegetative habitats, but of course to verify this, further investigations are required.

a) b)

200 µm

Figure 11. Unapertura latens sp.n. a) as an intact parthenogenetic female (holotype) and b) as a line drawing from the holotype (same scale). The picture is taken by the author.

The intact specimens of U. latens (Fig. 11) closely resemble Rhynchotalona falcata, but differ from it mainly in the shape of the carapace, the number of denticles on the postabdomen, the shape of the labrum, and in having a shorter rostrum. It very closely resembles another species of the Rhynchotalona genus, R. kistarae, which was described by Røen (1973) from Greenland. The body shape and postabdomen are quite similar, but U. latens differs in the shape of the rostrum and the shape of the headshield. Prior to the observations of Cotten (1985) and Korhola (1992), leading to the more exact description of subfossil U. latens remains by Sarmaja-Korjonen et al. (2000), there was only one refererence to an

28 a) b)

Figure 12. a) Photograph and b) line drawing of ephippium and resting egg of Unapertura latens (same scale). The picture is taken by the author. intact chydorid that closely resembles R. falcata, but has a shorter rostrum. This was made by Müller (1867), who described a new species from a single specimen and called this species Alona dentata. Over a hundred years later Frey (1989) reinvestigated Müller’s material, and, although A. dentata was not found preserved in the collection, concluded from the written description and illustrations that A. dentata is actually an R. falcata specimen with the rostrum broken off. The question now is, why has this evidently rather wide-spread (cf. Szeroczyńska and Sarmaja- Korjonen 2007) but scarce species not been encountered as intact specimens before this? The answer to this question may be the method by which samples are usually studied. In the present study a light microscope, with a magnification of 100-400, was used for precise identification of chydorids, whereas usually identification is performed using a stereo microscope with lower magnification.

3.2. Monitoring of modern sexual reproduction

3.2.1. Timing of gamogenesis (I)

The timing of gamogenesis during the open-water season (early May to mid-November) in 2005 was observed in the nine study lakes (Table 4, paper I). During the summer two gamogenetic Alona rectangula females were found in Hampträsk in late July and early August and one gamogenetic A. rectangula female was found in Kangaslampi in mid-July. Two gamogenetic Alona affinis females were encountered in Kalatoin in early summer. However, no males were recorded during the summer. These findings suggest that summer gamogenesis does occur in some lakes in southern Finland, although it is very weak and inconsistent. Apparently, summer gamogenesis is not triggered by climatic stimuli. The evidence for summer gamogenesis detected in Finland covers the time span from early June (Järnefelt 1956, paper I) to early August, and therefore climate as a stimulus may be ignored. These results may indicate that some ecological or limnological stressors may be able to induce gamogenesis in some chydorids during the summer months. The lakes in which summer gamogenesis was encountered are limnologically

29 very different; Hampträsk is mesotrophic with high pH, Kangaslampi is eutrophic with high pH, and Kalatoin is dystrophic with very low pH (Table 2), giving an inconsistent pattern. Crowding or predation pressure is known to induce gamogenesis in other cladoceran families (D’Abramo 1980; Kleiven et al. 1992; Ślusarczyk 1995, 2001; Pijanowska and Stolpe 1996), and therefore it can be assumed that these stimuli may induce summer gamogenesis in chydorids too. Usually chydorid populations are largest in midsummer (e.g. Goulden 1971; Whiteside 1974; Whiteside et al. 1978) when invertebrate predation may affect them most strongly (Goulden 1971; Williams 1982; Robertson 1990). The first sexual individual in the autumn of 2005, an Alonella excisa male, appeared in Iso Majaslampi in late August, while in the other lakes sexual reproduction began during the following two weeks up to mid-September. In Kangaslampi, sexual reproduction began mainly in late September and ended only a month later, as chydorids became very scarce and apparently died off. Kangaslampi is eutrophic and disturbed, with high human impact, and therefore it is possible that some limnological factor, e.g. poor oxygen conditions (Table 2), caused chydorids to die earlier there. In the other lakes the period of sexual reproduction continued until mid-November, when the lakes froze over. It has been reported that chydorid communities become entirely gamogenetic at the end of the sexual period (Flössner 1964; Kubersky 1977) but in the present study they never became entirely gamogenetic, except in Jousjärv. Parthenogenesis continued along with gamogenesis throughout the autumn and even after gamogenesis ended in some lakes. The results suggest that the general timing and duration of the autumnal sexual reproduction period of chydorids was rather synchronous in all the lakes studied, beginning mainly in mid-September and lasting for approximately two months. The lakes are situated in the same climate zone and gamogenesis apparently was induced by declining temperature and/or shortening photoperiod, as Shan (1974) and Frey (1982) suggested. The photoperiod length in the study area was approximately 12-14 h and water temperatures in the lakes varied between approximately 12 and 17 ºC when the first sexual individuals appeared. A quite similar pattern in the induction and duration of sexual reproduction in chydorids was detected in central Norway by Koksvik (1995). That the induction of gamogenesis is a response to certain temperatures and day lengths is most clearly evident in Alonella excisa, which was the first species to become gamogenetic in most lakes and showed the most uniform induction time. It also had the longest and most homogeneous sexual reproduction period among its populations, from mid-September to early November. The photoperiod length during the induction of A. excisa was 12-14 h and the water temperature 11-17 ºC. In contrast, for example, Acroperus harpae appeared to have slightly different preferences, since it initiated gamogenesis somewhat later, when the photoperiod was 10-13 h and the temperature 10-13 ºC. Acroperus harpae had uniform gamogenesis in October, and shorter periods of gamogenesis than A. excisa. These observations may suggest that some chydorid species have individual demands for photoperiod length and water temperature for the development of gamogenesis. Although the incidence of sexual individuals in Alonella nana populations was rather consistent in late September and mid-October, the induction time of gamogenesis among the populations ranged between late September and late October, water temperature and photoperiod ranging between ca. 8-13 ºC and 9-12 h respectively. The incidence of gamogenetic Alona affinis individuals in different populations also varied widely and over a large time range; between mid-September and late October, the water temperature and photoperiod were ca. 4-8 ˚C and 9-13 h and a uniform period of gamogenesis did not occur during the autumn. Thus, some chydorid species show considerable differences in the induction and timing of sexual reproduction among their populations in different environmental conditions. Such differences indicate that climate may not be the only cue for gamogenesis and that other environmental stimuli may also play a role. Shan (1974) suggested that local species populations may evolve different reproductive patterns to meet major environmental stressors in the region. Thus, the variance observed in the present

30 results suggests that chydorids have probably adapted to varying environmental conditions and that their sexual reproduction is optimized for these particular environments in southern Finland. However, it cannot be ruled out that the differences in timing of gamogenesis between the lakes reflect yearly variation (cf. Frey 1982).

3.2.2. Intensity of autumnal gamogenesis (II)

In 2005, the focus was on the autumnal sexual reproduction period (early September to mid-November) and intensity of gamogenesis. Only seven of the nine study lakes were examined (Table 4, paper II), because there were not enough chydorid individuals in the samples from Hauklampi and Kangaslampi. The total proportion of males increased from September towards the maximum in early and mid- October. Males peaked a few weeks earlier than gamogenetic females and after that their proportion began to decrease. The total proportion of gamogenetic females began to increase soon after the males, attaining a peak between late October and mid-November (Fig. 13). These results are in agreement with Frey (1982), who explained the progressive autumnal increase in chydorid gamogenesis as a response to progressive deterioration of environmental conditions (e.g. decreasing water temperature, day length, and food resources) associated with the onset of winter. Much research has been done on climate factors inducing gamogenesis in Daphnia species (Stross and Hill, 1965, 1968; Stross 1969a, 1969b) but similar accurate studies are lacking for chydorids, as

60 60

50 50

40 40

(%)

(%) 30 30

Males

Males 20 20

10 10

0 0 900 800 700 600 500 400 18 16 14 12 10 8 6 4 2 Photoperiod (min) Temperature C 50 50

(%) 40 (%) 40

females 30 30

females

20 20

10 10

Gamogenetic

Gamogenetic

0 0 900 800 700 600 500 400 18 16 14 12 10 8 6 4 2 Photoperiod (min) Temperature C

Figure 13. Relationship between autumnal intensity of males and gamogenetic females, and photoperiod and littoral water temperature during the autumn of 2005 in seven of the study lakes.

31 discussed earlier. In the present study, the relative proportion of males increased as photoperiod shortened (r = -0.30, p < 0.05) and as water temperature decreased (r = -0.34, p < 0.01). The proportion of gamogenetic females increased rather strongly and significantly with shortening photoperiod (r = -0.62, p < 0.001) and decreasing water temperature (r = -0.59, p < 0.001) (Fig. 13). This suggests that a relationship exists between water temperature, photoperiod and autumnal gamogenesis in chydorids in southern Finland, as the development and increase of intensity of gamogenetic individuals appeared to correspond to decreasing water temperature and photoperiod, as also shown by Shan (1969), Frey (1982) and Koksvik (1995). The intensity of gamogenetic individuals increased up to 10-50% when water temperature and photoperiod had decreased to 4-8 ºC and 400-600 min, respectively. The observations on chydorid gamogenesis suggest that after the induction of gamogenesis at a certain temperature and/or photoperiod, the abundance of gamogenetic individuals increases towards a maximum. Alekseev et al. (2006) stated that climate factors causing gamogenesis (photoperiod and temperature) should not be considered separately because they always operate in combination. However, during the gamogenetic reproduction period the mean proportion of males varied among the lakes from 5 to about 30% and that of gamogenetic females between 5 and 25%, indicating that there were considerable differences in the intensity of gamogenesis. Since the lakes are situated within a climatically restricted area (Fig. 6), the variation in intensity of gamogenesis cannot be explained by the forcing mechanisms of climate. Therefore, some other environmental forcing factors probably are behind the variation. The intensity of gamogenesis in chydorids was highest in fish-free Jousjärv and Kalatoin, and in Pieni Majaslampi, whose ecosystem has been recently disturbed (see above). Theoretically, fish fry could prey selectively on the dark ephippial females of littoral chydorids attached to plants or in the benthos, thus contributing to the low gamogenetic intensity in some lakes, because fish select their prey by eyesight (Lynch 1980, de Bernardi et al. 1987). However, chydorids, which are small, do not constitute a large part of the diet of fish (Pinder et al. 2006; Vašek et al. 2006) but are known to be an important prey of invertebrate predators that hunt with sensors, and therefore cannot choose their prey by color (Goulden 1971; Williams 1983). Therefore, it is unlikely that the low intensity was associated with elimination of dark ephippial females by fish predation. For cladocerans, production of resting eggs is a means of guaranteeing the survival of populations through stressful environmental conditions by dormancy and genetic variability (Frey 1982). However, since gamogenetic reproduction costs more in terms of resources than parthenogenetic reproduction, each population probably aims to produce only an optimally low number of resting eggs. Therefore, a large proportion of gamogenetic individuals in some chydorid communities in the present study may indicate a response to environmental stressors in these lakes. As mentioned above, Pieni Majaslampi had a high intensity of gamogenesis and is known to have experienced considerable food web changes during 2002-2005, and thus can be considered disturbed. The food web of this formerly fish-free lake was severely altered by perch introductions in 2002. These were unsuccessful, since the perch had nearly died out by the autumn of 2005 (K. Nyberg, pers. comm.). After the introductions, the number of benthic macroinvertebrates decreased markedly and later increased again as a consequence of diminished fish populations (P. Nummi and V.-M. Väänänen, pers. comm.). Apparently, these changes forced chydorids to undergo short-term adaptation in response to ecological stressors related to the changes. The intensity of gamogenesis was also high in lakes Jousjärv and Kalatoin, lakes in which invertebrate predation dominates, and this may have been a response to predation pressure or competion (III). In lakes Iso Lehmälampi and Tuhkuri, gamogenetic reproduction played a smaller role in the chydorid life cycle, the mean proportion of males being only 5-7% and gamogenetic females 5-9%. It is possible that the low gamogenetic intensity reflected a less stressful situation in which chydorids could rely more on parthenogenesis. If invertebrate predation pressure is an important stressor, as

32 discussed above, it is possible that in these lakes fish control the abundance of invertebrate predators, thus decreasing the need to produce large amounts of resting eggs. The chydorid community composition and intensity of gamogenesis in individual species varied considerably in the lakes (Fig. 14). Of the commonest species, Chydorus sphaericus s.l. was mostly parthenogenetic in lakes Tuhkuri, Hampträsk, and Jousjärv, exhibiting a rather low intensity of gamogenesis. Keen (1973) reported from North America that C. sphaericus (sensu stricto) had a very low production of resting eggs and males in the autumn, and overwintered as parthenogenetic individuals. de Eyto and Irvine (2001) found in Ireland that C. sphaericus had a faster egg development time than other chydorids in different temperatures and pH ranges, suggesting that it is very efficient in parthenogenetic reproduction. Alonella nana was also very common in the lakes, but there were considerable differences in its relative abundance among the lakes (Fig. 14). Alonella nana was very abundant in lakes Kalatoin, Tuhkuri, Jousjärv, and Iso Lehmälampi. It was also the dominant species in Hampträsk. The intensity of gamogenesis in A. nana also varied noticeably among the lakes. In lakes Kalatoin, Hampträsk, Tuhkuri, and Jousjärv it was high, the maximum proportion of gamogenetic females ranging between 15 and 30%. In contrast, the intensity was extremely low in Iso Lehmälampi, Iso Majaslampi, and Pieni Majaslampi, although the species was abundant in the first two. Heywood (1967) and Keen (1973) showed that some species may exist as parthenogenetic populations throughout the winter under the ice. Alonella nana is known to live perennially in

Figure 14. Proportions of chydorid species and their sexual individuals in the chydorid communities of the lakes a) Iso Majaslampi, b) Pieni Majaslampi, c) Iso Lehmälampi, d) Tuhkuri, e) Kalatoin, f) Jousjärv, and g) Hampträsk during the autumn of 2005. The legend is shown in Fig. 14a.

33 Iso Lehmälampi d) Tuhkuri c) 100 100

90 90

80 80

70 70

60 60 Alonella nana Alonella nana 50 50

40 40

30 30

20 20

10 10

30 20 20 Pleuroxus truncatus Alonella excisa 10 10

10 20 Alonella excisa Alonopsis elongata 10 10 Chydorus sphaericus

5 20 Alona affinis

Acroperus harpae 10 10 Alonella exigua

10 Camptocercus rectirostris 10 Acroperus harpae

5 Alona affinis Alonopsis elongata 5

5 10 Alona guttata var. t ub. Alona costata 5 Graptoleberis testudinaria 5 Alona guttata 5 Alona guttata var. t ub.

5 Chydorus sphaericus 10 Alona guttata 5 Anchistropus emarginatus

5 11.9. 19.9. 26.9. 3.10. 9.10. 17.10. 24.10. 30.10. 7.11. 14.11. Camptocercus rectirostris

11.9. 19.9. 25.9. 3.10. 9.10. 17.10. 24.10. 30.10. 7.11. 14.11. e) Kalatoin f) Jousjärv 100 50

90 40

80 30 Alona guttata var. t ub. 70 20

60 10 Alonella nana 50

40 50

30 40

20 30

10 Alonella nana 20

50 10

40 50 30 Chydorus sphaericus 40 20 30 10 Alona affinis 20

10 10 Alona guttata

30

10 Alonella excisa 20 Acroperus harpae 10

10 Alona guttata var. t ub.

20 5 Pleuroxus truncatus Chydorus sphaericus 10

10 Acroperus harpae 5 Alona guttata 5 Alona affinis Camptocercus rectirostris 5 5 Graptoleberis testudinaria Pleuroxus truncatus 5

5 11.9. 1 9.9. 25.9. 3 .10. 9.10. 1 7.10. 2 4.10. 30.10. 7 .11. 14.11. Graptoleberis testudinaria 5 Alonella excisa

12.9. 19.9. 26.9. 3.10. 10.10. 17.10. 24.10. 30.10. 7.11. 14.11. Figure 14. (Continued).

34 Norway and Estonia (Mäemets 1961; Koksvik g) Hampträsk 100 1995), and therefore most likely it is also 90 active throughout the winter in some lakes in 80 southern Finland. The very low intensity of 70 gamogenesis in A. nana in the oligotrophic 60 lakes Iso Majaslampi, Pieni Majaslampi, and Alonella nana 50

40 Iso Lehmälampi suggests that A. nana depends

30 mostly on parthenogenesis in these localities, 20 although Pieni Majaslampi`s food web is 10 considered disturbed. In addition, the relative proportion of A. nana increased in these lakes 10 Alonella excisa towards winter, suggesting that it thrives under

20 autumnal conditions, and probably continued

Chydorus sphaericus 10 its active period as parthenogenetic populations and was perennial. Those lakes in which a Dispalarona rostrata 5 high intensity of gamogenesis in A. nana was 5 Alona intermedia encountered had somewhat higher conductivity, 5 Alona rectangula 5 nutrient status, and pH (Hampträsk, Tuhkuri) Graptoleberis testudinaria 5 or water color (Kalatoin, Jousjärv) than the Alona guttata var. tub. other lakes (Fig. 14, Table 2). In addition, the 12.9. 19.9. 26.9. 3.10. 10.10. 17.10. 24.10. 30.10. 7.11. 14.11. high intensity of gamogenesis of A. nana was Figure 14. (Continued). associated with those chemical water properties (high pH, conductivity, nutrient status, and color) that prevail in dystrophic and meso-/eutrophic lakes. The high intensity of gamogenesis in A. nana indicates that the species was forced to rely more on gamogenetic reproduction in these lakes. It is possible that the limnological conditions were such in these lakes that it could not survive throughout the winter; e.g. in Jousjärv the oxygen concentration was low during winter (Table 2). It is also possible that in these lakes other environmental stressors may have caused A. nana to reproduce with abundant gamogenesis to ensure the survival of future populations with high genetic variability.

3.2.3. Population sizes and abundance of gamogenesis (III)

For quantitative observations three of the study lakes were monitored by activity traps during the open-water season (early May-late October) in 2006 (Table 4, paper III). The number of trapped parthenogenetic chydorid individuals during the season was clearly lower in lakes Tuhkuri and Iso Lehmälampi than in Kalatoin. Whiteside et al. (1978) indicated that the numbers of chydorids were much higher in vegetated habitats than in benthic habitats and, in addition Zingel et al. (2006) showed that the abundance of chydorids was higher in the littoral macrophyte habitat than in habitats without aquatic vegetation. Whiteside (1974) stated that the patchy distribution of chydorids may be due to many ecological interactions, such as heterogeneous habitats, aggregation of food, or predation. In the present study the habitat quality at the sampling sites varied from dense Sphagnum vegetation in Kalatoin to soft-sediment bottom with scarce macrophytes in the other two lakes. Therefore, Kalatoin probably provided the highest number of, as well as the most diverse, microhabitats for chydorids. The aquatic vegetation also provides shelter from predators and epiphytic algae for food, whereas the lack of appropriate microhabitats at the sampling sites in the other two lakes may have resulted in the lower numbers of chydorids. The number of chydorids sampled with the sweep net in 2005 was also very

35 high in other parts of Kalatoin (I) and, although exact abundances were not measured, suggests that abundance of chydorids during the summer in this lake can be very high. Only a few gamogenetic individuals were encountered during midsummer at the sampling site in Tuhkuri and none during the autumn, suggesting that autumnal gamogenesis did not occur at the sampling site during the autumn of 2006. In contrast to Tuhkuri, numerous gamogenetic individuals were trapped at the sampling sites in Kalatoin and Iso Lehmälampi during the autumn, from late August onwards, in similar proportions. It is very unlikely that autumnal gamogenesis was lacking entirely in Tuhkuri in 2006, since the intensity of autumnal gamogenetic individuals was 13% for males and 21% for gamogenetic females a year before (II). The activity traps used in the present study collect chydorids only from a very restricted area (cf. Fig. 8). Thus, the lack of gamogenesis in the Tuhkuri site, with its soft-sediment bottom and scarce vegetation, may imply that sexual individuals do not occur uniformly in the littoral zone but, instead, occur unevenly among habitat types. Recently, Örnólfsdóttir and Einarsson (2004) documented variation in the relative proportion of Eurycercus lamellatus males among different sampling sites of Lake Myvatn in Iceland. Their results may also suggest the presence of habitat-specific patterns in chydorid gamogenesis. Frey (1982) detected in November of five different years only slight variations (between 69 and 78%) in the percentages of gamogenetic chydorid populations in lakes in northern Indiana, suggesting small yearly differences in gamogenesis. Although the activity traps provided quantitative samples in the present study, relative proportions, i.e. intensities, of gamogenetic individuals were also calculated for each sampling site. The results suggest that yearly variation occurs in gamogenesis; e.g. in Kalatoin the maximum intensity of males during the autumn 2006 was 55% and that of gamogenetic females was ca. 13%, compared with 38% and 40%, respectively, in 2005 (II). The yearly differences were most striking in Iso Lehmälampi; the maximum intensity of males was ca. 60% and that of gamogenetic females was ca. 15% in 2006, while both were 12% in the 2005 sweep net samples. However, these differences may also be habitat-specific. Alonella nana was the most abundant species, although its population sizes and dynamics varied widely among the sampling sites. However, a distinct period for its autumnal gamogenesis occurred only in the sampling site of Kalatoin, whereas in Iso Lehmälampi only a few males were found. Its intensity of gamogenesis varied greatly in 2005 and high intensity was related to dystrophic and mesotrophic conditions (Fig. 14, paper II), and therefore it is possible that environmental conditions at the trap sampling sites were reflected in the reproductive patterns of the A. nana populations. Its intensive gamogenesis occurred in dystrophic Kalatoin, but only a few gamogenetic individuals of this species were encountered in the oligotrophic lakes Tuhkuri and Iso Lehmälampi, fitting well with the reproduction patterns observed during the year 2005 (Fig. 14, paper II). In addition, different Alona affinis populations showed variable gamogenetic reproduction patterns; two of the three populations showed signs of a slight summer gamogenesis, since early instar males were observed in low numbers in Kalatoin and Tuhkuri. Since the sampling interval was two weeks, it is not clear whether the presence of early instar males indicates that sexual reproduction between adult males and gamogenetic females eventually occurred. Since juvenile males of A. affinis were observed during the maximum chydorid abundance in the two lakes, and if their presence indicates eventual sexual reproduction, it may have been a response to crowding and competition as in Kalatoin in 2005 (I). Males were more abundant in the traps in lakes Iso Lehmälampi and Kalatoin. This phenomenon may be related to the suggestion that different environmental stimuli control the production of males and gamogenetic females, made by Shan (1974). However, this remains only a suggestion, and for an accurate conclusion, more laboratory and field experiments are needed. In the present study, the higher abundance of males during the autumn may also have been caused by the fact that males move more actively than gamogenetic females. Shan (1969) and Van Damme and Dumont (2006) showed that

36 mating in chydorids requires vigorous swimming of males and therefore they may be caught in the traps more easily than gamogenetic females.

3.3. Sexual reproduction reflected in sediments

3.3.1. Chydorid ephippia in the Hampträsk core (IV)

A short sediment sequence from Hampträsk was analysed for subfossil chydorid ephippia, among other paleolimnological proxies (Cladocera, Chironomidae, LOI), to test ephippium analysis under known historical warm and cold climate periods (IV). The results of ephippium analysis from the 46 cm long core showed that in the lower part of the core (46-24.5 cm, ca. 1300-1600 AD) chydorid ephippia occurred mostly at low proportions (TCE < 2%), indicating a relatively long parthenogenetic reproduction period and open-water season in southern Finland. Since the core does not extend to a time before the Medieval Warm Period (MWP) that is usually considered to have occurred ca. 1000- 1300 AD (Crowley and Lowery 2000; Moberg et al. 2005), it remains unknown whether the TCE was even lower before 1300 AD. A mild climate was also suggested by the other proxies, e.g. by the presence and relatively high abundance of warm-adapted chironomids and the high organic content of the sediment. This apparently warm period was interrupted for a short period during the 15th century, as the TCE increased suddenly from ca. 1% to 4-5%. The sudden change in the TCE was most likely associated with a cooling climate, suggesting that the open-water season became shorter. The occurrence of a short period of cooler climate was supported by chironomid and cladoceran data, which showed evidence of changes in community structure and diversity. There was also a change in the sediment properties, as the organic content slightly decreased, suggesting input of minerogenic material or lower organic production. Subfossil chydorid ephippia, together with the other proxies, indicated a considerable change thereafter which began in the 16th century and was most pronounced ca. 1700 AD, most probably reflecting the Little Ice Age (LIA). This period of climatic cooling was clearly indicated by the TCE record as a distinct increase, suggesting that the open-water season became considerably shorter. The TCE first increased to 4-5% (22-21 cm), and after a short decrease it reached a maximum, 5.5% (19 cm), thus having two distict maxima, suggesting that the LIA was not a uniformly cold event. Simultanously with the increased TCE, cold-adapted chironomid taxa increased markedly and thermophilous taxa were very rare. Also, the cladoceran community structure changed and the LOI decreased radically. Most probably the higher TCE around 1700 AD was the result of a shortening of the open-water season, changing the relative duration of the two chydorid reproductive periods. Sarmaja-Korjonen and Seppä (2007) found a similar scale of increase in TCE during the cold 8200 cal BP event in southern Finland. Traditionally, the LIA is considered to have covered the time period ca. 1500-1800 AD, although the evidence has varied greatly regionally and between different paleolimnological proxies. It has been stated that climatic cooling began in Europe in the mid-16th century and that the coolest period was the 17th century (Bradley and Jones 1993; Fischer et al. 1998; Jones et al. 1998; Jones and Briffa 2001). Tiljander et al. (2003), who examined annually laminated sediments from central Finland, found indications of cooler periods at approximately 1580-1630 and 1650-1710 AD. There is also historical data that the 17th century was cold in northern Europe (Grove 1988). In the years 1601-1602 and 1633-1635 AD almost the entire area of Finland experienced crop failures. Hampträsk is situated in the municipality of Sipoo in southern Finland and in the area several crop failures occurred in the late 17th century, and the harvest were also small during the entire 1720s (Rantanen and Kuvaja 1994).

37 The timing of the TCE maxima at ca. 1700 AD, as well as the other paleolimnological evidence from Hampträsk, fit quite well with the data mentioned above regarding climate cooling during this period. After the LIA, during the 18th century, the TCE decreased several percentage points to pre-LIA values, suggesting that the climate became warmer and that a longer open-water season prevailed, probably being of approximately the same length as presently. Similar changes were also indicated by the other proxies, as cold-adapted chironomids were replaced by warmer water taxa and the LOI increased. In addition, chironomid and cladoceran records suggest that after the LIA, during the 18th century, the nutrient status of the lake rose towards that of modern times. This most probably resulted from an increased input of nutrients as anthropogenic activities and land use increased in the catchment (Rantanen and Kuvaja 1994). In the mid-18th century the TCE increased from 1-2% to 3% and has since remained at that level. The chironomid record suggests that the climate warmed during the 18th century, and therefore it is unlikely that the increase in the TCE was caused by changes in the length of the open-water season. In addition, the increase in the TCE was due to ephippia of a single species, Alonella nana, whereas, with a changing climate and open-water season, ephippia of most species would be expected to increase (cf. Sarmaja-Korjonen 2003). In the sequence ca. 1700 AD onwards, ephippia of A. nana occurred consistently with proportions of 1-3%, whereas they were sporadic before the LIA. In this upper section, ephippia of other chydorid species occurred only sporadically. Sarmaja-Korjonen (2003) presented results according to which proportions of ephippia of a single species can increase markedly, and thus increase the TCE, as a result of some environmental stress irrelevant to the length of the open-water season. In such cases, chydorids apparently can somehow increase their sexual reproduction to ensure survival as resting eggs and adaptation by genetic variability. Thus, the continuous occurrence of A. nana ephippia in Hampträsk after the LIA suggests that it experienced a long period, from the mid-18th century until the present, of species-specific environmental stress. Since the other proxies from Hampträsk suggest that the productivity of the lake increased after 1700 AD, the high proportion of A. nana ephippia may be due to factors related to altered water chemistry inducing stress on the species. According to contemporary results (Fig. 14, papers II, III), Alonella nana appears to be dualistic in its reproduction patterns. In many oligotrophic lakes it produces very few ephippia, whereas in lakes with higher nutrient status and conductivity the intensity of its sexual reproduction is higher. It may possibly overwinter as asexual females in some lakes, but cannot survive in lakes with high nutrient status and conductivity, most likely due to low-oxygen conditions during winter, and therefore must rely on overwintering as ephippia (see above). The PCA results also indicate similar patterns in its sexual reproduction, as high proportions of ephippia in surface sediments were related to lakes with high pH, nutrient status and conductivity (Fig. 15), as is discussed below. In Hampträsk, the species apparently changed its reproduction mode from pre-LIA low intensity to after- LIA high intensity in response to the eutrophication process. This mode has prevailed until the present (II).

3.3.2. Chydorid ephippia in surface sediments (V)

Surface sediment samples from the nine lakes were analysed for subfossil chydorid ephippia to see whether there is variation in the TCE in the same climate region (V) and to see how contemporary sexual behavior is reflected in surface sediments (I, II, III). The TCE values were rather constant in six of the lakes, ranging from 3.2% to 4.4%. In Kalatoin the TCE was rather low (1.7%) and in Pieni Majaslampi anomalously high (8.9%). Since the lakes are limnologically and ecologically different (Table 2), but located in the same climate region (Fig. 6), the variation in the TCE is probably not due

38 to climate. According to Kultti et al. (2006) and Sarmaja-Korjonen (2007b), the usual TCE in surface sediments of lakes in southern Finland is ca. 2-4%, indicating a long parthenogenetic reproduction period. Thus, the TCE was rather similar (ca. 3-4%) in six of the nine lakes, and these results are in agreement with Kultti et al. (2006) and Sarmaja-Korjonen (2007b), specifically in that, although the majority of lakes in southern Finland have a TCE between 2-4%, divergent values occur. TCE in the surface sediments from nothern Finnish Lapland are clearly higher (Kultti et al. 2006; Sarmaja-Korjonen 2007b) than in southern Finland. According to Sarmaja-Korjonen (2007b), in a lake situated close to the pine limit, the TCE was 8.7% and in two lakes situated in very severe climate conditions above the altitudinal treeline, the TCEs were 26.2% and 30.0%. Therefore, the current result that a typical TCE for the study lakes situated in southern Finland was ca. 3-4%, whereas it is 26-30% in northernmost Finnish Lapland above the treeline (Sarmaja-Korjonen 2007b), clearly reflects the difference in climate, i.e. the length of the open-water season, which in southernmost Finland lasts ca. seven months and at high elevations in northernmost Finnish Lapland ca. three months. The chydorid species with the most variation in its ephippium proportions was Alonella nana, which was present in all the lakes and was abundant in most of them (Fig. 9), but its ephippia were found only in Kalatoin, Jousjärv, Hampträsk and Kangaslampi, i.e. in the dystrophic or meso-eutrophic/human- disturbed lakes. The PCA results (Fig. 15) and contemporary data (Fig. 14, paper II) showed that a high proportion of ephippia of A. nana was related to meso-/eutrophic and dystrophic status of the lakes and high water pH, conductivity and color. High proportions of ephippia of Alona affinis, Alonella excisa, and Acroperus harpae were related to acidic and oligotrophic conditions (Fig. 15), possibly indicating a similar dualism to that of A. nana. The contemporary samples also indicated that a high intensity of gamogenesis in A. excisa and A. harpae occurred in lakes with good oxygen conditions, low pH and low nutrients (II). In Kalatoin the TCE was very low indicating a low proportion of sexual reproduction in the chydorids. Because the nine lakes are located in the same climate region, other factors than the length of the open-water season appear to contribute to the TCE. The contemporary intensity of autumnal gamogenesis in the lake was observed to be

0 very high (II), although consisting mainly

1. KAL of gamogenesis in Alonella nana (Fig. 14),

Alo gut suggesting that gamogenesis is an important Kur lat reproduction mode during the autumn in the TUH Alo aff lake and could be expected to result in a high Rhy fal PML TCE. The abundance of chydorids, although Col not measured, was very high during the 2005 2 ILL TN Oxy monitoring. In addition, an extremely high axis HT abundance of parthenogenetic individuals Alo nan PCA TP Alo int Acr har was detected in the lake during the trap JJ IML Alo rus sampling in summer 2006 (III). Therefore, Alo exc the number of parthenogenetic individuals pH CondAlo tub Cam rec Chy sph and accumulated parthenogenetic remains

KL HL during the summer has apparently been so high that they have probably diluted the TCE. -1.0 In Iso Lehmälampi, in contrast to Kalatoin, -1.0 PCA axis 1 1.0 low gamogenetic intensity (II) and a low Figure 15. PCA ordination for subfossil chydorid abundance of parthenogenetic individuals ephippia in the surface sediments of the nine (III) could have caused the somewhat higher lakes and limnological variables. The codes are the same as in Fig. 10. TCE (5.4%) in surface sediment. Thus, it is

39 likely that summer population sizes can contribute to the TCE values. The very high TCE found in the surface sediment of Pieni Majaslampi is comparable to the TCE values in northern Finnish Lapland (Kultti et al. 2006), and therefore the high TCE cannot be caused by a shorter open-water season. Thus, the abnormally high TCE in the sediments of Pieni Majaslampi was either natural for this particular lake or due to some non-climatic environmental factor that had increased the TCE in the past. Since chydorids apparently can respond to environmental stress by increasing their sexual reproduction, resulting in a higher proportion of ephippia in sediments, the high TCE in Pieni Majaslampi could be the result of such stress. This is discussed in detail below together with the Pieni Majaslampi short core results. Although the contemporary sexual reproduction patterns showed much variation in timing and intensity between lakes, populations and years (papers I, II, III), surprisingly, the TCE appeared to be at a general level of 3-4% in six of the nine lakes. This suggests that climate and the length of the open-water season are the main regulators of the TCE in southern Finland, causing a low proportion of ephippia in surface sediments. The variation in the TCE between the six lakes was presumably due to local adaptations in reproduction patterns, e.g. in timing (I) and intensity of autumnal gamogenesis (II) or abundance of parthenogenetic and gamogenetic individuals during the open-water season (III).

3.3.3. Chydorid ephippia in the Pieni Majaslampi core (V)

A short sediment core (14 cm) from Pieni Majaslampi was analysed for subfossil chydorid ephippia to detect whether the high TCE value (8.9%) in its surface sediment was natural to this lake and had also prevailed in the recent past. This was apparently not the case, because in the lower samples, from 14 cm up to 6.5 cm the TCE was constantly ca. 3-5%. At 6.5 cm (the early 1980s), the TCE suddenly increased from 5.4% to ca. 10% and remained approximately at that level up to the top of the sequence. The increase was mainly due to an elevated proportion of ephippia of A. affinis and to a lesser extent of A. harpae. This change in the TCE occurred simultaneously with a change in the cladoceran community of the lake. The 3-5% TCE was comparable to the TCE values of the other lakes in the region before suddenly increasing twofold. This sudden increase from the TCE values typical to southern Finland to values found near the pine limit in Finnish Lapland (Sarmaja-Korjonen 2007b) evidently is due to some other forcing factor than sudden climate change, since ephippia of only two species increased. A similar sudden and considerable increase in TCE has also been found previously in a sediment sequence (Sarmaja-Korjonen 2003), and was probably due to the response of Alona affinis to environmental stressors following changes in the food web caused by an increased nutrient state (discussed above). A comparable change in sexual reproduction of Alonella nana was found in the sediment sequence of Hampträsk (discussed above, paper IV), as, before the LIA, its ephippia were rare but after the LIA they were consistently present up to the surface. During this time the nutrient status of the lake apparently rose due to increased land use and the resulting erosion and input of nutrients. These two results support the assumption that under some conditions chydorids can increase their sexual reproduction. It is therefore probable that Alona affinis, and to a lesser extent Acroperus harpae, also responded to some environmental stressor or threat by increased sexual reproduction in Pieni Majaslampi in order to better adapt to the situation genetically as well as provide more ephippia to ensure survival. It is probable that Alona affinis, a species with very divergent ecological affinities (e.g. Fryer 1968; Quade 1969; Whiteside 1970; Whiteside et al. 1978; Tremel et al. 2000), and apparently a wide tolerance for ecological conditions, has exceptional flexibility in its sexual reproduction, further enhancing its tolerance (Sebestyén 1946, paper I). It is also possible that local variation in sexual reproduction of all chydorids can further enhance their ecological flexibility in surviving under divergent environmental

40 conditions (Fig. 9, paper I). Unfortunately, the current sexual intensity of A. affinis in Pieni Majaslampi could not be studied reliably because presently it is very rare in the lake (I). Apparently, the aquatic food web was severely damaged by the perch introductions in 2002 and consequently, A. affinis was almost extirpated, as discussed above. Pieni Majaslampi has been very acidic since the early Holocene (Korhola and Tikkanen 1991), and received increasing atmospheric loading of trace elements during the 1960-80s (Virkanen et al. 1997). One possible explanation is that in the early 1980s this directly or indirectly caused the crossing of a threshold (Scheffer et al. 2001) for Alona affinis and to some extent also for Acroperus harpae, which responded with increased sexual reproduction. Other cladocerans can sense chemical information in their living environments and change their reproduction mode according to the changing chemical environment (e.g. Zadereev 2003; Zadereev and Lopatina 2007). It is possible that in Pieni Majaslampi chydorids sensed the changes in water chemistry e.g. the increase in harmful elements towards a physiologically damaging level, and therefore ensured survival by sexual reproduction. The contemporary sampling (II) and sediment results (Fig. 15, paper IV) showed that the intensity of gamogenesis in Alonella nana was forced by water chemistry, being more pronounced in lakes with high pH and nutrient status. The response to chemical changes in chydorids is also supported by the results of Kiser et al. (1963).

4. Conclusions

The results of this thesis demonstrate that chydorid gamogenesis is rather consistent among different lakes in southern Finland and that climate-related factors act as the main inducers and regulators of autumnal sexual reproduction. However, very weak and inconsistent summer gamogenesis occurred in some lakes and this is clearly not forced by climate cues, but is probably due to non-climatic local environmental factors, such as crowding or invertebrate predation. Chydorid species and populations among the lakes exhibited wide variation in the intensity, induction time and length of autumnal sexual reproduction. These variations apparently act as mechanisms for allowing local adaptations that enhance the ecological flexibility of chydorid species, allowing them to better inhabit a wide range of environments. Chydorid gamogenesis is much more complex than previously believed and therefore more detailed studies on contemporary chydorid gamogenesis in the laboratory and in the field are required from a wide limnological and geographical range. The general level of surface sediment TCE was ca. 3-4% in most of the lakes, reflecting an average proportion of sexual reproduction during the open-water season in this particular climate region. The variation in the TCE was much lower than could be expected on the basis of the monitoring results of two summers. This suggests that some of the variation detected by contemporary monitoring may derive from differences between sampling sites and years, which are smoothed out in the sediment samples, because they provide an average over the entire lake area and several years. In spite of the smoothing, some of the variation in contemporary sexual reproduction is a result of lake-specific factors and always causes some variation between lakes in the TCE value even in the same climate. This was most clearly observed in one of the lakes where the very high abundance of parthenogenetic individuals during summer diluted the high autumnal gamogenetic intensity in the resulting low TCE value. Accordingly, the TCE is always connected to various ecological interactions in lake ecosystems and therefore is always lake-specific. Hence, before the construction of a climate reconstruction model, the natural range of TCE in a certain climate area must be known. The study provided further proof that the TCE may also increase due to non-climatic factors. Hypothetically, worsening of climate conditions is detected in the TCE as an increase of ephippia of all chydorid species, since shortening of the open-water season affects the relative proportions of the

41 two reproduction modes and consequently the proportion of accumulating carapaces and ephippia in sediments. The sediment results indicated that the TCE can suddenly increase due to ephippia produced by only one or two species. This indicates that at least some chydorids, possibly all, can increase the production of resting eggs when under sufficient pressure from environmental conditions, thus ensuring the survival of their populations and gaining better fitness via genetic variability.Thus, some environmental factors may act as local and species-specific environmental stressors. The actual mechanism of the increased sexual reproduction seen as an anomalously high TCE in sediment is still not totally clear. However, the intensity of autumnal gamogenesis varied greatly in environmentally different lakes, being highest in disturbed and fish-free lakes, suggesting that it is a major adaptation mechanism for chydorids. Thus, it is probable that it is the increased intensity, i.e. the larger proportion of sexual individuals in autumnal populations, that results in a larger proportion of ephippia in sediments and a higher TCE. The sexual reproduction period may also continue longer into the autumn and contribute to the TCE. Although weak summer gamogenesis was observed, no indication of induction of the continuous gamogenetic period before its climate-forced time was observed. The results of this thesis show the utility of ephippium analysis, together with subfossil Cladocera analysis, as a paleoclimatological and paleolimnological tool. The method clearly detected the coldest period of the LIA, and it may also detect paleolimnological changes. For example, if species-specific environmental stressors can be identified with contemporary studies, they could be used as proxies of such environmental conditions in down-core studies. However, more ecological and paleolimnological research is required to detect the level of natural variation and existence of non-climatically forced gamogenesis in contemporary reproduction patterns and in TCE among different lakes and regions. To develop a quantitative TCE-based climate reconstruction model, the natural TCE in surface sediments in different climates must be further studied. The number of lakes in the present study was far too low for determining the exact range of the natural TCE, and therefore a much larger set of surface sediment samples is needed. It is also important to recognize those lakes where the TCE is not only a reflection of the length of the open-water season, but that are non-climatically forced and could interfere the model. Since the model will be used in down-core studies, it would be important to choose surface samples from lakes as pristine as possible because human impact may easily trigger environmental stressors, as seen in the present study. Furthermore, the results of ephippium analysis should always be interpreted individually for each lake and in the context of other paleoecological proxies.

42 Acknowledgements

The thesis was funded by the EPHIPPIUM-project (grant no. 1107062) of the Academy of Finland, the Finnish Concordia Fund, and the Finnish Graduate School of Geology, to whom I express my gratitude. I want to deeply thank my supervisor Dr. Kaarina Sarmaja-Korjonen for the knowledge and skills about cladocerans and science that she shared with me during this project. Besides her many professional qualities, I also truly acknowledge and respect her personal qualities: enthusiasm, humor, idealism, slight anarchy towards authorities and dog- and home-loving (just to mention some of the most important), that provided me a creative, free, and soft scientific work environment. I thank reviewers Prof. Krystyna Szeroczyńska and Dr. Milla Rautio for valuable and construcive comments on the manuscript. Additionally, I would like to warmly thank Prof. Veli-Pekka Salonen for supporting me in completing this thesis and my studies. The people at the Department of Geology that helped me in the field: Dr. Seija Kultti, Dr. Arto Miettinen and Dr. Anu Hakala; in the laboratory: Dr. Juhani Virkanen and Tuija Vaahtojärvi; with the bureaucracy: Kirsi-Marja Äyräs and Dr. Mia Kotilainen; with computers: M.Sc. Mikko Haaramo; and with work-related problems through discussions: M.Sc. Maija Heikkilä and M.Sc. Laura Arppe are all equally and warmly thanked. My dear parents Riitta and Reijo Nevalainen always supported me in following my dreams and thought that I would do fine in whatever I wanted to do, although my plans and dreams about my future were not always that realistic. Therefore, I thank them from my heart! Above all, I humbly thank my beloved soul mate and husband M.Sc.Tomi Luoto, and our precious dogs Onni and Helmi. Thank you for love, encouragement, strength and everyday life! In addition to being a true companion, Tomi has been a professional and hilarious colleague in science and helped me millions of times with things related to this thesis. I truly appreciate his efforts and ideas on my articles and this thesis! And, it was Tomi who unselfishly, with his magical technical skills, helped me in the field again and again and again…thank you so much! Perhaps next time by a lake we can just relax and enjoy!

43 References

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54 Appendix

Description of the holotype and ephippium of Unapertura latens sp. n.

Holotype, a parthenogenetic female (Fig. 11) from Kalatoin, southern Finland is preserved in glycerol gelatine and stored at the Department of Geology, University of Helsinki. The size of the holotype is ca. 250 µm in length and 180 µm in height. Carapace. In a lateral view the body shape is oval, resembling closely the Alona species. The anterior dorsal margin is convex and evenly curved. The ventral margin of the carapace is even and covered by marginal setae. The carapace is longitudinally striped with very faint lines which, in the holotype, are clearly visible only in the posterior part. The length of the carapace is ca. 200 µm. Head. In a lateral view the rostrum is relatively long, pointed and curved towards the body. The ocellus is situated at the base of the rostrum and is approximately of the same size as the compound (complex) eye. The anterior part of the labrum is evenly rounded and the posterior part is straight, without a clear ventral apex. The second antennae have three segments and end with long setae. Postabdomen. The dorsal margin of the postabdomen bears ca. 5 strong denticles, which enlarge towards the distal end, and groups of lateral setae. At the beginning of the preanal margin there are some long setae. The postabdominal claws are relatively strong with a basal spine. The length of the postabdomen and postabdominal claws together is ca. 100 µm. Ephippium. In lateral view most of the anterior and anterior-ventral margin is ripped away and the shape of the ephippium is therefore almost globular (Fig. 12). The dorsal margin and posterior-dorsal corner are thickened, which are visible as darker areas in the ephippium. The resting egg lies in the dorsal part of the ephippium near the anterior margin and it is oval-shaped. The size of the ephippium is ca. 200 µm and the resting egg ca. 100 µm

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