Comparison of five strains of a parthenogenetic species, Macrotrachela quadricornifera (Rotifera, Bdelloidea). I. Life history traits

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1 See discussions, stats, and author profiles for this publication at: Comparison of five strains of a parthenogenetic species, Macrotrachela quadricornifera (Rotifera, Bdelloidea). I. Life history traits ARTICLE in HYDROBIOLOGIA FEBRUARY 1991 Impact Factor: 2.28 DOI: /BF CITATIONS 36 READS 7 1 AUTHOR: Claudia Ricci University of Milan 102 PUBLICATIONS 2,192 CITATIONS SEE PROFILE All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately. Available from: Claudia Ricci Retrieved on: 13 January 2016

2 Hydrobiologia 211: , Kluwer Academic Publkhers. Printed in Belgium. 147 Comparison of five strains of a parthenogenetic species, Macrotrachela quadricornifera (Rotifera, Bdelloidea) I. Life history traits Claudia Ricci Dipartimento di Biologia Animale, Universit2 di Torino, via Accademia Albertina 17, Torino, Italy Received 25 September 1989; in revised form 31 January 1990; accepted 20 March 1990 Key words: Rotifera, bdelloids, life history, life tactics, parthenogenesis, intraspecies differences Abstract Five strains of the species Macrotrachela quadricornifra were cultured under experimental conditions. Four strains were collected from different environments, geographically paired, each pair consisting of an aquatic habitat (strains P and C) and a moss habitat (strains Ml and M2). Another population, F, was collected from an alpine stream and was added for comparison. In preliminary experiments food preference was evaluated by giving each strain five different food sources. Life table experiments were run at 16, 20 and 24 C. The strains differed in response to food types, to temperatures and in life history traits. In detail, while the aquatic populations responded to temperature by increasing or decreasing their life spans and their rates of increase as expected, the moss populations responded irregularly. Moreover, two types of life tactics could be identified: the aquatic strains, P, C and F invested maximally in reproduction, reducing their survival, while the moss strains, Ml and M2, were long-lived and devoted less resources to reproduction. A single link cluster analysis of some life-history parameters confirmed this divergence. The differences among the strains may easily be regarded as the results of adaptation to the different environmental conditions faced by the strains in nature. Whether the differences are genetic or phenotypic is still an open question. Introduction If a life-history tactic is a set of coadapted traits designed, by natural selection, to solve particular ecological problems (Stearns, 1976), given different ecological problems, the traits among the population facing each habitat are expected to differ in order to cope with specific environments. If the populations belong to different species or higher taxa, the differences in their life histories are likely to be the outcome of evolutionary processes and to represent genetic differences. But if the populations belong to the same species, the differences might be attributed to 1) genetic adaptation to local environmental pressures; or 2) phenotypic plasticity of a sort of general purpose genotype. Stearns (1980) questioned whether or not lifehistory tactics do exist and are discernible at the intraspecific level, and subsequently found that differences in life-history traits among stocks of mosquitofish are genetically based (Stearns, 1983). However, the differences in life-history tactics are much more evident when taxonomic entities higher than the species are considered, because of the phylogenetic constraints (Brown,

3 ). A life-history study of some species of bdelloid rotifers showed that the selective pressures exerted by different habitats may select and modify reproductive tactics of the different species in accord with the predictions of the stochastic theory formalized by Stearns (1977) (Ricci, 1983). But, when dealing with different species, the results may be attributed to the different evolutionary histories of the species, and therefore to genetic differences. In the present study life histories of live populations of the same species Macrotrachela quadricornifera Milne were investigated to ascertain whether the same lifehistory tactics are also maintained at the intraspecific level. The relatedness of the populations was obtained by examining life-history traits and isoenzymatic patterns, the latter being the subject of the following paper (Pagani et al., in press). Material and methods Macrotrachela quadricornifera is a very common species, widely distributed, inhabiting both aquatic and terrestrial habitats. Like all bdelloid rotifers, it reproduces by apomictic thelytoky only. The species concept is questionable for such a taxon (Koste & Shiel, 1989), only a morphological criterion can be used. M. quadricornifera is morphologically well recognizable because of two peculiar appendages dorsal to the spurs (Ricci & Melone, 1984). The populations were collected from different environments, geographically paired, each pair from an aquatic (P and C strains) and a moss (M 1 and M2 strains) habitat. One would expect the paired populations to share common founders sometimes. Another population (F) collected from Lombardia, but from a geographically distant habitat, was included for comparison. The animals were originally collected from five sampling sites: M2 from a wall moss near Milan, Ml from a wall moss in the Apennines, F from a cold stream in the Alps, P from a stream in the Apennines, C from a spring-fed pond near Milan. The rotifers of the strains varied in size: M2 400 pm, M 1400 pm, F 400 pm, P 600 pm and C 600 pm. Bottom samples from water courses and spring-fed ponds, and moss samples from walls were collected in the field and carried to the laboratory. Specimens of M. quadricomzjka were extracted from the samples, identified under a light microscope and cultured individually. The stemmed populations were kept in an incubator at 20 C, with photoperiod of 12L/12D. The culture conditions consisted of de-ionized water (Ricci, 1984) and a mixture of the algae Chlorella pyrenoidosa and Chlamydomonas reinhardti, the yeasts Rhodotorula rubra and Saccharomyces cerevisiae and the bacterium Escherichia coli. The rotifers of each strain with best reproductive performances were then selected and used to obtain clonal populations. From each clone thus established, three eggs were collected and placed individually in glass wells of about 3 ml capacity. The hatching rotifers were used to test the effects of food quality: three replicate populations were established and fed each food. Every other day food and medium were renewed and the living animals were counted under a dissecting microscope. For each experiment, the regression line for the natural logarithm of animal numbers versus days was calculated. The slope of the line represented the population growth rate (r) value, assuming an exponential growth model. The r values for the three replicates were averaged and their standard error calculated. The food supporting the greatest increase of the population was considered the best and used in subsequent experiments. Life table experiments were carried out at 16, 20 and 24 C on cohorts of about thirty individuals. The eggs were collected from the clonal populations, placed in glass wells of about 3 ml capacity and kept in the incubator. Daily, medium and food were renewed and the hatched rotifers were checked to register age-specific survival and fertility: eggs laid were counted and removed. From age-specific survival (1,) and fecundity (m,) tables, the following parameters were calculated: net reproductive rate (Ro = C m, l,), intrinsic rate of natural increase (r from 1 = X m, 1, e - ), mean generation time (T = lnro/r), reproductive effort (Ro/n reproductive days). In addition,

4 149 duration of development, age at first reproduction and mean life span were considered. To assess the effect of temperatures, the different parameters were compared by one-way ANOVA. Some parameters, the non-autocorrelated ones, were standardized by subtracting from each variable the mean of the parameter and the difference obtained was divided by its standard deviation. From this rectangular matrix a triangular matrix was then calculated through the Euclidean distances. On the latter dissimilarity matrix, single link cluster analysis was performed (NTSYS) (Rohlf, 1987). Results Food quality Each strain was fed each of the individual cultures of algae, yeasts and bacteria. In Table 1 the rate of increase (r) for each strain is the average value for three replicates. Algae were suitable food for most populations, but strain Ml was unable to east Chlorella pyrenoidosa. When fed with Chlamydomonas reinhardti, all the populations were able to reproduce, and attain a relatively high density. Escherichia coli was the best food for the M2 strain, but sustained only poor growth of the others. Of the yeasts, Rhodotorula rubra, which has a mannan-chitin cell wall, was unsuitable for all populations. Saccharomyces cerevisiae supported the growth of a couple of strains, namely P and C, but was unsuitable for strains Ml and M2 which did not survive when fed S. cerevisiae alone. In general, algae, and especially C. reinhardti, represented the best food among the ones tested for most populations, with the exceptions of M2 which preferred to feed on E. coli, and P which was sustained better by S. cerevisiae. Lif cycles The life spans of all strains were affected by temperature: the higher the temperature, the shorter the life time. However, while the decrease in longevity was significant (P < 0.05) for C, F and P strains, the difference was not as significant for the M 1 and M2 populations (Table 2). The age at first reproduction also decreased increasing temperatures and the decrement was significant for all strains. The reproductive effort, roughly estimated as number of eggs * female - * number of reproductive days -, increased linearly with temperature for strains C, P and F, but not Ml and M2. In fact, there was a significant difference in reproductive effort of M2 only at 24 C, and the Ml strain did not exhibit any different breeding efforts at the different temperatures. Total fecundity is related to the reproductive effort, of course. Table 1. Population growth rates (r + S.E.) for the strains fed with different foods. C F P Ml M2 Chlorella pyrenoidosa (0.006) (0.006) (0.005) no growth (0.003) Chlamydomonas reinhardti (0.007) (0.005) (0.008) (0.015) (0.013) Escherichia coli (0.003) (0.004) (0.006) (0.017) (0.016) Rhodotorula rubra i (0.003) (0.006) (0.003) no growth (0.003) Saccharomyces cerevisiae (0.007) (0.004) (0.017) no growth no growth

5 150 Table 2. Life table parameters ( + S.E.) for the five strains cultured at three temperatures. * Significance at F-test. # : Size of the cohort; developmental time, life span, age at first reproduction, mean generation time (T): days; net reproductive rate (Ro): eggs. female-. life time- ; reproductive effort: eggs. number of reproductive days- ; intrinsic rate of natural increase (r): days. # Develop- Life span Age at Reprod. r T ment (SE.) EE.) first repr. effort time (S.E.) (S.E.) C 16 20" 24" ** 10.43** 1.38 ** 0.45** (0.32) (0.61) (0.31) (0.03) 31.05** 22.33** 4.00** 0.93** (0.34) (0.10) (0.20) (0.04) 24.38** 21.43** 2.86** 1.30** (0.51) (0.41) (0.08) (0.02) F 16" 20" 24" ** 21.83** 6.11* 0.15 ** (0.36) (0.25) (0.13) (0.01) 36.08** 24.61** 6.13* 0.85** (0.42) (0.26) (0.14) (0.01) ** 21.96** 5.38* 0.94** (0.44) (0.32) (0.13) (0.02) P 16" 20" 24" * ** 0.11** (3.19) (0.90) (0.16) (0.04) 32.95* ** 1.01** (1.26) (0.61) (0.14) (0.04) 24.52* ** 1.43** (0.80) (0.81) (0.15) (0.05) Ml 16" 20" 24" * 0.41 (3.25) (1.61) (0.18) (0.03) * 0.52 (2.20) (1.14) (0.49) (0.03) * 0.41 (2.34) (1.41) (0.42) (0.04) M2 16" 20" 24" * 6.89* 13.25** 0.21** (1.93) (0.84) (1.20) (0.02) 45.18* 4.91* 1.83** 0.28** (2.10) (0.52) (0.56) (0.03) 40.10* 15.06* 5.30** 0.58** (2.52) (1.62) (0.25) (0.05) ** P < 0.05 * P = 0.05 This parameter was expected to be temperature- ferences by the F-test. Strictly related to the independent and to remain constant at the three previous parameters, the rate of natural increase temperatures, but this prediction fits P and Ml (Y) increased with temperature in all strains, strains only. Actually, the production of eggs reaching lower values in M 1, M2 and F increased significantly in C with temperature, ( ) than in P and C ( ) at the decreased significantly in F and produced one highest temperature. As a consequence, mean anomalous value in M2, causing significant dif- generation time (T) was longer in Ml, M2 and F

6 WC x C fi\,.,, Ix, F % I mx l!l!i mx , lliy!l I x.... P mx &;..._ I,,.., :, L. l!!l 1 Ix l....., Ml , 4, M2 '. 'A_ '...., ' a D Fig. 1. Age-specific fecundity (m,) (solid line) and age-specific survival (I,) (broken line) curves for the five strains of Macrotrachela quadricomifera at 16, 20 and 24 C. Age in days.

7 152 than in P and C at all temperatures. The duration of development which decreased with increasing temperature, differed among the strains: in general, it was longer for P and F than for C, M 1 and M2. The age-specific fecundity (m,) and survival (1,) were plotted against animal age to obtain the fecundity curves (solid line) and the survivorship curves (broken line) of the strains at the three temperatures (Fig. 1). At 16 C, the lowest temperature, the survivorship curves for C and F are very steep, denoting that the whole cohort died in a very short time at the end of its breeding period. On the contrary, P, Ml and M2 deaths were more scattered throughout the life span; their survival curves, in fact, were nearly diagonal but, while in P and M 1 the first deaths were recorded at a young age (younger than twenty), no death of M2 was registered until the age of 50. According to the fecundity curves, all rotifers at 16 C reproduced for quite a long time, starting around the age of ten and stopping when 70 days old. Only one strain, C, stopped reproduction much earlier. No maximum m, value can be recognized for any strain, in fact all fecundity curves are rather flattened; C, F and P animals laid about one egg every day on average, while Ml and M2 had m, values much lower than 1. At 20 o C, the aquatic populations (C, P, and F) had steep survivorship curves, while the moss strains (Ml and M2) maintained the diagonal pattern of survivorship. The same was true at 24 C. At 20 C, the fecundity curves of the aquatic populations tended to be triangular: a maximum m, (about 2 eggs day- female- ) at younger age was recognized, afterwards the reproductive output decreased but did not stop until the end of the life time. On the contrary, Ml reproduced with small effort throughout its life time, and M2 stopped its reproductive activity quite early, when 30 days old. At 24 C the triangular pattern of the fecundity curves of the aquatic strains, with evident early maxima (more than 2 eggs day- i female- ), was enhanced, but the flattened pattern in the moss populations was maintained, with the maxima (less or equal to 1 egg day - female - ) not easily recognizable. In order to understand better the effects of temperature on the life table parameters, the intrinsic rate of natural increase (I) and the net reproductive rate (Ro) are examined. In Fig. 2 the three values of r for each population are plotted against paired Ro. For each population, the set of points should be arranged linearly, with Ro invariant and Y increasing with temperature. This was true only for P, which could be defined as eurythermal under the test conditions. C performs well at the highest temperature, but badly at the lowest: it can be considered a warm stenothermal. On the other hand, the highest r value was coupled with the lowest Ro parameter of F strain at 16 C, with increasing temperature Ro increased but r decreased: the resulting line had a reverse slope and the population could be considered cold stenothermal. Ml and M2 strains behaved irregularly: neither set of paired values exhibited any linear configuration, but the maximum for M 1 and the minimum for M2 were at the intermediate temperature. To determine the degrees of dissimilarity among the strains a cluster analysis of some lifehistory traits was performed. These were duration of development, age at first reproduction, reproductive effort, net reproductive rate, rate of natural increase and mean life span. Not all the traits measured were included in the analysis 116 C 0 20 C A 24 C \I - Ml M net reproduction rate Fig. 2. Effects of temperature on intrinsic rate of natural increase (r) and on reproductive rate (Ro) for the five strains of M. quadricornifera. 30

8 153 because some, such as generation time and rate of increase, are clearly autocorrelated. The result of the analysis is given in Fig. 3. Two groups can be easily recognized: moss strains on one side and aquatic strains on the other. Ml and M2 are very similar, and among the aquatic strains, P and F are close and C seems to be a little apart from them. Discussion There were differences among strains with respect to food preference, response to growth temperatures and life-history traits. Among the five types of food, each strain seemed to prefer a different food : generally algae were the preferred food, but not all strains grew equally well when fed the same alga. It seems remarkable that there is variation among strains collected from the same habitat, although this has been reported for rotifers (Pourriot, 1977 ; Stemberger, 1981). All the foods tested are common in the habitats where the rotifers were collected, but they represent only a part of the available items that might be exploited by M. quadricomifera which, like most bdelloids, is a filterfeeder. Food size ranges between 2 and 15 pm, suitable for capture and ingestion by these small filter-feeders. The responses to the different temperatures differ greatly among the five strains: generally, two trends could be identified. With a few exceptions, when cultured at lower temperature, the aquatic populations lived longer and reproduced for _-i 0 Fig. 3. Dendrogram of the dissimilarities among the five strains of M. quadricornifera. See text for explanation. longer periods of time with lower reproductive efforts. At higher temperatures, they exhibited shorter life spans and increased their reproductive effort to about 1 egg day-, shortening the reproductive span. Only the P strain produced the same number of eggs at all three temperatures. C produced increasing numbers of propagules with increasing temperature and F exhibited the opposite. On the other hand, moss populations also change their life traits with temperature but the differences are much less evident: at increasing temperatures, life span is slightly shorter, reproductive effort is slightly increased but far from the 1 egg day- common to the aquatic strains, and the fecundity curve remained flattened rather than triangular. The three temperatures chosen more or less fall within the range of the environments the different strains inhabit: the only exception may be the highest temperature, 24 C, which is seldom reached by the alpine river from which the F population was collected. The variation of intrinsic rate of natural increase (r) and of net reproductive rate (Ro) at the three temperatures for each strain, given in Fig. 2, deserves a few comments. The two parameters are related and autocorrelated, but, while the rate of increase of poikilothermous animals depends on the metabolic rate, which increases with temperature, the net reproductive rate should be, in theory, unchanged by temperature (Fanestil & Barrows, 1965; Meadow & Barrows, 1971). Actually, two types of arrangement can be distinguished; the aquatic populations (P, C and F) reaction is dissimilar but linear, while the moss populations (Ml and M2) response seems to be at random. A possible explanation for this can be advanced in terms of the habitats the strains came from. P, C and F were collected from water bodies where temperature changes gradually and the animals can respond to it by lowering or increasing their metabolic rates. The F strain, collected from a rather cold environment, performed better at the lowest temperature than at the highest one. But the situation was very different for the moss inhabitants Ml and M2. For these, the values

9 154 were arranged randomly and no trend can be identified. A terrestrial moss changes its temperature according to the air conditions, and therefore its dwellers are used to sudden changes of the environmental temperature and probably do not behave according to it. In addition, temperature should not be regarded as the most effective limiting factor for moss dwellers: the availability of water and food are more important, Actually, terrestrial mosses dry out completely from time to time, unpredictably. Their inhabitants face such dramatic changes with interruption of active life and anhydrobiosis. Their mortality is likely to be due more to environmental shifts than to predation. Disregarding the effects of temperature, it seems that the different strains allocated their resources differently and two types of life tactics can be identified. The aquatic strain invest maximally in reproduction, reducing their survival, while the moss populations are longer-lived and devote lesser resources to reproductive activity. Also clustering the life-history traits (Fig. 3), the strains cannot be regarded as very close and perhaps at least two different species might be identified. The distinction, however, lacks any morphological support. The only morphological difference is in size, which is bigger for C and P, and smaller for F, Ml and M2, but F appears to be closer to P than to the Ml, M2 complex. The differences among the live strains may easily be regarded as the result of adaptation to the different environmental conditions faced by the strains in the field. Similar results were obtained when life-history tactics of different bdelloid species collected from the same kind of environments, water bodies and terrestrial mosses (Ricci, 1983) were studied. Two types of life history were identified by Ricci (1983) : species from aquatic environments, used to rather stable conditions, were characterized by triangular fecundity curves, greater reproductive effort (> 1) and shorter life span; species from terrestrial mosses, used to unstable and unpredictable conditions, were characterized by the opposite traits : flattened fecundity curve, reduced reproductive effort (< 1) and extended reproductive life. Senescent morphology could be recognised in the aquatic species, but was generally lacking in the moss species. 44. quadricornifera does not undergo senescence: only a few senescent rotifers were observed occasionally in the C strain. Both results lit quite well into the predictions of the bet-hedging hypothesis (Stearns, 1976) for strains with variable juvenile mortality. Moreover, life histories of terrestrial bdelloids are consistent with Murphy s (1968) prediction that variation in juvenile mortality can favor lesser reproductive efforts and longer reproductive age. Actually, bdelloids inhabiting terrestrial mosses are not equally resistant, if of different ages, to anhydrobiosis, which they have to face unpredictably from time to time. When cohorts of bdelloids of known age are desiccated, eggs and juveniles had lesser ability to recover than mature individuals (Ricci et al., 1987). As already stated, the animals of the five populations were ascribed to the species AI. quadricornifera on the basis of their morphology and this is the prevalent criterion applicable to parthenogenetic animals, but it is very limited. The analysis of life-history traits may be very helpful for detailing intraspecies differences (e.g. Stearns, 1980; Wilson, 1983; Dobson & Murie, 1987). The strains considered in the present study were sampled and cloned from natural populations and do not reflect the heterogeneity of the local populations, however their life traits detected under experimental conditions are consistent with the stability or unpredictability of their natural habitats. Different explanations might account for the intraspecific differences of life-history traits: 1) the populations are used to different habitats and perform differently, exhibiting a sort of phenotypic plasticity (Brown, 1983, 1985; Dobson & Murie, 1987; Baird et al., 1987). 2) they differ genetically (Stearns, 1983; and partly Lam & Calow 1989a, b). 3) in experiments done under artificial conditions, their adaptability to laboratory conditions can differ and bias the interpretation of the results.

10 155 Unhappily the last point cannot ever be evaluated because no carefully controlled field experiment of the life histories of small invertebrates like bdelloid rotifers can be run, nor can any laboratory data truly reflect the field situation. The present data Iit both explanations, further experiments should be run to see if the differences that have an adaptive significance can be ascribed to phenotypic plasticity or to genetic differences. However, it is important that the same life-history tactics of different species be maintained by strains of the same species. Just how similar the strains are is considered in the following paper (Pagani et al., 1991). Acknowledgements I thank Manuela Pagani, Umberto Fascia and Giulio Melone for their help in preparing the manuscript. References Baird, D. J., L. R. Linton & R. W. Davies, Life-history flexibility as a strategy for survival in a variable environment. Functional Ecol. 1: Brown, K. M., Do life history tactics exist at the intraspecific level? Data from freshwater snails. Am. Nat. 121: Brown, K. M., Intraspecific life history variation in a pond snail: the roles of population divergence and phenotipic plasticity. Evolution 39: Dobson, F. S. & J. 0. Murie, Interpretation of intraspecific life history patterns: evidence from columbian ground squirrels. Am. Nat. 129: Fanestil, D. D. & C. H. Barrows, Aging in the Rotifer. J. Gerontol. 20: Koste, W. & R. J. Shiel, Classical taxonomy and modern methodology. Hydrobiologia 186/187: Lam, P. K. S. & P. Calow, 1989a. Intraspecific life-history variation in Lymnaea peregra (Gastropoda: Pulmonata). I. Field study. J. anim. Ecol. 58: Lam, P. K. S. & P. Calow, 1989b. Intraspecific life-history variation in Lymnaea peregra (Gastropoda: Pulmonata). II. Environmental or genetic variance? J. anim. Ecol. 58: Meadow, N. D. & C. H. Barrows, Studies on aging in a bdelloid rotifer. II. The effects of various environmental conditions and maternal age longevity and fecundity. J. Gerontol. 26: Murphy, G. I., Pattern in life history and the environment. Am. Nat. 102: Pagani, M., C. Ricci & A. M. Bolzern, Comparison of five strains of a parthenogenetic species, Macrotrachelu quadricornifera (Rotifera, Bdelloidea). II. Isoenzymatic patterns. Hydrobiologia 211: Pourriot, R., Food and feeding habits of Rotifera. Arch. Hydrobiol. Beih., Ergebn. Limnol. 8: Ricci, C., Life histories of some species of Rotifera Bdelloidea. Hydrobiologia 104: Ricci, C., Culturing of some bdelloid rotifers. Hydrobiologia 112: Ricci, C. & G. Melone, Macrotrachela quadricornifera (Rotifera, Bdelloidea); a SEM study on active and cryptobiotic animals. Zoologica Scripta 13: Ricci, C., L. Vaghi & M. L. Manzini, Desiccation of rotifers (Macrotrachela quadricornifera): survival and reproduction. Ecology 68: Rohlf, F. J., Numerical taxonomy and multivariate analysis system for the IBM PC microcomputer. Metagraphics Software Corporation, California. 37 pp. Stearns, S. C., Life-history tactics: a review of the ideas. Q. Rev. Biol. 51: Stearns, S. C., The evolution of life history traits: a critique of the theory and a review of the data. Ann. Rev. Ecol. Syst. 8: Stearns, S. C., A new view of life-history evolution. Oikos 35: Stearns, S. C., The genetic basis of differences in lifehistory traits among six populations of mosquitoflsh (Gambusia allinis) that shared ancestors in Evolution 37: Stemberger, R. S., A general approach to the culture of planktonic rotifers. Can. J. Fish. aquat. Sci. 38: