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Density-dependent responses in some common lumbricid species
Accepted Manuscript Title: Density-dependent responses in some common lumbricid species Author: Alexei V. Uvarov PII: DOI: Reference:
S0031-4056(16)30113-5 http://dx.doi.org/doi:10.1016/j.pedobi.2017.01.002 PEDOBI 50478
To appear in: Received date: Revised date: Accepted date:
26-8-2016 28-12-2016 1-1-2017
Please cite this article as: Uvarov, Alexei V., Density-dependent responses in some common lumbricid species.Pedobiologia - International Journal of Soil Biology http://dx.doi.org/10.1016/j.pedobi.2017.01.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Density-dependent responses in some common lumbricid species\
Alexei V. Uvarov
A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninsky Prospect 33, 119071 Moscow, Russia;
e-mail address: [email protected]
Highlights 1. At field densities, populations of various earthworm species experience density dependence 2. In epigeic species, food competition was likely an important cause of density dependence 3. In endogeic species, density dependence was caused by spatial rather than food competition
ABSTRACT It is generally accepted that populations of most species experience some level of density dependence; however, this has rarely been shown for soil-dwelling invertebrate species, in particular for earthworms. Experimental data, mostly obtained at high laboratory densities, suggest intense intraspecific competition for resources and/or living space and density-dependent dynamics in earthworm populations, irrespective of their ecological group affiliation. In the present study performed in large field microcosms, I investigated whether density-dependent responses occur within the earthworm density gradients more realistic for the natural sites. Five lumbricid species from epigeic (Dendrobaena octaedra, Lumbricus rubellus), endogeic (Allolobophora chlorotica, Aporrectodea caliginosa) and anecic (Lumbricus terrestris)
2 ecological groups were tested. Soil systems populated with earthworm monocultures in large (20-L) microcosms were exposed in a beech-oak forest for 4.5 months; each species was represented by two (L. terrestris) or three treatments forming gradients of increasing density. In two endogeic species, manifold and generally similar density-dependent responses (a retardation of growth, maturation and reproduction rates, but higher mortality rates with density increase) were revealed, likely explained by spatial competition rather than by direct food competition. In each of epigeic species density-dependent responses were less variable and more speciesspecific. In contrast to endogeics, direct food competition was presumably a more important cause of density-dependence. In L. terrestris no significant density-dependent responses in adult earthworms were revealed; however, they need to be further investigated in relation to the age and territorial behaviour of individual earthworms. Importantly, in any earthworm species the density variations in the reproducing generation had significant consequences for the advancing generation, affecting either the numbers or/and the size (individual weight) of the cocoons produced. It is suggested that an underestimation of density-dependent processes may cause inaccurate estimates of the activities of local lumbricid populations.
Keywords: Lumbricidae, ecological groups, biomass, reproduction, cocoons, mortality
1. Introduction It is generally accepted that both density-independent and density-dependent processes operate in natural populations and that populations of most species experience some level of density dependence (Brook and Bradshaw 2006; Hixon and Johnson 2009). Having performed a multi-model inference analysis of a dataset of time-series observations for 1198 species, Brook and Bradshaw (2006) indicated a support of 74.7% for the density dependence, and even a higher one (78%) concerning invertebrates (639 species). This dataset, however, did not include soil
3 fauna (except for three soil surface-active carabid species); overall it may be noted that information on population dynamics of soil-dwelling invertebrate species, in particular of earthworms, is very scarce. After Hixon and Johnson (2009), competition (for food resources, living space, etc.) is one of the main causes of density dependence. Field densities of earthworm populations are extremely variable in space and time in species of any ecological group, ranging from single specimens to thousands of individuals m-2 (Petersen and Luxton 1982; Lee 1985; Uvarov 2009). Strong intrabiotopic density variations also occur across the seasons of the year and due to aggregative distribution of many lumbricid species (Phillipson et al. 1976; Holter 1983; Boag et al. 1994). This suggests an intense intraspecific competition in earthworm populations, at least during some periods and within the patches of higher densities, and potentially a densitydependent population dynamics. However, intraspecific responses of earthworms to population density variations are still insufficiently investigated. It may be suggested that an underestimation of density-dependent processes may cause inaccurate estimates of the local activities of lumbricid populations and, considering the significance of earthworms as engineering organisms (Eisenhauer et al. 2007; Briones 2014; Filser et al. 2016), of the functioning of detrital food webs and organic matter turnover in the soil. The majority of the experimental investigations of intraspecific relations in earthworms, as reviewed by Uvarov (2009), support density-dependent regulation of species populations, irrespective of their ecological group affiliation. Usually negative density-dependent population responses have been revealed, i.e. at higher density levels the growth, maturation and reproduction rates are retarded, whereas the mortality rates are increased. However, these results have almost exclusively been obtained in laboratory experiments mostly performed in small microcosms populated by very dense earthworm groups. The purpose of the present study was to investigate whether density-dependent responses are significant for earthworm species within the density gradients realistic in the natural sites.
4 Five common lumbricid species belonging to the main (epigeic, endogeic, anecic) ecological groups were chosen for a field experiment conducted in a mid-European beech-oak forest. The duration of the experiment (ca. 4.5 months in the summer/autumn season) was sufficient to estimate the response trends of the essential population parameters (changes of population biomass, mortality and reproduction rates, mean weights of earthworm individuals and cocoons). The hypotheses assumed (1) the presence of density-dependent responses in all of the species studied, and (2) a stronger expression of density dependence in the litter-feeding species compared to endogeic ones, due to more restricted amounts of organic matter resources (hence, a potentially more intense trophic competition) in the litter than in the soil.
2. Materials and Methods
Study site and weather conditions Litter and soil were collected in a beech-oak forest located in the Mazury Landscape Park (northern part of Piska Forest, NE Poland, 53°43 ́N and 21°36 ́E), in the beginning of June 2006. Undergrowth were lime, hazel and maple; soil plant cover was nearly absent. Overwintered litter 2-4 cm deep was weakly stratified (L+F/H) and mainly consisted of oak, beech and lime leaves markedly processed by soil biota. A weakly structured sandy soil was haplic arenosol (FAO/UNESCO classification); A horizon was 15 cm deep, pH(H2O) was 5.5. The carbon content of the litter and soil (A horizon) was 37.3 and 2.7%, while the nitrogen content 1.6 and 0.17%, respectively. The materials (FH-litter and soil from A horizon) were transferred 5 km N to the Mikolajki Hydrobiological Station (then belonging to the Centre for Ecological Research, Polish Academy of Sciences) where the experiment was conducted in a beech-oak grove. The summer–autumn season of 2006 was by 1.5-2o warmer (air temperature) and more rainy than the average weather conditions during the decade of 1996-2005 (as compiled using
5 the site www.weatheronline.co.nz for Mikolajki, Poland). The temperature regime in the microcosms was monitored at the litter/soil interface at 2 h intervals by means of data loggers. Monthly temperatures were 17.9, 16.0, 14.2, 10.5 and 5.2oC in July–November, respectively. The moisture conditions were ca. 65% of microcosm water holding capacity at the start and were kept favourable by regular watering (at 10-14 d intervals in the summer, rarer in the autumn). The amount of water added (totally 461 mm in July–November) corresponded to the precipitation of the rainy summer/autumn of 2006 (417 mm); excessive water was easily drained through the gauze bottom of the microcosms.
Microcosms Microcosms were plastic cylinders 60 cm high and 20 cm in inner diameter, with an air/water-tight lid; the lid had a 2 cm round hole for air exchange. Both the lid hole and the bottom of the cylinders were sealed with gauze to prevent earthworms from escape. In the microcosms, which were half-dug into the soil, soil columns were established resembling the soil structure in the field: 9.5 L of soil (30 cm deep) were covered with 100 g of overwintered litter. This corresponded to ca. 2.1 kg dwt m-2 and was at the higher limits of the range of litter supply reported for Eurasian broadleaved forests (1-2 kg dwt m-2, after Bazilevich 1993). The levels of the soil/litter interface inside and outside the microcosms corresponded to one another. The soil had been sieved (4 mm) for homogenization and the removal of roots, earthworms and macrofauna. The FH litter material had been sorted to remove earthworms and macrofauna predators, and mixed. To further equalize the starting conditions, and compensate for microbiota losses during the litter preparation, 50 mL of water suspension prepared from the untreated litter were added into each microcosm.
Treatments and sampling The prepared microcosms were kept for ca. 3 weeks in the field for soil system
6 establishment, and then populated by earthworms. Five common European lumbricid species were used in the experiment: Dendrobaena octaedra Savigny, 1826; Lumbricus rubellus Hoffmeister, 1843; L. terrestris L., 1758; Allolobophora chlorotica (Savigny, 1826); Aporrectodea caliginosa (Savigny, 1826). The species represented three main earthworm ecological groups: epigeic (D. octaedra and L. rubellus), anecic (L. terrestris) and endogeic (Al. chlorotica and A. caliginosa). Earthworms were collected during June – early July in Piska and Kampinos Forests, and kept in soil/litter containers at 4oC until the beginning of the experiment. Specimens of L. terrestris were expelled from the soil by syringe-injecting of a 0.15% formalin solution into their burrows; earthworms of other species were collected by handsorting of the litter or soil. At the start of the experiment, Lumbricus spp. and D. octaedra were represented by adult worms, A. caliginosa by large juveniles, and groups of Al. chlorotica contained 40% adults and 60% large juveniles. Before being placed into the microcosms, and during the terminal microcosm sampling (below), the living weight of earthworm individuals was determined after keeping for 24 h on wet filter paper to void their guts. The earthworms were introduced into the microcosms on 10.07.2006; the experiment was terminated (microcosms destructively sampled) after 130 d., on 16.11.2006. In each earthworm species, a gradient of density increase (minimum – medium – maximum treatments) was established in the microcosms; the actual densities depended on species size. Thus, for the smaller species (D. octaedra and Al. chlorotica) the respective densities comprised 5 – 15 – 25 ind. microcosm-1; for the medium-sized animals (L. rubellus and A. caliginosa) they were 3 – 9 – 15 ind. microcosm-1. In the largest species (L. terrestris), only two density levels were tested: 2 and 3 ind. microcosm-1. The earthworm densities in the microcosms generally corresponded to ranges lying between moderate/moderately high and high species densities in the natural sites (ca. 150-750 ind. m-2 in D. octaedra and Al. chlorotica, 90450 ind. m-2 in L. rubellus and A. caliginosa and 60-90 ind. m-2 in L. terrestris). Each treatment
7 had 4 microcosm replicates; thus, overall 56 microcosms and ca. 600 earthworms were used. During the terminal destructive sampling, microcosm substrates were checked for the initially settled earthworms; their numbers, individual (gut-free) weights and state of maturity were assessed. Half of the litter and ca.1 kg mixed soil samples (taken from various soil depths) were thoroughly hand-sorted for cocoons and young earthworms; based on these samples, offspring production (sum of cocoons and juveniles) per microcosm for any earthworm species was calculated. To compare offspring production across the density treatments in the species studied, data on offspring per microcosm were expressed per minimum earthworm groups, i.e. per 5 earthworms microcosm-1 for D. octaedra and Al. chlorotica, per 3 earthworms microcosm-1 for L. rubellus and A. caliginosa, per 2 earthworms microcosm-1 for L. terrestris. To discuss the significance of food deficit in explaining density-dependent responses, earthworm food consumption (as carbon) was approximately estimated using the equation: R/C = 11% (Hutchinson and King 1979), where R – respiration, C – consumption. Oxygen uptake at 20oC was assumed as 192, 89, 88, 78 and 74 mm3 O2 g-1h-1 for D. octaedra, L. rubellus, Al. chlorotica, A. caliginosa and L. terrestris, respectively (after Byzova 2007, with my alterations), temperature coefficient (q10) as 2.0 (Lee, 1985). Respiration (CO2-C production) for a given species was calculated on the basis of O2 uptake rates, biomass and monthly average temperatures; volumetric ratio (O2 consumed/CO2 produced) was assumed as 1.0. For July, August-October and November, starting, average and final earthworm biomass was used, respectively. Possible effects of density on the respiration rates (as shown by Uvarov and Scheu 2004) were ignored.
Statistics Assessing the influence of a density increase on the parameters estimated for a given earthworm species (biomass changes, maturity, mortality and reproduction rates), I first checked
8 the significance of the treatment effect. For reproduction rates (log-transformed where appropriate), biomass changes (percentages arcsin transformed) and cocoon weights one-way ANOVA was used, with subsequent Tukey test in the case of significant effects. For the data on mortality and maturation, as well as on individual earthworm weights, the significance of the treatment effects was assessed by means of nonparametric Kruskal–Wallis ANOVA (with subsequent nonparametric Tukey test).
3. Results
Mortality By the end of the experiment, many earthworms had died, but the mortality levels strongly differed across the species, comprising 36-76% in the epigeic D. octaedra and L. rubellus and ≤8% in the other species (Fig. 1). Only one L. terrestris individual died (in 3-ind. microcosms); in each of the endogeic species, Al. chlorotica and A. caliginosa, 7 inds. were lost at higher densities. Except for D. octaedra, the mortality levels tended to be higher with the density rise; this trend was significant for A. caliginosa and nearly significant for Al. chlorotica (Table 1).
Biomass Similarly to the data on mortality, earthworm species strongly differed in biomass change in the course of the experiment. Both epigeic species showed a drastic loss of the total biomass compared to its initial values. In D. octaedra, the biomass loss was not significantly different between the density treatments (Fig. 2; Table 1). Surviving specimens were on average significantly smaller by the end than at the start of the experiment (Table 2). Thus, the loss of the total biomass was due both to mortality (Table 1) and a weight loss of the individuals survived. In L. rubellus, the total biomass significantly decreased with density rise (Fig. 2; Table 1), Min
9 and Max values differed by Tukey test (P = 0.04). In the Min treatment the individuals survived were similar to those at the start (Table 2), thus the total biomass loss was due to the mortality. At higher densities the biomass loss was accounted to both mortality and individual weight loss (Fig. 1; Table 2). In contrast to the epigeics, the biomass of both endogeic species increased; however, this was negatively correlated with the treatment density (Fig. 2; Table 1). In the Min treatment of Al. chlorotica the total biomass increased due to the growth of juveniles, considering the lack of mortality (Fig. 1) and a similar body weight of the starting and final adult specimens (Table 2). At higher densities the total biomass was significantly lower than in the Min treatment (P = 0.010.04, by Tukey test); the biomass gain due to the growth of juveniles was nearly balanced by biomass loss due to mortality and the weight loss of adults (Figs. 1 and 2, Table 2). In the Min treatment of A. caliginosa, the highest total biomass was explained by the lack of mortality and the highest weights of mature specimens, which were by 1/3 lighter in the Med and Max treatments; in addition, in the Max treatment earthworm mortality was registered. The biomass changes in Min and Max treatments of L. terrestris were not significantly different (Fig. 2, Table 1), although earthworms in the Min treatment were on average by 8% heavier.
Maturation The maturation rates, which could only be checked in endogeic species, slowed down significantly with the density increase (Fig. 3; Table 1). In the Min treatments of both species all the juveniles reached maturity before the end of the experiment. In A. caliginosa, only 78% (Med treatment) and 70% (Max treatment, different from the Min treatment at P<0.01) of mature specimens were present at the terminal sampling. In Al. chlorotica, the retardation of the maturation with the rising density was even stronger (Fig. 3), with a significant difference between the Max and Min treatments (P=0.02).
10
Reproduction Both epigeic and endogeic species strongly reduced their offspring production with a density increase (Fig. 4; Table 1). The reproduction rates were significantly higher by Tukey test in the Min treatment than in the Max treatment (D. octaedra: P=0.027) or both in the Med and Max treatments (Al. chlorotica and A. caliginosa: P=0.001-0.024). Offspring production was considerably higher in epigeic species than in endogeic ones (Fig. 4). In pairs of species of similar body and population size, reproduction rates were significantly higher in D. octaedra over Al. chlorotica and in L. rubellus over A. caliginosa (both at P<0.001 by Tukey test). In contrast to the other species, offspring production of L. terrestris tended to grow (although not significantly) with the density increase (Fig. 4; Table 1). In all species, except for A. caliginosa, the weight of individual cocoons was decreased with an earthworm density increase; in A. caliginosa the smallest cocoons were recorded in the Med treatment (Table 3).
4. Discussion
Field densities of various lumbricid species representing any earthworm ecological group can be quite high (Lee 1985). E.g., 3218 ind. D. octaedra m-2 were recorded in a Canadian aspen forest (Dymond et al. 1997), and up to 600 ind. L. rubellus m-2 in peat meadows and floodplains (Makulec 2002; Zorn et al. 2005). Densities of up to 70-300 ind. m-2 (Edwards 1983; Daniel 1992) were reported for L. terrestris in grasslands and even higher in pasture soils under dung pats (30-40 ind. pat-1; Holter 1983). Baker (1999) reported up to 400-450 ind. m-2 of A. caliginosa in Australian pasture soils; in an old field near Moscow, 400-1500 ind. m-2
11 (Aporrectodea rosea dominated, A. caliginosa subdominated) were recorded for three consecutive years (Uvarov 2009). As compared to those estimates, earthworm density ranges in the present study can be qualified as moderate to high, as compared to the natural ones. In agreement with the first hypothesis, evidence for density-dependent effects was recorded in all of the species tested. However, earthworm responses to a density increase markedly varied and were species-specific and possibly ecological group-specific. In endogeic species most of the parameters measured were significantly negatively density-dependent. At an increased density, both in Al. chlorotica and A. caliginosa growth and maturation of juveniles were retarded (especially so in Al. chlorotica), up to 7% individuals died and the reproduction rates decreased. The size of cocoons produced also reacted to the density level, in Al. chlorotica decreasing in the Max treatment. The individual weight of adult earthworms was by 26-28% (Al. chlorotica) or 30-35% (A. caliginosa) lower in the Med and Max treatments than in the Min treatment. These results generally correspond to the literature data on these species, as obtained in laboratory experiments. Thus, density increase of Al. chlorotica juveniles retarded their growth and maturation, as well as induced their mortality (Butt 1997; Lowe and Butt 1999); also, in the presence of adult conspecifics their growth, maturation and consequently cocoon production slowed down (Lowe and Butt 2002, 2003). Similarly in A. caliginosa, with density increase the growth of juveniles was slower (Lowe and Butt 1999; Eriksen-Hamel and Whalen 2007), whereas the biomass of mixed-age groups tended to decline (Dalby et al. 1998; Baker et al. 2002). However, it should be noted that these density-dependent responses were documented at markedly higher density levels per unit substrate than those used in the present study (0.3–1.6 and 0.5–2.6 ind. L-1 for A. caliginosa and Al. chlorotica, respectively). In both endogeic species, the maximum alimentary demands (as approximately calculated on the basis of respiration rates) comprised up to 3.5-4 g C for the whole duration of the experiment. The total organic matter supply in the 10 cm topsoil of the microcosms was in a
12 strong excess (ca. 77 g C). Thus, the estimates do not suggest an overgrazing of food resources by endogeic earthworms. Hence, negative density-dependent responses recorded in both endogeic species could be accounted for intraspecific competition for space rather than for food. In contrast to the second hypothesis, the density-dependent responses in litter-feeding species were not necessarily stronger and appeared less variable than in endogeics. Thus, in both epigeic species the responses were markedly species-specific. In D. octaedra, the reproduction rates considerably decreased with a rising density, which phenomenon had earlier been shown in the lab, although at higher density levels (Uvarov and Scheu 2005; Uvarov 2009). In addition, in the Max treatment smaller cocoons were produced. However, the biomass loss and mortality rates in D. octaedra were not statistically different between the density treatments; moreover, these parameters tended to decrease with the density increase (cf. Figs. 1-2). The latter pattern can be explained by the fact that D. octaedra is a univoltine species in Central Europe, with the elimination of most of the adults after the reproduction (summer/autumn) season (Uvarov 1995; Uvarov et al. 2011). An increased reproductive effort in this species has also been shown to lead to enhanced biomass drop and mortality rates (Uvarov 1998, 2009). This is exactly the case as demonstrated in the present study, where a lower density is associated with a more intense reproduction and, consequently, with a trend of a higher mortality rate and a greater total biomass loss. Unlike D. octaedra, L. rubellus has a longer lifespan and lacks seasonal extinction (Klok and de Roos 1996; Uvarov et al. 2011). However, in the Med and Max treatments a substantial decrease in individual weights and total biomass, and up to a 2/3 reduction of L. rubellus numbers, were documented. The reproduction rates and cocoon size were also negatively correlated to the density increase. This agrees with laboratory data of Klok (2007) and Xia et al. (2011), indicating its density-dependent reduction of weight gain, survival and reproduction rates in the ranges of 300-1350 and 150-700 ind. m-2, respectively. An approximate estimate of epigeic earthworm consumption resulted in up to 4.5 and 6.5 g
13 C (in the Max treatments with D. octaedra and L. rubellus, respectively) for the whole duration of the experiment. Considering also the loss of litter carbon due to microbial respiration (I discuss these data elsewhere in more detail), a substantial part of the litter C (the total supply ca. 25 g per microcosm) in the epigeic (especially in L. rubellus) Max treatments can be concluded to have been processed by litter-consuming earthworms and microbial community. Indeed, the litter horizon in the microcosms was progressively depleted by the end of the experiment with the increase of L. rubellus density, indicating a strong decrease in the availability of food resources. Thus, food competition is presumably a significant factor affecting the intraspecific relationships in dense populations of epigeic earthworms in broadleaved forests, even at high levels of litter supply as used in this experiment. However, a deterioration of the litter environment was also evident. Klok (2007) suggested competition for space as a cause for a density-dependent lower food intake of individual L. rubellus juveniles, which reduced the intrinsic rate of the species population increase. However, this result was obtained at very high experimental densities (300-1350 ind. m-2) and with an excess of food supply. In contrast to the various density-dependent responses in L. rubellus, its anecic relative L. terrestris only showed a decrease in cocoon size by ca. 7% in the Max treatment; however, the cocoon numbers tended to be higher. In microcosms with L. terrestris, the litter was first concentrated in the middens around the earthworm burrows and then disappeared from the surface by the end of the experiment. However, no significant density-dependent differences in earthworm condition (biomass, mortality, reproduction rate) were revealed. Moreover, the final and starting weights of earthworm individuals failed to differ between the treatments. These results did not indicate an increased trophic deficit in the experimental animals at higher density level. The difference between the final condition of the two Lumbricus species looks surprising, considering that the maximum alimentary demands of L. terrestris (ca. 9 g C) exceeded those of L. rubellus. A possible explanation could be a stronger stimulation of soil community respiration by L. terrestris than by L. rubellus (the data on microcosm respiration are discussed elsewhere),
14 which suggests a more intense microbial biomass turnover and decomposition process, resulting in an additional supply of trophic resources for L. terrestris. In contrast to the present experiment conducted in the density/biomass range of 0.2-0.3 ind. L-1 or 0.9-1.3 g L-1, density-dependent responses have repeatedly been documented at higher densities of L. terrestris in the lab. In juveniles, clear negative responses (retardation of growth and maturation) have been shown with a density increase. The presence of adult conspecifics stimulated an early growth of juveniles (which gained from adults’ middens and casts), but later also retarded their growth and maturation (Butt et al. 1994ab; Lowe and Butt 1999, 2002, 2003; Eriksen-Hamel and Whalen 2007; Grigoropoulou et al. 2008, 2009). However, it should be noted that the behaviour of L. terrestris juveniles is closer to that of endogeic species and becomes anecic only in the late phase of their growth (Lowe and Butt 2002). In contrast, densitydependent responses of adults were not consequently negative. Thus, a slight increase in cocoon production and biomass (by 5 and 10%, respectively) in the density range of 1-2 ind. L-1 was reported by Butt (1998). The reproduction rates of L. terrestris pairs were higher at biomass level of 13.9 than of 8.7 or 26 g L-1 (Butt et al. 1994a). The rates of biomass production in the range of 4-20 ind. L-1 decreased at densities >8 ind. L-1 (Hartenstein and Amico 1983). A decline in cocoon production and earthworm survival in the range of 9.5-76 g L-1 was reported in the crowded laboratory cultures of L. terrestris (Butt et al. 1994a). In rare field manipulations (Grigoropoulou and Butt, 2010), density increase was accompanied by a higher dispersal intensity in L. terrestris populations. No doubt that the density-dependent responses in L. terrestris should further be studied at natural density levels, with consideration of the population age structure, complex adult behaviour and territorial habits. In conclusion, the discussion suggests that at very high densities (well-tested in laboratory experiments) negative density-dependent intraspecific relationships can be a significant regulation factor in lumbricid populations; this concerns various ecophysiological parameters in species representing any ecological group. At moderate to relatively high earthworm densities
15 (which are more realistic in natural sites), density-dependent responses are also characteristic, yet being more species- or ecological group-mediated. Thus, the present study revealed manifold and generally similar density responses in two endogeic species, presumably explained by spatial rather than direct food competition. In contrast, the latter factor was likely more important for the epigeic species characterized by species-specific density-dependent responses. Densitydependent relationships of L. terrestris in natural populations are presumably more complex and need to be further investigated in relation to the age structure and territorial behaviour of individual earthworms. It is important to note that in all the lumbricid species tested, density variations in the reproducing generation had significant consequences for the advancing generation, affecting either the numbers or/and the size (individual weight) of the cocoons produced.
Acknowledgements I cordially thank Janusz Uchmański and the colleagues from the Centre for Ecological Research PAS (CER) and Mikolajki Hydrobiological Station PAS (MHS) for encouragement and constant help. Józef Wróbel (MHS) and Maria Franczek (CER) strongly helped during the preparation of the experiment, whereas Ola Grabczyńska, Krassi Ilieva-Makulec, Piotr Ogrodowczyk, Julia Zielińska, Grzegorz Gryziak, Iza Olejniczak, Pawel Boniecki, Kamil Karaban (CER) assisted during the final destructive sampling. Soil description and analyses were performed by Jarosław Lasota and Maciej Zwydak (Uniwersytet Rolniczy w Krakowie). The local forestry (Nadłeśnictwo Maskulińskie) is thanked for the permission to take litter and soil substrates at the Mazursky Landscape Park. The comments of two anonymous referees strongly helped to improve the manuscript. Sergei Golovatch (Institute of Ecology and Evolution RAS, Moscow) kindly edited the English of the advanced draft. The study was supported by the grant 2P04F03030 of the Polish Ministry of Science and Higher Education, and by the Russian Foundation for Basic Research.
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References
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17 Byzova, Ju.B., 2007. Respiration of soil invertebrates. Moscow, KMK Publ. House, 328 pp. [in Russian]. Dalby, P.R., Baker, G.H., Smith, S.E., 1998. Competition and cocoon consumption by the earthworm Aporrectodea longa. Appl. Soil Ecol. 10, 127-136. Daniel, O., 1992. Population dynamics of Lumbricus terrestris L. (Oligochaeta: Lumbricidae) in a meadow. Soil Biol. Biochem. 24, 1425-1431. Dymond, P., Scheu, S., Parkinson, D., 1997. Density and distribution of Dendrobaena octaedra (Lumbricidae) in aspen and pine forests in the Canadian Rocky Mountains (Alberta). Soil Biol. Biochem. 29, 265-273. Edwards, C.A., 1983. Earthworm ecology in cultivated soils. In: Satchell, J.E. (Ed.), Earthworm Ecology – From Darwin to Vermiculture. Chapman & Hall, London, pp. 123-137. Eisenhauer, N., Partsch, S., Parkinson, D., Scheu, S., 2007. Invasion of a deciduous forest by earthworms: Changes in soil chemistry, microflora, microarthropods and vegetation. Soil Biol. Biochem. 39, 1099-1110. Eriksen-Hamel, N.S., Whalen, J.K., 2007. Competitive interactions affect the growth of Aporrectodea caliginosa and Lumbricus terrestris (Oligochaeta: Lumbricidae) in singleand mixed-species laboratory cultures. Eur. J. Soil Biol. 43, 142-150. Filser, J., Faber, J.H., Tiunov, A.V., Brussaard, L., Frouz, J., De Deyn, G., Uvarov, A.V., Berg, M.P., Lavelle, P., Loreau, M., Wall, D.H., Querner, P., Eijsackers, H., Jiménez, J.J., 2016. Soil fauna: key to new carbon models. Soil 2, 565-582, doi:10.5194/soil-2-565-2016. Grigoropoulou, N., Butt, K.R., 2010. Field investigations of Lumbricus terrestris spatial distribution and dispersal through monitoring of manipulated, enclosed plots. Soil Biol. Biochem. 42, 40-47. Grigoropoulou, N., Butt, K.R., Lowe, C.N., 2008. Effects of adult Lumbricus terrestris on cocoons and hatchlings in Evans’ boxes. Pedobiologia 51, 343-349. Grigoropoulou, N., Butt, K.R., Lowe, C.N., 2009. Interactions of juvenile Lumbricus terrestris
18 with adults and their burrow systems in a two-dimensional microcosm. Pesq. agropec. bras. 44, 964-968. Hartenstein, R., Amico, L., 1983. Production and carrying capacity for the earthworm Lumbricus terrestris in culture. Soil Biol. Biochem. 15, 51-54. Hixon, M.A., Johnson, D.W., 2009. Density Dependence and Independence. In: Encyclopedia of Life
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19 interactions of Allolobophora chlorotica (Savigny) and Lumbricus terrestris. Pedobiologia 47, 574-577. Makulec, G., 2002. The role of Lumbricus rubellus Hoffm. in determining biotic and abiotic properties of peat soils. Polish J. of Ecology 50, 301-339. Petersen H., Luxton M., 1982. A comparative analysis of soil fauna populations and their role in decomposition processes. Oikos 39: 287-388. Phillipson J., Abel R., Steel J., Woodell S.R.J., 1976. Earthworms and the factors governing their distribution in an English beechwood. Pedobiologia 16: 258-285. Uvarov, A.V., 1995. Response of an earthworm species to constant and diurnally fluctuating temperature regime in laboratory microcosms. Eur. J. Soil Biol. 31, 111-118. Uvarov, A.V., 1998. Respiration activity of Dendrobaena octaedra (Lumbricidae) under constant and diurnally fluctuating temperature regimes in laboratory microcosms. Eur. J. Soil Biol. 34, 1-10. Uvarov, A.V., 2009. Inter- and intraspecific interactions in lumbricid earthworms: Their role for earthworm performance and ecosystem functioning. Pedobiologia 53: 1-27. Uvarov, A.V., Scheu, S., 2004. Effects of developmental stage and temperature regime on respiration rate of Lumbricus rubellus (Lumbricidae). Pedobiologia 48: 365-371. Uvarov, A.V., Scheu, S., 2005. Effects of group maintenance and temperature regime on respiratory activity of epigeic earthworms, Dendrobaena octaedra and Lumbricus rubellus (Lumbricidae). Eur. J. Soil Biol. 40, 163-167. Uvarov, A.V., Tiunov, A.V., Scheu, S., 2011. Effects of seasonal and diurnal temperature fluctuations on population dynamics of two epigeic earthworm species in forest soil. Soil Biol. Biochem. 43, 559-570. Xia L., Szlavecz K., Swan C.M., Burgess J.L., 2011. Inter- and intra-specific interactions of Lumbricus rubellus (Hoffmeister, 1843) and Octolasion lacteum (Örley, 1881) (Lumbricidae) and the implication for C cycling. Soil Biol. Biochem. 43: 1584-1590.
20 Zorn, M.I., Van Gestel, C.A., Eijsackers, H., 2005. Species-specific earthworm population responses in relation to flooding dynamics in a Dutch floodplain soil. Pedobiologia 49: 189-198.
21 Figure captions:
Fig. 1. Mortality levels (% ± S.E.) in five earthworm species: monocultures with a density gradient. Symbols: Do – Dendrobaena octaedra, Lr – Lumbricus rubellus, Lt – L. terrestris, Acl – Allolobophora chlorotica, Aca – Aporrectodea caliginosa; Min, Med, Max – density treatments.
Fig. 2. Biomass changes (% ± S.E.) in five earthworm species: monocultures with a density gradient. Symbols: Do – Dendrobaena octaedra, Lr – Lumbricus rubellus, Lt – L. terrestris, Acl – Allolobophora chlorotica, Aca – Aporrectodea caliginosa; Min, Med, Max – density treatments.
Fig. 3. Proportion of mature specimens (% ± S.E.) in Allolobophora chlorotica (Acl) and Aporrectodea caliginosa (Aca) monocultures. Min, Med, Max – density treatments.
Fig. 4. Offspring production, ind. ± S.E. (expressed per minimum earthworm group, see Methods) in five earthworm species: monocultures with a density gradient. Symbols: Do – Dendrobaena octaedra, Lr – Lumbricus rubellus, Lt – L. terrestris, Acl – Allolobophora chlorotica, Aca – Aporrectodea caliginosa; Min, Med, Max – density treatments.
22
100 90
%.
80
MIN
70
MED
60
MAX
50 40 30 20 10 0
Do
Lr
Lt
Acl
Aca
Fig. 1. Mortality levels (% ± S.E.) in monocultures of five earthworm species in the gradient of density increase. Symbols: Do – Dendrobaena octaedra, Lr – Lumbricus rubellus, Lt – L. terrestris, Acl – Allolobophora chlorotica, Aca – Aporrectodea caliginosa; Min, Med, Max – density treatments.
23
60 50 40 30 20 10 0
%.
-10
Do
Lr
Lt
Acl
Aca
-20 -30 -40 -50
MIN
-60
MED
-70
MAX
-80 -90 -100
Fig. 2. Biomass changes (% ± S.E.) in monocultures of five earthworm species in the gradient of density increase. Symbols: Do – Dendrobaena octaedra, Lr – Lumbricus rubellus, Lt – L. terrestris, Acl – Allolobophora chlorotica, Aca – Aporrectodea caliginosa; Min, Med, Max – density treatments.
24
100 90 80 70
%.
60 50 40
Acl
30 Aca 20 10 0
MIN
MED
MAX
Fig. 3. Proportion of mature specimens (% ± S.E.) in monocultures of Allolobophora chlorotica (Acl) and Aporrectodea caliginosa (Aca) in the gradient of density increase. Min, Med, Max – density treatments.
25
100
MIN MED
80
MAX
Ind.
60
40
20
0
Do
Lr
Lt
Acl
Aca
Fig. 4. Offspring production, ind. ± S.E., (expressed per minimum earthworm group, see Methods) in monocultures of five earthworm species in the gradient of density increase. Symbols: Do – Dendrobaena octaedra, Lr – Lumbricus rubellus, Lt – L. terrestris, Acl – Allolobophora chlorotica, Aca – Aporrectodea caliginosa; Min, Med, Max – density treatments.
26 Table 1.Significance of density-dependent changes in mortality, biomass, maturation and reproduction rates in five earthworm species, estimated by one-way ANOVA or Kruskal–Wallis ANOVA. Probability levels are indicated by *** (<0.001), ** (<0.01), * (<0.5) and ^ (<0.1).
Parameter Mortality Biomass changes Maturation Offspring
D. octaedra H2,12=0.27; P=0.873 F2,9=0.09; P=0.914 – F2,9=5.08; P=0.033*
L. rubellus H2,12=2.18; P=0.337 F2,9=4.82; P=0.038* – F2,9=3.89; P=0.061^
L. terrestris H1,8=1.00; P=0.317 H1,8=2.08; P=0.149 – F1,6=1.72; P=0.238
Al. chlorotica H2,12=4.93; P=0.085^ F2,9=7.51; P=0.012* H2,12=10.24; P=0.006** F2,9=16.57; P=0.001***
A. caliginosa H2,12=7.17; P=0.028* F2,9=3.41; P=0.079^ H2,12=8.04; P=0.018** F2,9=8.75; P=0.008**
27 Table 2. Starting and final individual weights of adult earthworms (g). Significant differences in the line Start – Min – Med – Max treatments (estimated by Kruskal–Wallis ANOVA, with subsequent nonparametric Tukey test in the case of significant effects) are indicated by different letters.
Earthworm species D. octaedra ind. weight L. rubellus ind. weight L. terrestris ind. weight Al. chlorotica ind. weight A. caliginosa ind. weight
Start MIN Treatment effect: H3,193=98.18; P<0.001*** 0.202 ± 0.004 (141) a 0.111 ± 0.016 (6) b Treatment effect: H3,131=68.82; P<0.001*** 0.904 ± 0.021 (86) a 1.011 ± 0.107 (7) a No treatment effect: H2,39=1.98; P=0.372 4.240 ± 0.116 (20) 4.642 ± 0.252 (8) Treatment effect: H3,120=38.58; P<0.001*** 0.292 ± 0.005 (51) a 0.319 ± 0.015 (18) a Treatment effect: H2,77=15.30; P<0.001*** – 0.783 ± 0.069 (12) a
MED
MAX
0.098 ± 0.020 (16) b
0.079 ± 0.007 (30) b
0.449 ± 0.047 (17) b
0.369 ± 0.040 (21) b
–
4,259 ± 0,208 (11)
0.238 ± 0.014 (23) b
0.232 ± 0.009 (28) b
0.552 ± 0.031 (28) b
0.503 ± 0.020 (37) b
28 Table 3. Weight of individual cocoons (mg) produced by earthworms in the MIN, MED and MAX treatments (one-way ANOVA, with subsequent Tukey test). Significant differences in the line Min – Med – Max treatments are indicated by different letters.
Earthworm species D. octaedra cocoon weight L. rubellus cocoon weight L. terrestris cocoon weight Al. chlorotica cocoon weight A. caliginosa cocoon weight
MIN MED Treatment effect: F2,651=51.7; P<0.001*** 4.43 ± 0.03 (126) a 4.43 ± 0.02 (271) a Treatment effect: F2,368=32.9; P<0.001*** 13.19 ± 0.16 (110) a 12.65 ± 0.09 (121) b Treatment effect: F1,77=14.67; P=0.001*** 63.71 ± 0.96 (38) a – Treatment effect: F2,191=4.44; P=0.013** 13.08 ± 0.10 (83) a 13.05 ± 0.12 (57) a Treatment effect: F2,67=8.25; P=0.001*** 25.41 ± 0.39 (35) a 23.08 ± 0.60 (20) b
MAX 4.13 ± 0.02 (257) b 11.97 ± 0.07 (140) c 59.34 ± 0.64 (41) b 12.64 ± 0.12 (54) b 26.23 ± 0.66 (15) a
S0031-4056(16)30113-5 http://dx.doi.org/doi:10.1016/j.pedobi.2017.01.002 PEDOBI 50478
To appear in: Received date: Revised date: Accepted date:
26-8-2016 28-12-2016 1-1-2017
Please cite this article as: Uvarov, Alexei V., Density-dependent responses in some common lumbricid species.Pedobiologia - International Journal of Soil Biology http://dx.doi.org/10.1016/j.pedobi.2017.01.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Density-dependent responses in some common lumbricid species\
Alexei V. Uvarov
A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninsky Prospect 33, 119071 Moscow, Russia;
e-mail address: [email protected]
Highlights 1. At field densities, populations of various earthworm species experience density dependence 2. In epigeic species, food competition was likely an important cause of density dependence 3. In endogeic species, density dependence was caused by spatial rather than food competition
ABSTRACT It is generally accepted that populations of most species experience some level of density dependence; however, this has rarely been shown for soil-dwelling invertebrate species, in particular for earthworms. Experimental data, mostly obtained at high laboratory densities, suggest intense intraspecific competition for resources and/or living space and density-dependent dynamics in earthworm populations, irrespective of their ecological group affiliation. In the present study performed in large field microcosms, I investigated whether density-dependent responses occur within the earthworm density gradients more realistic for the natural sites. Five lumbricid species from epigeic (Dendrobaena octaedra, Lumbricus rubellus), endogeic (Allolobophora chlorotica, Aporrectodea caliginosa) and anecic (Lumbricus terrestris)
2 ecological groups were tested. Soil systems populated with earthworm monocultures in large (20-L) microcosms were exposed in a beech-oak forest for 4.5 months; each species was represented by two (L. terrestris) or three treatments forming gradients of increasing density. In two endogeic species, manifold and generally similar density-dependent responses (a retardation of growth, maturation and reproduction rates, but higher mortality rates with density increase) were revealed, likely explained by spatial competition rather than by direct food competition. In each of epigeic species density-dependent responses were less variable and more speciesspecific. In contrast to endogeics, direct food competition was presumably a more important cause of density-dependence. In L. terrestris no significant density-dependent responses in adult earthworms were revealed; however, they need to be further investigated in relation to the age and territorial behaviour of individual earthworms. Importantly, in any earthworm species the density variations in the reproducing generation had significant consequences for the advancing generation, affecting either the numbers or/and the size (individual weight) of the cocoons produced. It is suggested that an underestimation of density-dependent processes may cause inaccurate estimates of the activities of local lumbricid populations.
Keywords: Lumbricidae, ecological groups, biomass, reproduction, cocoons, mortality
1. Introduction It is generally accepted that both density-independent and density-dependent processes operate in natural populations and that populations of most species experience some level of density dependence (Brook and Bradshaw 2006; Hixon and Johnson 2009). Having performed a multi-model inference analysis of a dataset of time-series observations for 1198 species, Brook and Bradshaw (2006) indicated a support of 74.7% for the density dependence, and even a higher one (78%) concerning invertebrates (639 species). This dataset, however, did not include soil
3 fauna (except for three soil surface-active carabid species); overall it may be noted that information on population dynamics of soil-dwelling invertebrate species, in particular of earthworms, is very scarce. After Hixon and Johnson (2009), competition (for food resources, living space, etc.) is one of the main causes of density dependence. Field densities of earthworm populations are extremely variable in space and time in species of any ecological group, ranging from single specimens to thousands of individuals m-2 (Petersen and Luxton 1982; Lee 1985; Uvarov 2009). Strong intrabiotopic density variations also occur across the seasons of the year and due to aggregative distribution of many lumbricid species (Phillipson et al. 1976; Holter 1983; Boag et al. 1994). This suggests an intense intraspecific competition in earthworm populations, at least during some periods and within the patches of higher densities, and potentially a densitydependent population dynamics. However, intraspecific responses of earthworms to population density variations are still insufficiently investigated. It may be suggested that an underestimation of density-dependent processes may cause inaccurate estimates of the local activities of lumbricid populations and, considering the significance of earthworms as engineering organisms (Eisenhauer et al. 2007; Briones 2014; Filser et al. 2016), of the functioning of detrital food webs and organic matter turnover in the soil. The majority of the experimental investigations of intraspecific relations in earthworms, as reviewed by Uvarov (2009), support density-dependent regulation of species populations, irrespective of their ecological group affiliation. Usually negative density-dependent population responses have been revealed, i.e. at higher density levels the growth, maturation and reproduction rates are retarded, whereas the mortality rates are increased. However, these results have almost exclusively been obtained in laboratory experiments mostly performed in small microcosms populated by very dense earthworm groups. The purpose of the present study was to investigate whether density-dependent responses are significant for earthworm species within the density gradients realistic in the natural sites.
4 Five common lumbricid species belonging to the main (epigeic, endogeic, anecic) ecological groups were chosen for a field experiment conducted in a mid-European beech-oak forest. The duration of the experiment (ca. 4.5 months in the summer/autumn season) was sufficient to estimate the response trends of the essential population parameters (changes of population biomass, mortality and reproduction rates, mean weights of earthworm individuals and cocoons). The hypotheses assumed (1) the presence of density-dependent responses in all of the species studied, and (2) a stronger expression of density dependence in the litter-feeding species compared to endogeic ones, due to more restricted amounts of organic matter resources (hence, a potentially more intense trophic competition) in the litter than in the soil.
2. Materials and Methods
Study site and weather conditions Litter and soil were collected in a beech-oak forest located in the Mazury Landscape Park (northern part of Piska Forest, NE Poland, 53°43 ́N and 21°36 ́E), in the beginning of June 2006. Undergrowth were lime, hazel and maple; soil plant cover was nearly absent. Overwintered litter 2-4 cm deep was weakly stratified (L+F/H) and mainly consisted of oak, beech and lime leaves markedly processed by soil biota. A weakly structured sandy soil was haplic arenosol (FAO/UNESCO classification); A horizon was 15 cm deep, pH(H2O) was 5.5. The carbon content of the litter and soil (A horizon) was 37.3 and 2.7%, while the nitrogen content 1.6 and 0.17%, respectively. The materials (FH-litter and soil from A horizon) were transferred 5 km N to the Mikolajki Hydrobiological Station (then belonging to the Centre for Ecological Research, Polish Academy of Sciences) where the experiment was conducted in a beech-oak grove. The summer–autumn season of 2006 was by 1.5-2o warmer (air temperature) and more rainy than the average weather conditions during the decade of 1996-2005 (as compiled using
5 the site www.weatheronline.co.nz for Mikolajki, Poland). The temperature regime in the microcosms was monitored at the litter/soil interface at 2 h intervals by means of data loggers. Monthly temperatures were 17.9, 16.0, 14.2, 10.5 and 5.2oC in July–November, respectively. The moisture conditions were ca. 65% of microcosm water holding capacity at the start and were kept favourable by regular watering (at 10-14 d intervals in the summer, rarer in the autumn). The amount of water added (totally 461 mm in July–November) corresponded to the precipitation of the rainy summer/autumn of 2006 (417 mm); excessive water was easily drained through the gauze bottom of the microcosms.
Microcosms Microcosms were plastic cylinders 60 cm high and 20 cm in inner diameter, with an air/water-tight lid; the lid had a 2 cm round hole for air exchange. Both the lid hole and the bottom of the cylinders were sealed with gauze to prevent earthworms from escape. In the microcosms, which were half-dug into the soil, soil columns were established resembling the soil structure in the field: 9.5 L of soil (30 cm deep) were covered with 100 g of overwintered litter. This corresponded to ca. 2.1 kg dwt m-2 and was at the higher limits of the range of litter supply reported for Eurasian broadleaved forests (1-2 kg dwt m-2, after Bazilevich 1993). The levels of the soil/litter interface inside and outside the microcosms corresponded to one another. The soil had been sieved (4 mm) for homogenization and the removal of roots, earthworms and macrofauna. The FH litter material had been sorted to remove earthworms and macrofauna predators, and mixed. To further equalize the starting conditions, and compensate for microbiota losses during the litter preparation, 50 mL of water suspension prepared from the untreated litter were added into each microcosm.
Treatments and sampling The prepared microcosms were kept for ca. 3 weeks in the field for soil system
6 establishment, and then populated by earthworms. Five common European lumbricid species were used in the experiment: Dendrobaena octaedra Savigny, 1826; Lumbricus rubellus Hoffmeister, 1843; L. terrestris L., 1758; Allolobophora chlorotica (Savigny, 1826); Aporrectodea caliginosa (Savigny, 1826). The species represented three main earthworm ecological groups: epigeic (D. octaedra and L. rubellus), anecic (L. terrestris) and endogeic (Al. chlorotica and A. caliginosa). Earthworms were collected during June – early July in Piska and Kampinos Forests, and kept in soil/litter containers at 4oC until the beginning of the experiment. Specimens of L. terrestris were expelled from the soil by syringe-injecting of a 0.15% formalin solution into their burrows; earthworms of other species were collected by handsorting of the litter or soil. At the start of the experiment, Lumbricus spp. and D. octaedra were represented by adult worms, A. caliginosa by large juveniles, and groups of Al. chlorotica contained 40% adults and 60% large juveniles. Before being placed into the microcosms, and during the terminal microcosm sampling (below), the living weight of earthworm individuals was determined after keeping for 24 h on wet filter paper to void their guts. The earthworms were introduced into the microcosms on 10.07.2006; the experiment was terminated (microcosms destructively sampled) after 130 d., on 16.11.2006. In each earthworm species, a gradient of density increase (minimum – medium – maximum treatments) was established in the microcosms; the actual densities depended on species size. Thus, for the smaller species (D. octaedra and Al. chlorotica) the respective densities comprised 5 – 15 – 25 ind. microcosm-1; for the medium-sized animals (L. rubellus and A. caliginosa) they were 3 – 9 – 15 ind. microcosm-1. In the largest species (L. terrestris), only two density levels were tested: 2 and 3 ind. microcosm-1. The earthworm densities in the microcosms generally corresponded to ranges lying between moderate/moderately high and high species densities in the natural sites (ca. 150-750 ind. m-2 in D. octaedra and Al. chlorotica, 90450 ind. m-2 in L. rubellus and A. caliginosa and 60-90 ind. m-2 in L. terrestris). Each treatment
7 had 4 microcosm replicates; thus, overall 56 microcosms and ca. 600 earthworms were used. During the terminal destructive sampling, microcosm substrates were checked for the initially settled earthworms; their numbers, individual (gut-free) weights and state of maturity were assessed. Half of the litter and ca.1 kg mixed soil samples (taken from various soil depths) were thoroughly hand-sorted for cocoons and young earthworms; based on these samples, offspring production (sum of cocoons and juveniles) per microcosm for any earthworm species was calculated. To compare offspring production across the density treatments in the species studied, data on offspring per microcosm were expressed per minimum earthworm groups, i.e. per 5 earthworms microcosm-1 for D. octaedra and Al. chlorotica, per 3 earthworms microcosm-1 for L. rubellus and A. caliginosa, per 2 earthworms microcosm-1 for L. terrestris. To discuss the significance of food deficit in explaining density-dependent responses, earthworm food consumption (as carbon) was approximately estimated using the equation: R/C = 11% (Hutchinson and King 1979), where R – respiration, C – consumption. Oxygen uptake at 20oC was assumed as 192, 89, 88, 78 and 74 mm3 O2 g-1h-1 for D. octaedra, L. rubellus, Al. chlorotica, A. caliginosa and L. terrestris, respectively (after Byzova 2007, with my alterations), temperature coefficient (q10) as 2.0 (Lee, 1985). Respiration (CO2-C production) for a given species was calculated on the basis of O2 uptake rates, biomass and monthly average temperatures; volumetric ratio (O2 consumed/CO2 produced) was assumed as 1.0. For July, August-October and November, starting, average and final earthworm biomass was used, respectively. Possible effects of density on the respiration rates (as shown by Uvarov and Scheu 2004) were ignored.
Statistics Assessing the influence of a density increase on the parameters estimated for a given earthworm species (biomass changes, maturity, mortality and reproduction rates), I first checked
8 the significance of the treatment effect. For reproduction rates (log-transformed where appropriate), biomass changes (percentages arcsin transformed) and cocoon weights one-way ANOVA was used, with subsequent Tukey test in the case of significant effects. For the data on mortality and maturation, as well as on individual earthworm weights, the significance of the treatment effects was assessed by means of nonparametric Kruskal–Wallis ANOVA (with subsequent nonparametric Tukey test).
3. Results
Mortality By the end of the experiment, many earthworms had died, but the mortality levels strongly differed across the species, comprising 36-76% in the epigeic D. octaedra and L. rubellus and ≤8% in the other species (Fig. 1). Only one L. terrestris individual died (in 3-ind. microcosms); in each of the endogeic species, Al. chlorotica and A. caliginosa, 7 inds. were lost at higher densities. Except for D. octaedra, the mortality levels tended to be higher with the density rise; this trend was significant for A. caliginosa and nearly significant for Al. chlorotica (Table 1).
Biomass Similarly to the data on mortality, earthworm species strongly differed in biomass change in the course of the experiment. Both epigeic species showed a drastic loss of the total biomass compared to its initial values. In D. octaedra, the biomass loss was not significantly different between the density treatments (Fig. 2; Table 1). Surviving specimens were on average significantly smaller by the end than at the start of the experiment (Table 2). Thus, the loss of the total biomass was due both to mortality (Table 1) and a weight loss of the individuals survived. In L. rubellus, the total biomass significantly decreased with density rise (Fig. 2; Table 1), Min
9 and Max values differed by Tukey test (P = 0.04). In the Min treatment the individuals survived were similar to those at the start (Table 2), thus the total biomass loss was due to the mortality. At higher densities the biomass loss was accounted to both mortality and individual weight loss (Fig. 1; Table 2). In contrast to the epigeics, the biomass of both endogeic species increased; however, this was negatively correlated with the treatment density (Fig. 2; Table 1). In the Min treatment of Al. chlorotica the total biomass increased due to the growth of juveniles, considering the lack of mortality (Fig. 1) and a similar body weight of the starting and final adult specimens (Table 2). At higher densities the total biomass was significantly lower than in the Min treatment (P = 0.010.04, by Tukey test); the biomass gain due to the growth of juveniles was nearly balanced by biomass loss due to mortality and the weight loss of adults (Figs. 1 and 2, Table 2). In the Min treatment of A. caliginosa, the highest total biomass was explained by the lack of mortality and the highest weights of mature specimens, which were by 1/3 lighter in the Med and Max treatments; in addition, in the Max treatment earthworm mortality was registered. The biomass changes in Min and Max treatments of L. terrestris were not significantly different (Fig. 2, Table 1), although earthworms in the Min treatment were on average by 8% heavier.
Maturation The maturation rates, which could only be checked in endogeic species, slowed down significantly with the density increase (Fig. 3; Table 1). In the Min treatments of both species all the juveniles reached maturity before the end of the experiment. In A. caliginosa, only 78% (Med treatment) and 70% (Max treatment, different from the Min treatment at P<0.01) of mature specimens were present at the terminal sampling. In Al. chlorotica, the retardation of the maturation with the rising density was even stronger (Fig. 3), with a significant difference between the Max and Min treatments (P=0.02).
10
Reproduction Both epigeic and endogeic species strongly reduced their offspring production with a density increase (Fig. 4; Table 1). The reproduction rates were significantly higher by Tukey test in the Min treatment than in the Max treatment (D. octaedra: P=0.027) or both in the Med and Max treatments (Al. chlorotica and A. caliginosa: P=0.001-0.024). Offspring production was considerably higher in epigeic species than in endogeic ones (Fig. 4). In pairs of species of similar body and population size, reproduction rates were significantly higher in D. octaedra over Al. chlorotica and in L. rubellus over A. caliginosa (both at P<0.001 by Tukey test). In contrast to the other species, offspring production of L. terrestris tended to grow (although not significantly) with the density increase (Fig. 4; Table 1). In all species, except for A. caliginosa, the weight of individual cocoons was decreased with an earthworm density increase; in A. caliginosa the smallest cocoons were recorded in the Med treatment (Table 3).
4. Discussion
Field densities of various lumbricid species representing any earthworm ecological group can be quite high (Lee 1985). E.g., 3218 ind. D. octaedra m-2 were recorded in a Canadian aspen forest (Dymond et al. 1997), and up to 600 ind. L. rubellus m-2 in peat meadows and floodplains (Makulec 2002; Zorn et al. 2005). Densities of up to 70-300 ind. m-2 (Edwards 1983; Daniel 1992) were reported for L. terrestris in grasslands and even higher in pasture soils under dung pats (30-40 ind. pat-1; Holter 1983). Baker (1999) reported up to 400-450 ind. m-2 of A. caliginosa in Australian pasture soils; in an old field near Moscow, 400-1500 ind. m-2
11 (Aporrectodea rosea dominated, A. caliginosa subdominated) were recorded for three consecutive years (Uvarov 2009). As compared to those estimates, earthworm density ranges in the present study can be qualified as moderate to high, as compared to the natural ones. In agreement with the first hypothesis, evidence for density-dependent effects was recorded in all of the species tested. However, earthworm responses to a density increase markedly varied and were species-specific and possibly ecological group-specific. In endogeic species most of the parameters measured were significantly negatively density-dependent. At an increased density, both in Al. chlorotica and A. caliginosa growth and maturation of juveniles were retarded (especially so in Al. chlorotica), up to 7% individuals died and the reproduction rates decreased. The size of cocoons produced also reacted to the density level, in Al. chlorotica decreasing in the Max treatment. The individual weight of adult earthworms was by 26-28% (Al. chlorotica) or 30-35% (A. caliginosa) lower in the Med and Max treatments than in the Min treatment. These results generally correspond to the literature data on these species, as obtained in laboratory experiments. Thus, density increase of Al. chlorotica juveniles retarded their growth and maturation, as well as induced their mortality (Butt 1997; Lowe and Butt 1999); also, in the presence of adult conspecifics their growth, maturation and consequently cocoon production slowed down (Lowe and Butt 2002, 2003). Similarly in A. caliginosa, with density increase the growth of juveniles was slower (Lowe and Butt 1999; Eriksen-Hamel and Whalen 2007), whereas the biomass of mixed-age groups tended to decline (Dalby et al. 1998; Baker et al. 2002). However, it should be noted that these density-dependent responses were documented at markedly higher density levels per unit substrate than those used in the present study (0.3–1.6 and 0.5–2.6 ind. L-1 for A. caliginosa and Al. chlorotica, respectively). In both endogeic species, the maximum alimentary demands (as approximately calculated on the basis of respiration rates) comprised up to 3.5-4 g C for the whole duration of the experiment. The total organic matter supply in the 10 cm topsoil of the microcosms was in a
12 strong excess (ca. 77 g C). Thus, the estimates do not suggest an overgrazing of food resources by endogeic earthworms. Hence, negative density-dependent responses recorded in both endogeic species could be accounted for intraspecific competition for space rather than for food. In contrast to the second hypothesis, the density-dependent responses in litter-feeding species were not necessarily stronger and appeared less variable than in endogeics. Thus, in both epigeic species the responses were markedly species-specific. In D. octaedra, the reproduction rates considerably decreased with a rising density, which phenomenon had earlier been shown in the lab, although at higher density levels (Uvarov and Scheu 2005; Uvarov 2009). In addition, in the Max treatment smaller cocoons were produced. However, the biomass loss and mortality rates in D. octaedra were not statistically different between the density treatments; moreover, these parameters tended to decrease with the density increase (cf. Figs. 1-2). The latter pattern can be explained by the fact that D. octaedra is a univoltine species in Central Europe, with the elimination of most of the adults after the reproduction (summer/autumn) season (Uvarov 1995; Uvarov et al. 2011). An increased reproductive effort in this species has also been shown to lead to enhanced biomass drop and mortality rates (Uvarov 1998, 2009). This is exactly the case as demonstrated in the present study, where a lower density is associated with a more intense reproduction and, consequently, with a trend of a higher mortality rate and a greater total biomass loss. Unlike D. octaedra, L. rubellus has a longer lifespan and lacks seasonal extinction (Klok and de Roos 1996; Uvarov et al. 2011). However, in the Med and Max treatments a substantial decrease in individual weights and total biomass, and up to a 2/3 reduction of L. rubellus numbers, were documented. The reproduction rates and cocoon size were also negatively correlated to the density increase. This agrees with laboratory data of Klok (2007) and Xia et al. (2011), indicating its density-dependent reduction of weight gain, survival and reproduction rates in the ranges of 300-1350 and 150-700 ind. m-2, respectively. An approximate estimate of epigeic earthworm consumption resulted in up to 4.5 and 6.5 g
13 C (in the Max treatments with D. octaedra and L. rubellus, respectively) for the whole duration of the experiment. Considering also the loss of litter carbon due to microbial respiration (I discuss these data elsewhere in more detail), a substantial part of the litter C (the total supply ca. 25 g per microcosm) in the epigeic (especially in L. rubellus) Max treatments can be concluded to have been processed by litter-consuming earthworms and microbial community. Indeed, the litter horizon in the microcosms was progressively depleted by the end of the experiment with the increase of L. rubellus density, indicating a strong decrease in the availability of food resources. Thus, food competition is presumably a significant factor affecting the intraspecific relationships in dense populations of epigeic earthworms in broadleaved forests, even at high levels of litter supply as used in this experiment. However, a deterioration of the litter environment was also evident. Klok (2007) suggested competition for space as a cause for a density-dependent lower food intake of individual L. rubellus juveniles, which reduced the intrinsic rate of the species population increase. However, this result was obtained at very high experimental densities (300-1350 ind. m-2) and with an excess of food supply. In contrast to the various density-dependent responses in L. rubellus, its anecic relative L. terrestris only showed a decrease in cocoon size by ca. 7% in the Max treatment; however, the cocoon numbers tended to be higher. In microcosms with L. terrestris, the litter was first concentrated in the middens around the earthworm burrows and then disappeared from the surface by the end of the experiment. However, no significant density-dependent differences in earthworm condition (biomass, mortality, reproduction rate) were revealed. Moreover, the final and starting weights of earthworm individuals failed to differ between the treatments. These results did not indicate an increased trophic deficit in the experimental animals at higher density level. The difference between the final condition of the two Lumbricus species looks surprising, considering that the maximum alimentary demands of L. terrestris (ca. 9 g C) exceeded those of L. rubellus. A possible explanation could be a stronger stimulation of soil community respiration by L. terrestris than by L. rubellus (the data on microcosm respiration are discussed elsewhere),
14 which suggests a more intense microbial biomass turnover and decomposition process, resulting in an additional supply of trophic resources for L. terrestris. In contrast to the present experiment conducted in the density/biomass range of 0.2-0.3 ind. L-1 or 0.9-1.3 g L-1, density-dependent responses have repeatedly been documented at higher densities of L. terrestris in the lab. In juveniles, clear negative responses (retardation of growth and maturation) have been shown with a density increase. The presence of adult conspecifics stimulated an early growth of juveniles (which gained from adults’ middens and casts), but later also retarded their growth and maturation (Butt et al. 1994ab; Lowe and Butt 1999, 2002, 2003; Eriksen-Hamel and Whalen 2007; Grigoropoulou et al. 2008, 2009). However, it should be noted that the behaviour of L. terrestris juveniles is closer to that of endogeic species and becomes anecic only in the late phase of their growth (Lowe and Butt 2002). In contrast, densitydependent responses of adults were not consequently negative. Thus, a slight increase in cocoon production and biomass (by 5 and 10%, respectively) in the density range of 1-2 ind. L-1 was reported by Butt (1998). The reproduction rates of L. terrestris pairs were higher at biomass level of 13.9 than of 8.7 or 26 g L-1 (Butt et al. 1994a). The rates of biomass production in the range of 4-20 ind. L-1 decreased at densities >8 ind. L-1 (Hartenstein and Amico 1983). A decline in cocoon production and earthworm survival in the range of 9.5-76 g L-1 was reported in the crowded laboratory cultures of L. terrestris (Butt et al. 1994a). In rare field manipulations (Grigoropoulou and Butt, 2010), density increase was accompanied by a higher dispersal intensity in L. terrestris populations. No doubt that the density-dependent responses in L. terrestris should further be studied at natural density levels, with consideration of the population age structure, complex adult behaviour and territorial habits. In conclusion, the discussion suggests that at very high densities (well-tested in laboratory experiments) negative density-dependent intraspecific relationships can be a significant regulation factor in lumbricid populations; this concerns various ecophysiological parameters in species representing any ecological group. At moderate to relatively high earthworm densities
15 (which are more realistic in natural sites), density-dependent responses are also characteristic, yet being more species- or ecological group-mediated. Thus, the present study revealed manifold and generally similar density responses in two endogeic species, presumably explained by spatial rather than direct food competition. In contrast, the latter factor was likely more important for the epigeic species characterized by species-specific density-dependent responses. Densitydependent relationships of L. terrestris in natural populations are presumably more complex and need to be further investigated in relation to the age structure and territorial behaviour of individual earthworms. It is important to note that in all the lumbricid species tested, density variations in the reproducing generation had significant consequences for the advancing generation, affecting either the numbers or/and the size (individual weight) of the cocoons produced.
Acknowledgements I cordially thank Janusz Uchmański and the colleagues from the Centre for Ecological Research PAS (CER) and Mikolajki Hydrobiological Station PAS (MHS) for encouragement and constant help. Józef Wróbel (MHS) and Maria Franczek (CER) strongly helped during the preparation of the experiment, whereas Ola Grabczyńska, Krassi Ilieva-Makulec, Piotr Ogrodowczyk, Julia Zielińska, Grzegorz Gryziak, Iza Olejniczak, Pawel Boniecki, Kamil Karaban (CER) assisted during the final destructive sampling. Soil description and analyses were performed by Jarosław Lasota and Maciej Zwydak (Uniwersytet Rolniczy w Krakowie). The local forestry (Nadłeśnictwo Maskulińskie) is thanked for the permission to take litter and soil substrates at the Mazursky Landscape Park. The comments of two anonymous referees strongly helped to improve the manuscript. Sergei Golovatch (Institute of Ecology and Evolution RAS, Moscow) kindly edited the English of the advanced draft. The study was supported by the grant 2P04F03030 of the Polish Ministry of Science and Higher Education, and by the Russian Foundation for Basic Research.
16
References
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19 interactions of Allolobophora chlorotica (Savigny) and Lumbricus terrestris. Pedobiologia 47, 574-577. Makulec, G., 2002. The role of Lumbricus rubellus Hoffm. in determining biotic and abiotic properties of peat soils. Polish J. of Ecology 50, 301-339. Petersen H., Luxton M., 1982. A comparative analysis of soil fauna populations and their role in decomposition processes. Oikos 39: 287-388. Phillipson J., Abel R., Steel J., Woodell S.R.J., 1976. Earthworms and the factors governing their distribution in an English beechwood. Pedobiologia 16: 258-285. Uvarov, A.V., 1995. Response of an earthworm species to constant and diurnally fluctuating temperature regime in laboratory microcosms. Eur. J. Soil Biol. 31, 111-118. Uvarov, A.V., 1998. Respiration activity of Dendrobaena octaedra (Lumbricidae) under constant and diurnally fluctuating temperature regimes in laboratory microcosms. Eur. J. Soil Biol. 34, 1-10. Uvarov, A.V., 2009. Inter- and intraspecific interactions in lumbricid earthworms: Their role for earthworm performance and ecosystem functioning. Pedobiologia 53: 1-27. Uvarov, A.V., Scheu, S., 2004. Effects of developmental stage and temperature regime on respiration rate of Lumbricus rubellus (Lumbricidae). Pedobiologia 48: 365-371. Uvarov, A.V., Scheu, S., 2005. Effects of group maintenance and temperature regime on respiratory activity of epigeic earthworms, Dendrobaena octaedra and Lumbricus rubellus (Lumbricidae). Eur. J. Soil Biol. 40, 163-167. Uvarov, A.V., Tiunov, A.V., Scheu, S., 2011. Effects of seasonal and diurnal temperature fluctuations on population dynamics of two epigeic earthworm species in forest soil. Soil Biol. Biochem. 43, 559-570. Xia L., Szlavecz K., Swan C.M., Burgess J.L., 2011. Inter- and intra-specific interactions of Lumbricus rubellus (Hoffmeister, 1843) and Octolasion lacteum (Örley, 1881) (Lumbricidae) and the implication for C cycling. Soil Biol. Biochem. 43: 1584-1590.
20 Zorn, M.I., Van Gestel, C.A., Eijsackers, H., 2005. Species-specific earthworm population responses in relation to flooding dynamics in a Dutch floodplain soil. Pedobiologia 49: 189-198.
21 Figure captions:
Fig. 1. Mortality levels (% ± S.E.) in five earthworm species: monocultures with a density gradient. Symbols: Do – Dendrobaena octaedra, Lr – Lumbricus rubellus, Lt – L. terrestris, Acl – Allolobophora chlorotica, Aca – Aporrectodea caliginosa; Min, Med, Max – density treatments.
Fig. 2. Biomass changes (% ± S.E.) in five earthworm species: monocultures with a density gradient. Symbols: Do – Dendrobaena octaedra, Lr – Lumbricus rubellus, Lt – L. terrestris, Acl – Allolobophora chlorotica, Aca – Aporrectodea caliginosa; Min, Med, Max – density treatments.
Fig. 3. Proportion of mature specimens (% ± S.E.) in Allolobophora chlorotica (Acl) and Aporrectodea caliginosa (Aca) monocultures. Min, Med, Max – density treatments.
Fig. 4. Offspring production, ind. ± S.E. (expressed per minimum earthworm group, see Methods) in five earthworm species: monocultures with a density gradient. Symbols: Do – Dendrobaena octaedra, Lr – Lumbricus rubellus, Lt – L. terrestris, Acl – Allolobophora chlorotica, Aca – Aporrectodea caliginosa; Min, Med, Max – density treatments.
22
100 90
%.
80
MIN
70
MED
60
MAX
50 40 30 20 10 0
Do
Lr
Lt
Acl
Aca
Fig. 1. Mortality levels (% ± S.E.) in monocultures of five earthworm species in the gradient of density increase. Symbols: Do – Dendrobaena octaedra, Lr – Lumbricus rubellus, Lt – L. terrestris, Acl – Allolobophora chlorotica, Aca – Aporrectodea caliginosa; Min, Med, Max – density treatments.
23
60 50 40 30 20 10 0
%.
-10
Do
Lr
Lt
Acl
Aca
-20 -30 -40 -50
MIN
-60
MED
-70
MAX
-80 -90 -100
Fig. 2. Biomass changes (% ± S.E.) in monocultures of five earthworm species in the gradient of density increase. Symbols: Do – Dendrobaena octaedra, Lr – Lumbricus rubellus, Lt – L. terrestris, Acl – Allolobophora chlorotica, Aca – Aporrectodea caliginosa; Min, Med, Max – density treatments.
24
100 90 80 70
%.
60 50 40
Acl
30 Aca 20 10 0
MIN
MED
MAX
Fig. 3. Proportion of mature specimens (% ± S.E.) in monocultures of Allolobophora chlorotica (Acl) and Aporrectodea caliginosa (Aca) in the gradient of density increase. Min, Med, Max – density treatments.
25
100
MIN MED
80
MAX
Ind.
60
40
20
0
Do
Lr
Lt
Acl
Aca
Fig. 4. Offspring production, ind. ± S.E., (expressed per minimum earthworm group, see Methods) in monocultures of five earthworm species in the gradient of density increase. Symbols: Do – Dendrobaena octaedra, Lr – Lumbricus rubellus, Lt – L. terrestris, Acl – Allolobophora chlorotica, Aca – Aporrectodea caliginosa; Min, Med, Max – density treatments.
26 Table 1.Significance of density-dependent changes in mortality, biomass, maturation and reproduction rates in five earthworm species, estimated by one-way ANOVA or Kruskal–Wallis ANOVA. Probability levels are indicated by *** (<0.001), ** (<0.01), * (<0.5) and ^ (<0.1).
Parameter Mortality Biomass changes Maturation Offspring
D. octaedra H2,12=0.27; P=0.873 F2,9=0.09; P=0.914 – F2,9=5.08; P=0.033*
L. rubellus H2,12=2.18; P=0.337 F2,9=4.82; P=0.038* – F2,9=3.89; P=0.061^
L. terrestris H1,8=1.00; P=0.317 H1,8=2.08; P=0.149 – F1,6=1.72; P=0.238
Al. chlorotica H2,12=4.93; P=0.085^ F2,9=7.51; P=0.012* H2,12=10.24; P=0.006** F2,9=16.57; P=0.001***
A. caliginosa H2,12=7.17; P=0.028* F2,9=3.41; P=0.079^ H2,12=8.04; P=0.018** F2,9=8.75; P=0.008**
27 Table 2. Starting and final individual weights of adult earthworms (g). Significant differences in the line Start – Min – Med – Max treatments (estimated by Kruskal–Wallis ANOVA, with subsequent nonparametric Tukey test in the case of significant effects) are indicated by different letters.
Earthworm species D. octaedra ind. weight L. rubellus ind. weight L. terrestris ind. weight Al. chlorotica ind. weight A. caliginosa ind. weight
Start MIN Treatment effect: H3,193=98.18; P<0.001*** 0.202 ± 0.004 (141) a 0.111 ± 0.016 (6) b Treatment effect: H3,131=68.82; P<0.001*** 0.904 ± 0.021 (86) a 1.011 ± 0.107 (7) a No treatment effect: H2,39=1.98; P=0.372 4.240 ± 0.116 (20) 4.642 ± 0.252 (8) Treatment effect: H3,120=38.58; P<0.001*** 0.292 ± 0.005 (51) a 0.319 ± 0.015 (18) a Treatment effect: H2,77=15.30; P<0.001*** – 0.783 ± 0.069 (12) a
MED
MAX
0.098 ± 0.020 (16) b
0.079 ± 0.007 (30) b
0.449 ± 0.047 (17) b
0.369 ± 0.040 (21) b
–
4,259 ± 0,208 (11)
0.238 ± 0.014 (23) b
0.232 ± 0.009 (28) b
0.552 ± 0.031 (28) b
0.503 ± 0.020 (37) b
28 Table 3. Weight of individual cocoons (mg) produced by earthworms in the MIN, MED and MAX treatments (one-way ANOVA, with subsequent Tukey test). Significant differences in the line Min – Med – Max treatments are indicated by different letters.
Earthworm species D. octaedra cocoon weight L. rubellus cocoon weight L. terrestris cocoon weight Al. chlorotica cocoon weight A. caliginosa cocoon weight
MIN MED Treatment effect: F2,651=51.7; P<0.001*** 4.43 ± 0.03 (126) a 4.43 ± 0.02 (271) a Treatment effect: F2,368=32.9; P<0.001*** 13.19 ± 0.16 (110) a 12.65 ± 0.09 (121) b Treatment effect: F1,77=14.67; P=0.001*** 63.71 ± 0.96 (38) a – Treatment effect: F2,191=4.44; P=0.013** 13.08 ± 0.10 (83) a 13.05 ± 0.12 (57) a Treatment effect: F2,67=8.25; P=0.001*** 25.41 ± 0.39 (35) a 23.08 ± 0.60 (20) b
MAX 4.13 ± 0.02 (257) b 11.97 ± 0.07 (140) c 59.34 ± 0.64 (41) b 12.64 ± 0.12 (54) b 26.23 ± 0.66 (15) a