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Do cryptic species of earthworms affect soil arthropods differently? The case of the Carpetania elisae complex in the center of the Iberian Peninsula
European Journal of Soil Biology 96 (2020) 103147
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European Journal of Soil Biology journal homepage: www.elsevier.com/locate/ejsobi
Do cryptic species of earthworms affect soil arthropods differently? The case of the Carpetania elisae complex in the center of the Iberian Peninsula
T
Mónica Gutiérrez López∗, María Isla García de Leaniz, Dolores Trigo Aza Department of Biodiversity, Ecology and Evolution, Complutense University of Madrid, Faculty of Biology, C/José Antonio Novais s/n, 28040, Madrid, Spain
A R T I C LE I N FO
A B S T R A C T
Handling editor: Stefan Schrader
In recent years, it has been discovered that the endemic earthworm Carpetania elisae (formerly Hormogaster elisae) consists of a complex of at least six cryptic species. Studying the relationships between these new cryptic species and other soil animal communities may be important to understand their functional role in the soil, and eventually to detect possible differences that may help in the delimitation of these species. We have studied the effects of two of these cryptic species (species I from El Molar [Ce1] and species II from El Tomillar [Ce2]) on soil microarthropod communities. Several laboratory experiments were performed with both cryptic species, in the soils of both localities inhabited by these earthworm species, to determine their effects on soil microarthropod communities. Both earthworm species grew quite well throughout the experiment in their original soil, but while Ce1 grew up well when cultivated in the opposite soil from El Tomillar, Ce2 cultivated in the soil from El Molar survived but did not grow. Ce1 from El Molar showed a clear negative effect on microarthropods from El Molar and El Tomillar, whereas Ce2 from El Tomillar had a clear negative effect on soil microarthropods from its original soil but not on the ones from the opposite soil of El Molar. This paper demonstrates differences of both cryptic species on their effects on soil community, which suggest that they may play different roles in the soil system. Several mechanisms likely involved on the effect of earthworms on soil arthropods are discussed, being the competition for organic matter the most probable one in the case of these endogeic earthworms.
Keywords: Earthworms Cryptic species Growth Organic matter Mites Springtails
1. Introduction Numerous invertebrates inhabit the soil, arthropods being one of the most representative groups, mainly dominated by Collembola and Oribatida [1,2]. On the other hand, earthworms represent the highest animal biomass in most temperate terrestrial ecosystems [3]. Due to the action of earthworms, new habitats are continually created, which can be occupied by different groups of organisms as the microarthropods, so it is logical to assume that this activity influences their richness and abundance [4]. Several researches have studied the effect of earthworms on edaphic microarthropods but the results found are very variable. Some authors [5–8] have found positive relationships between both groups due to the creation of new microhabitats interconnected by their galleries and to the fragmentation of fresh organic matter, which improves the availability of nutrients and stimulates microbial activity. The enzymatic activity of soil and microorganisms is increased in the galleries generated by the earthworms (drilosphere) which is important for springtails, since edaphic microorganisms are their main source of food.
Adejuyigbe et al. [8] claim that the action of earthworms and soil microarthropods may even be synergistic, contributing significantly to accelerate the nutrient cycles together. Other authors have found negative relationships between earthworms and microarthropods [9–12], being the hypothesis of competition for food between both groups the most accepted. For example, some earthworms selectively feed on bacteria and fungi in the same way as Collembola and Oribatida do. Other authors [4], however, point to mechanical disturbances through the conversion of litter into earthworm casts and middens as the main cause of the negative effect of earthworms on microarthropods. Finally, other studies did not find a clear effect of one group on the other or observed variable effects depending on the species or the systems considered [4,8,13–16]. This shows the complexity of the interactions between soil fauna and the difficulty of predicting their effects on certain processes such as the decomposition of leaf litter and the nitrogen cycles [14]. The review of Eisenhauer [9] about the influence of earthworms on microarthropods, determine that their effect probably varies according to their ecological category. While epigeic
Abbreviations: Ce1, cryptic species of Carpetania elisae from El Molar; Ce2, cryptic species of Carpetania elisae from El Tomillar ∗ Corresponding author. Department of Biodiversity, Ecology and Evolution, C/ José Antonio Novais, 2, 28040, Madrid, Spain. E-mail address: [email protected] (M. Gutiérrez López). https://doi.org/10.1016/j.ejsobi.2019.103147 Received 14 June 2019; Received in revised form 19 November 2019; Accepted 19 November 2019 1164-5563/ © 2019 Elsevier Masson SAS. All rights reserved.
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earthworms show variable effects according to their density, endogeics usually have negative effects due to competition for trophic resources and yet anecic earthworms tend to exert a positive microhabitat effect through their galleries (burrows and middens), rich in nutrients and microorganisms. Earthworm responses to manipulations of a mesofauna community has also been documented with both, negative and positive mesofauna effects on earthworms, with species-specific and ecological group-specific responses mediated by competition for available resources or by their facilitated exploitation due to mesofauna activities [17]. Novo et al. [18] have shown that the endogeic earthworm Carpetania elisae [19] (formerly Hormogaster elisae, see Marchán et al. [20]) constitutes a monophyletic group separated from the rest of species of the genus Carpetania, with a high genetic diversity that suggests the presence of at least six cryptic allopatric species [21,22]. These authors found a high degree of isolation and a lack of gene flow between populations, concluding that the current distribution of C. elisae could be due to a colonization and later fragmentation of the populations, which have been genetically modified while remaining morphologically the same. To date, only the shape of the genital chaetae seems to distinguish cryptic linages using geometric morphometric analysis, helping in species delimitation and taxonomic description [21]. Most of the studies carried out so far are focused on the lineage I from El Molar (Madrid). This endogeic species has shown a clear negative effect on most microarthropods of this soil, probably due to competition for the same resources [23–25]. It is known that this species has a high resistance to drought and adaptation to semi-arid soils [26], so it is likely to benefit from climate change, which is expected to be highly pronounced in the Mediterranean Basin, with an increase in average temperatures and a decrease in average annual precipitation in the Iberian Peninsula [27]. With the advance of climate change, changes in the distribution and the expansion of some of these cryptic species to the soil that inhabit the others may occur. This fact could have border conflicts and important consequences if the cryptic species show a different effect on soil properties and communities from the occupied territories. Marchán et al. [28] point out the importance of looking for biologically relevant differences between cryptic lineages. Additional traits can help to reveal the functional meaning of cryptic taxa and to define new differential characters that can be used in their delimitation. Among the characters listed are the ecological preferences, behavior and associated biota. The functional role of cryptic lineages may be different in relation to some compartments of the soil ecosystem and, in this case, it may eventually help to better describe and understand these cryptic lineages. Microarthropods are a basic component of soil biota and their relationships with earthworms can modulate the function of important processes such as the organic matter cycle. For this reason, this study is not only interesting itself, but may also help to better understand the phenomenon of cryptic lineages and eventually help in their delimitation. Given these antecedents it seems interesting to study if the negative effect observed for lineage I from El Molar on the microarthropods from this soil is maintained for the other cryptic species, as well as if these species can affect the microarthropods from other soils where they could expand. The objective of this work is to study, in laboratory experiments, the effect of two cryptic species of the C. elisae complex on microarthropod communities from the soils that they inhabit, as well as from soils that they could colonize. The selected cryptic species are lineage I (Ce1), located in the municipality of El Molar, and lineage II (Ce2), located in a plot of El Tomillar in the municipality of Gargantilla de Lozoya, both areas situated at northeast of Madrid Community and separated around 30 km from each other.
Fig. 1. Map of Central Iberian Peninsula showing the location of the two study zones where both earthworm populations inhabit.
2. Material and methods 2.1. Description of the species and the study area C. elisae complex is composed of at least six endemic cryptic species from the central area of the Iberian Peninsula belonging to the family Hormogastridae [29]. They are characterized by having a relatively large size, ecologically classified as endogeic, geophagous and oligohumic and by usually inhabiting areas poor in organic matter, subject to erosion and summer aridity [26,30]. To resist unfavorable environmental conditions, they make vertical migrations depending on the moisture of the soil [31] and may enter paradiapause [32]. The two cryptic species studied come from two localities located in the northeast of Madrid separated about 30 km from each other (Fig. 1). The cryptic species I (Ce1) is located in the municipality of El Molar, 40 km from Madrid (GMS 404422 N, 033350 W) and at an approximate altitude of 800 m. This zone presents a sandy-loam texture, formed by materials from the Tertiary, represented mainly by clays, arches and silts of the Middle Miocene on sandstones. The climate is semi-arid Mediterranean type with an average annual precipitation of 438 mm distributed irregularly throughout the year and an average temperature of 14 °C. The vegetation consists of an abandoned pasture with a high percentage of terophyte plants [33]. The cryptic species II (Ce 2) is located in a plot of El Tomillar area, in the municipality of Gargantilla de Lozoya (GMS 405640 N, 034313 W), 80 km from Madrid and at an average altitude of 1140 m. This zone displays a soil mainly formed by granitic rocks dating from the Cambrian-Precambrian. The climate is oceanic-mediterranean type with an annual average temperature of 10.4 °C and an average annual rainfall of 515 mm. It is a prairie dominated mainly by herbaceous plants, surrounded by forest of Quercus coccifera. 2.2. Laboratory methodology A methodology similar to that used by Gutiérrez López et al. [23] was used. Four types of microcosms were assembled with five replicates each for a total of 20 microcosms. Each microcosm consisted of rectangular plastic boxes of 2 L capacity and a size of 19 × 14 × 7 cm, divided into two compartments separated by a grid of 2 mm of mesh size to allow the microarthropods to move freely from an area to another but making impossible for earthworms to move from one side to the other. In each compartment of the microcosm 500 g of moist soil was introduced with its natural microarthropod community, measuring the initial humidity of each type of soil and adding the distilled water 2
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Fig. 2. Diagram of the four types of microcosms used in the experiment. Ce1: species from El Molar; Ce2: species from El Tomillar.
the specimens. Springtails were identified at the family level and mites at the suborder level, using Jordana and Arbea's classification [34] for springtails and Krantz [35] and Dindal's [36] for mites. The other groups of arthropods were identified at the order or suborder level. With the objective of evaluating the natural community of microarthropods of the study areas not subjected to any type of treatment or culture, total microarthropods were extracted, identified and counted from the same amount of fresh soil used in each experimental sample (500 g at 20% soil humidity) with the same methods explained above, before performing the experiments. These samples were called “time 0”, and 5 replicates were taken for each soil types, from El Tomillar and El Molar. The chemical properties of the original soils used in the experiment, both, from El Molar and El Tomillar, were analyzed. Total organic carbon was determined according to the Anne [37] method adapted to the microplates and total organic nitrogen was analyzed by the Kjeldahl method as indicated in Page et al. [38]; pH was measured according to the method described in Guitián and Carballas [39] and the calcium following the complexometric method proposed in Porta [40].
needed to reach 20% humidity. Twenty-four hours after their assembly, two adult earthworms of similar weight were introduced into one of the compartments of each microcosm. The earthworms used were previously sampled in their respective areas by manual excavation and hand shorting. The sampling was made after spring rains, which is the better season to sample this species in the field, when soil has optimal conditions of moisture in both areas (between 18% and 20%). They were kept in optimal conditions for the cultivation of these species (15 °C and 20% soil moisture) in dark growth chambers for at least one month before their use in the experiments, with the aim of allowing them to acclimatize to the experimental conditions. The soils needed to assemble the microcosms were collected at each area and were crumbled to remove any other earthworm unrelated to the experiment, but they were not dried or sieved to maintain their natural microarthropod community. The four types of microcosms were as follows (Fig. 2): (A) control microcosms were made with soil from El Tomillar, without earthworms in any of the compartments (called A and B), to see if any other factor other than the earthworms is influencing the distribution of microarthropods; (B) microcosms with soil from El Molar and compartments with or without earthworms Ce2 from El Tomillar; (C) microcosms with soil from El Tomillar and compartments with or without earthworms Ce2 from El Tomillar; (D) microcosms with soil from El Tomillar and compartments with or without earthworms Ce1 from El Molar. Experiments with soil from El Molar and the species Ce1 from El Molar had previously been performed and published by Gutiérrez López et al. [23]. The experiments were carried out in spring and the microcosms were maintained in a culture chamber in darkness at constant and controlled temperature and humidity (15 °C and 20% respectively) for 21 days which, given the rate of cast production in this earthworm species, was enough time to affect soil, as made in Gutiérrez et al. [23–25]. After this time, the microcosms were dismantled, and the earthworms were weighted to obtain the percentage of weight increase [%ΔWeight = (Final Weight - Initial Weight) x 100/Initial Weight]. The microarthropods were extracted separately from the soil of each compartment following Tullgren modification of Berlese's method for 10 days. The separation and counting of each of the groups of microarthropods were carried out by means of stereomicroscope observation for which Scheerpeltz liquid was used in order to preserve and maintain
2.3. Statistical analysis Firstly, we compared the abundances of each group of arthropods and the chemical properties between both types of soil studied at time 0 (El Molar and El Tomillar). The weight increase of the earthworms during the experiment was compared among the different types of microcosms which housed earthworms. These data were compared by simple ANOVA, when were normally distributed, or Kruskal-Wallis when were not normally distributed and, when necessary, the multiplerank test was performed. The abundances of each group of microarthropod obtained at the end of the experiment were compared by paired t-tests for paired samples using the presence or absence of earthworms, or the compartment A or B in the controls, as independent variable (Fig. 2) for each type of microcosm. The statistical program Statgraphics (Centurion XVI version) was used for all the analyses.
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Table 1 Abundances per microcosm of each arthropod group (mean ± standard deviation) in samples at time 0, chemical properties and edaphic description of these soils from El Tomillar and El Molar. The p-value and the statistics F (ANOVA) or E (Kruskal-Wallis) are indicated. %C: percentage of organic carbon; %N: percentage of total nitrogen; C/N: carbon/nitrogen ratio; Ca2+ (meq/100 g): content of calcium.
Hypogastruridae Neanuridae Isotomidae Total Arthropleona Sminthuridae Total Collembola Oribatida Actinedida Gamasida Total Acari Other arthropods %C %N C/N Ca
2+
(meq/100 g)
pH
El Tomillar soil
El Molar soil
61.8 ± 22.30 6.8 ± 4.87 34.8 ± 15.55 104.6 ± 38.95 2.4 ± 1.14 107.4 ± 38.06 38.6 ± 3.91 31.6 ± 8.65 5.6 ± 3.85 76.0 ± 14.44 9.8 ± 5.67 2.51 ± 0.10 Normal content 0.21 ± 0.01 Slightly high content 11.95 ± 0.79 Slightly high 3.03 ± 0.23 Low content 5.97 ± 0.24 Moderately acidic
10.2 ± 10.62 2.6 ± 2.611 9.4 ± 8.17 22.4 ± 13.76 0.2 ± 0.45 23.0 ± 13.38 17.4 ± 4.77 26.2 ± 5.49 0.6 ± 0.55 45.8 ± 10.08 1.4 ± 0.55 1.46 ± 0.39 Very poor content 0.13 ± 0.02 Normal content 10.87 ± 2.26 Slightly high 3.83 ± 0.30 Low content 6.05 ± 0.10 Moderately acidic
F = 21.83; P < 0.01 F = 2.89; P > 0.05 F = 10.46; P < 0.05 F = 19.80; P < 0.01 E = 6.77; P < 0.01 F = 21.89; P < 0.01 F = 58.98; P < 0.001 F = 1.39; P > 0.05 E = 5.65; P < 0.05 F = 14.70; P < 0.01 F = 36.81; P < 0.001 E = 11.29; P < 0.001 E = 11.29; P < 0.001 F = 1.63; P > 0.05 F = 32.03; P < 0.001 E = 0.01; P > 0.05
3. Results
3.3. Experiments performed with the soil from El Tomillar
3.1. Microarthropod community and chemical properties of the studied soils
As seen in Fig. 3 (A), which shows the results obtained in the control microcosms, no significant differences were observed between the two compartments (A and B) for the abundances of any group of microarthropods. In the experiments carried out with soil and earthworm species Ce2 from El Tomillar (Fig. 3 (C)), almost all arthropod groups were more abundant in compartments without earthworms, significantly for Isotomidae springtails (T = 2.61, P < 0.05) and Oribatida (T = 8.75, P < 0.001), Actinedida (T = 2.70, P < 0.05) and Total Acari (T = 3.67, P < 0.05). The abundances of Hypogastruridae and Sminthuridae springtails were very similar between compartments, independently of the presence or absence of earthworms. In the experiments performed with soil from El Tomillar and the earthworm species Ce1 from El Molar (Fig. 3 (D)), most arthropod groups were also more abundant in the absence of earthworms, significantly for Hypogastruridae (T = 4.23, P < 0.01), Isotomidae (T = 2.99, P < 0.05), total Arthropleona (T = 3.13, P < 0.05), total Collembola (T = 3.16, P < 0.05) and Total Acari (T = 2.70, P < 0.05). Oribatida mites presented similar trends with values very close to significance (T = 2.58; P = 0.06).
The microarthropod community of the studied soils was formed mainly by two large groups, springtails (Collembola) and mites (Acari). Within the order Collembola, the suborder Arthropleona was dominant over the rest of groups, the family Isotomidae being the most numerous, followed by Hypogastruridae and Neanuridae, although Entomobryidae were also found in smaller quantities. There were also individuals of Symphypleona, all of them from the family Sminthuridae. As for mites, the four main edaphic suborders appeared: Oribatida (the most numerous group of mites), Actinedida, Gamasida and the smallest Acaridida. In addition to mites and springtails other groups of Hexapoda and Myriapoda were found and grouped under “Other arthropods”. Within Hexapoda the orders Protura, Psocoptera, Hymenoptera, Diplura (Japygida), Heteroptera, Homoptera and larvae of Diptera, Lepidoptera and Coleoptera were found. Within Myriapoda, Symphyla, Pauropoda and Chilopoda were found. Table 1 represents the microarthropod community found in samples taken at time 0, before the beginning of the experiment in both soil types. All the groups found were more abundant in the original soils from El Tomillar than in those from El Molar. No significant differences were observed for Neanuridae springtails and Actinedida mites, although both groups were also more abundant in the soils from El Tomillar. Chemical analysis (Table 1) show that the soil from El Tomillar had higher carbon and nitrogen content than the one from El Molar. Both soils presented low content of calcium, which was slightly higher in the soil from El Molar. C/N ratio value was slightly high in both soils and pH values were moderately acidic and very similar in both soils.
3.4. Experiments performed with the soil from El Molar Fig. 3 (B) shows the results of the experiments performed with soil from El Molar and the earthworm species Ce2 from El Tomillar. The decrease in the abundance of all the groups of microarthropods with respect to those made in the soils from El Tomillar is quite striking, as observed in the soils analyzed at time 0. In any case, there were no significant differences for any group of microarthropod between compartments with and without earthworms, so the presence of the species Ce2 does not seem to affect the microarthropod populations of the soil from El Molar.
3.2. Earthworm growth
4. Discussion
The earthworms started the experiment with a very similar weight in each type of microcosm (Table 2). At the end of the experiment individuals of both species grown in the soil from El Tomillar increased their weight significantly, being the weight increase very similar. However, individuals of Ce2 cultivated in the soil from El Molar maintained or even slightly decreased their weight.
Several studies have shown that earthworms are important biological invaders causing effects in invaded communities [13,16,41]. A possible scenario of climate change, characterized by an increase in average temperatures as well as a decrease in average rainfall in the 4
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Table 2 Initial and final earthworm weights per microcosm and percentage of weight increase (% ΔWeight) (mean ± standard deviation) in each type of microcosm. The pvalue and the F-statistic (ANOVA) are given. Different letters in the same line indicate significant differences. Ce1: species from El Molar; Ce2: species from El Tomillar.
Initial weight (g) Final weight (g) % ΔWeight
Ce2 El Tomillar soil
Ce1 El Tomillar soil
Ce 2 El Molar soil
5.75 ± 0.28 7.13 ± 0.84b 23.74 ± 11.13b
5.91 ± 0.1 7.10 ± 0.65b 20.04 ± 12.14b
5.79 ± 0.12 5.74 ± 0.71a −0.90 ± 13.02a
F = 1.03; P > 0.05 F = 5.82; P < 0.05 F = 6.01; P < 0.05
Fig. 3. Mean abundances of each arthropod group comparing both compartments in each experiment type. (A): control microcosms made with the soil from El Tomillar in both compartments (A and B); (B): experiments made in soil from El Molar with and without the species from El Tomillar (Ce2); (C): experiments made in soil from El Tomillar with or without the species from El Tomillar (Ce2); (D): experiments made in soil from El Tomillar with or without the species from El Molar (Ce1). The results of the statistical analysis are also shown when differences between compartments were significant (*p < 0.05; **p < 0.01; ***p < 0.001).
effects of species Ce2 on some groups of microarthropods such as springtails, as significant effects were observed not only on the Isotomidae, but also on Hypogastruridae and the total Arthropleona and Collembola. However, in the case of mites, the negative effect was less evident as only were observed on Total Acari, although similar trends were observed for some individual taxa as Oribatida. In any case, the effect of both cryptic species seems to be quite similar, exerting a clearly negative effect on the arthropod communities from El Tomillar. Considering the ease of growth of Ce1 in the soil from El Tomillar, a likely outcome of a hypothetical range expansion and colonization of this soil would be its establishment and modification of the arthropod communities of said soil, in a similar way that cryptic species Ce2 already does.
Iberian Peninsula [27], could lead to fundamental changes in habitat conditions for the different cryptic species of C. elisae, which could determine a possible expansion of these species to other areas. In the experiments developed in this study, we have observed that cryptic species of C. elisae could have variable effects on soil microarthropod communities, which highlight the importance of a deeper understanding of these species. This fact may help to describe and delimit these two cryptic species and understand how borders are produced between them [22].
4.1. What could happen in the soil from El Tomillar? In the experiments performed with soil from El Tomillar both cryptic species showed a clear negative effect on the main microarthropod groups found in this soil. The species Ce2, which grew perfectly when cultivated in its original soil, showed a clear negative effect on Isotomidae Springtails and on Oribatida and Actinedida mites. The species Ce1 from El Molar also grew perfectly in the soil from El Tomillar, which could be favored by the bigger content in organic matter in this soil. The effects of Ce1 seem even more evident than the
4.2. What could happen in the soil from El Molar? The species Ce2 from El Tomillar did not show any effect on the microarthropods from El Molar. This could be surprising at first, since a negative effect had been observed on the microarthropods from its own soil. However, it should be noted that this species does not appear to 5
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of earthworms [9], with high densities causing a drastic change of the physical structure of the soil and negatively affecting microarthropods [10,13,43]. The long-term negative effects and high densities (or low earthworm/soil ratios) are consistent with our study, where earthworms had processed all the soil within their reach by the time the experiments ended.
grow up optimally in the soil from El Molar, so their action on this soil would be much weaker. In the case of a possible expansion and colonization of this species to the soils from El Molar, it appears unlikely that it could be easily established or could alter the communities of arthropods that inhabit this soil. We must remember that the soil from El Molar is arid [33] and presents less organic matter than the soil from El Tomillar. The species Ce1 that inhabits El Molar is very well adapted to this type of soil through various mechanisms such as the processes of aestivation or food selection [32,42]. However, the species Ce2 from El Tomillar is adapted to soils much richer in organic matter, thus it may not have such adaptation mechanisms, making their expansion towards these areas improbable. We must also consider that, since the soil from El Molar is less favorable than the soil from El Tomillar, the period of acclimation to the new conditions could be longer for Ce2 to El Molar soil than for Ce1 to El Tomillar soil. In any case, in the face of the advance of climate change, it seems very unlikely the expansion of the Ce2 species to El Molar area, since this zone is much more arid, and the soil is much poorer than in its area of origin. Additional studies on their biology and ecology as well as on its capacity to expand and colonize new habitats should be done to verify it.
5. Conclusions The study of the biology and ecology of new cryptic species is fundamental since, as we have seen in these experiments, their effect on other soil communities could be very different. The two cryptic species studied have shown a clear negative effect on microarthropods from the soils they inhabit. However, while the species Ce1 from El Molar could colonize and benefit from El Tomillar soil aggravating the negative effect and altering the microarthropod communities from this area, it is not clear that the species Ce2 from El Tomillar could colonize the soil from El Molar and affect their microarthropod community. Soil organic matter is suggested as one of the most probable mechanisms for understanding the interrelationships between these earthworm species and the microarthropods. However, it is important to underline that the results of these experiments are preliminary as have been performed in laboratory microcosms, in limiting conditions. Therefore, it would be interesting to carry out further field studies that allow extrapolation of these results and the prediction of the possible patterns of expansion and colonization of these species as well as their effects on the new soils.
4.3. Main mechanisms driving earthworms and microarthropods relationship Eisenhauer [9] stated that positive effect of earthworms on microarthropods occurs mainly in the case of anecics, provided their densities are not very high. However, the effect of endogeic earthworms on microarthropods is generally negative, but its causes are not yet clearly known. One of the most probable mechanisms could be a competitive relationship between both groups [13,17,24]. Due to their largest size and highest agility, earthworms can access soil resources more effectively by hurting other edaphic groups, being more efficient competitors than microarthropods; thus, their relationship should really be considered amensalism rather than competition [9]. In the case of the experimental conditions of this study, in which the amount of available soil is limited, it is possible that earthworms consume a significant part of the useable organic matter, not being available to microarthropods, which would tend to migrate to compartments without earthworms. In fact, Gutiérrez López et al. [24], had already determined in laboratory, by means of a supply of organic matter, that the negative effect observed for C. elisae species from El Molar on microarthropods from this area is probably due to competition for food resources. The results obtained in this study with the species Ce2 from El Tomillar also point in this direction, as the negative effect is only clearly observed on microarthropods from their own soil, but not on the microarthropods from El Molar. This result suggests that this species, adapted to richer soils, is not able to make an optimal use of the poorer organic matter soil from El Molar and it frees up a resource that microarthropods can take advantage of. However, we must consider that these results, obtained in the laboratory, could be mitigated in the field, where both, earthworms and microarthropods, have the possibility of moving around looking for other trophic resources and thus avoid competition. Another mechanism to be considered is the disturbing effect of earthworms on the soil; for example, distributing organic matter more homogeneously in the soil layers [23]. Many microarthropods are sensitive to this disturbance of the soil [10,43], which would force them to look for trophic resources in deeper layers. Earthworms also modify the soil, producing biogenic structures such as galleries and accumulations of casts in which chemical changes occur, increasing the amount of organic matter and changing the structure of soils [3]. In this sense, Straube et al. [16], found similar results for the invasive, and endogeic species, Octolasion tyrtaeum, which at low densities altered the soil moderately, increasing the number of microhabitats, the microbial biomass and favoring microarthropods, but that positive effect disappeared with increasing earthworm density. Therefore, the negative effects of endogeic species would depend on the biomass or the density
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Do cryptic species of earthworms affect soil arthropods differently? The case of the Carpetania elisae complex in the center of the Iberian Peninsula
T
Mónica Gutiérrez López∗, María Isla García de Leaniz, Dolores Trigo Aza Department of Biodiversity, Ecology and Evolution, Complutense University of Madrid, Faculty of Biology, C/José Antonio Novais s/n, 28040, Madrid, Spain
A R T I C LE I N FO
A B S T R A C T
Handling editor: Stefan Schrader
In recent years, it has been discovered that the endemic earthworm Carpetania elisae (formerly Hormogaster elisae) consists of a complex of at least six cryptic species. Studying the relationships between these new cryptic species and other soil animal communities may be important to understand their functional role in the soil, and eventually to detect possible differences that may help in the delimitation of these species. We have studied the effects of two of these cryptic species (species I from El Molar [Ce1] and species II from El Tomillar [Ce2]) on soil microarthropod communities. Several laboratory experiments were performed with both cryptic species, in the soils of both localities inhabited by these earthworm species, to determine their effects on soil microarthropod communities. Both earthworm species grew quite well throughout the experiment in their original soil, but while Ce1 grew up well when cultivated in the opposite soil from El Tomillar, Ce2 cultivated in the soil from El Molar survived but did not grow. Ce1 from El Molar showed a clear negative effect on microarthropods from El Molar and El Tomillar, whereas Ce2 from El Tomillar had a clear negative effect on soil microarthropods from its original soil but not on the ones from the opposite soil of El Molar. This paper demonstrates differences of both cryptic species on their effects on soil community, which suggest that they may play different roles in the soil system. Several mechanisms likely involved on the effect of earthworms on soil arthropods are discussed, being the competition for organic matter the most probable one in the case of these endogeic earthworms.
Keywords: Earthworms Cryptic species Growth Organic matter Mites Springtails
1. Introduction Numerous invertebrates inhabit the soil, arthropods being one of the most representative groups, mainly dominated by Collembola and Oribatida [1,2]. On the other hand, earthworms represent the highest animal biomass in most temperate terrestrial ecosystems [3]. Due to the action of earthworms, new habitats are continually created, which can be occupied by different groups of organisms as the microarthropods, so it is logical to assume that this activity influences their richness and abundance [4]. Several researches have studied the effect of earthworms on edaphic microarthropods but the results found are very variable. Some authors [5–8] have found positive relationships between both groups due to the creation of new microhabitats interconnected by their galleries and to the fragmentation of fresh organic matter, which improves the availability of nutrients and stimulates microbial activity. The enzymatic activity of soil and microorganisms is increased in the galleries generated by the earthworms (drilosphere) which is important for springtails, since edaphic microorganisms are their main source of food.
Adejuyigbe et al. [8] claim that the action of earthworms and soil microarthropods may even be synergistic, contributing significantly to accelerate the nutrient cycles together. Other authors have found negative relationships between earthworms and microarthropods [9–12], being the hypothesis of competition for food between both groups the most accepted. For example, some earthworms selectively feed on bacteria and fungi in the same way as Collembola and Oribatida do. Other authors [4], however, point to mechanical disturbances through the conversion of litter into earthworm casts and middens as the main cause of the negative effect of earthworms on microarthropods. Finally, other studies did not find a clear effect of one group on the other or observed variable effects depending on the species or the systems considered [4,8,13–16]. This shows the complexity of the interactions between soil fauna and the difficulty of predicting their effects on certain processes such as the decomposition of leaf litter and the nitrogen cycles [14]. The review of Eisenhauer [9] about the influence of earthworms on microarthropods, determine that their effect probably varies according to their ecological category. While epigeic
Abbreviations: Ce1, cryptic species of Carpetania elisae from El Molar; Ce2, cryptic species of Carpetania elisae from El Tomillar ∗ Corresponding author. Department of Biodiversity, Ecology and Evolution, C/ José Antonio Novais, 2, 28040, Madrid, Spain. E-mail address: [email protected] (M. Gutiérrez López). https://doi.org/10.1016/j.ejsobi.2019.103147 Received 14 June 2019; Received in revised form 19 November 2019; Accepted 19 November 2019 1164-5563/ © 2019 Elsevier Masson SAS. All rights reserved.
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earthworms show variable effects according to their density, endogeics usually have negative effects due to competition for trophic resources and yet anecic earthworms tend to exert a positive microhabitat effect through their galleries (burrows and middens), rich in nutrients and microorganisms. Earthworm responses to manipulations of a mesofauna community has also been documented with both, negative and positive mesofauna effects on earthworms, with species-specific and ecological group-specific responses mediated by competition for available resources or by their facilitated exploitation due to mesofauna activities [17]. Novo et al. [18] have shown that the endogeic earthworm Carpetania elisae [19] (formerly Hormogaster elisae, see Marchán et al. [20]) constitutes a monophyletic group separated from the rest of species of the genus Carpetania, with a high genetic diversity that suggests the presence of at least six cryptic allopatric species [21,22]. These authors found a high degree of isolation and a lack of gene flow between populations, concluding that the current distribution of C. elisae could be due to a colonization and later fragmentation of the populations, which have been genetically modified while remaining morphologically the same. To date, only the shape of the genital chaetae seems to distinguish cryptic linages using geometric morphometric analysis, helping in species delimitation and taxonomic description [21]. Most of the studies carried out so far are focused on the lineage I from El Molar (Madrid). This endogeic species has shown a clear negative effect on most microarthropods of this soil, probably due to competition for the same resources [23–25]. It is known that this species has a high resistance to drought and adaptation to semi-arid soils [26], so it is likely to benefit from climate change, which is expected to be highly pronounced in the Mediterranean Basin, with an increase in average temperatures and a decrease in average annual precipitation in the Iberian Peninsula [27]. With the advance of climate change, changes in the distribution and the expansion of some of these cryptic species to the soil that inhabit the others may occur. This fact could have border conflicts and important consequences if the cryptic species show a different effect on soil properties and communities from the occupied territories. Marchán et al. [28] point out the importance of looking for biologically relevant differences between cryptic lineages. Additional traits can help to reveal the functional meaning of cryptic taxa and to define new differential characters that can be used in their delimitation. Among the characters listed are the ecological preferences, behavior and associated biota. The functional role of cryptic lineages may be different in relation to some compartments of the soil ecosystem and, in this case, it may eventually help to better describe and understand these cryptic lineages. Microarthropods are a basic component of soil biota and their relationships with earthworms can modulate the function of important processes such as the organic matter cycle. For this reason, this study is not only interesting itself, but may also help to better understand the phenomenon of cryptic lineages and eventually help in their delimitation. Given these antecedents it seems interesting to study if the negative effect observed for lineage I from El Molar on the microarthropods from this soil is maintained for the other cryptic species, as well as if these species can affect the microarthropods from other soils where they could expand. The objective of this work is to study, in laboratory experiments, the effect of two cryptic species of the C. elisae complex on microarthropod communities from the soils that they inhabit, as well as from soils that they could colonize. The selected cryptic species are lineage I (Ce1), located in the municipality of El Molar, and lineage II (Ce2), located in a plot of El Tomillar in the municipality of Gargantilla de Lozoya, both areas situated at northeast of Madrid Community and separated around 30 km from each other.
Fig. 1. Map of Central Iberian Peninsula showing the location of the two study zones where both earthworm populations inhabit.
2. Material and methods 2.1. Description of the species and the study area C. elisae complex is composed of at least six endemic cryptic species from the central area of the Iberian Peninsula belonging to the family Hormogastridae [29]. They are characterized by having a relatively large size, ecologically classified as endogeic, geophagous and oligohumic and by usually inhabiting areas poor in organic matter, subject to erosion and summer aridity [26,30]. To resist unfavorable environmental conditions, they make vertical migrations depending on the moisture of the soil [31] and may enter paradiapause [32]. The two cryptic species studied come from two localities located in the northeast of Madrid separated about 30 km from each other (Fig. 1). The cryptic species I (Ce1) is located in the municipality of El Molar, 40 km from Madrid (GMS 404422 N, 033350 W) and at an approximate altitude of 800 m. This zone presents a sandy-loam texture, formed by materials from the Tertiary, represented mainly by clays, arches and silts of the Middle Miocene on sandstones. The climate is semi-arid Mediterranean type with an average annual precipitation of 438 mm distributed irregularly throughout the year and an average temperature of 14 °C. The vegetation consists of an abandoned pasture with a high percentage of terophyte plants [33]. The cryptic species II (Ce 2) is located in a plot of El Tomillar area, in the municipality of Gargantilla de Lozoya (GMS 405640 N, 034313 W), 80 km from Madrid and at an average altitude of 1140 m. This zone displays a soil mainly formed by granitic rocks dating from the Cambrian-Precambrian. The climate is oceanic-mediterranean type with an annual average temperature of 10.4 °C and an average annual rainfall of 515 mm. It is a prairie dominated mainly by herbaceous plants, surrounded by forest of Quercus coccifera. 2.2. Laboratory methodology A methodology similar to that used by Gutiérrez López et al. [23] was used. Four types of microcosms were assembled with five replicates each for a total of 20 microcosms. Each microcosm consisted of rectangular plastic boxes of 2 L capacity and a size of 19 × 14 × 7 cm, divided into two compartments separated by a grid of 2 mm of mesh size to allow the microarthropods to move freely from an area to another but making impossible for earthworms to move from one side to the other. In each compartment of the microcosm 500 g of moist soil was introduced with its natural microarthropod community, measuring the initial humidity of each type of soil and adding the distilled water 2
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Fig. 2. Diagram of the four types of microcosms used in the experiment. Ce1: species from El Molar; Ce2: species from El Tomillar.
the specimens. Springtails were identified at the family level and mites at the suborder level, using Jordana and Arbea's classification [34] for springtails and Krantz [35] and Dindal's [36] for mites. The other groups of arthropods were identified at the order or suborder level. With the objective of evaluating the natural community of microarthropods of the study areas not subjected to any type of treatment or culture, total microarthropods were extracted, identified and counted from the same amount of fresh soil used in each experimental sample (500 g at 20% soil humidity) with the same methods explained above, before performing the experiments. These samples were called “time 0”, and 5 replicates were taken for each soil types, from El Tomillar and El Molar. The chemical properties of the original soils used in the experiment, both, from El Molar and El Tomillar, were analyzed. Total organic carbon was determined according to the Anne [37] method adapted to the microplates and total organic nitrogen was analyzed by the Kjeldahl method as indicated in Page et al. [38]; pH was measured according to the method described in Guitián and Carballas [39] and the calcium following the complexometric method proposed in Porta [40].
needed to reach 20% humidity. Twenty-four hours after their assembly, two adult earthworms of similar weight were introduced into one of the compartments of each microcosm. The earthworms used were previously sampled in their respective areas by manual excavation and hand shorting. The sampling was made after spring rains, which is the better season to sample this species in the field, when soil has optimal conditions of moisture in both areas (between 18% and 20%). They were kept in optimal conditions for the cultivation of these species (15 °C and 20% soil moisture) in dark growth chambers for at least one month before their use in the experiments, with the aim of allowing them to acclimatize to the experimental conditions. The soils needed to assemble the microcosms were collected at each area and were crumbled to remove any other earthworm unrelated to the experiment, but they were not dried or sieved to maintain their natural microarthropod community. The four types of microcosms were as follows (Fig. 2): (A) control microcosms were made with soil from El Tomillar, without earthworms in any of the compartments (called A and B), to see if any other factor other than the earthworms is influencing the distribution of microarthropods; (B) microcosms with soil from El Molar and compartments with or without earthworms Ce2 from El Tomillar; (C) microcosms with soil from El Tomillar and compartments with or without earthworms Ce2 from El Tomillar; (D) microcosms with soil from El Tomillar and compartments with or without earthworms Ce1 from El Molar. Experiments with soil from El Molar and the species Ce1 from El Molar had previously been performed and published by Gutiérrez López et al. [23]. The experiments were carried out in spring and the microcosms were maintained in a culture chamber in darkness at constant and controlled temperature and humidity (15 °C and 20% respectively) for 21 days which, given the rate of cast production in this earthworm species, was enough time to affect soil, as made in Gutiérrez et al. [23–25]. After this time, the microcosms were dismantled, and the earthworms were weighted to obtain the percentage of weight increase [%ΔWeight = (Final Weight - Initial Weight) x 100/Initial Weight]. The microarthropods were extracted separately from the soil of each compartment following Tullgren modification of Berlese's method for 10 days. The separation and counting of each of the groups of microarthropods were carried out by means of stereomicroscope observation for which Scheerpeltz liquid was used in order to preserve and maintain
2.3. Statistical analysis Firstly, we compared the abundances of each group of arthropods and the chemical properties between both types of soil studied at time 0 (El Molar and El Tomillar). The weight increase of the earthworms during the experiment was compared among the different types of microcosms which housed earthworms. These data were compared by simple ANOVA, when were normally distributed, or Kruskal-Wallis when were not normally distributed and, when necessary, the multiplerank test was performed. The abundances of each group of microarthropod obtained at the end of the experiment were compared by paired t-tests for paired samples using the presence or absence of earthworms, or the compartment A or B in the controls, as independent variable (Fig. 2) for each type of microcosm. The statistical program Statgraphics (Centurion XVI version) was used for all the analyses.
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Table 1 Abundances per microcosm of each arthropod group (mean ± standard deviation) in samples at time 0, chemical properties and edaphic description of these soils from El Tomillar and El Molar. The p-value and the statistics F (ANOVA) or E (Kruskal-Wallis) are indicated. %C: percentage of organic carbon; %N: percentage of total nitrogen; C/N: carbon/nitrogen ratio; Ca2+ (meq/100 g): content of calcium.
Hypogastruridae Neanuridae Isotomidae Total Arthropleona Sminthuridae Total Collembola Oribatida Actinedida Gamasida Total Acari Other arthropods %C %N C/N Ca
2+
(meq/100 g)
pH
El Tomillar soil
El Molar soil
61.8 ± 22.30 6.8 ± 4.87 34.8 ± 15.55 104.6 ± 38.95 2.4 ± 1.14 107.4 ± 38.06 38.6 ± 3.91 31.6 ± 8.65 5.6 ± 3.85 76.0 ± 14.44 9.8 ± 5.67 2.51 ± 0.10 Normal content 0.21 ± 0.01 Slightly high content 11.95 ± 0.79 Slightly high 3.03 ± 0.23 Low content 5.97 ± 0.24 Moderately acidic
10.2 ± 10.62 2.6 ± 2.611 9.4 ± 8.17 22.4 ± 13.76 0.2 ± 0.45 23.0 ± 13.38 17.4 ± 4.77 26.2 ± 5.49 0.6 ± 0.55 45.8 ± 10.08 1.4 ± 0.55 1.46 ± 0.39 Very poor content 0.13 ± 0.02 Normal content 10.87 ± 2.26 Slightly high 3.83 ± 0.30 Low content 6.05 ± 0.10 Moderately acidic
F = 21.83; P < 0.01 F = 2.89; P > 0.05 F = 10.46; P < 0.05 F = 19.80; P < 0.01 E = 6.77; P < 0.01 F = 21.89; P < 0.01 F = 58.98; P < 0.001 F = 1.39; P > 0.05 E = 5.65; P < 0.05 F = 14.70; P < 0.01 F = 36.81; P < 0.001 E = 11.29; P < 0.001 E = 11.29; P < 0.001 F = 1.63; P > 0.05 F = 32.03; P < 0.001 E = 0.01; P > 0.05
3. Results
3.3. Experiments performed with the soil from El Tomillar
3.1. Microarthropod community and chemical properties of the studied soils
As seen in Fig. 3 (A), which shows the results obtained in the control microcosms, no significant differences were observed between the two compartments (A and B) for the abundances of any group of microarthropods. In the experiments carried out with soil and earthworm species Ce2 from El Tomillar (Fig. 3 (C)), almost all arthropod groups were more abundant in compartments without earthworms, significantly for Isotomidae springtails (T = 2.61, P < 0.05) and Oribatida (T = 8.75, P < 0.001), Actinedida (T = 2.70, P < 0.05) and Total Acari (T = 3.67, P < 0.05). The abundances of Hypogastruridae and Sminthuridae springtails were very similar between compartments, independently of the presence or absence of earthworms. In the experiments performed with soil from El Tomillar and the earthworm species Ce1 from El Molar (Fig. 3 (D)), most arthropod groups were also more abundant in the absence of earthworms, significantly for Hypogastruridae (T = 4.23, P < 0.01), Isotomidae (T = 2.99, P < 0.05), total Arthropleona (T = 3.13, P < 0.05), total Collembola (T = 3.16, P < 0.05) and Total Acari (T = 2.70, P < 0.05). Oribatida mites presented similar trends with values very close to significance (T = 2.58; P = 0.06).
The microarthropod community of the studied soils was formed mainly by two large groups, springtails (Collembola) and mites (Acari). Within the order Collembola, the suborder Arthropleona was dominant over the rest of groups, the family Isotomidae being the most numerous, followed by Hypogastruridae and Neanuridae, although Entomobryidae were also found in smaller quantities. There were also individuals of Symphypleona, all of them from the family Sminthuridae. As for mites, the four main edaphic suborders appeared: Oribatida (the most numerous group of mites), Actinedida, Gamasida and the smallest Acaridida. In addition to mites and springtails other groups of Hexapoda and Myriapoda were found and grouped under “Other arthropods”. Within Hexapoda the orders Protura, Psocoptera, Hymenoptera, Diplura (Japygida), Heteroptera, Homoptera and larvae of Diptera, Lepidoptera and Coleoptera were found. Within Myriapoda, Symphyla, Pauropoda and Chilopoda were found. Table 1 represents the microarthropod community found in samples taken at time 0, before the beginning of the experiment in both soil types. All the groups found were more abundant in the original soils from El Tomillar than in those from El Molar. No significant differences were observed for Neanuridae springtails and Actinedida mites, although both groups were also more abundant in the soils from El Tomillar. Chemical analysis (Table 1) show that the soil from El Tomillar had higher carbon and nitrogen content than the one from El Molar. Both soils presented low content of calcium, which was slightly higher in the soil from El Molar. C/N ratio value was slightly high in both soils and pH values were moderately acidic and very similar in both soils.
3.4. Experiments performed with the soil from El Molar Fig. 3 (B) shows the results of the experiments performed with soil from El Molar and the earthworm species Ce2 from El Tomillar. The decrease in the abundance of all the groups of microarthropods with respect to those made in the soils from El Tomillar is quite striking, as observed in the soils analyzed at time 0. In any case, there were no significant differences for any group of microarthropod between compartments with and without earthworms, so the presence of the species Ce2 does not seem to affect the microarthropod populations of the soil from El Molar.
3.2. Earthworm growth
4. Discussion
The earthworms started the experiment with a very similar weight in each type of microcosm (Table 2). At the end of the experiment individuals of both species grown in the soil from El Tomillar increased their weight significantly, being the weight increase very similar. However, individuals of Ce2 cultivated in the soil from El Molar maintained or even slightly decreased their weight.
Several studies have shown that earthworms are important biological invaders causing effects in invaded communities [13,16,41]. A possible scenario of climate change, characterized by an increase in average temperatures as well as a decrease in average rainfall in the 4
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Table 2 Initial and final earthworm weights per microcosm and percentage of weight increase (% ΔWeight) (mean ± standard deviation) in each type of microcosm. The pvalue and the F-statistic (ANOVA) are given. Different letters in the same line indicate significant differences. Ce1: species from El Molar; Ce2: species from El Tomillar.
Initial weight (g) Final weight (g) % ΔWeight
Ce2 El Tomillar soil
Ce1 El Tomillar soil
Ce 2 El Molar soil
5.75 ± 0.28 7.13 ± 0.84b 23.74 ± 11.13b
5.91 ± 0.1 7.10 ± 0.65b 20.04 ± 12.14b
5.79 ± 0.12 5.74 ± 0.71a −0.90 ± 13.02a
F = 1.03; P > 0.05 F = 5.82; P < 0.05 F = 6.01; P < 0.05
Fig. 3. Mean abundances of each arthropod group comparing both compartments in each experiment type. (A): control microcosms made with the soil from El Tomillar in both compartments (A and B); (B): experiments made in soil from El Molar with and without the species from El Tomillar (Ce2); (C): experiments made in soil from El Tomillar with or without the species from El Tomillar (Ce2); (D): experiments made in soil from El Tomillar with or without the species from El Molar (Ce1). The results of the statistical analysis are also shown when differences between compartments were significant (*p < 0.05; **p < 0.01; ***p < 0.001).
effects of species Ce2 on some groups of microarthropods such as springtails, as significant effects were observed not only on the Isotomidae, but also on Hypogastruridae and the total Arthropleona and Collembola. However, in the case of mites, the negative effect was less evident as only were observed on Total Acari, although similar trends were observed for some individual taxa as Oribatida. In any case, the effect of both cryptic species seems to be quite similar, exerting a clearly negative effect on the arthropod communities from El Tomillar. Considering the ease of growth of Ce1 in the soil from El Tomillar, a likely outcome of a hypothetical range expansion and colonization of this soil would be its establishment and modification of the arthropod communities of said soil, in a similar way that cryptic species Ce2 already does.
Iberian Peninsula [27], could lead to fundamental changes in habitat conditions for the different cryptic species of C. elisae, which could determine a possible expansion of these species to other areas. In the experiments developed in this study, we have observed that cryptic species of C. elisae could have variable effects on soil microarthropod communities, which highlight the importance of a deeper understanding of these species. This fact may help to describe and delimit these two cryptic species and understand how borders are produced between them [22].
4.1. What could happen in the soil from El Tomillar? In the experiments performed with soil from El Tomillar both cryptic species showed a clear negative effect on the main microarthropod groups found in this soil. The species Ce2, which grew perfectly when cultivated in its original soil, showed a clear negative effect on Isotomidae Springtails and on Oribatida and Actinedida mites. The species Ce1 from El Molar also grew perfectly in the soil from El Tomillar, which could be favored by the bigger content in organic matter in this soil. The effects of Ce1 seem even more evident than the
4.2. What could happen in the soil from El Molar? The species Ce2 from El Tomillar did not show any effect on the microarthropods from El Molar. This could be surprising at first, since a negative effect had been observed on the microarthropods from its own soil. However, it should be noted that this species does not appear to 5
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of earthworms [9], with high densities causing a drastic change of the physical structure of the soil and negatively affecting microarthropods [10,13,43]. The long-term negative effects and high densities (or low earthworm/soil ratios) are consistent with our study, where earthworms had processed all the soil within their reach by the time the experiments ended.
grow up optimally in the soil from El Molar, so their action on this soil would be much weaker. In the case of a possible expansion and colonization of this species to the soils from El Molar, it appears unlikely that it could be easily established or could alter the communities of arthropods that inhabit this soil. We must remember that the soil from El Molar is arid [33] and presents less organic matter than the soil from El Tomillar. The species Ce1 that inhabits El Molar is very well adapted to this type of soil through various mechanisms such as the processes of aestivation or food selection [32,42]. However, the species Ce2 from El Tomillar is adapted to soils much richer in organic matter, thus it may not have such adaptation mechanisms, making their expansion towards these areas improbable. We must also consider that, since the soil from El Molar is less favorable than the soil from El Tomillar, the period of acclimation to the new conditions could be longer for Ce2 to El Molar soil than for Ce1 to El Tomillar soil. In any case, in the face of the advance of climate change, it seems very unlikely the expansion of the Ce2 species to El Molar area, since this zone is much more arid, and the soil is much poorer than in its area of origin. Additional studies on their biology and ecology as well as on its capacity to expand and colonize new habitats should be done to verify it.
5. Conclusions The study of the biology and ecology of new cryptic species is fundamental since, as we have seen in these experiments, their effect on other soil communities could be very different. The two cryptic species studied have shown a clear negative effect on microarthropods from the soils they inhabit. However, while the species Ce1 from El Molar could colonize and benefit from El Tomillar soil aggravating the negative effect and altering the microarthropod communities from this area, it is not clear that the species Ce2 from El Tomillar could colonize the soil from El Molar and affect their microarthropod community. Soil organic matter is suggested as one of the most probable mechanisms for understanding the interrelationships between these earthworm species and the microarthropods. However, it is important to underline that the results of these experiments are preliminary as have been performed in laboratory microcosms, in limiting conditions. Therefore, it would be interesting to carry out further field studies that allow extrapolation of these results and the prediction of the possible patterns of expansion and colonization of these species as well as their effects on the new soils.
4.3. Main mechanisms driving earthworms and microarthropods relationship Eisenhauer [9] stated that positive effect of earthworms on microarthropods occurs mainly in the case of anecics, provided their densities are not very high. However, the effect of endogeic earthworms on microarthropods is generally negative, but its causes are not yet clearly known. One of the most probable mechanisms could be a competitive relationship between both groups [13,17,24]. Due to their largest size and highest agility, earthworms can access soil resources more effectively by hurting other edaphic groups, being more efficient competitors than microarthropods; thus, their relationship should really be considered amensalism rather than competition [9]. In the case of the experimental conditions of this study, in which the amount of available soil is limited, it is possible that earthworms consume a significant part of the useable organic matter, not being available to microarthropods, which would tend to migrate to compartments without earthworms. In fact, Gutiérrez López et al. [24], had already determined in laboratory, by means of a supply of organic matter, that the negative effect observed for C. elisae species from El Molar on microarthropods from this area is probably due to competition for food resources. The results obtained in this study with the species Ce2 from El Tomillar also point in this direction, as the negative effect is only clearly observed on microarthropods from their own soil, but not on the microarthropods from El Molar. This result suggests that this species, adapted to richer soils, is not able to make an optimal use of the poorer organic matter soil from El Molar and it frees up a resource that microarthropods can take advantage of. However, we must consider that these results, obtained in the laboratory, could be mitigated in the field, where both, earthworms and microarthropods, have the possibility of moving around looking for other trophic resources and thus avoid competition. Another mechanism to be considered is the disturbing effect of earthworms on the soil; for example, distributing organic matter more homogeneously in the soil layers [23]. Many microarthropods are sensitive to this disturbance of the soil [10,43], which would force them to look for trophic resources in deeper layers. Earthworms also modify the soil, producing biogenic structures such as galleries and accumulations of casts in which chemical changes occur, increasing the amount of organic matter and changing the structure of soils [3]. In this sense, Straube et al. [16], found similar results for the invasive, and endogeic species, Octolasion tyrtaeum, which at low densities altered the soil moderately, increasing the number of microhabitats, the microbial biomass and favoring microarthropods, but that positive effect disappeared with increasing earthworm density. Therefore, the negative effects of endogeic species would depend on the biomass or the density
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