1. Introduction
Invasive earthworms have caused significant effects on local biota and ecosystem processes (such as nutrient dynamics) in the invaded areas, e.g., European Lumbricids in North America [
1,
2,
3]. Population declines of native earthworms, particularly in remote and non-fragmented forests, have contributed to a result of competitive exclusion by expanding invasive earthworm populations [
2,
4,
5]. Lachnicht et al. [
6] observed that invasive
Pontoscolex corethrurus (Müller, 1856) earthworms, when incubated with native
Estherella sp., utilized different N resources, possibly avoiding direct competition on food resource. Winsome et al. [
7] found that invasive
Aporrectodea trapezoides (Dugès, 1828) lost its competition advantage when co-existing with native
Argilophilus marmoratus (Eisen, 1893) in the resource-poor habitat of a Californian grassland. Interactions between native and invasive earthworms varied with resource utilization of earthworm species and resource availability [
6,
7]. Earthworms are categorized into three ecological groups, epigeic, endogeic, and anecic, based on their preferences on space and food resources [
8]. Epigeic earthworms mainly consume leaf litter (and microbial populations colonizing on it) and inhabit the litter layer, while endogeic earthworms occupy mineral soils and use soil organic matter as their main food resources. Anecic earthworms utilize mainly leaf litter but with the ability to build burrows deep in the soil [
8]. Earthworms with same feeding strategies are expected to evolve stronger competitive interactions because they share the same food resources [
2,
9,
10]. Hence, resource utilization of earthworms could serve as a determinant for the success of earthworm invasions and its effects on the native earthworm community [
7].
Earthworm invasions have significantly altered nutrient dynamics (e.g., carbon (C) and nitrogen (N)) in invaded soils [
1,
11,
12]. A mixed-species of European Lumbricid earthworm assemblage has been documented to lessen organic layers and relocate leaf litter and humus fragments (C) into the deeper mineral soils, as well as to cause an increase of N loss in the soil adjacent to plant roots in the temperate forests of North America [
1]. The effects of earthworms on soil C and N dynamics may vary with the feeding strategies of earthworms and composition of earthworm assemblages [
13]. For example, epigeic earthworms may have stronger effects on nutrient fluxes between leaf litter layers and microbial populations that colonized on it (detritusphere) from their comminution and digestion of the leaf litter substrate [
1,
11,
12]. Endogeic/anecic earthworms, on the other hand, may play a significant role in regulating nutrient dynamics in mineral soil and plant root zones (rhizosphere) by their consumption of soil organic matter and root exudates (and depositions) and their active burrowing activity [
14,
15,
16]. In an area inhabited by a mixture of earthworms (either different feeding strategies or native co-existing with invasive worms), whether earthworm effects on soil nutrient dynamics can be explained by a summation of individual earthworm effects or disproportionally dominated by one aggressive earthworm species is a topic of interest, yet still in need of more research.
Stable isotope
13C and
15N techniques, including
13C- and
15N-labeled plant materials and a natural abundance of
13C and
15N isotopes, have recently provided invaluable information for studying earthworm feeding strategies and their effects on soil C and N dynamics [
6,
17,
18,
19,
20]. For example, Hendrix et al. [
17] suggested an inter-specific competition for N resources based on their observation of overlapped natural abundance
15N in both
Estherella sp. and
P. corethrurus in a lower altitude tabonuco forest, Puerto Rico. Neilson et al. [
18] found that a natural abundance of
13C and
15N in earthworms can be used to assess the availability and diversity of food resources in the environment. With the application of
13C- and
15N-enriched plant materials, how earthworms utilize different type of food resources and the corresponding effects on soil C and N dynamics can be evaluated by tracking changes of δ
13C and δ
15N associated with
13C and
15N-labeled plant materials in soils, earthworms, and the microbial populations. In this study, we applied
13C-labeled
Tabebuia heterophylla (DC.) Britton leaves and
15N-labeled
Andropogon glomeratus (Walter) Britton, Sterns, & Poggenb. grass to investigate resource utilization of three earthworm species from Puerto Rico (invasive
Pontoscolex corethrurus, native
Estherella spp., and native
Onychochaeta borincana (Borges, 1994) and their effects on soil C and N dynamics in Puerto Rican soils.
Pontoscolex corethrurus has invaded multiple habitats in Puerto Rico, in contrast to the restricted distribution of the native earthworms in mature forests [
21,
22]. Competition pressure from invasive
P. corethrurus to native earthworms has been suggested to be responsible for the absence of native earthworms in most disturbed areas, i.e., pasture and young forests [
22,
23,
24]. Lachnicht et al. [
6] observed that endogeic
P. corethrurus and anecic
Estherella sp. showed resource partitioning (in terms of space and food) to avoiding direct competition in a 19-day laboratory experiment. The interactions observed between
P. corethrurus and
Estherella sp. have also caused differential influences on soil C and N mineralization [
6]. In this study, we investigated feeding strategies of endogeic
P. corethrurus, anecic
Estherella sp., and endogeic
Onychochaeta borincana (single-species earthworm treatments) on
13C-labeled
Tabebuia leaves and
15N-labeled
Andropogon grass. Changes in resource utilization of individual earthworm species would be evaluated by comparing earthworm tissue
13C and
15N of single-species earthworm treatments to those of mixed-species earthworm treatments (co-existed with other anecic/endogeic earthworms). Influences of individual earthworm species and inter-specific earthworm interactions on soil C and N dynamics would be assessed by tracking the changes of
13C and
15N in soils, earthworms, and microbial populations in single- and mixed-species earthworm treatments. Anecic
Estherella spp. was expected to utilize more
13C-labeled
Tabebuia leaves, as compared with endogeic
O. borincana and endogeic
P. corethrurus. Given that
P. corethrurus is believed to exhibit flexible feeding behaviors and enhance soil mineralization [
6,
17], we expected that
P. corethrurus would utilize more leaf litter (detritusphere) than plant roots (rhizosphere) resources, when incubated with endogeic
O. borincana, to avoid competition with
O. borincana. Higher population growth would be observed in a
P. corethrurus population, which would enhance soil C and N mineralization. However, the presence of anecic
Estherella sp. and endogeic
O. borincana would weaken enhanced soil mineralization caused by
P. corethrurus.
4. Discussion
In this study, newly added
13C-labeled leaf litter and
15N-labeled grass were sufficiently incorporated into 10 cm of top soil, soil microbial biomass, and earthworm tissue. Natural abundance of δ
13C in earthworms was suggested to be 1−3‰ heavier than its dietary sources (such as leaf litter, root exudates, and microbial populations in the soil) [
18,
35]. In this study, earthworm δ
13C showed on average 1.4‰ heavier in
Estherella spp., 3.5‰ heavier in
P. corethrurus, and 5‰ heavier in
O. borincana, with respect to the soil δ
13C, while earthworm tissue showed on average 5.9‰ heavier δ
13C in
Estherella spp., 7.2‰ heavier in
P. corethrurus, and 8.5‰ heavier in
O. borincana than the microbial biomass δ
13C in which they inhabited (
Table 2,
Table 3,
Table 4 and
Table 5).
We did not observe any competition exclusion among three earthworm species based on the survivorship and biomass gain among the single-species and the mixed-species treatments for each individual species. However, anecic
Estherella spp., when cultivated alone, did show higher tissue—
13C (%) and δ
13C—compared with when it was cultivated with other earthworm species. This suggested that
Estherella spp. might change its feeding strategy by reducing its utilization of
13C-labeled litter materials and/or the microbial community that was related to the
13C-labeled litter when cultivated with
P. corethrurus or both
P. corethrurus and
O. borincana. Lachnicht et al. [
6] observed that
P. corethrurus and
Estherella spp., while cultivated together, excluded each other in the bottom and upper layers of soil, respectively, in a 19-day laboratory experiment in Puerto Rican soils. The authors also found that
P. corethrurus acquired more
15N-labeled leaf litter when co-occurring with
Estherella spp. [
6]. We did not find that
P. corethrurus changed its feeding preference in this 22-day experiment based on worm tissue
13C and δ
13C as well as tissue
15N and δ
15N between the single-species and mixed-species earthworm treatments, nor did
O. borincana. In this study, cultivating live
A. glomeratus grass plants could provide a steady, continuous supply of root exudates and rhizodeposits for soil microbes and earthworms, as compared to the one-time application of
13C-labeled glucose and
15N-labeled leaf litter adopted by Lachnicht et al. [
6]. Such a continuous supply of food resources might relieve potential inter-specific competitive pressure derived from limited food resources in short-term experiments, especially for endogeic earthworms like
O. borincana and
P. corethrurus that strongly rely on rhizosphere resources.
Both endogeic
O. borincana and
P. corethrurus showed 5‰ or higher δ
13C signature than their food resources (soil organic matter and soil microbial biomass). Higher δ
13C signature in both endogeic earthworms could be explained by their utilization on soil microbial populations (i.e., bacteria and fungi) as food resources. Fungal species (such as mycorrhizal and saprotrophic fungi) have been reported to have a higher
13C enrichment than plant foliage, fine roots, and soils because of fungal biochemical synthesis and transport between plant parts [
36]. Microbial activity releases the lighter
12C in respiration and gradually results in an increase of
13C concentration in humified residues and its own population [
37,
38]. As a result, endogeic earthworms (active in rhizosphere and the mineral soils),
P. corethrurus and
O. borincana in this study, showed higher δ
13C signature and tissue
13C (%) than anecic
Estherella spp. due to their preferential consumption of
13C-enriched decayed/humified debris in the mineral soil layer, to a significant portion of
13C-enriched microbial (higher microbial δ
13C observed in P, O + P and E + O + P earthworm treatments;
Table 5) and fungal populations, or to both [
6,
36,
37]. The possibility that both endogeic
O. borincana and
P. corethrurus consumed the microbial populations in the mineral soil, the rhizosphere, or both is also confirmed by their heavier δ
15N signatures (0.6‰ and 2.7‰ δ
15N heavier, respectively) compared with the soil δ
15N (
Table 2,
Table 3 and
Table 4).
We found that soil microbial-δ
15N was on average 6.1‰ heavier than
Estherella spp., 5.8‰ heavier than
P. corethrurus, and 2.6‰ heavier than
O. borincana (
Table 3,
Table 4 and
Table 6). The stronger
15N enrichment in endogeic
O. borincana could be derived from its utilization of
15N-labeled rhizosphere (plant roots, root exudates, and rhizosphere-related microbes). Even though no study has yet investigated the feeding behavior of
O. borincana, some endogeic earthworms (e.g.,
P. corethrurus) are often found aggregated in the root zones utilizing living root fragments and dead root cells, or as response to enhanced microbial activities in the rhizosphere [
35,
39]. In this study, the presence of
O. borincana seemed to relate to higher microbial biomass
15N and δ
15N (in the E + O earthworm mesocosms) and higher DIN and higher
15N-DIN (%) in the O + P treatment (although not statistically significant), as compared with other earthworm treatments (
Table 6). The potential effect of endogeic
O. borincana on rhizospheric microbial populations and activities is a topic of interest, yet in need for further research.
Pontoscolex corethrurus showed a prolific reproduction (a total of eight juvenile
P. corethrurus) within the 22-day soil mesocosm experiment. The stronger δ
15N signal observed in juvenile
P. corethrurus, as compared with the adults, might be explained by (1) the possibility that adult
P. corethrurus allocated its assimilated
15N into cocoon reproduction, which later integrated into the tissue of juvenile
P. corethrurus, and (2) a higher soil consumption and biomass increase in relation to overall biomass by juvenile worms than the adult worms [
6].
Pontoscolex corethrurus has been described as one of the cosmopolitan earthworm species that has aggressively invaded many regions in the tropics, including Puerto Rico, Central Amazonian, and Peruvian soils [
40,
41,
42,
43]. Exceptional reproductive strategies of
P. corethrurus, such as a high rate of cocoon production and hatching success, a short development time, and the ability of parthenogenesis, critically influence the local native earthworm community in the invaded soils [
2]. The rapid population growth of
P. corethrurus may increase competition pressure on food resources to the local native earthworm community [
22]. The relevance of resource availability to the population growth of
P. corethrurus and its significance in a
P. corethrurus invasion is certainly a topic in need of future research.
Earthworms showed differential effects on soil mineralization processes in this study. All earthworm treatments along with the control (no worms) had higher soil respiration C-CO
2 at Day 21, especially in the P, E + O, and O + P treatments, as compared with other control treatments (Soil Only, Grass, and Litter mesocosms). There were higher
13C-CO
2 (%) and δ
13C from the P mesocosms (Tukey’s HSD,
p < 0.001) and the mixed E + O mesocosms (marginally significant;
p = 0.06) compared with those from the O + P treatments. The effects on soil microbial activities by earthworms could be explained by earthworms’ direct grazing behavior on soil microbial community or indirect burrowing and casting activities [
11,
14]. Whether the higher soil respiration C-CO
2 from the control (no worms) mesocosms was due to the release from earthworms’ grazing activity is uncertain. However, the significantly higher soil respiration
13C-CO
2 (%) and δ
13C from
P. corethrurus (includes P and E + P) were an indicator of facilitating effects of earthworms on the enriched soil microbial biomass δ
13C from the same mesocosms.
Pontoscolex corethrurus might cause an increase in soil respiration via its simulation on the activity of the
13C-labeled microbial population. However, the lower soil respiration
13C-CO
2 (%) and δ
13C in the mixed
P. corethrurus and
O. borincana treatments (i.e., O + P) suggested that the presence of
O. borincana and its interaction with
P. corethrurus might have a negative effect on the
13C-labeled microbial community and facilitate the
15N-labeled microbial communities in the rhizosphere. Such a possibility is supported by the observation of the slightly increased
15N (%) in the soil DIN from the increased microbial activity related to the
15N-labeled rhizosphere in the O + P treatment (
Table 6).
The individual stimulation on soil N mineralization by
Estherella spp. and
O. borincana was slightly reduced when they were incubated with other earthworm species (mixed-species earthworm treatments;
Table 6). No significant change was observed in microbial biomass (C and N) between treatments, thus the changes shown in soil respiration δ
13C and DIN could be explained by the changed activities from the microbial population or possibly alternation of microbial community induced by the inter-specific earthworm interactions from the mixed earthworm treatments. Studies have suggested that the preference of earthworms on utilizing different food resources can reshape microbial communities in the detritusphere and the rhizosphere [
44,
45]. Native
Estherella spp. and
O. borincana may individually sustain a microbial community that specialized on N mineralization in the rhizopshere, yet the microbes switched to those which utilized a labile, newly added
13C-labeled resource when sharing resources with the other species. Earthworm effect on either microbial activities or microbial community by individual species is confounded when inter-specific interactions are considered, and the individual effect on microbial activities and communities was not additive. Furthermore, changes in microbial activities and alterations to the microbial community by earthworms could gradually alter soil nutrient dynamics and availability of labile C and N over time [
46], which later has an effect on habitat suitability for other species. For example, invasive
Amynthas agrestis (Goto and Hatai, 1899) was documented to change soil microbial communities, which positively affected the habitat invasibility for another invasive species,
Lumbricus rubellus (Hoffmeister, 1843) [
47]. Many studies have focused on the earthworm effects on soil microbial biomass and soil mineralization [
11,
47,
48,
49,
50,
51,
52,
53]; however, research investigating the effects of earthworms with different feeding strategies (i.e., epigeic, anecic, and endogeic) on soil microbial activities and communities in terms of functional groups is still limited.