Invasive lumbricid earthworms in North America—Different life histories but common dispersal?

Lumbricid earthworms are invasive across northern North America, causing notable changes in forest ecosystems. During their range expansion, they encountered harsher climatic conditions compared to their native ranges in short time (~400 years). This study investigated if (a) dispersal barriers, (b) climatic selection or (c) anthropogenic activities, that is fishing bait disposal, structure the dispersal of free‐living earthworm populations.

Due to their ability to tolerate disturbances, they also occur in agricultural fields and meadows, with varying frequencies and abundances (Hendrix et al., 1992). In general, earthworms are susceptible to prolonged freezing periods, drought and geographic barriers like mountain ranges and large water bodies, which usually restrict their natural dispersal pattern (Eggleton, Inward, Smith, Jones, & Sherlock, 2009;Reynolds, 1994). However, they recently were recorded from interior Alaska and Fennoscandia suggesting that they can also withstand very low temperatures (Booysen, Sikes, Bowser, & Andrews, 2018;Wackett, Yoo, Olofsson, & Klaminder, 2018). Active dispersal of earthworms is slow, but they were able to spread across northern North America within a few hundred years by passive dispersal or repeated introductions, and today they are present in large areas from the east coast to the Midwest, east of the Rocky Mountains in Canada, and the Pacific coast (Hale et al., 2005;Holdsworth, Frelich, & Reich, 2007;Reynolds, 1977Reynolds, , 1994Reynolds, , 2016Reynolds, Linden, & Hale, 2002;Scheu & Parkinson, 1994). The pronounced ecological consequences of earthworm invasions in North America are well documented, making earthworms one of the best-studied invasive animal species living below the ground (Wardle, Bardgett, Callaway, & Putten, 2011) and thus, a unique model system for biological invasion and accompanying effects (Hendrix et al., 2008).
During their expansion across northern North America, European earthworms established in distinct climate zones that differ in the amount and distribution of precipitation across the year, as well as frost intensity and duration, two abiotic factors that are known to drive earthworm distribution (Curry, 2004;Fisichelli et al., 2013;Holmstrup, 2003;Uvarov, Tiunov, & Scheu, 2011). At the west coast, precipitation is high (1,200 mm/year), mild frost occurs sporadically and lasts for only few weeks between December and January. By contrast, in the central plains of North America, precipitation is low (400-600 mm/year), and strong frost conditions typically persist between November and March, with occasional night frost already starting in late August and extending into early June. In the east, precipitation is intermediate (800-1,000 mm/year), and frost conditions typically last from December to February. Given this wide range of climatic conditions, knowledge on genetic diversity and relationships among populations across North America is needed for a better understanding of dispersal mechanisms and population establishment.
We investigated the genetic structure of Lumbricus rubellus and L. terrestris, two exotic earthworm species that are widespread and common across northern North America. Both feed on litter but have distinct ecological preferences and life histories (Sims & Gerard, 1999). Lumbricus rubellus is an epi-endogeic species, living in horizontal burrows up to 30-cm deep and moves freely within the litter layer for foraging. It prefers neutral to slightly acidic soils and generally has a higher pH and frost tolerance than L. terrestris (Addison, 2009;Tiunov, Hale, Holdsworth, & Vsevolodova-Perel, 2006). In contrast, L. terrestris prefers neutral to slightly alkaline soils, lives in permanent, vertical burrows of up to 2-m depth, and collects litter in the vicinity of its burrow entrance (Addison, 2009;Sims & Gerard, 1999;Tiunov et al., 2006). Active dispersal rates of the two earthworm species range between 2-4 m/year for L. terrestris and 10-14 m/year for L. rubellus (Marinissen & van den Bosch, 1992). As epi-endogeic species, passive dispersal of L. rubellus by human activities may be more likely by moving animals and cocoons in surface soils, such as through activities related to forestry or tourism (i.e., by hiking or wheels of vehicles). In contrast, L. terrestris lives in permanent, vertical burrows that are only left for foraging and mating (usually at night), making it less likely to be passively transported by human activities above the ground. Lumbricus terrestris is commonly used as fishing bait and sold in bait shops, which likely facilitates its dispersal. By contrast, L. rubellus is rarely sold in bait shops (A. Klein, pers. obs.). Disposal of fishing baits contributes substantially to the introduction and establishment of earthworm populations in recreational and fishing areas (Holdsworth et al., 2007;Keller et al., 2007), but the long-term establishment of these populations and further dispersal in the field remain unclear.
We sampled earthworms from five transects of ~150-to 300km length (north-south orientation) in three climatic regions in two provinces in Canada and three states in the USA: the warm and moist region of British Columbia, Canada (BC), the cold and dry regions of Alberta, Canada (AL) and Minnesota, USA (MN), and the cold and moderately moist regions of Michigan, USA (MI) and New York State, USA (NY), respectively. This is the first study investigating the invasion of detritivorous soil animals on continental scale, including two different dispersal barriers and distinct climate zones in its sampling design.
We tested three hypotheses to understand if climate (H1), dispersal barriers (H2), and/or human migrations and transport (H3) predominantly structured the distribution and establishment of European earthworm species in northern North America: We tested, if (H1) distinct genetic clades from genetically diverse source populations established in the different climate zones.
Due to environmental filtering we expected to find monophyletic clades in the different regions, if only individuals survived that were better adapted to regional drought or cold conditions.  (Hale, 2008;Holdsworth et al., 2007;Seidl & Klepeis, 2011), we purchased earthworms from bait shops near sampling locations in all transects to test if bait genotypes contribute to free-living populations, thereby increasing local diversity.
A mcmc run of 4 million generations with default settings was performed. We analysed the North American haplotype identities with European earthworms using Bayesian phylogenetic trees of the COI and H3 datasets and included sequences available from NCBI. A list of the data sources is found in Appendix S1 Table S4. Parameter settings were nst = 6, rates = invgamma and default settings for the mcmc run.

| Phylogeography and genetic differentiation across putative dispersal barriers
Spatial distribution of genetic clades was analysed with haplotype networks and constructed for 16S rDNA, which provided the most informative resolution. Median-joining (MJ) networks (Bandelt, Forster, & Röhl, 1999)

| Climate data
The responses of genetically diverse earthworms to ecological factors were inspected using a multiple regression matrix (MRM).
Bioclimatic data were retrieved from WorldClim v2 bioclimatic variables database (Fick & Hijmans, 2017) and had a spatial resolution of ~5 km 2 . The response matrix compared genetic pairwise differences of the COI sequence data and was calculated with the Analysis of Phylogenetics and Evolution (ape) package (Paradis, Claude, & Strimmer, 2004) in R (http://www.R-proje ct.org). Tested factors were (a) environmental abiotic parameters, that is, annual mean temperature (BIO01), maximum temperature of the warmest month (BIO05), minimum temperature of the coldest month (BIO06), mean temperature of the wettest quarter (BIO08), mean temperature of the driest quarter (BIO09), annual precipitation (BIO12), precipitation of the driest month (BIO14), precipitation seasonality (BIO15), and (b) the geographical parameter spatial distance and elevation.
Data were first transformed into scaled explanatory distance matrices using Euclidean distances for standardization and then normalized. Information on the correlation of the environmental variables is provided in Appendix S2 (Table S6). With the present dataset from the sampling locations provided in

| Sampling and genetic diversity
In total, 120 L. rubellus (LR) and 122 L. terrestris (LT) individuals were sampled from the 25 locations. The number of individuals per transect varied from 12 to 48 for L. terrestris and from 12 to 37 for L.
rubellus (Appendix S1 Figure S1). Nucleotide (NUD) and haplotype diversity (HTD) was greater in L. rubellus and decreased in both species from COI to 16S rDNA to 12S rDNA to H3. Overall, nucleotide diversity of COI was two or three times higher in L. rubellus than in L. terrestris and varied among transects (Appendix S1 Tables S2, S3).
F I G U R E 1 Bayesian phylogenetic tree based on a supermatrix of four genes (COI, 16S, 12S and H3) of 120 individuals of Lumbricus rubellus (a), and distribution and abundance of the four genetic clades in the five transects across northern North America (b). The corresponding clades of the haplotype network analysis based on 16S are provided next to each clade, the area of each circle is proportional to the numbers of individuals for each haplotype, the colour code refers to the five transects British Columbia (BC, red), Alberta (AL, orange), Minnesota (MN, green), Michigan (MI, violet) and New York (NY, blue). For abbreviations of sampling locations see Table 1, posterior probabilities of well-supported clades are highlighted in bold

| Relatedness and spatial distribution
In both species, earthworms were closely related resulting in phylogenetic trees with a weakly supported backbone and clades with mixed geographic origin. Accordingly, phylogenetic and geographic structure was generally weak, in particular in L. rubellus. However, in both species, some populations formed well-supported clades (posterior probabilities: 0.95-1; Figure 1a) that were also recovered by haplotype network analyses. In L. rubellus, two clades comprised closely related individuals from all transects (mixed clades 1 and 4 with 37 and 60 individuals, respectively). However, five individuals from Minnesota (clade 2, green) and 18 individuals from New York (clade 3, blue) were distinct and did not occur in other transects (Figure 1b). All North American COI haplotypes of L. rubellus could be assigned to lineages from Europe (Giska, Sechi, & Babik, 2015;Sechi, 2013 Genetic distances among populations of L. terrestris were less distinct but had more haplotypes separating into more clades than L. rubellus (Figure 2a Most haplotypes of L. terrestris from bait shops were identical to common and widespread haplotypes from field populations ( Figure 3). Only few haplotypes formed separate clades (mainly AL and BC) or were related to rare field haplotypes (BC) from the same sampling region. The North American COI and H3 haplotypes of L.
terrestris were closely related or identical to haplotypes described from Europe or North America in previous studies (Appendix S1 Table S4, Appendix S3 Figures S4-S5).
F I G U R E 2 Bayesian phylogenetic tree based on a supermatrix of four genes (COI, 16S, 12S and H3) of 122 individuals of Lumbricus terrestris (a), and distribution and abundance of the seven genetic clades in the five transects across northern North America (b). The corresponding clades of the haplotype network analysis based on 16S are provided next to each clade, the area of each circle is proportional to the numbers of individuals for each haplotype, the colour code refers to the five transects as in Figure 1. For abbreviations of sampling locations see Table 1, posterior probabilities of well-supported clades are highlighted in bold

| Genetic differentiation across putative barriers
Analysis of molecular variance (AMOVA) across all four genes showed that most of the molecular variance was at local scale (within sampling points = populations, Appendix S2

| Importance of bioclimatic factors
The MRM showed contrasting results for the two earthworm species; the permutation test indicated that 22% and 4% of the variance were explained by climatic variables for the complete datasets of L. rubellus and L. terrestris, respectively (Appendix S2

| Genetic diversity
This study shows that northern North American populations of the two earthworm species L. rubellus and L. terrestris share the same genetic lineages with populations of their native range in Europe. However, genetic diversity is lower in North America than in Europe, which is typical for invasive species (Sakai et al., 2001;Allendorf & Lundquist, 2003;King, Tibble, & Symondson, 2008;Gailing et al., 2012;Donnelly et al., 2013;Donnelly, Harper, Morgan, Pinto-Juma, & Bruford, 2014;Giska et al., 2015).
In North America, common and widespread haplotypes dominated in both species, but genetic and geographic structure differed.
Among populations of L. rubellus, haplotypes divided into two genetic lineages that predominantly occurred in all sampling regions (except in New York), which belonged to common and widespread lineages from Europe (Sechi, 2013). Future studies with higher spatial resolution sampling should explore if different nationalities of European settlers are mirrored by the genetic structure of earthworm populations.
The co-occurring pattern of omnipresent lineages of L. rubellus and L. terrestris across northern North America suggests a common origin and mode of dispersal for both species. In particular road constructions, traffic, logging, fishing and agriculture have been identified as main drivers of earthworm range expansion in North America (Marinissen & van den Bosh, 1992, Dymond, Scheu, & Parkinson, 1997Casson et al., 2002;Holdsworth et al., 2007;Gundale, Jolly, & Deluca, 2005;Cameron, Bayne, & Coltman, 2008;Cameron & Bayne, 2009) and certainly also apply here. Human-mediated long-range dispersal by passive transport is more likely for L. rubellus, which frequently moves in leaf litter near or on the soil surface than for soil-dwelling anecic species (Terhivuo & Saura, 1997). However, the presence of locally occurring lineages in L. rubellus and the distinct genetic assembly of L.
terrestris in Alberta and Minnesota indicate that additional factors affected the dispersal and introduction of these two earthworm species.
The relevance of bait abandonment for the distribution of L.
rubellus is difficult to assess. This species has been commonly used as fishing bait (Reynolds, 1977), but was not sold in any bait shops we purchased earthworms from. However, dispersal via bait abandonment in the past cannot be excluded. In contrast, L.
terrestris is the most commonly sold live fishing bait in northern North America today, and a large fraction of individuals from bait shops and the field shared identical or closely related haplotypes, indicating that bait abandonment contributes significantly to the TA B L E 2 Analyses of molecular variance (AMOVA) of Lumbricus rubellus and L. terrestris assigned a priori into populations separated by climate regions (Climate), geographic barriers (Great Plains and Rocky Mountains) or by geographic distance between transects (Transect)

| Climate and dispersal barriers
Genetic variation among regions was very low for L. rubellus, and bioclimatic factors or dispersal barriers did not explain the distribution of common lineages, which agrees with its higher tolerance to frost (Fisichelli et al., 2013;Sims & Gerard, 1999;Tiunov et al., 2006). The ability of epi-endogeic earthworms to quickly adapt to cold and fluctuating temperatures through behavioural and physiological changes (Holmstrup, 2003), and their persistence to perturbations, such as heavy metal pollution by fertilizers and intoxication by pesticides, are well known (Edwards & Bohlen, 1996;Kruse & Barrett, 1985;Levine, Hall, Barrett, & Taylor, 1989).
In contrast to L. rubellus, genetic variance in the common lineages of L. terrestris in part was related to climate factors, in particular frost, drought and seasonality. These results corresponded to findings that anecic earthworm species are negatively affected by prolonged drought periods, high frequency of freeze-thaw cycles and low soil moisture during their prime reproductive periods in spring and autumn (Addison, 2009;Curry, 2004;Sims & Gerard, 1999).
Conform to these findings, the distinct genetic composition of populations in Alberta and Minnesota correlated with the continental climate in both transects. However, both species were recorded from areas with harsh frost conditions (Booysen et al., 2018;Wackett et al., 2018). These areas were associated with recent human introductions and human land use indicating potential new stepping stones of earthworm invasions. If the more severe frost and drought periods in these regions facilitated genetic diversity by continuous extinctions and reintroductions, or if only climatically pre-adapted lineages were able to establish viable populations in these areas needs to be investigated under controlled experimental conditions (Holmstrup, 2003).
Additionally, joined analyses of more molecular datasets from North from which we can infer dispersal barriers and migration routes or the extent of genetic bottlenecks earthworms experienced during their invasion.

| CON CLUS IONS
Genetic diversity and structure of the two invasive earthworm species L. rubellus and L. terrestris was homogenous across all regions indicating a dominant common dispersal vector and the ability to adjust to most environmental conditions in northern North America.
However, L. terrestris was genetically more structured, and here its genetic variance positively correlated with harsh climatic conditions in central North America. In contrast to L. rubellus, this species is common in arable fields with frequent disturbances, and distinctness of genetic lineages occurring predominantly in transects of Alberta and Minnesota could be explained by their position at the edges of the North American corn belt. Genetic patterns indicate that both species have common long-distance distribution vector(s).
For L. terrestris, nation-wide bait distributors potentially play a major role as dispersal agent of field populations. In the past two decades, the globalization of economy has changed infrastructure, intensity and range of traffic including commercial distribution of soil-related goods, and potentially will increase dispersal of L. rubellus and L. terrestris.
Our present study exemplifies how earthworms as belowground invaders with substantial differences in life history traits can be used to test broad questions in invasion ecology, such as the genetic underpinnings of successful invasion events, geographic and climatic dispersal barriers, as well as the human role in ecologically relevant invasions. Thus, the present results may inspire future work on the role of different hypothesized main drivers of invasive species that can be explored with comparative genetic analyses (Sovic, Carstens, & Gibbs, 2016).

ACK N OWLED G EM ENTS
We gratefully acknowledge the following people. For collections: Author contributions: A.K., N.E. and I.S. conceived the original idea. A.K. conducted the field work, collected and analysed the data, and wrote the manuscript. All authors contributed to this study and the manuscript in form of discussions, suggestions and revisions, and approved the final manuscript.

S U PP O RTI N G I N FO R M ATI O N
Additional supporting information may be found online in the Supporting Information section.