Invertebrate Biology x(x): 1–14. © 2016 The Authors. Invertebrate Biology published by Wiley Periodicals, Inc. on behalf of American Microscopical Society. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. DOI: 10.1111/ivb.12145 Phylogeographic analysis of invasive Asian earthworms (Amynthas) in the northeast United States Nancy Schult, Kelly Pittenger, Sam Davalos, and Damhnait McHugha Department of Biology, Colgate University, Hamilton, New York 13346, USA Abstract. Phylogeographic studies are useful in reconstructing the history of species inva- sions, and in some instances can elucidate cryptic diversity of invading taxa. This can help in predicting or managing the spread of invasive species. Among terrestrial invasive species in North America, earthworms can have profound ecological effects. We are familiar with the centuries-old invasions of European earthworms (Lumbricidae) and their impacts on nutrient cycling in soils. More recent invasions by Asian earthworms of the family Megascolecidae are less fully understood. We used data for two mitochondrial gene frag- ments, cytochrome oxidase I (COI) and 16S rRNA, to examine the relationships among populations of Asian earthworms in the megascolecid genus Amynthas in the northeast Uni- ted States. Recent reports have indicated that one species in particular, Amynthas agrestis, is having detrimental effects in mixed forest ecosystems, and we were interested in under- standing the invasion history for this species. We were surprised to discover three divergent mitochondrial lineages of Amynthas occurring sympatrically in upstate New York. Given the gap between intra- and inter-lineage sequence divergences, we propose that these three lineages represent cryptic species of Amynthas, one of which is A. agrestis. For each of the three lineages of Amynthas, we observed shared haplotypes across broad geographic dis- tances. This may reflect common origins for populations in each lineage, either by direct routes from native ranges or through post-introduction spread by natural dispersal or human-mediated transport within North America. Management efforts focused on horticul- tural imports from Asia, commercial nurseries within the USA, and on prohibition of bait disposal may help to reduce the further invasion success of Amynthas. Additional key words: 16S rRNA, cytochrome oxidase I, haplotype frequency, barcode, Megascolecidae With enhanced globalization, the spread of inva- the ecosystem in ways that are detrimental to native sive species has increased dramatically over the past species. With the ability to disrupt natural ecosys- several decades as non-native species are being tems, invasive species are regarded as a leading transported across the world either unintentionally threat to biodiversity, the environment, and the or deliberately. International trade provides human- economy (Tsutsui et al. 2000; Pejchar & Mooney mediated pathways for the transmission of organ- 2009; Vila et al. 2011; Qiu 2015). If we are to isms across bodies of water and great distances over address the effects of invasive species, we need to land, with the consequence that many species understand their establishment, dynamics, and rela- become established beyond their natural ranges tionship with invaded ecosystems. However, we are (Hulme 2009). Once introduced, invasive species can often poorly informed about the history of a species act as agents of change that threaten biological invasion, and know even less about the ecology, life diversity by outcompeting native species or altering history, or physiology of the species itself. In some instances we do not even understand the systematics aAuthor for correspondence. of the invading taxa (e.g., Yassin et al. 2008; E-mail: [email protected] Folino-Rorem et al. 2009; Bastos et al. 2011). This 2 Schult, Pittenger, Davalos, & McHugh makes it very difficult to predict or manage the Metaphire, and the fact that many recognized spe- spread of invasive species. cies in the Amynthas-Metaphire complex are Among invasive species, those that engineer parthenogenetic lineages. Furthermore, Blakemore ecosystems to alter the physical environment and (2013) suggests that a proliferation of taxonomic limit the quantity and quality of resources available descriptions of new species without due attention to to native species have the biggest impacts. Invasive ICZN rules has created a situation where molecular earthworms are among such species in North Amer- analyses are needed to help bring clarity to the ica, where most earthworm species are non-native. In validity of Japanese and Korean species. For this North America, recession of the Wisconsin glacial reason, many ecological studies of Amynthas do not shield ~10–12,000 bp exposed defaunated landscapes identify which species is under investigation, (Tiunov et al. 2006; Archer 2012), and subsequent although it is assumed that populations being stud- evolution of northern forests proceeded with few ied include just a single species. native earthworm species. The dominant earthworm Field studies of invasive Amynthas spp. have indi- species currently inhabiting northern forests and agri- cated that they alter the forest floor habitat by reduc- cultural lands were introduced by settlers from Eur- ing organic matter within the soil horizon and ope over the past few hundred years (Hendrix et al. increasing soil aggregation and nitrification, thus lim- 2006). The ecological impacts of these invasions iting available food resources for other fauna (Stein- include the physical alteration of soil layers, the inter- berg et al. 1997; Zhang et al. 2010). The worms can ruption of nutrient cycling, and competition with cause destruction of soil surface layers (O-horizon native invertebrates and plants (Bohlen et al. 2004a, and A-horizon) until only the castings of the worm b). Over the past few decades, with the increase in remain, and by creating significantly larger soil aggre- trade across the Pacific, the number of Asian invasive gates that are stable even over months, Amynthas earthworm species has been increasing in North worms can have a lasting impact on soil dynamics America and among these, species of the genus that may reduce biodiversity by minimizing soil Amynthas Kinberg 1867 (Megascolecidae) have been heterogeneity (Gorres€ & Melnichuk 2012; Greiner of particular concern (Hendrix & Bohlen 2002). et al. 2012). The invasion of Amynthas into forested These epi-endogeic worms live beneath leaf litter or habitats has also been reported to negatively impact mulch or in topsoil layers, where they alter the soil native millipede species richness and density, likely structure and remove organic materials from the soil due to the rapid removal of the top-most organic soil surface (Archer 2012; Gorres€ & Melnichuk 2012; horizons (Snyder 2008; Snyder et al. 2009, 2013), and Greiner et al. 2012; Ikeda et al. 2015). The relatively the worms detrimentally affect the behavior of native large size of some Amynthas worms (up to 25 cm), salamanders (Ziemba et al. 2015). their violent defensive behavior, and their serpentine To reconstruct the invasion of Amynthas in North locomotion have earned them the common names America, we can draw on published survey records “Crazy Worm,” “Jumping Worm,” and “Snake and online reports by environmental agencies to Worm.” The reproductive mode in species of Amyn- provide estimates of earliest introduction to particu- thas, as inferred from the degree of functionality or lar areas. The presence of Amynthas was recorded in degradation of testes, spermathecae, etc., can be sex- Mississippi as early as ~1900 (Gates 1958), and sub- ual or parthenogenetic; in either case, the worms have sequent reports of worms in this genus in northeast- an annual life cycle in temperate regions, with fast- ern US forests suggest that it has been spreading growing juveniles hatching from over-wintering egg into this region over the past several decades. Most cases in late spring (Reynolds 2010; Gorres€ et al. of these reports have been attributed to Amynthas 2014). The worms are found in natural forests and agrestis (Goto & Hatai 1899), which according to are often locally patchy, reaching densities up to 50 Gates (1982) was first recorded in Maryland, USA individuals mÀ2 (Gorres€ & Melnichuk 2012). in 1939, with subsequent records in arboreta, nurs- Members of the genus Amynthas are easily recog- eries, and green houses in Massachusetts in 1953, nizable by a light-colored, smooth clitellum that New York in 1954, Louisiana and Connecticut in fully encircles the body. However, distinctive mor- 1958, Missouri in 1962, and Maine in 1966. Later phological characteristics are not apparent at the reports indicate A. agrestis invading natural habitats species level. In his review of Japanese earthworms, ranging from Alabama, Connecticut, Georgia, Blakemore (2003) reports that the taxonomy of Louisiana, Maryland, Massachusetts, Missouri, New Amynthas is complicated by the lack of diagnostic Jersey, New York, North Carolina, Oklahoma, characters, the interrelationships among species South Carolina, and Tennessee (Reynolds 1978; attributed to Amynthas and another putative genus, Gates 1982; Callaham et al. 2003; Reynolds & Invertebrate Biology vol. x, no. x, xxx 2016 Phylogeographic analysis of invasive