Biol Invasions DOI 10.1007/s10530-017-1607-x INVASION NOTE Co-invasion of three Asian earthworms, Metaphire hilgendorfi, Amynthas agrestis and Amynthas tokioensis in the USA Chih-Han Chang . Marie R. Johnston . Josef H. Go¨rres . Andrea Da´valos . Damhnait McHugh . Katalin Szlavecz Received: 29 March 2017 / Accepted: 27 October 2017 Ó Springer International Publishing AG, part of Springer Nature 2017 Abstract Earthworm invasions are one of the most is more common than having only one species. In serious causes of ecological deterioration in the addition, A. tokioensis was dominant in many of these temperate deciduous forests of North America. Non- earthworm communities. The status change in species native earthworms impact understory vegetation, leaf composition from only one species to two or three and litter layer, carbon dynamics, nutrient availability, and the shift in dominance are most likely caused by the associated food webs. Here we report a significant previous incorrect species identification. Our results status change and confirm expansion of known range support expansion of known range of A. tokioensis and of Amynthas agrestis, one of the most serious invasive M. hilgendorfi northward and westward into states species in North America, and two of its close with colder winters. This range expansion may have relatives, A. tokioensis and Metaphire hilgendorfi. taken place alongside that of A. agrestis in the last The three species have never been confirmed to co- 10–20 years, but has long been overlooked. Alto- occur in North American ecosystems. We examined gether, results highlight an urgent need for correct 1760 earthworms collected from 30 sites across species identification. The recognition of an expand- northeastern USA, and identified them using a new ing multi-species system represents a unique oppor- morphological key. Our data show that sympatric tunity to further evaluate complex interactions among occurrence of at least two, and often all three, species C.-H. Chang (&) Á K. Szlavecz A. Da´valos Department of Earth and Planetary Sciences, Johns Department of Natural Resources, Cornell University, Hopkins University, Baltimore, MD, USA Ithaca, NY, USA e-mail: [email protected]; [email protected] Present Address: A. Da´valos C.-H. Chang Biological Sciences, SUNY Cortland, Cortland, NY, USA Department of Environmental Science and Technology, University of Maryland, College Park, MD, USA D. McHugh Department of Biology, Colgate University, Hamilton, M. R. Johnston NY, USA Department of Soil Science, University of Wisconsin- Madison, Madison, WI, USA J. H. Go¨rres Plant and Soil Science Department, University of Vermont, Burlington, VT, USA 123 C.-H. Chang et al. co-invading and resident species, and to investigate specimens to understand the extent of co-occurrence whether interspecific interactions have unexpected of the three species. non-additive impacts on ecological processes. We examined 1224 specimens previously reported from 15 sites as Amynthas spp. (Da´valos et al. 2015; Keywords Earthworm invasion Á Amynthas Schult et al. 2016; Table 1) and 536 newly collected agrestis Á Amynthas tokioensis Á Metaphire specimens from another 15 sites where Asian earth- hilgendorfi Á Co-invasion worms have never been reported (Wilmette, IL) or only one of A. agrestis or M. hilgendorfi was previously reported or observed (Szlavecz et al. 2006, 2014;Go¨rres and Melnichuk 2012; Chang Introduction et al. 2016a). All samples were collected from 2002 to 2016 with a variety of techniques, including hand Invasive earthworms are one of the most important sorting, digging, and extraction by formalin or mus- ecological drivers currently changing forest ecosys- tard solutions. Species identification followed Chang tems in North America (Bohlen et al. 2004;Da´valos et al. (2016b). et al. 2015; Craven et al. 2017). Of particular concern are three Asian species in the genera Amynthas and Metaphire due to their impacts on forest floor, soil Results structure, carbon dynamics, nutrient concentration, and other soil organisms (Zhang et al. 2010; Snyder A total of 1760 specimens (1124 identifiable) belong- et al. 2011, 2013; Greiner et al. 2012; Qiu and Turner ing to three species, M. hilgendorfi, A. agrestis and A. 2017). In the scientific and popular literature these tokioensis, were collected from 30 sites across eight species are frequently referred to as ‘‘Amynthas spp.’’ states in the Midwest and northeastern USA (Fig. 1; (e.g. Da´valos et al. 2015) and nicknamed ‘‘crazy Table 1). Amynthas tokioensis, M. hilgendorfi and A. worm’’ or ‘‘jumping worm’’. They invade remote agrestis were found in 25, 23 and 22 sites, respec- forests in the United States (Callaham et al. 2003), and tively. Of the 30 sites, 27 had at least two species, and one species in particular, Amynthas agrestis (Goto & 13 had all three. In addition to being the most common Hatai, 1899), was recently discovered spreading north species, A. tokioensis was also dominant in 11 of the and west from the Mid-Atlantic into New England and 25 sites based on relative abundance, whereas A. the Midwest (Go¨rres and Melnichuk 2012; Reynolds agrestis was only dominant in nine sites. Among 51 et al. 2015; Schult et al. 2016). specimens that were analyzed genetically in Schult So far most of the Amynthas and Metaphire et al. (2016) and re-examined morphologically per sightings in the northeastern United States involved Chang et al. (2016b), 22 were unidentifiable, but 29 only A. agrestis (Callaham et al. 2003; Snyder et al. were identified as A. tokioensis (8), A. agrestis (12), or 2011;Go¨rres and Melnichuk 2012;Go¨rres et al. 2014; M. hilgendorfi (9), and all species identifications Reynolds et al. 2015; Schult et al. 2016), with corresponded unequivocally to the three species-level occasional reports of Metaphire hilgendorfi (Michael- genetic lineages, A, B and C reported in Schult et al. sen, 1892), often as Amynthas hilgendorfi (e.g. Chang (2016), respectively. et al. 2016a; see Chang et al. 2016b for details), a species closely related to A. agrestis. The two species have never been reported at the same locality. Discussion However, recent genetic analysis indicated sympatric occurrence of three species-level cryptic lineages Our data highlight widespread species co-occurrence within invading Amynthas in upstate New York in the current range of Amynthas and Metaphire in (Schult et al. 2016). Meanwhile, at several locations northeastern USA, and provide the first confirmed where A. agrestis or M. hilgendorfi was reported, we reports of A. tokioensis in Maryland, Delaware, also found a previously overlooked species, Amynthas Vermont, Illinois, and Wisconsin and M. hilgendorfi tokioensis (Beddard, 1892) (Chang et al. 2016b). in New Hampshire. We herein emphasize that co- These findings led us to re-examine archived and new occurrence of two or three species is what should be 123 Co-invasion of three Asian earthworms, Table 1 Sampling locations in northeastern USA and numbers of Metaphire hilgendorfi, Amynthas agrestis and Amynthas tokioensis recorded Location State Label GPS-N GPS-W Collection Total NID Metaphire Amynthas Amynthas References date hilgendorfi* agrestis tokioensis* Flintwood Preserve DE DE19 39°49031.000N75°36018.900W 7/2013 81 32 33 – 16 a Blowdown Flintwood Preserve DE DE20 39°49026.000N75°36013.200W 7/2013 63 38 9 – 16 a Diversity Flintwood Preserve Former DE DE21 39°48046.000N75°36016.600W 7/2013 51 12 18 – 21 a Field Wilmette IL WMT 42°0403800N87°4302500W 10/2016 112 – 42 2 68 – Cub Hill, Parkville MD CUH 39°24045.200N76°31014.900W 7/2002, 36 8 13 5 10 b 10/2003 Metaphire hilgendorfi Johns Hopkins Univ., MD JHU 39°19045.900N76°37028.600W 8/2015 61 – 8 16 37 b Baltimore Stony Run, Baltimore MD STR 39°21018.000N76°37047.800W 7/2015 31 13 2 12 4 b Oregon Ridge Park, MD ORP 39°28043.000N76°41021.400W 9/2015 32 – 6 7 19 b Cockeysville Plummers Island, Cabin MD PIS 38°58013.200N77°10035.100W 9/2012 11 – 7 – 4 b , John Amynthas agrestis Treefall, SERC, Edgewater MD SERC2 38°53030.700N76°33056.400W 10/2016 9 – 9 – – b Triangle, SERC, Edgewater MD SERC1 38°53028.200N76°33043.900W 9/2015 18 4 – 14 – b Hanover NH HNV 43°41049.500N72°17022.100W 10/2015 11 – 9 2 – b Clinton NY BCA8/ 43°02057.200N75°24000.700W 9/2014, 30 17 – 7 6 c 24 10/2015 Hamilton NY BCA22 42°47049.000N75°30020.000W 10/2015 10 3 – 5 2 c Millbrook NY BCA21 41°52034.000N73°40033.000W 11/2015 9 – 9 – – c Mundy Wildflower Preserve NY NY3 42°27000.700N76°28008.200W 7/2013 159 125 9 24 1 a West Point NY WP5 41°23026.700N74°00036.300W 7/2011 160 78 30 13 39 a West Point NY WP7 41°22015.200N74°02020.500W 7/2011 180 41 62 45 32 a West Point NY WP9 41°20018.900N74°04040.500W 7/2011 162 67 39 42 14 a Kendal-Crosslands PA PA16 39°52038.400N75°39037.900W 7/2013 84 48 9 7 20 a Communities Longwood For. PA PA17 39°52034.300N75°40038.500W 7/2013 84 36 18 28 2 a Longwood Garden PA PA18 39°52025.100N75°40029.800W 7/2013 50 33 6 3 8 a 123 Waterloo Mills Preserve PA PA15 40°01032.700N75°25025.100W 7/2013 14 – 9 – 5 a Welkinweir Old PA PA14 40°09008.500N75°41006.900W 7/2013 87 44 40 – 3 a Audubon, Huntington VT ADB 44°20049.600N72°59046.500W 10/2015 49 25 – 18 6 b 123 Table 1 continued Location State Label GPS-N GPS-W Collection Total NID Metaphire Amynthas Amynthas References date hilgendorfi* agrestis tokioensis* Duxbury VT DXB 44°22030.700N72°57017.200W 10/2015 6 1 – 4 1 b Centennial Woods, South VT CTW 44°28033.200N73°11016.400W 9/2015 18 – 2 16 – b Burlington HRC, South Burlington VT HRC 44°25052.500N73°11058.900W 9/2015 103 – – 15 88 b UVM Nursery, South VT UVM 44°25045.300N73°12009.100W 9/2010 23 11 1 3 8 b Burlington UW Arboretum, Madison WI UWA 43°02036.600N89°25030.800W 9/2015 16 – – 5 11 b Total 1760 636 390 293 441 Total total number of specimens examined, NID number of individuals that are not identifiable due to parthenogenesis or immaturity *First state records of M.