Northeastern Naturalist 536 J.T. Aman, M.F. Docker, and K. Wilson Grimes 22001177 NORTHEASTERN NATURALIST 2V4(o4l). :2543,6 N–5o4. 34 New England Range Extension of American Brook Lamprey (Lethenteron appendix), as Confirmed by Genetic Analysis Jacob T. Aman1,*, Margaret F. Docker2, and Kristin Wilson Grimes1,3 Abstract- Genetic analysis (i.e., DNA sequence data from the mitochondrial cytochrome b and cytochrome oxidase subunit I genes) confirms the presence of Lethenteron appendix (American Brook Lamprey) in Shorey’s Brook, a small stream located in the towns of Eliot and South Berwick, ME. This study documents the easternmost record of this species in New England, and the first known occurrence of the American Brook Lamprey in Maine. Introduction Lethenteron appendix (DeKay) (American Brook Lamprey) inhabit streams in the Great Lakes basin, the St. Lawrence and Mississippi River drainages, and the eastern United States (Jacobs and O’Donnell 2009, Potter et al. 2015, Renaud et al. 2009). In the New England states, prior to this report, the documented eastern limit of the American Brook Lamprey was in the Oyster River in New Hampshire (New Hampshire Fish and Game Department 2010). It is state-listed as a threatened species in Massachusetts (Massachusetts Division of Fisheries and Wildlife 2015), Rhode Island (Rhode Island Department of Environmental Management 2015), and Vermont (Vermont Wildlife Action Plan Team 2015) and as an endangered species in Connecticut (Connecticut Department of Energy and Environmental Protection 2015) and New Hampshire (Normandeau 2015). However, this species has no current state conservation status in Maine due to lack of confirmed records. It is probably not uncommon, however, for freshwater, non-parasitic brook lampreys, which spend the majority of their life cycle as blind, filter-feeding larvae (ammocoetes) burrowed in the sediment of their natal streams (Potter et al. 2015), to go undetected. Furthermore, since lampreys possess few taxonomically informative morphological characters, particularly during the larval stage, species identification can be problematic (Docker et al. 2009, Potter et al. 2015); American Brook Lamprey larvae are very similar in appearance to Petromyzon marinus L. (Sea Lamprey) larvae, which are known to occur in Maine and occupy similar larval habitat (Vladykov 1960, Vladykov and Kott 1980). We therefore use genetic analysis to assist in species identification of larval lampreys collected in Maine, a method that has proven to be successful in lampreys (e.g., Docker et al. 2016, Neave et al. 2007) and 1Wells National Estuarine Research Reserve, 342 Laudholm Farm Road, Wells, ME 04090. 2Department of Biological Sciences, University of Manitoba, 50 Sifton Road, Winnipeg, MB R3T 2N2, Canada. 3Center for Marine and Environmental Studies, University of the Virgin Islands, 2 John Brewers Bay, St. Thomas 00802. *Corresponding author - jacobaman@ wellsnerr.org. Manuscript Editor: David Halliwell Northeastern Naturalist Vol. 24, No. 4 J.T. Aman, M.F. Docker, and K. Wilson Grimes 2017 537 other field-collected marine specimens (e.g., Sweijd et al. 2000), providing useful information for species management. Study Sites and Methods Resident fish specimens were collected from 8 randomly selected study sites on Shorey’s Brook in the Piscataqua River watershed in southwestern Maine during August (30–31), September (9, 12, 21, 27), and October (6) 2011 using backpack electrofishing methods (Fig. 1). All sites were located in free-flowing reaches upstream of an impoundment created by a historic dam at the head of tide, and a perched culvert where State Route 101 crosses the brook. Backpack electrofishing surveys consisted of a single upstream pass sampling all areas likely to provide shelter for resident fish species for a standardized longitudinal distance of ~34 m. Due to the small size of the stream, we sampled the majority of each study reach. Stunned fish were collected with 6.35-mm–mesh dip nets. We identified all captured fish to species in the field prior to releasing them alive at the point of capture, except for 7 voucher specimens of what we initially thought were Sea Lamprey larvae that we collected and stored on ice during transport to the lab for further identification. Each voucher specimen was labeled with a unique letter from A to G and preserved in 90% Figure 1. (A) Regional location of study sites, (B) geographic distribution of known American Brook Lamprey occurrence within the Piscataqua River watershed, and (C) location of study sites in Shorey’s Brook. Northeastern Naturalist 538 J.T. Aman, M.F. Docker, and K. Wilson Grimes 2017 Vol. 24, No. 4 ethanol solution for genetic analysis. Information as to which study site each voucher specimen originated from was not retained, though anecdotally, most voucher specimens were collected at site LB02, where lamprey were most abundant. We used 4 specimens (Maine A, B, D, and F) to confirm species identification through genetic analyses performed at the University of Manitoba. DNA was extracted from muscle tissue, and the mitochondrial cytochrome b (cytb) and cytochrome oxidase subunit I (COI) genes were amplified by polymerase chain reaction (PCR) and sequenced in 2 (Maine A and B) and 3 (Maine B, D, and F) individuals using the primers and protocols outlined by Li (2014) and Boguski (2009), respectively. Results We caught a total of 62 lamprey larvae at 6 of the 8 study sites (Fig. 1). We identified the voucher specimens in the lab as freshwater American Brook Lamprey, rather than Sea Lamprey, based on their lack of pigmentation around the nostril and along the side of the body above the gill openings (Hartel et al. 2002). Further support for this determination was the presence of 2 assumed physical barriers (a head-of-tide dam with a head of ~3 m, and a perched culvert with an outlet drop of 0.5 m at State Route 101) to upstream migration by anadromous Sea Lamprey (Reinhardt et al. 2009; Fig. 1). It should be noted the head-of-tide dam has since been removed and the perched culvert replaced at stream grade (see Discussion). Many of the larger individuals appeared to be in various stages of metamorphosis (Manzon et al. 2015), though none had fully completed the over-winter transition to the adult stage. Genetic analysis of tissue samples from the 4 lampreys confirmed that all samples were American Brook Lamprey. Cytochrome b DNA sequences (1191 bp) were identical in Maine A and B (GenBank Accession Numbers KJ684702 and KJ6847003, respectively) and were compared to a taxonomically comprehensive cytb gene sequence database derived from Lang et al. (2009) and Li (2014). Maine A and B were genetically indistinguishable from an American Brook Lamprey from Delaware (Li 2014) and differed by 0.3–0.4% (Kimura 2-parameter distance, K2P) from American Brook Lamprey from the Great Lakes basin and 0.8% from specimens of this species caught in Tennessee (Li 2014 and Lang et al. 2009, respectively). When compared to Sea Lamprey, the only other lamprey species reported from Maine, these 2 specimens differed by 14.8%. All other lamprey species from eastern North America (Least Brook Lamprey Lampetra aepyptera (Abbott) and the 6 species from genus Ichthyomyzon) differed by 6.2–15.1%. Other closely related species in the Lethenteron genus (L. camtschaticum (Tilesius von Tilenau) [Arctic Lamprey], L. alaskense Vladykov and Kott [Alaskan Brook Lamprey], L. reissneri (Dybowski) [Far Eastern Brook Lamprey], L. kessleri (Anikin) [Siberian Brook Lamprey]) differed by just 0.2–0.4% but are found only in Arctic and Pacific drainages (Renaud 2011). Similarly, COI DNA sequences (588 bp) were identical in Maine B, D, and F (GenBank Accession Numbers KX938428– KX938430) and were compared to the Northeastern Naturalist Vol. 24, No. 4 J.T. Aman, M.F. Docker, and K. Wilson Grimes 2017 539 taxonomically comprehensive COI gene sequence database derived from Hubert et al. (2008) and April et al. (2011); COI is generally recognized as the “DNA barcode” for the animal kingdom. Maine B, D, and F were genetically indistinguishable from 9 American Brook Lamprey from tributaries to the St. Lawrence River in Quebec (Hubert et al. 2008) and differed by 0.3–0.5% K2P from 15 American Brook Lamprey from other locations. These 3 specimens differed by 18.8–19.4% from Sea Lamprey and 7.7–18.4% from other lamprey species from eastern North America. Other closely related species in the Lethenteron genus differed by 0–0.5% (including one Arctic Lamprey from the Yukon River in Alaska that was genetically indistinguishable from the 3 Maine specimens), but none of these species occur in North America east of the Northwest Territories and northern Alberta in Canada (Renaud et al. 2009). Low levels of genetic variation among “paired” or “satellite” lamprey species is common (see April et al. 2011, Docker 2009). Discussion The results of the genetic analysis confirm tentative species identification based on body pigmentation and represent the first-documented record of American Brook Lamprey in the State of Maine. Cytochrome b and COI gene sequences from these specimens were only 0–0.8% and 0–0.5% different, respectively, from other known American Brook Lamprey specimens, well within the range of observed intraspecific variation (Hubert et al. 2008, Li 2014), but 14.8% and 18.8–19% different, respectively, from Sea Lamprey. Although it should be noted that mitochondrial DNA is maternally inherited (Gyllensten et al. 1985) and thus, on its own, cannot detect recent or ancient hybridization events (see Wilson and Bernatchez 1998), hybridization is unlikely in this case. Hybrids between closely related lamprey species have been shown to be viable, but hybrids between species in different genera were not (Piavis et al. 1970). Thus, we are confident that the lamprey specimens collected in Maine were not Sea Lamprey bearing American Brook Lamprey mitochondrial DNA, but rather American Brook Lamprey. Review of the available literature shows no previous records of the American Brook Lamprey in the State. Limited historical surveys for this species have occurred in southern Maine, though not resulting in positive identifications (D.B. Halliwell, Maine Department of Environmental Protection, Augusta, ME, pers. comm.). Reports from New Hampshire limit the species’ documented occurrences to the upper Oyster River (NHFG 2010), which is also part of the Piscataqua River watershed (Fig. 1). Given the cryptic freshwater nature of larval lampreys and the difficulty in distinguishing them from Sea Lamprey based on morphology alone, we suspect that this species has long been present in Maine but not documented. Thus, this current study likely documents an eastern range extension, rather than a range expansion, in the northeastern United States. Though Shorey’s Brook and the Oyster River both occur in the Piscataqua River watershed, they are separated by the Great Bay and Piscataqua River estuaries (see Fig. 1). Given the distance separating these systems, and that the American Brook Lamprey is a freshwater fish, there is little likelihood of recent exchange between the 2 systems, except, potentially, by stocking. American Brook Lamprey larvae Northeastern Naturalist 540 J.T. Aman, M.F. Docker, and K. Wilson Grimes 2017 Vol. 24, No. 4 have been used as bait in Canada (Renaud 2011), but given that its occurrence has been so rare in New Hampshire and Maine, and that Maine law prohibits the use of live lampreys for bait (MDIFW 2017), stocking by recreational fishermen is an unlikely source. A more plausible explanation is that these nearby populations have become isolated over time as sea level has risen and saltwater has intruded further up the rivers (Belknap et al. 1987). The absence of metamorphosed adult American Brook Lamprey in our samples is likely due to the timing of sampling. Like all lampreys, the American Brook Lamprey is semelparous; adults spawn in late March to early April over areas of gravel and cobble substrate, after which they die (Werner 2004). Our sampling occurred in late summer and early fall when larvae would still be undergoing their putative metamorphosis and no adults would be present (Seagle and Nagel 1982). This documentation of American Brook Lamprey in Maine will likely have significance for its conservation status in the state, where it could become a candidate for state endangered or threatened species listing. In 2015, the Maine Department of Inland Fisheries and Wildlife included American Brook Lamprey in updates to the Maine Wildlife Action Plan, as a Priority 3 Species of Greatest Conservation Need, based on data shared with state biologists from this study (MDIFW 2015). However, information on the status of this species in Maine and New Hampshire is extremely limited due primarily to the lack of effort to locate existing populations. In Shorey’s Brook, removal of the head-of-tide dam in November 2011, and replacement of the perched culvert at State Route 101 in 2014 (Aman 2013, 2016), now allows sea run species, inclusive of Sea Lamprey, access to freshwater habitats where the American Brook Lamprey currently resides. Alterations to stream characteristics have been shown to affect this sensitive species (Moerke and Lamberti 2003), but it is unclear to what extent this non-migratory species will be affected by the removal of the stream barriers. Additional surveys in Shorey’s Brook and elsewhere in southern Maine and New Hampshire should be conducted to document this rare fish and to better understand how human actions and climate change may affect its populations in the future. Acknowledgments The authors thank Dr. Michele Dionne and Emily Thornton for their contributions to the design and implementation of the field study. We are also grateful to the Maine Outdoor Heritage Fund for funding the field portion of the study. An award from the US National Oceanic and Atmospheric Administration to the Wells National Estuarine Research Reserve (Award NA15NOS4200115) provided support for data analysis and publication. Literature Cited Aman, J.A. 2013. Monitoring of post-restoration habitat conditions in Shorey’s Brook. Wells National Estuarine Research Reserve, Wells, ME. 18 pp. Aman, J.A. 2016. Assessing fish usage of restored habitat in two southern Maine watersheds, Shorey’s Brook. Report to the Maine Outdoor Heritage Fund. Wells National Estuarine Research Reserve, Wells, ME. 8 pp. Northeastern Naturalist Vol. 24, No. 4 J.T. Aman, M.F. Docker, and K. Wilson Grimes 2017 541 April, J., R.L. Mayden, R.H. Hanner, and L. Bernatchez. 2011. Genetic calibration of species diversity among North America’s freshwater fishes. Proceedings of the National Academy of Sciences of the United States of America 108:10602–10607. Belknap, D. F., B.G. Anderson, W.A. Anderson, H.W. Borns Jr., G.L. Jacobson, J.T. Kelley, R.C. Shipp, D.C. Smith, R. Stuckenrath Jr., W.B. Thompson, and D.A. Tyler. 1987. Late Quaternary sea-level changes in Maine. Pp. 71–85, In D. Nummedal, O.H. Pilkey Jr., and J.D. Howard (Eds.). Sea-level fluctuation and coastal evolution: Society of Economic Paleontologists and Mineralogists Special Publication 41. Tulsa, OK. 276 pp. Boguski, D.A. 2009. The genetic diversity of brook lampreys genus Lampetra (Petromyzontidae) along the Pacific coast of North America. M.Sc. Thesis. University of Manitoba, Winnipeg, MB, Canada. 144 pp. Connecticut Department of Energy and Environmental Protection. 2015. Connecticut wildlife action plan 2015 (CWCS). Bureau of Natural Resources, Hartford, CT. Available online at http://www.ct.gov/deep/cwp/view.asp?a=2723&q=329520&deep. Accessed 15 February 2016. Docker, M.F. 2009. A review of the evolution of nonparasitism in lampreys and an update of the paired species concept. Pp. 71–114, In L.R. Brown, S.D. Chase, M.G. Mesa, R.J. Beamish, and P.B. Moyle (Eds). Biology, Management, and Conservation of Lampreys in North America. American Fisheries Society, Symposium 72. Bethesda, MD. 321 pp. Docker, M.F., G.S. Silver, J.C. Jolley, and E.K. Spice. 2016. Simple genetic assay distinguishes lamprey genera Entosphenus and Lampetra: Comparison with existing genetic and morphological identification methods. North American Journal of Fisheries Management 36:780–787. Gyllensten, U., D. Wharton, and A.C. Wilson. 1985. Maternal inheritance of mitochondrial DNA during backcrossing of two species of mice. Journal of Heredity 76:321–324. Hartel, K.E., D.B. Halliwell, and A.E. Launer. 2002. Inland fishes of Massachusetts. Massachusetts Audubon Society, Lincoln, MA. 328 pp. Hubert, N., R. Hanner, E. Holm, N.E. Mandrak, E. Taylor, M. Burridge, D. Watkinson, P. Dumont, A. Curry, P. Bentzen, J. Zhang, J. April, and L. Bernatchez. 2008. Identifying Canadian freshwater fishes through DNA barcodes. PLoS ONE 3:e2490. Jacobs, R.P., and E.B. O’Donnell. 2009. A Pictorial Guide to Freshwater Fishes of Connecticut. Connecticut Department of Environmental Protection, Hartford, CT. 242 pp. Lang, N.J., K.J. Roe, C.B. Renaud, H.S. Gill, I.C. Potter, J. Freyhof, A.M. Naseka, P. Cochran, H.E. Perez, E.M. Habit, B.R. Kuhajda, D.A. Neely, Y.S. Reshetnikov, V.B. Salnikov, M.T. Stoumboudi, and R.L. Mayden. 2009. Novel relationships among lampreys (Petromyzontiformes) revealed by a taxonomically comprehensive molecular data set. Pp. 41–55, In L.R. Brown, S.D. Chase, M.G. Mesa, R.J. Beamish, and P.B. Moyle (Eds). Biology, Management, and Conservation of Lampreys in North America. American Fisheries Society, Symposium 72. Bethesda, MD. 321 pp. Li, Y. 2014. Phylogeny of the lamprey genus Lethenteron Creaser and Hubbs 1922 and closely related genera using the mitochondrial cytochrome b gene and nuclear gene introns. M.Sc. Thesis. University of Manitoba, Winnipeg, MB, Canada. 228 pp. Maine Department of Inland Fisheries and Wildlife (MDIFW). 2015. Maine’s Wildlife Action Plan. Augusta, ME. Available online at http://www.maine.gov/ifw/fish-wildlife/ wildlife/wildlife-action-plan.html. Accessed 6 December 2017. MDIFW. 2017. Open Water and Ice Fishing Laws. Augusta, ME. Available online athttp:// www.maine.gov/ifw/fishing-boating/fishing/laws-rules/baitfish-dealers.html. Accessed 6 December 2017. Northeastern Naturalist 542 J.T. Aman, M.F. Docker, and K. Wilson Grimes 2017 Vol. 24, No. 4 Manzon, R.G., J.H. Youson, and J.A. Holmes. 2015. Lamprey metamorphosis. Pp. 139– 214. In M.F. Docker (Ed.), Lampreys: Biology, Conservation, and Control. Springer, Dordrecht, The Netherlands. 438 pp. Massachusetts Division of Fisheries and Wildlife. 2015. Massachusetts State Wildlife Action Plan 2015. Westborough, MA. Available online at https://www.mass.gov/eea/ agencies/dfg/dfw/wildlife-habitat-conservation/state-wildlife-conservation-strategy. html. Accessed 15 February 2016. Moerke, A.H., and G.A. Lamberti. 2003. Responses in fish community structure to restoration of two Indiana streams. North American Journal of Fisheries Management, 23(3):748–759. Neave, F.B., N.E. Mandrak, M.F. Docker, and D.L. Noakes. 2007. An attempt to differentiate sympatric lchthyomyzon ammocoetes using meristic, morphological, pigmentation, and gonad analyses. Canadian Journal of Zoology 85:549–560. New Hampshire Fish and Game Department. 2010. 2010 operational master plan. Inland Fisheries Division. Concord, NH. Available online at https://www3.epa.gov/region1/ npdes/merrimackstation/pdfs/ar/AR-620.pdf. Accessed 6 December 2017. Normandeau, G. 2015. New Hampshire Wildlife Action Plan. New Hampshire Department of Fish and Game. Concord, NH. Available online at Available online at http://www. wildlife.state.nh.us/wildlife/wap.html. Accessed 6 December 2017. Piavis, G.W., J.H. Howell, and A.J. Smith. 1970. Experimental hybridization among five species of lampreys from the Great Lakes. Copeia 1970:29-37. Potter, I.C., H.S. Gill, C.B. Renaud, and D. Haoucher. 2015. The taxonomy, phylogeny, and distribution of lampreys. Pp. 35–73, In M.F. Docker (Ed.), Lampreys: Biology, Conservation, and Control. Springer, Dordrecht, The Netherlands. 438 pp. Reinhardt U.G., T.H.O Binder, D.G. McDonald. 2009. Ability of adult Sea Lamprey to climb inclined surfaces. American Fisheries Society, Symposium 72:125–138. Renaud, C.B. 2011. Lampreys of the World: An Annotated and Illustrated Catalogue of Lamprey Species Known to Date. Food and Agriculture Organization of the United Nations, Rome, Italy. 110 pp. Renaud, C.B., M.F. Docker, and N.E. Mandrak. 2009. Taxonomy, distribution, and conservation of lampreys in Canada. Pp. 293–309, In L.R. Brown, S.D. Chase, M.G. Mesa, R.J. Beamish, and P.B. Moyle (Eds). Biology, Management, and Conservation of Lampreys in North America. American Fisheries Society, Symposium 72. Bethesda, MD. 321 pp. Rhode Island Department of Environmental Management. 2005. Rhode Island’s comprehensive wildlife conservation strategy. Division of Fish and Wildlife, Wakefield, RI. Available online at http://www.dem.ri.gov/programs/bnatres/fishwild/pdf/swgplan.pdf. Accessed 15 February 2016. Seagle, H.H. and J.W. Nagel. 1982. Life cycle and fecundity of the American Brook Lamprey, Lampetra appendix, in Tennessee. Copeia, 2:362–366. Sweijd, N.A., R.C.K. Bowie, B.S. Evans and A.L. Lopata. 2000. Molecular genetics and the management and conservation of marine organisms. Hydrobiologia, 420(1):153–164. Vermont Wildlife Action Plan Team. 2015. Vermont wildlife action plan 2015. Vermont Fish and Wildlife Department. Montpelier, VT. Available online at http://www. vtfishandwildlife.com/about_us/budget_and_planning/revising_vermont_s_wildlife_ action_plan/draft_2015_wildlife_action_plan_for_public_review/. Accessed 15 February 2016. Vladykov, V.D. 1960. Description of young ammocoetes belonging to two species of lampreys: Petromyzon marinus and Entosphenus lamottenii. Journal of the Fisheries Research Board of Canada 17:267–288. Northeastern Naturalist Vol. 24, No. 4 J.T. Aman, M.F. Docker, and K. Wilson Grimes 2017 543 Vladykov, V.D., and E. Kott. 1980. Description and key to metamorphosed specimens and ammocoetes of Petromyzonidae found in the Great Lakes region. Canadian Journal of Fisheries and Aquatic Sciences 37:1616–1625. Werner, R.G. 2004. Freshwater Fishes of the Northeastern United States: A Field Guide. Syracuse University Press, Syracuse, NY. 280 pp. Wilson, C.C. and L. Bernatchez. 1998. The ghost of hybrids past: Fixation of Arctic Charr (Salvelinus alpinus) mitochondrial DNA in an introgressed population of Lake Trout (S. namaycush). Molecular Ecology 7:127–132.