New England Range Extension of American Brook Lamprey
(Lethenteron appendix), as Confirmed by Genetic Analysis
Jacob T. Aman, Margaret F. Docker, and Kristin Wilson Grimes
Northeastern Naturalist, Volume 24, Issue 4 (2017): 536–543
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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
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J.T. Aman, M.F. Docker, and K. Wilson Grimes
2017
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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.
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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
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J.T. Aman, M.F. Docker, and K. Wilson Grimes
2017
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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
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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.
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