North American Journal of Fisheries Management 36:780–787, 2016 © American Fisheries Society 2016 ISSN: 0275-5947 print / 1548-8675 online DOI: 10.1080/02755947.2016.1167146

MANAGEMENT BRIEF

Simple Genetic Assay Distinguishes Genera Entosphenus and : Comparison with Existing Genetic and Morphological Identification Methods

Margaret F. Docker* Department of Biological Sciences, University of Manitoba, 50 Sifton Road, Winnipeg, Manitoba R3T 2N2, Canada Gregory S. Silver and Jeffrey C. Jolley U.S. Fish and Wildlife Service, Columbia River Fisheries Program Office, 1211 Southeast Cardinal Court, Suite 100, Vancouver, Washington 98683, USA Erin K. Spice Department of Biological Sciences, University of Manitoba, 50 Sifton Road, Winnipeg, Manitoba R3T 2N2, Canada

Along the West Coast of North America, the lamprey genera Abstract Entosphenus and Lampetra co-occur from Alaska to California Several species of lamprey belonging to the genera (Potter et al. 2015). Six species have been described in the genus Entosphenus Lampetra and , including the widely distributed Entosphenus (including the widely distributed PacificLamprey PacificLampreyE. tridentatus and Western L. richardsoni, co-occur along the West Coast of North E. tridentatus). In the genus Lampetra, four species are formally America. These genera can be difficult to distinguish morpho- recognized in North America (including the widely distributed logically during their first few years of larval life in freshwater, Western Brook Lamprey L. richardsoni), although the presence thus hampering research and conservation efforts. However, of genetically distinct populations of Lampetra spp. in Oregon fi existing genetic identi cation methods are time consuming or and California suggests the occurrence of additional, currently expensive. Here, we describe a simpler genetic assay using the fi Pacific Lamprey microsatellite locus Etr-1; the assay was found unidenti ed, species (Boguski et al. 2012). Several species in to be 100% reliable in distinguishing Entosphenus from these lamprey genera are of conservation and management con- Lampetra, even in genetically divergent Lampetra populations. cern (Maitland et al. 2015; Wang and Schaller 2015). However, Using a sample of 244 larvae (18–136 mm TL) from the research and conservation efforts are hampered by the fact that Columbia River basin, we tested the accuracy with which during the long larval stage, Entosphenus and Lampetra live in previously described differences in caudal fin pigmentation can distinguish these genera. Attempts at morphological iden- similar habitats (sediment in freshwater streams and rivers) and tification were abandoned for 50-mm and smaller larvae can sometimes be difficult to distinguish morphologically because differences in caudal fin pigmentation were very diffi- (Goodman et al. 2009). To rectify information deficits and to cult to discern. However, morphological identification was cor- develop appropriate conservation strategies for West Coast lam- – rect for 81.8% of 51 70-mm larvae and 100% of 71-mm and prey species, accurate methods of larval identification are larger larvae, which roughly corresponds with the results of previous studies. In agreement with previous work using mito- necessary. chondrial DNA, our assay also supported placement of the Morphological methods for distinguishing between larval L. hubbsi (formerly E. hubbsi)intothe Entosphenus and Lampetra have been developed but generally genus Lampetra. are only applied to larger larvae. Richards et al. (1982) reared larvae through metamorphosis to confirm species identity and

*Corresponding author: [email protected] Received November 23, 2015; accepted March 9, 2016

780 MANAGEMENT BRIEF 781 found that the pigmentation pattern in the tail region was the on an agarose gel (Goodman et al. 2009). However, this only reliable external morphological character for distinguish- RFLP assay only interrogates the cytochrome-b PCR product ing Pacific Lampreys or Vancouver Lampreys E. macrostomus for sequence differences at the enzyme’s recognition site from Western Brook Lampreys in British Columbia. However, (detecting, in this case, only two nucleotide differences within differences in this character were reported only for 96-mm TL the 432-bp cytochrome-b fragment), which might not accu- and larger larvae. Using a genetic assay for verification of rately represent genus-level differences across the entire gene. genus identity (see below), Goodman et al. (2009) were able The discovery of several genetically distinct Lampetra popu- to distinguish between Entosphenus and Lampetra larvae in lations in Oregon and California (Boguski et al. 2012) sug- Washington, Oregon, and California by examining pigmenta- gests that these RFLP assays may be unable to distinguish tion on the caudal fin, caudal ridge, and ventral aspect of the such populations from Entosphenus. One single-nucleotide body, but only 60-mm and larger larvae were included in that polymorphism (SNP) locus identified by Hess et al. (2015) study. The general rule of thumb applied by many field biol- can distinguish Pacific Lamprey from Lampetra species ogists is that caudal fin pigmentation cannot reliably be used (Western Brook Lamprey, Western River Lamprey, and to distinguish these two genera for larvae smaller than Pacific Brook Lamprey L. pacifica) in the Pacific Northwest approximately 60 mm (e.g., Reid and Goodman 2015)or based on TaqMan SNP genotyping assays; however, this locus 70 mm (e.g., Hayes et al. 2013). However, to the best of our has not yet been tested in divergent Lampetra populations or knowledge, the ability to use caudal fin pigmentation to iden- in other species of Entosphenus. Reagents for these assays are tify smaller larvae has not been explicitly tested or reported in much more expensive than those for conventional PCR, and the literature. Based on a multivariate analysis of morpho- access to a real-time PCR thermal cycler is required. metric characteristics (e.g., oral hood, branchial region, A reliable, efficient, inexpensive method for genetic identi- trunk region, and tail region), Meeuwig et al. (2006) were fication of larval lampreys would be an asset for research and able to identify prolarval and larval Pacific Lampreys and conservation. During the initial development of microsatellite Western Brook Lampreys (up to 11 mm TL) from the loci for Pacific Lampreys, Spice et al. (2011) found that two Columbia River, but this identification method requires time- microsatellite loci (Etr-1 and Etr-6) produced fragments of consuming analysis (i.e., truss and discriminant function ana- different sizes in Entosphenus and Lampetra, albeit among a lyses). Furthermore, these characteristics have not been eval- limited number of samples (e.g., Lampetra was represented by uated for potential geographic variability and applicability to only 10 Western Brook Lamprey individuals from Lockwood larger larvae. If morphological identification methods are used Creek, Washington). The Etr-1 and Etr-6 assays require only during situations in which their effectiveness is not known PCR amplification and gel electrophoresis and are thus much (e.g., larvae of different sizes or from different geographic more efficient and cost effective relative to direct cytochrome- areas), some individuals may be misidentified, negatively b gene sequencing or the TaqMan SNP genotyping assays impacting the quality of data that are collected. described above. Therefore, the first objective of the present Despite the high degree of morphological similarity study was to test whether the Etr-1 and Etr-6 loci are reliable between Entosphenus and Lampetra during the larval stage, for distinguishing between Lampetra and Entosphenus these two genera are genetically very distinct (e.g., Docker throughout their North American range, even in highly diver- et al. 1999; Lang et al. 2009). Genetic identification methods gent populations, and to compare their reliability to that of the can be applied to any size and developmental stage of lam- HaeIII PCR-RFLP assay used by Goodman et al. (2009). The prey, but existing genetic methods are time consuming, expen- second objective was to use genetic identification to test the sive, or insufficient to permit identification. Sanger sequencing accuracy of existing morphological identification methods of the cytochrome-b gene can unambiguously distinguish (namely, caudal fin pigmentation) for lamprey larvae over a Entosphenus and Lampetra, as the DNA sequence of this wide range of sizes (18–136 mm TL). 1,191-base-pair (bp) gene differs between the genera by 9.4– 12.7% (Boguski et al. 2012). Cytochrome-b sequencing can distinguish among all but the closely related “paired species” METHODS within each genus (e.g., Western Brook Lamprey and Western For evaluation of the accuracy and general applicability of the River Lamprey L. ayresii; Docker 2009); however, this Etr-1 and Etr-6 assays, individuals (total n =208)werechosen method would be cost prohibitive for routine discrimination from four of the six known Entosphenus species (as well as one of Entosphenus and Lampetra. A more cost-effective restric- potentially undescribed species from Upper Klamath Lake), all tion fragment length polymorphism (RFLP) assay was devel- four recognized Lampetra species in North America, and four oped that screens the cytochrome-b gene for diagnostic genetically divergent Lampetra populations examined by sequence differences between the genera without requiring Boguski et al. (2012). Samples were included from throughout direct sequencing; this assay amplifies the cytochrome-b each species’ range (Table 1); all individuals were identified to gene by using PCR, digests it with the restriction enzyme species based on complete cytochrome-b gene sequencing and, HaeIII, and visualizes the genus-specific fragment patterns when possible, based on morphologically identifiable adults. The 782 DOCKER ET AL.

TABLE 1. Amplification of the microsatellite loci Etr-1 and Etr-6 in 208 specimens of Entosphenus and Lampetra from throughout North America, including four potential new Lampetra spp. from Oregon and California (see Spice et al. 2011; Boguski et al. 2012). Fragment sizes (base pairs [bp]) of Etr-1, Etr-6, and the cytochrome-b gene (cyt b) after digestion with the restriction enzyme HaeIII are given (n = number of specimens tested; dashes indicates failure to amplify).

Etr-1 Etr-6 Cyt-b HaeIII Species n Locations (bp) (bp) (bp) Genus Entosphenus Pacific Lamprey E. tridentatus 16 Oregon (OR), Washington (WA), and 230 285 92, 166, 174 California (CA) Klamath Lamprey E. similis 15 OR 230 285 92, 166, 174 Miller E. minimus 5 OR 230 285 92, 166, 174 Pit–Klamath Brook Lamprey 19 OR and CA 230 285 92, 166, 174 E. lethophagus Entosphenus sp. 6 OR (Upper Klamath Lake) 230 285 92, 166, 174 Genus Lampetra Western River Lamprey L. ayresii 10 British Columbia (BC) and CA 265 175 166, 266 Western Brook Lamprey 97 Alaska, BC, WA, OR, and CA 265 175 166, 266 L. richardsoni Pacific Brook Lamprey L. pacifica 7 OR 265 175 166, 266 Kern Brook Lamprey L. hubbsi 16 CA 265 175 166, 266 Lampetra species 1 4 OR (Fourmile Creek) 265 175 166, 266 Lampetra species 2 5 OR (North Fork Siuslaw River) 265 175 166, 266 Lampetra species 3 4 CA (Mark West Creek) 265 – 92, 341 Lampetra species 4 4 CA (Kelsey Creek) 265 – 92, 166, 174

cytochrome-b DNA sequence in all Lampetra species differs by Goodman et al. (2009; Figure 1); however, differences in at least 9.4% from that of Entosphenus lampreys (Boguski et al. pigmentation were very difficult to discern in 50-mm and 2012), thus permitting unambiguous identification of genus. The smaller larvae (Figure 2; see Results). Identification was per- results of the three more cost-effective genetic assays (the Etr-1 formed in the field under ambient light conditions by biolo- and Etr-6 assays and the HaeIII PCR-RFLP assay) were com- gists with at least 7 years of experience in larval lamprey pared with the known identifications of these individuals. The identification. DNA was extracted by using the Wizard Genomic DNA For genetic analysis of the samples from the Columbia Purification Kit (Promega). The Etr-1 and Etr-6 loci were ampli- River basin, PCR was performed by using the Phire fied as described by Spice et al. (2011), and PCR products were Tissue Direct PCR Kit (Thermo Scientific) in accordance with visualized on a 1.5% agarose gel under ultraviolet (UV) light. the manufacturer’s instructions (annealing temperature = The HaeIII PCR-RFLP assay was performed in accordance with 63.5°C). This permitted Etr-1 amplification directly from lam- the methods of Goodman et al. (2009). prey fin clips without the need for a separate DNA extraction Accuracy of morphological identification was tested by step, and only a very small tissue sample (0.5-mm diameter) comparing morphological and genetic (Etr-1, which was was required. The PCR products were visualized on a 1.5% found to be as reliable as direct cytochrome-b gene sequen- agarose gel under UV light, and genus identification was cing; see Results) identification of Entosphenus and Lampetra determined based on fragment size (Table 1). larvae (n = 244) from the Columbia River basin. Larvae were Binomial confidence intervals (CIs) depicting the accuracy collected by electrofishing from the Klickitat, White Salmon, of morphological identification were calculated using JavaStat and Wind rivers, Washington; Drano Lake, Washington; the (statpages.org/confint.html). Fisher’sexacttests(GraphPad main-stem Columbia River below Bonneville Dam; Lake QuickCalcs; graphpad.com/quickcalcs/contingency1.cfm) were Celilo (the reservoir created by The Dalles Dam); and Lake used to make several comparisons of morphological assignment Bonneville (the reservoir created by Bonneville Dam; Jolley accuracy in different groups: (1) all 51–70-mm larvae versus all et al. 2012, 2013, 2014). Total length was measured to the 71-mm and larger larvae; (2) all 51–60-mm larvae versus all nearest millimeter, and nonlethal fin clips were taken and 61–70 mm larvae; (3) all Entosphenus larvae versus all preserved in a 95% solution of ethanol. Morphological identi- Lampetra larvae; (4) 51–70-mm Entosphenus larvae versus fication to genus was based on pigmentation of the caudal fin 51–70-mm Lampetra larvae; and (5) 71-mm and larger and caudal ridge according to Richards et al. (1982) and Entosphenus larvae versus 71-mm and larger Lampetra larvae. MANAGEMENT BRIEF 783

FIGURE 1. Caudal fin and caudal ridge pigmentation in large (>70-mm TL) Entosphenus and Lampetra larvae: (a) a Pacific Lamprey (99 mm TL) with a dark caudal fin and light pigmentation at the posterior tip of the caudal ridge; and (b) a Western Brook Lamprey (85 mm TL) with a lightly pigmented or peppered caudal fin and a uniformly dark caudal ridge.

RESULTS The Etr-1 and Etr-6 assays each produced fragments of dif- ferent sizes from Entosphenus and Lampetra; however, Etr-1 was more broadly reliable and in fact was as reliable (100%) as direct DNA sequencing of the cytochrome-b gene. The Etr-1 FIGURE 2. Caudal fin and caudal ridge pigmentation in small (≤50-mm TL) assay produced approximately 265-bp fragments from all Entosphenus and Lampetra larvae: (a) a Pacific Lamprey (36 mm TL); (b) a Lampetra samples and 230-bp fragments from all Entosphenus Western Brook Lamprey (46 mm TL); and (c) a Western Brook Lamprey samples (Table 1). The Etr-6 assay produced approximately 285- (46 mm TL). Identification was determined genetically. In the present study, it fi bp fragments from all Entosphenus samples and approximately was dif cult to discern differences in the degree of pigmentation on the caudal fin and the caudal ridge in most (e.g., panels a, b) but not all (e.g., panel c) 175-bp fragments from all Lampetra samples except those of the larvae that were 50 mm TL or smaller. two genetically divergent populations in Mark West and Kelsey creeks, California (the primers failed to amplify in those cases). Size differences were clearly evident on an agarose gel. The Kern Brook Lamprey L. hubbsi (formerly E. hubbsi) was genotyped as attempted for one 22-mm individual, which was correctly Lampetra by both Etr-1 and Etr-6.IntheHaeIII PCR-RFLP identified as belonging to the genus Entosphenus. Of the 24 assay, all Entosphenus species and unidentified Lampetra species larvae that were 41–50 mm in length, morphological identifi- 4 from Kelsey Creek showed the cut pattern that was previously cation was only attempted with nine individuals: four considered diagnostic for Entosphenus (i.e., three bands that Entosphenus larvae and two Lampetra larvae were correctly were 92, 166, and 174 bp in length); two Lampetra-specific identified, but three Lampetra larvae were incorrectly assigned patterns were evident in all other Lampetra samples (Table 1). to the genus Entosphenus. Given that morphological identifi- Differences in caudal fin and caudal ridge pigmentation cation was attempted in so few (6.9%) larvae that were 50 mm were very difficult to discern in 40-mm and smaller larvae (n TL or less, these smaller larvae were excluded from all sub- = 120), and morphological identification was in fact only sequent analyses. Morphological identification was attempted 784 DOCKER ET AL. for 19 of 20 individuals that were 51–60 mm and for 23 of 24 identified by Boguski et al. (2012) as exhibiting very diver- larvae that were 61–70 mm; accuracy of this method was 80% gent cytochrome-b sequences. Microsatellite loci designed and 83%, respectively (Table 2). Considering both genera for one species often do not amplify in more distantly together, morphological identification of 51–70-mm larvae related organisms (Selkoe and Toonen 2006); thus, it is not was correct 81.8% of the time (95% CI = 67.3–91.8%). surprising that Etr-6, which was designed for the Pacific Morphological identification was correct for all 71-mm and Lamprey, failed to amplify in these divergent Lampetra larger larvae (n = 56; 100% reliable; 95% CI = 93.6–100%) specimens. The Etr-1 assay was also more reliable than and was significantly more accurate for those individuals than the HaeIII PCR-RFLP assay in distinguishing Lampetra for 51–70-mm lampreys (Fisher’s exact test: P = 0.0010); from Entosphenus; the latter would have misidentified the however, there was no significant difference in the accuracy Kelsey Creek Lampetra population as Entosphenus sp. of assignment for 51–60-mm larvae versus 61–70-mm larvae (Table 1). A different RFLP assay may correctly identify (P = 1.0000). The accuracy of assignment did not significantly this population; computer-simulated digests of the 432-bp differ between Entosphenus versus Lampetra for all larvae (P cytochrome-b sequences from each of these species (using = 0.4772), for 51–70-mm larvae (P = 0.6895), or for 71-mm RestrictionMapper version 3) indicated that the restriction and larger larvae (P = 1.0000). enzyme BsaAI would produce wholly diagnostic patterns for distinguishing between Lampetra (187 and 245 bp) and Entosphenus (432 bp). However, the BsaAI assay would DISCUSSION be slower and more expensive than the Etr-1 assay (see Use of the microsatellite marker Etr-1 is a simple, inexpen- below). sive, and accurate method for distinguishing between the co- When choosing a method for routine genetic identification occurring lamprey genera Lampetra and Entosphenus. For the of lamprey larvae, two primary concerns are the cost per taxonomically and geographically diverse lamprey specimens sample and the time required for sample processing and ana- we examined, the Etr-1 assay was 100% accurate—that is, it lysis. Although these factors will vary between laboratories always agreed with identification based on the more labor- (e.g., both cost and time will be lower in high-throughput intensive direct DNA sequencing of the cytochrome-b gene genetics facilities relative to standard research laboratories), (see below). The Etr-1 assay can be applied nonlethally by approximate costs (for consumables) can be compared by using the DNA extracted from very small fin clips (0.5-mm using methods similar to those described in the present diameter). It allowed for the identification of larvae of all study. All of the previously used methods (cytochrome-b sizes, whereas morphological identification was rarely possi- sequencing, RFLP assays, and TaqMan assays) require DNA ble for larvae that were 50 mm TL or smaller. Although this to be extracted from tissue (50–100 samples/d at US$1–3 per genetic assay cannot be applied streamside during field sam- sample). After DNA is extracted, identification via DNA pling, it may help to improve the accuracy of morphological sequencing is relatively slow and expensive (30–50 samples/ identifications made in the field. For example, it could be used dat$5–8 per sample for PCR and sequencing) and requires to verify the accuracy of identifications made by inexper- more processing and interpretation of the sequence data that ienced personnel, thus indicating whether additional training are generated. The PCR-RFLP assays are less expensive and is necessary. Alternatively, the assay could be employed to test slightly faster (80–100 samples/d at $2–3 per sample for PCR, whether additional refinements of existing methods (e.g., the enzyme digestion, and gel electrophoresis). TaqMan assays use of improved magnification or lighting) could allow experi- (e.g., for genotyping SNP loci) allow for very efficient sample enced personnel to accurately identify smaller larvae. The Etr- processing (200–400 samples/d); however, this method 1 assay does not distinguish among species within each genus, requires an initial outlay of several hundred dollars for but it will be particularly useful for differentiating between the TaqMan primers and probes, and the reagent costs are high Pacific Lamprey and Western Brook Lamprey, which are the ($10 per sample). Furthermore, TaqMan assays require the use two most widely distributed lamprey species on the West of a real-time PCR system, which can cost approximately Coast of North America. For species with more restricted $25,000 (versus $3,000–8,000 for a standard thermocycler). geographic distributions, combining genus identification with The SNP locus that distinguishes the Pacific Lamprey from distributional information (Goodman et al. 2009), supplemen- Lampetra species in the Pacific Northwest is useful for rapid ted where necessary with complete cytochrome-b gene confirmation of identification when used alongside a suite of sequence data (Boguski et al. 2012), will allow for the identi- Pacific Lamprey-specific SNPs developed for other applica- fication of most individuals to species. tions (e.g., parentage analysis; characterization of neutral and The Etr-1 locus was a more suitable marker than Etr-6 adaptive variation), but employing that locus is not cost effec- because Etr-1 distinguished Entosphenus from Lampetra for tive when the main purpose or only purpose is species identi- all individuals and populations examined, whereas Etr-6 fication. When the Etr-1 method is used with the Phire Animal failed to amplify in two Lampetra populations: Mark West Tissue Direct PCR Kit, as described here, only PCR and gel and Kelsey creeks. These two populations are among those electrophoresis are necessary. The initial DNA extraction step TABLE 2. Accuracy of morphological identification for Entosphenus and Lampetra larvae (n = 100) from the Columbia River basin, evaluated against genetic identification using the microsatellite locus Etr-1. The 95% confidence interval for the accuracy of identification is shown in parentheses. Differences in caudal fin and caudal ridge pigmentation were very difficult to discern in 50-mm TL and smaller larvae (n = 140); therefore, the accuracy of morphological identification was not evaluated for individuals smaller than 51 mm TL.

All larvae Entosphenus Lampetra n n % % % % % % % % % TL (mm) n Entosphenus Lampetra Correct Incorrect Uncertain Correct Incorrect Uncertain Correct Incorrect Uncertain AAEETBRIEF MANAGEMENT 51–60 20 6 14 80 15 5 83.3 16.7 0 78.6 14.3 7.1 (56.3–94.3) (35.9–99.6) (49.2–95.3) 61–70 24 10 14 83.3 12.5 4.2 90 10 0 78.6 14.3 7.1 (62.6–95.3) (55.5–99.8) (49.2–95.3) 71–80 14 6 8 100 0 0 100 0 0 100 0 0 (75.3–100) (54.1–100) (63.1–100) 81–90 14 6 8 100 0 0 100 0 0 100 0 0 (78.2–100) (54.1–100) (63.1–100) 91–100 11 4 7 100 0 0 100 0 0 100 0 0 (63.1–100) (39.8–100) (59.0–100) 101–120 10 4 6 100 0 0 100 0 0 100 0 0 (69.2–100) (39.8–100) (54.1–100) 121–140 7 3 4 100 0 0 100 0 0 100 0 0 (59.0–100) (29.2–100) (39.8–100) 785 786 DOCKER ET AL.

(50–100 samples/d at $1–3 per sample) is omitted, and about genes (particularly nuclear genes) are needed to resolve the 100–200 samples can easily be processed per day at an differences between the morphological and molecular phylo- approximate cost of $0.75 per sample. This method combines genies (Potter et al. 2015). The microsatellite loci Etr-1 and reliability, efficiency, and cost effectiveness, and it can be Etr-6 as nuclear markers provide additional support for the re- applied to lamprey larvae of any size. classification of the Kern Brook Lamprey as L. hubbsi. In contrast, we found that morphological identification using caudal fin and caudal ridge pigmentation was generally not possible for 50-mm and smaller larvae. No other studies ACKNOWLEDGMENTS have reported the use of this character in larvae 60 mm TL or This study was funded by the Natural Sciences and — fi Engineering Research Council of Canada and the U.S. Fish and smaller presumably due to the same dif culty we encoun- fi tered in discerning caudal fin pigmentation differences in Wildlife Service. Samples for initial veri cation of the assays were small larvae. However, we did find that morphological identi- kindly provided by David Boguski, Damon Goodman, and fication was correct in 80% of the 51–60-mm larvae, suggest- Stewart Reid. Samples from Lake Bonneville and Lake Celilo ing that this character could be used in slightly smaller larvae on the Columbia River were part of a U.S. Army Corps of than previously reported if a lower level of accuracy is accep- Engineers-funded study. Karen Bailey, Meagan Robidoux, and fi table (e.g., when multiple individuals are being examined to Tim Whitesel provided technical assistance. The ndings and establish a species’ presence at a site and accurate identifica- conclusions in this paper are those of the authors and do not tion of each individual is not required). We found that the necessarily represent those of the U.S. Fish and Wildlife Service. accuracy of morphological identification was not significantly Reference to trade names does not imply endorsement by the U.S. different in 61–70-mm larvae (only 83% were identified cor- Government. rectly); accuracy was significantly higher (100%) only in larvae that were larger than 70 mm TL. Goodman et al. REFERENCES (2009) evaluated the accuracy of morphological identification Boguski, D. A., S. B. Reid, D. H. Goodman, and M. F. Docker. 2012. Genetic in 60-mm and larger lamprey larvae from 15 coastal rivers of diversity, endemism and phylogeny of lampreys within the genus California, Oregon, and Washington; those authors found that Lampetra sensu stricto (Petromyzontiformes: Petromyzontidae) in western pigmentation of the caudal fin and caudal ridge allowed for North America. Journal of Fish Biology 81:1891–1914. fi Docker, M. F. 2009. A review of the evolution of nonparasitism in lampreys proper genus identi cation in 100% of Entosphenus larvae and and an update of the paired species concept. Pages 71–114 in L. R. Brown, at least 97.3% of Lampetra larvae. Notably, although the S. D. Chase, M. G. Mesa, R. J. Beamish, and P. B. Moyle, editors. Entosphenus larvae examined by Goodman et al. (2009) Biology, management, and conservation of lampreys in North America. were as small as 60 mm TL, none of the Lampetra larvae in American Fisheries Society, Symposium 72, Bethesda, Maryland. that study was smaller than 70 mm TL. Collectively, our Docker, M. F., J. H. Youson, R. J. Beamish, and R. H. Devlin. 1999. fi Phylogeny of the lamprey genus Lampetra inferred from mitochondrial results suggest that caudal n pigmentation characters should cytochrome b and ND3 gene sequences. Canadian Journal of Fisheries and not be used in 50-mm and smaller lampreys, should be used Aquatic Sciences 56:2340–2349. with caution in 51–70-mm individuals, and are very reliable Goodman, D. H., A. P. Kinziger, S. B. Reid, and M. F. Docker. 2009. for 71-mm and larger larvae. This technique takes minimal Morphological diagnosis of Entosphenus and Lampetra ammocoetes training and, with experience, it can be performed rapidly in (Petromyzontidae) in Washington, Oregon, and California. Pages 223–232 fi fi in L. R. Brown, S. D. Chase, M. G. Mesa, R. J. Beamish, and P. B. Moyle, the eld. However, for studies in which any misidenti cation editors. Biology, management, and conservation of lampreys in North based on morphology would be unacceptable—and especially America. American Fisheries Society, Symposium 72, Bethesda, Maryland. when the identification of 50-mm and smaller larvae is neces- Hayes, M. C., R. Hays, S. P. Rubin, D. M. Chase, M. Hallock, C. Cook-Tabor, sary—the PCR-based genetic assay described and tested here C. W. Luzier, and M. L. Moser. 2013. Distribution of Pacific Lamprey offers the simplest, most reliable method to date for distin- Entosphenus tridentatus in watersheds of Puget Sound based on smolt monitoring data. Northwest Science 87:95–105. guishing these two widespread North American lamprey Hess, J. E., N. R. Campbell, M. F. Docker, C. Baker, A. Jackson, R. Lampman, genera. B. McIlraith, M. L. Moser, D. P. Statler, W. P. Young, A. J. Wildbill, and S. As an additional point of interest, the Etr-1 and Etr-6 R. Narum. 2015. Use of genotyping by sequencing data to develop a high- assays both placed the Kern Brook Lamprey into the genus throughput and multifunctional SNP panel for conservation applications in fi – Lampetra (reviewed by Docker et al. 1999). The Kern Brook Paci c Lamprey. Molecular Ecology Resources 15:187 202. fi Jolley, J. C., G. S. Silver, J. J. Skalicky, and T. A. Whitesel. 2014. Evaluation Lamprey was originally classi ed as belonging to the genus of larval Pacific Lamprey rearing in mainstem areas of the Columbia and Entosphenus based on morphology (Vladykov and Kott 1976), Snake rivers impacted by dams. U.S. Fish and Wildlife Service, 2013 but subsequent mitochondrial (cytochrome-b) DNA sequence Annual Report, Vancouver, Washington. data suggested that this species was better classified as Jolley, J. C., G. S. Silver, and T. A. Whitesel. 2012. Occurrence, detection, and Lampetra (Docker et al. 1999; Lang et al. 2009; Boguski habitat use of larval lamprey in Columbia River mainstem environments: fi Bonneville tailwater and tributary mouths. U.S. Fish and Wildlife Service, et al. 2012). Lampetra hubbsi has now been recon rmed by 2011 Annual Report, Vancouver, Washington. the American Fisheries Society as the official scientific name Jolley, J. C., G. S. Silver, and T. A. Whitesel. 2013. Occurrence, detection, and for the Kern Brook Lamprey (Page et al. 2013), but more habitat use of larval lamprey in the lower White Salmon River and mouth: post- MANAGEMENT BRIEF 787

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