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 Lamprey Genera Entosphenus and Lampetra: 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 Brook Lamprey 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 Kern Brook Lamprey 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).