Mitochondrial DNA variation of an isolated population of the Adriatic brook lamprey Lampetra zanandreai (Agnatha: Petromyzontidae): phylogeographic and phylogenetic inferences

Two mitochondrial genes were examined to compare an isolated population of the Adriatic brook lamprey Lampetra zanandreai in central Italy with other populations in the species range (Po plain) and with parasitic and freshwater lampreys. A single haplotype, identical to one in a Venetian sample, was found in 10 individuals from the isolated population. The reduced variability is consistent with a history of dispersal after the Pleistocene expansion of the Po basin. The results support the hypothesis of an origin of L. zanandreai and L. fluviatilis–L. planeri from a common anadromous ancestor.     

Genetic diversity,endemism and phylogeny of lampreys within the genus Lampetra sensu stricto (Petromyzontiformes: Petromyzontidae) in western North America

Phylogenetic structure of four Lampetra species from the Pacific drainage of North America (western brook lamprey Lampetra richardsoni, Pacific brook lamprey Lampetra pacifica, river lamprey Lampetra ayresii and Kern brook lamprey Lampetra hubbsi) and unidentified Lampetra specimens (referred to as Lampetra sp.) from 36 locations was estimated using the mitochondrial cytochrome b gene. Maximum parsimony and Bayesian inferences did not correspond with any taxonomic scheme proposed to date. Rather, although L. richardsoni (from Alaska to California) and L. ayresii (from British Columbia to California) together constituted a well‐supported clade distinct from several genetically divergent Lampetra populations in Oregon and California, these two species were not reciprocally monophyletic. The genetically divergent populations included L. pacifica (from the Columbia River basin) and L. hubbsi (from the Kern River basin) and four Lampetra sp. populations in Oregon (Siuslaw River and Fourmile Creek) and California (Kelsey and Mark West Creeks). These four Lampetra sp. populations showed genetic divergence between 2·3 and 5·7% from any known species (and up to 8·0% from each other), and may represent morphologically cryptic and thus previously undescribed species. A fifth population (from Paynes Creek, California) may represent a range extension of L. hubbsi into the Upper Sacramento River.     

Morphological abnormalities in a population of Lampetra planeri,with a short review of petromyzontid teratologies

Reported here are several deviations (n = 15; 1·96%) from typical morphology in a large sample (n = 767) of European brook lamprey Lampetra planeri from a single population in the Loch Lomond catchment; this includes one specimen bearing a true anal fin. A brief review of petromyzontid teratology is provided.     

Isolation and characterization of eight microsatellite loci in the brook lamprey Lampetra planeri (Petromyzontiformes) using 454 sequence data

Eight polymorphic microsatellite loci were developed for the brook lamprey Lampetra planeri through 454 sequencing and their usefulness was tested in 45 individuals of both L. planeri and the river lamprey Lampetra fluviatilis. The number of alleles per loci ranged between two and five; the Italian and Irish populations had a mean expected heterozygosity of 0·388 and 0·424 and a mean observed heterozygosity of 0·418 and 0·411, respectively.     

Phylogeographical analysis reveals multiple conservation units in brook lampreys Lampetra planeri of Portuguese streams

The populations of brook lamprey Lampetra planeri of Portuguese Rivers were analysed phylogeographically using a fragment of 644 bp of the mitochondrial control region of 158 individuals from six populations. Samples representing L. planeri and migratory lampreys Lampetra fluviatilis of rivers draining to the North Sea and the Baltic Sea were also included to assess the relationships of Portuguese samples. The data support a clear differentiation of all the populations studied. Several populations, which are isolated among themselves and also from the migratory lampreys, proved to be entirely composed of private haplotypes, a finding that supports some time of independent evolutionary history for these populations. This, combined with the geographic confinement to small water bodies, justifies the recognition of at least four conservation units in the Portuguese rivers Sado, São Pedro, Nabão and Inha.     

Phylogeography of the European brook lamprey (Lampetra planeri) and the European river lamprey (Lampetra fluviatilis) species pair based on mitochondrial data

The European river lamprey Lampetra fluviatilis and the European brook lamprey Lampetra planeri (Block 1784) are classified as a paired species, characterized by notably different life histories but morphological similarities. Previous work has further shown limited genetic differentiation between these two species at the mitochondrial DNA level. Here, we expand on this previous work, which focused on lamprey species from the Iberian Peninsula in the south and mainland Europe in the north, by sequencing three mitochondrial marker regions of Lampetra individuals from five river systems in Ireland and five in southern Italy. Our results corroborate the previously identified pattern of genetic diversity for the species pair. We also show significant genetic differentiation between Irish and mainland European lamprey populations, suggesting another ichthyogeographic district distinct from those previously defined. Finally, our results stress the importance of southern Italian L. planeri populations, which maintain several private alleles and notable genetic diversity.     

HPLC and ELISA analyses of larval bile acids from Pacific and western brook lampreys

Comparative studies were performed on two native lamprey species, Pacific lamprey (Lampetra tridentata) and western brook lamprey (Lampetra richardsoni) from the Pacific coast along with sea lamprey (Petromyzon marinus) from the Great Lakes, to investigate their bile acid production and release. HPLC and ELISA analyses of the gall bladders and liver extract revealed that the major bile acid compound from Pacific and western brook larval lampreys was petromyzonol sulfate (PZS), previously identified as a migratory pheromone in larval sea lamprey. An ELISA for PZS has been developed in a working range of 20 pg-10 ng per well. The tissue concentrations of PZS in gall bladder were 127.40, 145.86, and 276.96 micro g/g body mass in sea lamprey, Pacific lamprey, and western brook lamprey, respectively. Releasing rates for PZS in the three species were measured using ELISA to find that western brook and sea lamprey released PZS 20 times higher than Pacific lamprey did. Further studies are required to determine whether PZS is a chemical cue in Pacific and western brook lampreys.     

Spawning ecology of the American brook lamprey, <Emphasis Type="Italic">Lampetra appendix</Emphasis>

Synopsis We observed spawning American brook lamprey, Lampetra appendix, in coldwater streams in Minnesota to assess various aspects of their spawning behavior and spawning habitat requirements. Spawning occurred during April and May, at water temperatures ranging from 8.7 to 15.5°C. Average adult lamprey length and mass differed significantly among streams, but there were no significant differences in length or mass between males and females. Overall sex ratio was 1:1, although one stream had significantly more males than females and one stream held significantly more females. Lampreys spawned in groups of 2–14 individuals, averaging 4.2 adults per nest across all streams. Nests were constructed in gravel and cobble substrate just upstream of riffles, spaced at an average density of three nests m⁻². The typical nest was 16 cm in diameter in water 31 cm deep with a bottom current velocity of 14 cm s⁻¹, and was excavated to a depth of 4 cm below the stream bottom; however, some nest characteristics varied significantly in a few streams. Nests were larger in streams with larger spawning groups, deeper water, and slower current velocities. American brook lamprey exhibited spawning behaviors and spawning habitat requirements similar to those of other species of lamprey in North America.     

Enzyme variation in the brook lamprey,Lampetra planeri (Bloch), a member of the vertebrate group Agnatha

A natural population of the brook lamprey, Lampetra planeri, was assayed for electrophoretically detectable variation at 30 enzyme loci. The mean heterozygosity per locus of this primitive vertebrate, a member of the Agnatha, was found to be 0.076 ± 0.031, a value similar to those recorded for other vertebrates. The high chromosome numbers recorded for this and related species have been attributed to polyploidy, but our studies do not indicate the existence of large numbers of duplicated loci. Indeed, several enzymes that are encoded by duplicate loci in other vertebrate species appear to be encoded by single loci in the lamprey. It is suggested that studies on the biology and taxonomy of lampreys will benefit greatly from an electrophoretic approach.     

Characterization of 12 microsatellite loci for the Pacific lamprey, Entosphenus tridentatus (Petromyzontidae), and cross-amplification in five other lamprey species

The Pacific lamprey (Entosphenus tridentatus) is an anadromous fish that is of conservation concern in North America and Asia. Data on Pacific lamprey population structure are scarce and conflicting, impeding conservation efforts. We optimized 12 polymorphic microsatellite loci for the Pacific lamprey. Three to 13 alleles per locus were observed in a sample of 51 fish collected from the West Fork Illinois River, Oregon. Observed heterozygosity ranged from 0.235 to 0.902 and expected heterozygosity ranged from 0.214 to 0.750. Cross-species amplification produced 8 to 12 polymorphic loci in four other Entosphenus species and in the western brook lamprey (Lampetra richardsoni). Two loci appear to be diagnostic for distinguishing Entosphenus from Lampetra. These markers will be valuable for evaluating population structure and making conservation decisions for E. tridentatus and other lamprey species.     

Variations in the presence of chloride cells in the gills of lampreys (Petromyzontiformes) and their evolutionary implications

Although confined to fresh water, non‐parasitic species of lampreys and the landlocked parasitic sea lamprey, all of which were derived relatively recently from anadromous ancestors, still develop chloride cells, whose function in their ancestors was for osmoregulation in marine waters during the adult parasitic phase. In contrast, such cells are not developed by the non‐parasitic least brook lamprey Lampetra aepyptera, which has been separated from its ancestor for >2 million years, nor by the freshwater parasitic species of the genus Ichthyomyzon. The length of time that a non‐parasitic species or landlocked parasitic form or species has spent in fresh water is thus considered the overriding factor determining whether chloride cells are developed by those lampreys.     

Low reproductive isolation and highly variable levels of gene flow reveal limited progress towards speciation between European river and brook lampreys

Ecologically based divergent selection is a factor that could drive reproductive isolation even in the presence of gene flow. Population pairs arrayed along a continuum of divergence provide a good opportunity to address this issue. Here, we used a combination of mating trials, experimental crosses and population genetic analyses to investigate the evolution of reproductive isolation between two closely related species of lampreys with distinct life histories. We used microsatellite markers to genotype over 1000 individuals of the migratory parasitic river lamprey (Lampetra fluviatilis) and freshwater‐resident nonparasitic brook lamprey (Lampetra planeri) distributed in 10 sympatric and parapatric population pairs in France. Mating trials, parentage analyses and artificial fertilizations demonstrated a low level of reproductive isolation between species even though size‐assortative mating may contribute to isolation. Most parapatric population pairs were strongly differentiated due to the joint effects of geographic distance and barriers to migration. In contrast, we found variable levels of gene flow between sympatric populations ranging from panmixia to moderate differentiation, which indicates a gradient of divergence with some population pairs that may correspond to alternative morphs or ecotypes of a single species and others that remain partially isolated. Ecologically based divergent selection may explain these variable levels of divergence among sympatric population pairs, but incomplete genome swamping following secondary contact could have also played a role. Overall, this study illustrates how highly differentiated phenotypes can be maintained despite high levels of gene flow that limit the progress towards speciation.     

Alternative migration and host parasitism strategies and their long-term stability in river lampreys from the River Endrick, Scotland

The stability of a discrete body size dimorphism of sexually mature river lamprey Lampetra fluviatilis from the River Endrick, Scotland, was examined over a 21 year period. Stable isotope analysis was used to test the hypothesis that the two size forms comprise individuals with differing migration and parasitic foraging strategies. Maturing river lamprey and the brook lamprey Lampetra planeri were trapped over 3 months each year in the periods 1983–1984 and 2004–2005. Brook lamprey catches and catches of both species combined showed no significant trend in catch rate with time. The catch rate of small body size river lamprey declined between 1983–1984 and 2004–2005 (although the difference did not reach statistical significance; P = 0·055). In contrast, there was a significant increase in the catch rate of the large body size river lamprey and as a consequence, a significant change in the relative proportion of each of the two river lamprey morphs over the study period. Analysis of the stable isotopes of C and N in muscle tissue showed that brook lamprey tissue derived its carbon from a freshwater source and had a δ¹³C more consistent with that of the River Endrick than with Loch Lomond. δ¹⁵N values for this species showed it to be feeding at the base of the food chain, consistent with filter feeding as an ammocoete. The large body size and the small body size river lamprey adults differed substantially in their δ¹³C values, with the small body size δ¹³C signature indicative of a freshwater carbon source and the large body size morph of a marine source. The small body size morph had a δ¹³C signature that was consistent with that of Loch Lomond powan Coregonus lavaretus suggesting that they share a common carbon source. The large body size morph was clearly feeding at a higher trophic level than the small body size morph. A single small body size river lamprey individual with typical morphology for that group, however, had C and N signatures that clustered with those of the large body size morphs. This individual had either migrated to sea to forage, as is typical for the species, or had been feeding on an anadromous fish with a strong marine C signature in fresh water. It is concluded that the body size dimorphism is indicative of a differential migration and foraging strategy in the parasitic phase of the life cycle of river lamprey at this site.     

Contrasting population genetic structure among freshwater‐resident and anadromous lampreys: the role of demographic history,differential dispersal and anthropogenic barriers to movement

The tendency of many species to abandon migration remains a poorly understood aspect of evolutionary biology that may play an important role in promoting species radiation by both allopatric and sympatric mechanisms. Anadromy inherently offers an opportunity for the colonization of freshwater environments, and the shift from an anadromous to a wholly freshwater life history has occurred in many families of fishes. Freshwater‐resident forms have arisen repeatedly among lampreys (within the Petromyzontidae and Mordaciidae), and there has been much debate as to whether anadromous lampreys, and their derived freshwater‐resident analogues, constitute distinct species or are divergent ecotypes of polymorphic species. Samples of 543 European river lamprey Lampetra fluviatilis (mostly from anadromous populations) and freshwater European brook lamprey Lampetra planeri from across 18 sites, primarily in the British Isles, were investigated for 13 polymorphic microsatellite DNA loci, and 108 samples from six of these sites were sequenced for 829 bp of mitochondrial DNA (mtDNA). We found contrasting patterns of population structure for mtDNA and microsatellite DNA markers, such that low diversity and little structure were seen for all populations for mtDNA (consistent with a recent founder expansion event), while fine‐scale structuring was evident for nuclear markers. Strong differentiation for microsatellite DNA loci was seen among freshwater‐resident L. planeri populations and between L. fluviatilis and L. planeri in most cases, but little structure was evident among anadromous L. fluviatilis populations. We conclude that postglacial colonization founded multiple freshwater‐resident populations with strong habitat fidelity and limited dispersal tendencies that became highly differentiated, a pattern that was likely intensified by anthropogenic barriers.     

Allozyme Polymorphisms and Biochemical-Genetic Taxon Markers in Lampreys (Lampetra, Eudontomyzon, Petromyzon)

Variation at 23 putative enzyme-coding loci was scored in 424 lampreys, including 321 European brook lampreys (Lampetra planeri), 83 European river lampreys (L. fluviatilis), 11 Ukrainian brook lampreys (Eudontomyzon mariae), and nine sea lampreys (Petromyzon marinus). Twelve polymorphisms are described for Lampetra species (LDH*, SOD-2*, PNP*, AAT-1*, AK-1*, ES-2*, LGL*, MPI*, GPI-1*, GPI-2*, PGM*, IDHP-2*), and two each for E. mariae (GPI-1*, ME-2*) and P. marinus (MDH-1*, ME-2*). Diagnostic allozymes are presented for the discrimination of lamprey taxa, some of which are difficult to recognize from the morphology of ammocoetes larvae, the life stage usually encountered when collecting cyclostomes. The allelic markers described permit the clear allocation to a genus, except for the species L. fluviatilis and L. planeri, which are not differentiated by qualitative allozyme analysis.     

Selection and preference of benthic habitat by small and large ammocoetes of the least brook lamprey (<Emphasis Type="Italic">Lampetra aepyptera</Emphasis>)

In this laboratory study, we quantified substrate selection by small (<50 mm) and large (100–150 mm) ammocoetes of the least brook lamprey (Lampetra aepyptera). In aquaria, ammocoetes were given a choice to burrow into six equally-available substrate types: small gravel (2.360–4.750 mm), coarse sand (0.500–1.400 mm), fine sand (0.125–0.500 mm), organic substrate (approximately 70% decomposing leaves/stems and organic sediment particles, and 30% silt and fine sand), an even mixture of silt, clay, and fine sand, and silt/clay (<0.063 mm). Fine sand was selected with a significantly higher probability than any other substrate. Fine sand habitat is limited in many streams, in part owing to geology, but also as a result of channelization and excessive silt/clay sedimentation, which is a conservation concern. Our results indicate that ammocoetes of least brook lampreys are habitat specialists that prefer fine sand habitat. Hence, availability of fine sand habitat may limit distributions and population sizes.     

Habitat selection of larval brook lampreys (Lampetra planeri,Bloch) in a South Swedish stream

Summary The burrowing larvae of the brook lamprey Lampetra planeri (Bloch) were shown by discriminant analysis to inhabit stream sites having certain qualities. Although a few larvae occurred in far-from-optimal habitats, most larvae selected those with low current velocity, low water depth, a low number of particles in the 0.5–1 mm range, and with low chlorophyll content. Other factors normally positively attributed to larval lamprey distribution such as organic content, shade, and presence of algae did not improve the discriminant model. The last factor even showed a negative correlation. Stream bed stability was considered to be of great importance although it was not possible to estimate quantitatively.     

Using stable isotopes and C:N ratios to examine the life‐history strategies and nutritional sources of larval lampreys

Natural abundance stable‐isotope analysis (δ¹³C and δ¹⁵N) and C:N ratios were used to study the ammocoete phase of two common non‐parasitic lamprey species (least brook lamprey Lampetra aepyptera and American brook lamprey Lethenteron appendix) in two tributaries of the Ohio River (U.S.A.). The C:N ratios suggest that each species employs different lipid accumulation strategies to support its metamorphosis and recruitment into an adult animal. Ammocoete δ¹³C values generally increased with increasing C:N values. In contrast to δ¹³C, ammocoete δ¹⁵N values were weakly related to the total length (L_T) in L. aepyptera, but positively correlated to both L_T and C:N ratios in L. appendix. In L. appendix, C:N also correlated positively with L_T, and presumably age. A Bayesian mixing model using δ¹³C and δ¹⁵N was used to estimate nutritional subsidies of different potential food resources to ammocoetes at each site. The models suggested that although nutritional subsidies to ammocoetes varied as a function of site, ammocoetes were generally reliant on large contributions (42–62% at three sites) from aquatic plants. Contributions from aquatic sediment organic matter were also important at all sites (32–63%) for ammocoetes, with terrestrially derived plant materials contributing smaller amounts (4‐33%). These findings provide important insights into the feeding ecology and nutrition of two species of lampreys. They also suggest that similar and other quantitative approaches are required to (1) fully understand how the observed stable‐isotopes ratios are established in ammocoetes and (2) better assess ammocoete nutritional subsidies in different natal streams.     

Disturbance of the indigenous gene pool of the threatened brook lamprey Lethenteron sp. S by intraspecific introgression and habitat fragmentation

Artificial introduction and habitat fragmentation affect the indigenous gene pools of fluvial animals. To investigate the effect of human activities on the genetic population structure of vulnerable brook lamprey Lethenteron sp. S in a single river system, samples from 12 tributaries of the Jinzu River, Japan, were analyzed using mitochondrial and microsatellite DNA markers. Exogenous Lake Biwa (Japan) haplotypes and alleles were detected in lampreys from several Jinzu River tributaries. Since Lake Biwa is the source of the commercial ayu fish Plecoglossus altivelis that is introduced in the Jinzu River, the exogenous Lake Biwa lamprey genotypes in the Jinzu River probably originated from the Lake Biwa lampreys that were unintentionally introduced along with the ayu fish. Bayesian admixture and mitochondrial DNA analyses revealed various genetic disturbance phases of the exogenous genotypes in the Jinzu River, such as the six indigenous populations, four admixed populations with low frequencies (average admixture proportion = 0.02–0.04; exogenous haplotype proportion <0.01), one introgressed population (0.71 and 0.57) and one population almost displaced by exogenous genotypes (0.93 and 0.96). Samples from three tributaries with weirs were genetically differentiated from the others by using pairwise F _ST and Bayesian analyses; the results suggested isolation by the weirs. Reduced mitochondrial DNA diversity was observed in 1 of the 3 samples probably due to reduced population size. These findings indicate that the indigenous lamprey populations in the Jinzu River are seriously affected by introgression with exogenous genotypes via unintentional introduction and habitat fragmentation by weirs.     

Contemporary gene flow between “paired” silver (<Emphasis Type="Italic">Ichthyomyzon unicuspis</Emphasis>) and northern brook (<Emphasis Type="Italic">I. fossor</Emphasis>) lampreys: implications for conservation

In most lamprey genera, “paired” species exist in which the larvae are morphologically similar or indistinguishable but, following metamorphosis, one species becomes parasitic while the other bypasses the adult feeding phase and rapidly becomes sexually mature. Since DNA barcoding and similar studies using short segments of the mitochondrial genome do not provide sufficient resolution to distinguish between recent divergence and a lack thereof, the current study examined the relationship between the parasitic silver lamprey (Ichthyomyzon unicuspis) and nonparasitic northern brook lamprey (I. fossor) using up to 10,230 bp from the mitochondrial genome to make phylogenetic inferences and mitochondrial restriction fragment length polymorphism (RFLP) and microsatellite markers to test for significant differences in allele frequencies. We found that silver and northern brook lampreys are not reciprocally monophyletic; two lineages were observed but each occurred within both species. There also were no significant range-wide differences in RFLP haplotype or microsatellite allele frequencies between the species, nor were there significant differences where they occurred sympatrically within the Lake Huron basin. Significant genetic differentiation was found only within the Lake Michigan basin where the results were potentially confounded by geographic separation. Our results thus support suggestions that silver and northern brook lampreys represent ecotypes of a single species since, where they are sympatric, they appear to be experiencing ongoing gene flow. However, alternative life history strategies can be important for a species’ long-term persistence, and critical data are needed to decide whether these two feeding types should be managed as a single unit.