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.     

Lethal temperatures of ammocoetes of the Southern Hemisphere lamprey,Geotria australis Gray

Synopsis The resistance time of ammocoetes ofGeotria australis was related to the time of emergence from the substrate. The incipient lethal temperatures of animals acclimated at 5, 15 and 25° C were 27.3, 27.5 and 28.2° C respectively, the ultimate incipient lethal being 28.3° C. This value, which is similar to that of Northern Hemisphere lampreys such asLampetra planeri, almost certainly accounts for the restriction of this species to the southernmost rivers in Western Australia.     

Genetic relationships of brook lamprey of the genus <Emphasis Type="Italic">Lampetra</Emphasis> in a Pyrenean stream in Spain

The genetic relationships of a population of brook lamprey Lampetra planeri from Spain were analysed using a fragment of the mitochondrial non-coding region in order to describe its relationships with other European Lampetra populations. DNA sequences were obtained from ten larvae, all corresponding to a single private haplotype. Its closest haplotype differed by five mutations and was found in several samples of Lampetra fluviatlis, both from the North Sea and the Baltic. These results argue for the great conservation value of this brook lamprey population, likely originated from L. fluviatilis ancestors.     

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.     

Lethal Temperatures in Ammocoetes of Four Species of Lampreys

Abstract An investigation has been made of the resistance time and upper lethal temperature of ammocoetes of four species of lampreys provided with a substrate into which they could readily burrow. In general, ammocoetes burrowed after transfer from the acclimation to the experimental temperature baths and later came out of the substrate only in lethal temperatures. A relationship was observed between the resistance time and the time taken to emerge, with the resistance time increasing exponentially with decreasing experimental temperature. In Ichthyomyzon fossor, landlocked Petromyzon marinus, Lampetra (Lethenteron) Lamottenii and in Lampetra (Lampetra) planeri from two different times of the year, the incipient lethal levels over a two week experimental period for larvae acclimated to 15° G were respectively 30.5, 30, 29.5, 28.5 and 28° C. Values for P. marinus acclimated to 5 and 25° C were respectively 29.5 and 31° C, whereas in L. planeri they were 28 and 29° C in April/May and 27 and 29° C in July/August. Extrapolation of the results for the three acclimation temperatures yielded ultimate incipient lethal levels of 31.4° G in P. marinus and 29.2 and 29.4° C for L. planeri examined in the spring and summer respectively.     

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.     

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.     

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.     

The ecology of lampreys (Petromyzonidae) in the Loch Lomond area

All three lamprey species occurring in western Europe are found in the Loch Lomond area: Sea Lamprey Petromyzon marinus L., River Lamprey Lampetra fluviatilis (L.) and Brook Lamprey Lampetra planeri (Bloch). There are major differences in ecology among the three species and in each some features seem to be unique to the Lomond populations. Available data on the ecology of the three species in the area are presented and existing threats reviewed. Future research and conservation requirements are outlined.     

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.     

Different stocks of brook lamprey in Spain and their origin from Lampetra fluviatilis at two distinct times and places

Using the mitochondrial non‐coding region I, it was shown that the two Spanish Lampetra planeri populations (Cares–Deva and Olabidea–Ugarana) correspond to different genetic units. The Cares–Deva population is probably a recent offshoot of Lampetra fluviatilis, being the Olabidea–Ugarana population less diverse and of older origin.     

Quantitative studies on the skin of the paired species of lampreys,Lampetra fluviatilis (L.) and Lampetra planeri (BLOCH)

The number of mucous, club, and granular cells in the epidermis, and the number of rows of subcutaneous adipose cells, as well as the thickness of the epidermis and the dermal collagen layer, have been recorded for the larval and metamorphosing stages of the anadromous parasitic lamprey, Lampetra fluviatilis, and for the larval, metamorphosing, and adult stages of the nonparasitic lamprey, Lampetra planeri. In L. fluviatilis, the mucous cells predominated in all stages but were more abundant in fully metamorphosed individuals than in larvae. During metamorphosis, the number of granular cells increased continuously, whereas the club cells showed little change. Although lampreys do not feed during metamorphosis, there was an increase in the thickness of the epidermis and in the dermal collagen sheath; the latter increase probably foreshadows the increase in activity by the adults. Simultaneously, there is a reduction in the subcutaneous fat layer, which can be attributed to mobilization of lipid as an energy source. Changes similar to those just described for L. fluviatilis were also found in metamorphosing L. planeri. However, the pattern altered markedly during adult stages in this nonparasitic species. There were marked declines in the number of cells, in the thickness of the epidermis, in the width of the collagen sheath, and in the quantity of subcutaneous fat.     

Ultrastructure of the presumed ion-transporting cells in the gills of ammocoete lampreys,Lampetra fluviatilis (L.) and Lampetra planeri (Bloch)

Summary Mitochondria-rich cells were located in the interplatelet area of gill filaments from ammocoete Lampetra fluviatilis and L. planeri. The ultrastructure of this cell type differs from typical teleost chloride cells by the absence of a tubular, smooth endoplasmic reticulum (SER). This difference is discussed in relation to the presumed functions of the cell and to the evolutionary histories of lampreys and teleosts. It is concluded that the mitochondria-rich cell is responsible for the active uptake of ions by the ammocoete gill.     

The reticular formation of lampreys (Petromyzonidae) — A target area for exohypothalamic vasotocinergic fibres

Summary The octapeptide vasotocin, which is formed in the classical neurosecretory nuclei of lampreys (Petromyzonidae), is transported, bound to the carrier protein neurophysin, not only to the neurohypophysis but also to various other regions of the brain via exohypothalamic fibres. A target area of this exohypothalamic vasotocinergic system is, in the brook lamprey (Lampetra planeri Bloch), a relatively well circumscribed area in the isthmus region of the rhombencephalic tegmentum motoricum, which is called area lateralis tegmenti. In this area, which belongs to the reticular formation, the vasotocinergic fibres form synaptic contacts with nerve cell perikarya and processes. The vesicles contained in the fibres were identified, ultrahistochemically, as neurophysin vesicles. They correspond to the neurophysin vesicles observed in the neurohypophysis of the same species. The functional significance of the vasotocinergic supply to portions of the reticular formation in lampreys is open to discussion.Supported by grants from the Ministry for Science and Technology of the German Democratic Republic. Acknowledgements: For skilled technical assistance the authors are indebted to their colleagues Mrs. S. Mehnert, E. Siebert, C. Schneider, and I. Seifert.     

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.     

Riverscape genetics in brook lamprey: genetic diversity is less influenced by river fragmentation than by gene flow with the anadromous ecotype

Understanding the effect of human-induced landscape fragmentation on gene flow and evolutionary potential of wild populations has become a major concern. Here, we investigated the effect of riverscape fragmentation on patterns of genetic diversity in the freshwater resident European brook lamprey (Lampetra planeri) that has a low ability to pass obstacles to migration. We tested the hypotheses of (i) asymmetric gene flow following water current and (ii) an effect of gene flow with the closely related anadromous river lamprey (L. fluviatilis) ecotype on L. planeri genetic diversity. We genotyped 2472 individuals, including 225 L. fluviatilis, sampled from 81 sites upstream and downstream barriers to migration, in 29 western European rivers. Linear modelling revealed a strong positive relationship between genetic diversity and the distance from the river source, consistent with expected patterns of decreased gene flow into upstream populations. However, the presence of anthropogenic barriers had a moderate effect on spatial genetic structure. Accordingly, we found evidence for downstream-directed gene flow, supporting the hypothesis that barriers do not limit dispersal mediated by water flow. Downstream L. planeri populations in sympatry with L. fluviatilis displayed consistently higher genetic diversity. We conclude that genetic drift and slight downstream gene flow drive the genetic make-up of upstream L. planeri populations whereas gene flow between ecotypes maintains higher levels of genetic diversity in L. planeri populations sympatric with L. fluviatilis. We discuss the implications of these results for the design of conservation strategies of lamprey, and other freshwater organisms with several ecotypes, in fragmented dendritic river networks.Subject terms: Conservation biology, Ecological genetics, Evolutionary genetics, Genetic variation     

The systematics of British larval lampreys

Several taxonomic characters, including the number of trunk myomeres, proportional body measurements and patterns of external and internal pigmentation, have been investigated in populations of ammocoetes belonging to the three species of lampreys found in British rivers. Petromyzon marinus can be readily distinguished by the pigmentary pattern in the tail and on the oral hood, and by the much higher number of trunk myomeres. Although no character can be used to separate with certainty the larvae of Lampetra fluviatilis and Lampetra planeri , significant differences were found between the mean number of trunk myomeres in the two species. Moreover, the values for ammocoetes were virtually identical to those of the respective post-larval stages, at which phase in the life cycle the two Lampetra species are clearly distinguishable. The incidence of pigmentation on the tongue precursor was much higher in larval populations of L. fluviatilis than in those of L. planeri and there was also a difference in the rate of change in the relative length of the trunk and tail. Where comparisons can be made, the results in this paper are in agreement with those of most other European and North American workers. The data differ markedly, however, from that contained in the only other previous detailed systematic study on British larval lampreys, which was subsequently used as the basis for taxonomic keys.     

Gill Morphometrics of the Lampreys Lampetra fluviatilis (L.) and Lampetra planeri (Bloch)

Morphometric measurements have been made on various gill components of different stages in the life cycle of the anadromous parasitic lamprey, Lampetra fluviatilis, and its nonparasitic derivative Lampetra planeri. The total gill area, expressed in terms of body weight, of both larval (1462–2717 mm² g^–1) and adult (1402–2337 mm² g^–1) L. fluviatilis are greater than those previously recorded in the rather meagre literature on lamprey gill measurements and are comparable with those found in the most active teleosts. The gills of the two Lampetra species are apparently identical in the larval stages and those of metamorphosing and adult L. planeri are similar to those of metamorphosing L. fluviatilis. Although the pharyngeal arrangement of lampreys differs greatly from that of teleosts, there are many features of the gills indicative of convergence between the two groups. Thus, in a given stage in the life cycle of lampreys, the secondary lamellae on either side of the filaments also alternate, become more widely spaced as the filament length increases and increase in area as the body weight becomes greater. Furthermore, the fractional cumulative increase in secondary lamellae area along a line following the presumed direction of water flow is also represented by a sigmoid curve. While at metamorphosis the pharynx becomes considerably modified to accommodate the change from a unidirectional to a tidal respiratory water flow, the total gill areas of the ammocoete are similar to those of metamorphosing stages which have attained adult characteristics. However, there are clearly differences in some of the components that influence and contribute towards the total gill area. Thus, in terms of body weight, the number and total length of the filaments and the total number of secondary lamellae, together with the number of secondary lamellae found on a given distance of filament, are greater in late metamorphosing stages, while the reverse is true for the average bilateral area of the secondary lamellae which is considerably greater in ammocoetes.     

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.     

European Lampreys: New Insights on Postglacial Colonization,Gene Flow and Speciation

Ice ages are known to be the most dominant palaeoclimatic feature occurring on Earth, producing severe climatic oscillations and consequently shaping the distribution and the population structure of several species. Lampreys constitute excellent models to study the colonization of freshwater systems, as they commonly appear in pairs of closely related species of anadromous versus freshwater resident adults, thus having the ability to colonize new habitats, through the anadromous species, and establish freshwater resident derivates. We used 10 microsatellite loci to investigate the spatial structure, patterns of gene flow and migration routes of Lampetra populations in Europe. We sampled 11 populations including the migratory L. fluviatilis and four resident species, L. planeri, L. alavariensis, L. auremensis and L. lusitanica, the last three endemic to the Iberian Peninsula. In this southern glacial refugium almost all sampled populations represent a distinct genetic cluster, showing high levels of allopatric differentiation, reflecting long periods of isolation. As result of their more recent common ancestor, populations from northern Europe are less divergent among them, they are represented by fewer genetic clusters, and there is evidence of strong recent gene flow among populations. These previously glaciated areas from northern Europe may have been colonized from lampreys expanding out of the Iberian refugia. The pair L. fluviatilis/L. planeri is apparently at different stages of speciation in different locations, showing evidences of high reproductive isolation in the southern refugium, and low differentiation in the north.