A New Nonparasitic Species of the Holarctic Lamprey Genus Lethenteron Creaser and Hubbs, 1922, (Petromyzonidae) from Northwestern North America with Notes on Other Species of the Same Genus

A new nonparasitic lamprey, Lethenteron alaskense from Alaska and Northwest Territories is described and illustrated. The holotype (No. NMC 76-614) is deposited in the National Museum of Natural Sciences, Ottawa, Canada. The study was based on 67 metamorphosed specimens. The species, by its permanently non-functional intestinal tract and weak dentition, smaller disc and much smaller size (maximum 188 mm), is easily separable from the parasitic Lenthenteron japonicum (maximum length 625 mm) found in the same areas. It is distinguishable from nonparasitic L. lamottenii, found in eastern and southern North America, by 1) a generally weaker dentition but possessing more anterials and supplementary marginals; 2) typically with five velar tentacles as opposed to seven in L. lamottenii; 3) differences in pigmentation pattern of the second dorsal fin and a lack of dark pigmentation on the gular region; 4) smaller size in comparison to 299 mm maximum length in L. lamottenii; and 5) distinct areas of geographical distribution separated from each other by 2400 km. All three, L. alaskense, L. lamottenii, and L. japonicum have usually 66 to 72 trunk myomeres. L. alaskense, by its higher number of myomeres is separable from two other nonparasitic species: L. reissneri from Asia with less than 64 myomeres and L. meridionale from eastern tributaries of the Gulf of Mexico with 50 to 58 myomeres

Characterizing the diet and population structure of lampreys Lethenteron spp. using molecular techniques

Thesis (M.S.) University of Alaska Fairbanks, 2017Lampreys contribute to the health of aquatic ecosystems and are targeted in both subsistence and commercial fisheries. Despite their ecological and commercial importance, the management and conservation of native lampreys have been largely overlooked. The goal of this study was to close current knowledge gaps of lamprey biology through the examination of Lethenteron spp. in Alaska. This study applied two molecular techniques, DNA metabarcoding and microsatellite genotyping, to (1) characterize the diet of marine-phase Arctic lamprey Lethenteron camtschaticum (N = 250) in the eastern Bering Sea and (2) investigate the population structure of larval lampreys Lethenteron spp. (N = 120) within and among three Yukon River tributaries. A combination of visual observations and DNA metabarcoding revealed the presence of diagnostic structures/tissues (i.e., eggs, fin[s], internal organs, otoliths, and vertebrae) and detected DNA sequences of ten ray-finned fishes in the diets of L. camtschaticum. The most frequent prey taxa were Pacific sand lance Ammodytes hexapterus, Pacific herring Clupea pallasii, gadids, and capelin Mallotus villosus. Five of the ten taxa identified in this study were reported for the first time as prey for L. camtschaticum. To investigate the genetic diversity of larval lampreys, a recognized knowledge gap for populations in Alaska, a total of 81 larval lampreys were successfully genotyped at all loci. Global FST of larvae was 0.074 (95% CI: 0.042 - 0.110), while pairwise FST values among the three localities examined ranged from 0.066 - 0.081. Hierarchical model-based Bayesian clustering analyses detected three genetic clusters (K = 3) among all larval lampreys and two genetic clusters (K = 2) among Chena River larvae; no further genetic clustering was identified within the remaining two tributaries. Estimates of contemporary gene flow indicated reciprocal migration among sites. The diet analyses indicated anadromous L. camtschaticum function as flesh-feeding predators that prey upon pelagic fishes in the eastern Bering Sea, while genetic analyses suggested that larval lamprey aggregations within three Yukon River tributaries exhibited higher levels of genetic diversity than are typically found among broad-ranging populations of anadromous lamprey species. Ultimately, this study highlighted the value of molecular techniques to improve our understanding of the biology of a poorly studied fish species in Alaska.Chapter 1: Utilizing DNA metabarcoding to characterize the diet of marine-phase Arctic lamprey Lethenteron camtschaticum) in the eastern Bering Sea -- Chapter 2: Characterizing the genetic variation among larval populations of Lethenteron spp. within the Yukon River drainage, Alaska, using microsatellite markers -- General conclusions

Habitat selection and predation risk in larval lampreys

This thesis examines habitat preference and the influence of habitat on predation of larvae (ammocoetes) of the least brook lamprey (Lampetra aepyptera). The thesis comprises three chapters: (1) an introduction and literature review on the general life history of lampreys and on studies related to ammocoetes and their habitat, (2) an experimental study of habitat preference in ammocoetes of the least brook lamprey, and (3) an experimental study of the relationship between habitat availability and predation risk in ammocoetes. For the first study, we quantified substrate selection in small (\u3c 50 mm) and large (100-150 mm) ammocoetes of the least brook lamprey. In aquaria, ammocoetes were given a choice to burrow into six equally-available substrate types: small gravel (2.36-4.75 mm), coarse sand (0.5-1.4 mm), fine sand (0.125-0.5 mm), organic debris (approximately 70% decomposing leaves and stems, 15% silt, and 15% sand), an even mixture of silt, clay, and fine sand, and silt/clay (\u3c 0.063 mm). Fine sand was selected with a significantly higher probability than any other substrate. In the second study, we experimentally examined the influence of habitat availability on predation risk of ammocoetes. Ammocoetes were placed in aquaria containing a predator species (yellow bullhead, Ameiurus natalis) and one of 3 substrates: fine sand (0.125-0.5 mm), coarse sand (0.5-1.4 mm), or silt/clay (\u3c0.063 mm). Use of the three substrate types was based on a previous experiment where fine sand was determined to be the preferred benthic habitat of least brook lamprey. Based on 10 trials with each habitat type, survival of ammocoetes was highest in aquaria with fine sand (mean = 80%), and lower in those with coarse sand (mean = 58%) and silt/clay (mean = 4%). The results of both studies conducted indicate that populations of least brook lamprey ammocoetes may be limited by the availability of fine sand habitat. The first study indicated that least brook lamprey ammocoetes are habitat specialists, preferring substrates composed primarily of fine sand. The second study showed that the availability of fine sand habitat may influence the predation risk of ammocoetes, as ammocoete survival from predation was highest in fine sand, and lower in other substrates

Three new cryptic species of the lamprey genus Lampetra Bonnaterre, 1788 (Petromyzontiformes: Petromyzontidae) from the Iberian Peninsula.

The Iberian Peninsula is a repository for biodiversity, presenting high levels of endemism in both plants and animals. In this peninsular region, brook lampreys confined to small, isolated river basins evolved in allopatry giving rise to evolutionary lineages, as revealed by mitochondrial DNA markers. For a better understanding of the taxonomic status and relationships of Iberian populations of the genus Lampetra, we combined previous data from genetics and morphological analysis (assessed here), and describe three new species of the lamprey genus Lampetra Bonnaterre, 1788 in Portugal. In this region L. planeri actually represent a complex of cryptic species, each having smaller geographic ranges than L. planeri, and consequently, greater vulnerability to extinction. The description of Lampetra alavariensis sp. nov. is based on 36 specimens collected on Ribeira de Mangas, a tributary of river Esmoriz, in Northern Portugal. Lampetra auremensis sp. nov. is described on the basis of 31 specimens collected on Ribeira do Olival, a small tributary of river Nabão (Tagus basin). Finally, Lampetra lusitanica sp. nov. is described based on 38 specimens from Ribeira da Marateca, Sado river basin, the southernmost distribution of the genus Lampetra. The recognition of these new species will contribute to the conservation of these already imperilled taxa and will help prevent the extinction of three important evolutionary lineages

Novel Relationships among Lampreys (Petromyzontiformes) Revealed by a Taxonomically Comprehensive Molecular Data Set

The systematics of lampreys was investigated using complete mitochondrial cytochrome b sequences from all genera and nearly all recognized species. The families Geotriidae and Petromyzontidae are monophyletic, but the family Mordaciidae was resolved as two divergent lineages at the base of the tree. Within Petromyzontidae, the nonparasitic Lethenteron sp. S and Okkelbergia aepyptera were recognized as distinct lineages, Lethenteron morii and Lampetra zanandreai were moved to new genera, a sister species relationship was recovered between Caspiomyzon wagneri and Eudontomyzon hellenicus, and a clade was recovered inclusive of Entosphenus hubbsi and western North American Lampetra (L. ayresii and L. richardsoni). The placement of E. hellenicus as the sister species to C. wagneri reduces the number of genera comprised entirely of parasitic species to two, Geotria and Petromyzon. The recognition of distinct lineages for O. aepyptera and Lethenteron sp. S recognizes, for the first time, lineages comprised entirely of nonparasitic species. Apart from the results mentioned above, monophyly was supported for the multispecific genera Entosphenus, Eudontomyzon, Ichthyomyzon, Lampetra (restricted to European species), and Lethenteron. Intergeneric relationships within Petromyzontidae were poorly resolved, but separate clades inclusive of Entosphenus and Tetrapleurodon (subfamily Entospheninae) and one comprised of Eudontomyzon, Lampetra, and Okkelbergia were recovered

Ecological notes on an endemic freshwater lamprey, Lampetra zanandreai (Vladykov, 1955)

Lampetra zanandreai (Vladykov, 1955) is a non-parasitic, freshwater lamprey endemic to the ancient Po basin. A few, mostly very dated studies have investigated some aspect of the biology of this lamprey, but surprisingly, despite it being considered a threatened species, information on its ecology is practically absent. Specifically, information about habitat preferences is generic and qualitative. Since most of the life cycle is spent in the fossorial larval stage, which is also the only one in which organisms feed, information about ecological requirements of ammocoetes is essential for any conservation strategy. In this study we provide the first data about physical habitat preferences for lamprey ammocoetes by analyzing their presence within sampled hydromorphological units (HMUs), following the approach of habitat attribute description of the MesoHABSIM (MesoHABitat SImulation Model) methodology. To explore the relationship between lamprey presence and HMU characteristics, a random forest (RF) model was developed and tested using data collected in five stream reaches of the Po basin (NW Italy). The final parsimonious RF model performed well in terms of accuracy (95.2%) and true skill statistic (90.4%), allowing us to identify the most significant mesohabitat attributes for the considered species. Furthermore, in the Ghiandone River, where the highest density and number of individuals were found, a granulometric analysis of the riverbed material was carried out. Results showed that selected strains of sand and fine gravel, with low organic content, are preferred by ammocoetes. To our knowledge, this is the first study exploring the habitat preference of this endangered species, listed in Annex II of the European Habitats Directive

Population structure in anadromous lampreys: Patterns and processes

Population structure can reveal the diversity, gene flow, and dispersal of a species. This information can be used to make management decisions and reveal fundamental aspects of an organism’s biology. Distinct intrinsic (e.g., biological characteristics) and extrinsic (e.g., geographical and historical events, environment, human pressures) factors can influence population structure, with significant differences among species. However, detection of population structure in migratory lamprey species can be difficult to detect due to their lack of natal homing; this is particularly the case for anadromous lampreys, with their potential for wide dispersal at sea during their parasitic feeding stage. We review phenotypic and genetic markers, as well as the methods that have been used to assess population structure in lampreys, and discuss the relative strengths and limitations of each. Structure has been detected in several anadromous species using some of these methods, even without homing in these species, but we briefly contrast the weak population structure observed in anadromous species with the stronger structure observed in freshwater-resident lamprey species (particularly non-migratory brook lampreys). We relate lamprey population structure to species-specific ecological traits, such as juvenile dispersal tendencies, and provide case studies of six species. Delineation of appropriate management units in migratory lamprey species is important for conservation and management

The evolutionary ecology of lampreys (Petromyzontiformes)

Lampreys (Petromyzontiformes) are an ancient vertebrate group, comprising 40 currently recognised species that range throughout the Northern and Southern Hemispheres. Despite a conserved morphology, lampreys nevertheless express a diverse range of life history strategies. Unusually for vertebrates larval lampreys are filter-feeding organisms prior to undergoing an extensive anatomical reorganisation, and the adoption of either a parasitic or a non-parasitic adult life. Parasitic lampreys consume the flesh and blood of actinopterygian fishes, either in marine or freshwater environments, while non-parasitic lampreys do not feed following their metamorphosis from the larval form. Morphological and genetic similarities between pairs of parasitic and non-parasitic lampreys have led to taxonomic confusion regarding the specific status of many non-parasitic forms, and the suggestion that the loss of the trophic adult phenotype is the result of a single species capable of producing alternative life history strategies. In this thesis it is argued that at least some paired species of lampreys do not comprise two distinct evolutionary lineages; rather, that non-parasitic lampreys represent one extreme in a continuum of life history variation expressed by a parasitic species. Some lamprey species, such as the European river lamprey Lampetra fluviatilis, are morphologically variable, exhibiting divergent phenotypes in response to ecological pressures, such as alternative foraging environments. Loch Lomond, Scotland contains a population of L. fluviatilis that feeds exclusively in the lake and exhibits a reduced body size and an overall morphology distinct from the typical anadromous form. Its foraging strategy indicates that it may be capable of switching hosts in the face of declining numbers of a presumed favoured and formerly abundant host, suggesting a certain amount of plasticity in its trophic ecology that may have ensured its survival in this freshwater lake. This freshwater-resident form, as well as anadromous L. fluviatilis and the non-parasitic species L. planeri, were found to spawn in a single river system within the Loch Lomond basin, and this site is crucial for the continued presence of this life history variant in Loch Lomond. The appearance of sexually mature specimens of three discrete phenotypes in this river, each representing an alternative life history strategy that may, or may not, belong to a single species, provides a crucial opportunity to test the strength of assortative mating between lamprey species pairs. Within this system the strength of assortative mating was found to be weak, and points to the possibility that freshwater-resident L. fluviatilis are mitigating gene flow between large anadromous parasitic L. fluviatilis, and small, non-parasitic L. planeri. As well as weak behavioural isolation, inter-specific sneak male mating tactics were documented among these populations, and represents the first time this phenomenon has been observed between paired lamprey species. Such behaviour indicates a lack of species-specific cues acting between L. fluviatilis and L. planeri, and suggests that hybrid offspring could be common in some systems. Testing hybrid viability (survivorship) between Loch Lomond’s two L. fluviatilis life history strategies and the sympatric L. planeri revealed no post-zygotic barriers to gene flow, at least in the form of gamete incompatibility. Perhaps more convincingly though, when comparing traditional morphometrics and body shape variation, as well as mitochondrial DNA sequences, between L. fluviatilis expressing different foraging strategies with populations of L. planeri, no robust species specific differentiation was observed. In fact, species delimitation between L. fluviatilis and L. planeri appears to be related solely to overall body size, which is itself a function of life history strategy. However, life history strategy was not correlated with current species designation as relationships among mtDNA haplotypes indicate non-parasitic populations have evolved independently multiple times throughout the geographic range of L. fluviatilis in Europe. Therefore, L. planeri should not be considered as a distinct species, either morphologically or genetically. Instead, L. fluviatilis appears capable of expressing a range of life history strategies; from parasitic anadromous populations through to non-parasitic stream-resident populations. The overall research approach employed in this thesis, i.e., the combination of ecological, behavioural, taxonomic and molecular studies, could be used to robustly examine the evolutionary ecology of parasitic and non-parasitic lampreys elsewhere

The complete mitogenome of two Australian lampreys: mordacia mordax and mordacia praecox

As one of only two surviving outgroups to all jawed vertebrates, lampreys (Petromyzontiformes) can provide important information in our understanding of early vertebrate evolution. However, with many phylogenetic aspects of lamprey evolution still uncertain, the ability to use contemporary lampreys in this role depends on robust phylogenetic hypotheses regarding the interrelationship of the three lamprey families, as well as the relationship between lampreys and hagfishes. To achieve this, complete mitogenome data of Southern Hemisphere lampreys is required. Another contentious issue in lamprey taxonomy is the status of paired species. Whilst many studies have focused on Northern Hemisphere species pairs, this study is the first to compare Mordacia mordax and Mordacia praecox, two lamprey species endemic to Australia and the only species pair in the Southern Hemisphere. The complete mitochondrial genome of Mordacia mordax and Mordacia praecox was determined twice independently, in a single shotgun sequencing run on an Ion Torrent PGM, and using a combination of Sanger sequencing of short range PCR products and Roche 454 GS Junior pyrosequencing of long range PCR products. Both of the mitogenomes contain the 37 typical vertebrate genes. Their gene order and contents are identical to those of previously described lamprey mitogenomes, with the exception of a novel tandem repeat array located between Cyt b and tRNA proline. The tandem repeat array, referred to as NCIII, contains pseudogenes of tRNA proline and phenylalanine, indicating that it has arisen by tandem duplication of the tRNA proline – phenylalanine region. Characterisation of NCIII revealed that the number of repeat copies was polymorphic between individuals of both species, and was a source of both intra-individual and inter-individual variation. Consistent with other studies of lamprey species pairs, the mitogenome of M. mordax and M. praecox are nearly identical. Phylogenetic analyses were carried out using the newly determined mitogenomes, together with five additional lamprey species and two hagfishes. Most tree topologies obtained strongly support the hypothesis that Petromyzontidae plus Geotriidae are a clade whose sister group is Mordaciidae. Additionally, lamprey divergence times were estimated by a temporally-calibrated phylogenetic analysis that included 20 vertebrate mitogenomes and was done using nine well-established fossil calibration points. The recovered topology strongly supported the hypothesis that lampreys separated from hagfishes about 409 MYA, and that lamprey divergence involved the early radiation of Mordaciidae (about 132 MYA), followed by the monophyletic divergence of Geotriidae plus Petromyzontidae about 85 MYA. Taken together, the results in this study provide robust hypotheses regarding the interrelationship of lamprey families and the relationship between hagfish and lampreys, whilst providing an estimation of their divergence times

Gill Structure & Function in Parasitic and Non-parasitic Lampreys: The Effects of Metamorphosis and Freshwater-Seawater Transfer

Lamprey (Petromyzontiformes) are a phylogenetically ancient group of jawless fishes that begin their lives as filter-feeding larvae (ammocoetes) before undergoing a complex metamorphosis into juvenile lamprey that involves major changes to their internal and external body plan. Some parasitic species, such as the sea lamprey (Petromyzon marinus), migrate to sea following metamorphosis, where they use their oral discs and rasping tongue to attach to and ingest vast quantities of blood from fishes. Thus, sea lamprey have to counter the simultaneous challenges of hyposmoregulation in sea water and the generation of large quantities of ammonia due to the catabolism of protein-rich blood. A goal of this study was to characterize how changes in the structure and function of the gills facilitated osmoregulation and nitrogenous waste (N-waste) excretion by sea lamprey following metamorphosis, particularly after acclimation to sea water and the ingestion of blood from teleost fishes. Accordingly, key features of the lamprey gill including the distribution and abundance of Na+/K+-ATPase (NKA) and H+-ATPase (V-ATPase) pumps involved in ion regulation, and ammonia transporting Rhesus glycoproteins and urea transporting proteins, were investigated using through immunohistochemical staining and Western blotting techniques. In contrast to the sea lamprey, there are other species of lamprey that remain in fresh water following metamorphosis. Many of these species are non-parasitic including the northern brook lamprey (Ichthyomyzon fossor), but some such as the closely related silver lamprey (Ichthymyzon unicuspis) are parasitic. To learn more about how an exclusively FW existence affected ion transport and ammonia excretion by lampreys, the gills of post-metamorphic (juvenile) northern and silver lamprey were compared to those of larval and juvenile sea lamprey. As in sea lamprey, the gills of both species were characterized by the presence of Rhesus c-like glycoprotein (Rhcg-like) and urea transport (UT) protein but, the distribution of these proteins more closely resembled those of larval sea lamprey than juvenile sea lamprey. In both the silver and northern brook lamprey, Rhcg-like protein co-localized with V-ATPase, suggesting that H+ excretion was coupled with Rhcg-like protein mediated diffusion trapping of NH3. Similarly, UT abundance in both species was comparable to that of the larval sea lamprey. I conclude that in freshwater lampreys, NH3 extrusion via apical Rhcg-like proteins is coupled to V-ATPase mediated H+ excretion, which maintains favourable diffusion gradients by trapping NH3 as NH4+. Given that the lampreys and teleosts have evolved along separate lineages for at least 360 million years, I propose that this method of ammonia excretion is an ancient strategy used by aquatic organisms to facilitate ammonia excretion across the gills in fresh water. In contrast, the need for V-ATPase trapping of NH3 as NH4+ is not required in sea water, in which the Rhcg-like proteins were restricted to the basolateral membrane and co-localized with NKA in sea water mitochondrion-rich cells (SW MRCs). These findings suggest that Rhcg-like protein may mediate ammonia excretion by loading the SW MRC with ammonia, with the resulting NH4+ pumped out of the cell via substitution for H+ on an apical Na+/H+ exchanger, or via an outwardly directed NH4+ electrochemical gradient that favours excretion via paracellular junctions