Introduction

Anadromous lampreys, which feed parasitically on teleost fishes in estuaries or at sea in the adult trophic phase of their life cycle, develop, in their gill epithelium during metamorphosis, a special type of mitochondria-rich cells (MRC) or ionocyte, previously called “chloride cells” (Peek and Youson 1979; Bartels and Potter 2004). In teleost fishes, such cells have been shown to secrete the excess of intestinally resorbed Na+ and Cl−, thereby helping maintain the osmolality of their internal milieu far below that of the environment and thus acting as the main effector cells in marine osmoregulation in this group of fishes (Karnaky 1986; Hiroi and McCormick 2012). Since this type of MRC is found in anadromous lampreys only during the period when these animals are able to osmoregulate efficiently in a hypertonic environment (Bartels and Potter 1991; Reis-Santos et al. 2008; Ferreira-Martins et al. 2016), it has been concluded that it is only involved in osmoregulation in sea water (Bartels and Potter 2004) and is thus now termed seawater-type MRC (SW-MRC).

The larvae (ammocoetes) of all lamprey species are microphagous and live in burrows in the soft substrate of streams and rivers, filtering their food from the overlying water (Dawson et al. 2015). Eighteen of the 41 recognized species are parasitic as adults, nine of which undergo an anadromous migration, whereas another nine remain in fresh water, feeding on other fishes in rivers and lakes (Potter et al. 2015). The remaining 23 species remain in their natal streams for the whole of their lifecycle and do not feed as adults, with each of these so-called “brook” lampreys assumed to have evolved from a parasitic ancestor (Potter 1980; Docker 2009; Potter et al. 2015). The majority of the non-parasitic species are “paired” with an extant parasitic species with which they are morphologically and genetically very similar (Potter 1980; Docker 2009; Potter et al. 2015; Rougemont et al. 2015, 2017, b; Mateus et al. 2016). There are in addition to these recent non-parasitic derivatives, six non-parasitic species for which there is no obvious parasitic counterpart, either morphologically or genetically (Potter et al. 2015). The dentition of each of these “relict” species is degenerate, implying that these brook lampreys have diverged markedly from their parasitic ancestor and are thus older non-parasitic derivatives (Docker et al. 1999; Docker 2009; Potter et al. 2015).

Previous studies have shown that the various non-parasitic species that have been derived recently from their anadromous parasitic ancestor still develop the SW-MRC during metamorphosis. These include the European brook lamprey Lampetra planeri, the western brook lamprey Lampetra richardsoni and the American brook lamprey Lethenteron appendix (Youson and Beamish 1991; Bartels et al. 2011, 2015). The landlocked form of the parasitic sea lamprey Petromyzon marinus likewise still develops the SW-MRC during metamorphosis (Youson and Freeman 1976), which is consistent with a derivation from the anadromous form in post-glacial or historical times (Waldman et al. 2004; Bryan et al. 2005; Eshenroder 2014). In contrast, a SW-MRC is developed neither by the relict least brook lamprey Lampetra aepyptera (Bartels et al. 2015), nor by the exclusively freshwater parasitic silver lamprey Ichthyomyzon unicuspis and chestnut lamprey Ichthyomyzon castaneus (Bartels et al. 2012, 2015). The lack of SW-MRCs in these latter three species is consistent with the assumption that they have long been confined to fresh water (Bartels et al. 2012, 2015).

Molecular phylogenetic analyses have significantly advanced our understanding of the interrelationships and evolutionary history of extant lamprey taxa (Docker et al. 1999; Lang et al. 2009), but there are still gaps in our knowledge (Potter et al. 2015). While, for example, the lack of fixed mitochondrial DNA sequence differences between the members of most paired species studied indicates a very close evolutionary relationship, such data are not sufficient to distinguish between recent divergence and ongoing gene flow (Espanhol et al. 2007; Docker 2009; April et al. 2011). Recent population genetic and genomic analyses, using microsatellite loci or Restriction-site Associated DNA (RAD) sequencing, are helping to shed light on the demographic history of the species pair that comprises the European river lamprey Lampetra fluviatilis and the European brook lamprey Lampetra planeri (Bracken et al. 2015; Rougement et al. 2016, 2017; Mateus et al. 2016), but are also revealing a much more complex situation than previously appreciated (Rougemont et al. 2016, 2017).

The evolutionary history of other paired lamprey species is even more poorly understood. For example, there is debate over the direct ancestor of the Ukrainian brook lamprey Eudontomyzon mariae, the most widespread species of its genus and of all non-parasitic species in eastern Europe (Holčík and Renaud 1986; Renaud 2011; Levin et al. 2016). While E. mariae is generally considered a derivative of the Carpathian lamprey Eudontomyzon danfordi, an exclusively freshwater parasitic species (Docker 2009; Renaud 2011; Potter et al. 2015), it has alternatively been proposed that E. mariae is a relatively young species, which evolved from a now extinct anadromous Black Sea ancestor as recently as the beginning of the last century (Holčík and Renaud 1986). The IUCN Red List of Threatened Species includes the extinct Eudontomyzon sp. nov. ‘migratory’ from the Dniester, Dnieper and Don drainages in the Black Sea basin. Although its adults were the target of fisheries during their autumn and spring migrations, it is not known whether this species spent a period feeding at sea, i.e., underwent an anadromous migration (Freyhof and Kottelat 2008). Molecular phylogenetic analyses indicate that the European river lamprey is the closest extant anadromous relative of E. mariae and E. danfordi (Blank et al. 2008). There is, however, no known tissue of Eudontomyzon sp. nov. ‘migratory’ from which DNA could be extracted to test the relatedness between this species and E. mariae.

In this study, two independent but complementary lines of evidence are thus used to test whether E. mariae likely descended recently from an anadromous ancestor or one that was an exclusively freshwater resident. The first aim of this study was to determine if the SW-MRC is present in the gills of E. mariae; against the background that previous studies found SW-MRCs in the gills of four freshwater lampreys that are recently derived from anadromous species and absent in three species with a long history in fresh water (see above). The results will provide strong indications of whether or not E. mariae evolved recently from an anadromous species. The second aim was to use mitochondrial DNA sequence data to determine the relationships among freshwater populations of E. mariae and E. danfordi and anadromous L. fluviatilis. Although mitochondrial DNA sequence alone is not sufficient to fully resolve the evolutionary history of E. mariae, our analysis of publicly-available sequence data will shed additional light on the transition from anadromy to freshwater residency in Eudontomyzon.

Materials and methods

Five E. mariae, which had completed metamorphosis but were still sexually immature, were caught in early November 2015, in the River Gać, a small tributary of the Pilica River (west tributary of the Vistula River) close to Lódz, Poland (Fig. 1). Eudontomyzon mariae is readily distinguishable from the sympatric L. planeri by the contrast between the strong pigmentation of its caudal fin (Holčík and Renaud 1986) and the virtual absence of such pigmentation in L. planeri (Hardisty 1986). They were maintained for 1 week in freshwater aquaria until sacrifice in a solution of clove oil in water (Stetter 2001). Transverse sections were cut through the gill region and immediately fixed in 1.5% glutaraldehyde and 1.5% formaldehyde in 0.15 M HEPES-buffer, pH 7.3. Small pieces of the gills, containing 3–6 filaments, were dissected out under a microscope, thoroughly rinsed in buffer and postfixed in 2% OsO4 in 0.1 M Na-cacodylate-HCl-buffer and thereafter in uranyl acetate overnight. These samples were then dehydrated in ethanol and embedded in Epon. Thick sections (0.5 μm) were stained with toluidine blue for light microscopy, while thin sections (ca 60 nm) were stained with uranyl acetate and lead citrate and examined in a FEI Morgagni 268 Electron Microscope (FEI Europe B.V., Eindhoven, Netherlands) at 80 kV. Images were processed by sharpening and adapting contrast.

Fig. 1
figure 1

Map showing the sample site of E. mariae in the Gac River, a tributary of the Pilica River which belongs to the Vistula River system (inset)

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Cytochrome b sequence data from previous studies were acquired from the NCBI Nucleotide Database (GenBank): with the E. mariae coming from the Black, Baltic and Caspian Sea basins (n = 23), E. danfordi from the Black Sea basin (n = 5) and L. fluviatilis from the Black, Baltic, and North Sea basins (n = 8; Table 1).

Table 1 Cytochrome b sequence data acquired from the NCBI Nucleotide Database (GenBank) for Eudontomyzon mariae (n = 23), Eudontomyzon danfordi (n = 5), and Lampetra fluviatilis (n = 8) from the Black, Baltic, Caspian, and North Sea basins. The river drainage for E. mariae within each basin is provided
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Median-joining network analysis was performed on 812 bp of the cytochrome b gene using DNAsp 5.10.1 (Librado and Rozas 2009) and Network 4.613 (Bandelt et al. 1999; www.fluxus-engineering.com). Thirty-six sequences, containing 68 variable sites, were included in the analysis and 14 haplotypes were resolved from the dataset. The resulting network was color coded to reflect the geographical origin of the specimens sampled. Interspecific and intraspecific genetic distances were calculated using the Kimura-2-parameter method in MEGA6 (Tamura et al. 2013).

Results

Cellular composition of the gills

The epithelium of the gill filaments and lamellae of fully-metamorphosed E. mariae is two-layered and consists of a basal layer of undifferentiated basal cells and a superficial cell layer of pavement cells and intercalated mitochondria-rich cells (IMRC) (Fig. 2). Both cell types have a distribution on the filaments and the lamellae and an ultrastructural appearance very similar to those described for other non-parasitic species and for parasitic species when in fresh water (Fig. 2) (Bartels and Potter 2004; Bartels et al. 2015). Squamous pavement cells, which are the most abundant cell type, cover the surface of the lamellae, whereas IMRCs are few in number and occur singly between pavement cells, mostly in the interlamellar region and at the base of the filament (Figs. 2 and 3). SW-MRCs were not observed in any section of three different gills of each of the five E. mariae examined. Secretory cells, similar to those found in the gill epithelium of L. appendix, L. planeri and L. aepyptera (Bartels et al. 2011, 2015), are present between and at the base of the gill filaments (Fig. 2).

Fig. 2
figure 2

Representative survey electron micrograph of a gill filament in metamorphosed Eudontomyzon mariae showing several interlamellar regions. The gill surface is mostly covered by pavement cells. Single intercalated mitochondria-rich cells are indicated by arrowheads, and a group of secretory cells by an arrow. Note that seawater mitochondria-rich cells are absent in the interlamellar regions

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Fig. 3
figure 3

An intercalated mitochondria-rich cell between two pavement cells (arrows). BC, basal cell

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Molecular studies

The median-joining haplotype network analysis revealed a total of 14 haplotypes, of which three were in L. fluviatilis, three in E. danfordi and eight in E. mariae (Fig. 4). The E. mariae haplotypes, which differed from each other by up to 29 out of 812 nucleotides or 3.83% Kimura-2-parameter genetic distance (K2P), comprised four distinct groups. One group, which included two individuals from the Danube River, differed from E. danfordi by only 6–9 nucleotides (0.74–1.12% K2P), whereas the remaining three groups differed from E. danfordi by 16–28 nucleotides (2.01–3.7% K2P). Lampetra fluviatilis differed from all E. danfordi and E. mariae by 30–42 nucleotides (3.82–5.01% K2P). Another E. mariae group, which included two E. mariae from the Vistula River basin, showed 5 nucleotide substitutions (0.62%) relative to the hypothetical Eudontomyzon ancestor. All other E. mariae and E. danfordi differed from the hypothetical Eudontomyzon ancestor by 11–15 nucleotide substitutions (1.37–1.87%).

Fig. 4
figure 4

Median-joining network colored by species for Eudontomyzon danfordi (n = 5) and Lampetra fluviatilis (n = 8) and by drainage basin for Eudontomyzon mariae (n = 23) using 812 bp of cytochrome b gene sequence; Eudontomyzon danfordi and E. mariae are entirely freshwater residents; L. fluviatilis is anadromous. The size of each of the 14 colored nodes is proportional to the frequency of the observed haplotype; the median vectors represent haplotypes not recovered in any of the included sequences. The number of substitutions between haplotypes is given; where no number appears on the line linking two haplotypes, the number of substitutions is 1

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Discussion

Absence of seawater-type mitochondria-rich cells

The observation that SW-MRCs are not present in the gill epithelium of E. mariae which had metamorphosed but not yet become sexually mature has important implications for discussions of the ability to osmoregulate in hypertonic media and of the potential longevity and origin of this non-parasitic lamprey.

The SW-MRC type develops in anadromous lampreys during metamorphosis and disappears during the upstream spawning migration, when the lampreys become sexually mature (Bartels and Potter 2004). The period between the development and subsequent loss of this cell type, which encompasses the marine trophic phase, is the only one during which the lamprey is able to osmoregulate efficiently in sea water (Morris 1972; Beamish 1980; Bartels and Potter 2004). The SW-MRC is thus assumed to play the same crucial role in osmoregulation in the marine phase of the anadromous species of lampreys (Bartels and Potter 2004; Reis-Santos et al. 2008; Ferreira-Martins et al. 2016), as shown to be performed by very similar cells in marine teleosts (Karnaky 1986; Hiroi and McCormick 2012).

The absence of SW-MRCs in E. mariae implies that metamorphosed individuals of this brook lamprey are able to osmoregulate only in hypotonic, i.e., freshwater environments. Such an absence contrasts with the situation recorded for corresponding stages in the post-larval life of three other non-parasitic species of lampreys, i.e., L. planeri, L. appendix, and L. richardsoni (Youson and Beamish 1991; Bartels et al. 2011, 2015). Eudontomyzon mariae has a similar life cycle to those three species, and, in general, of non-parasitic lampreys in the northern hemisphere, with metamorphosis commencing in the summer and completed during the autumn, and spawning occurring in the following spring/early summer (Renaud 2011; Manzon et al. 2015). Morphological similarities between these species and their presumed anadromous parasitic ancestors suggest that they have evolved relatively recently (Potter 1980; Docker 2009). The SW-MRCs in metamorphosed individuals of L. planeri, L. appendix, and L. richardsoni, and in landlocked P. marinus in the Great Lakes of North America (Youson and Freeman 1976; Bartels et al. 2011, 2015) are thus considered remnants of those cells present in the ancestral anadromous parasitic lampreys, and which are retained for some time after the species had become confined to fresh water. Some molecular studies suggest that the separation of both L. planeri in northern Europe (Mateus et al. 2016) and of L. richardsoni in North America occurred post-glacially (Docker et al. 1999; Boguski et al. 2012). Recent genetic analyses in replicate L. fluviatilis–L. planeri pairs using advanced computational techniques indicate older divergence times (201,000–257,000 years) followed by introgression during secondary contact starting around 92,000 year ago (Rougemont et al. 2017). Divergence between L. appendix and the anadromous Arctic lamprey Lethenteron camtschaticum may also predate the most recent glacial retreat (i.e., up to 130,000 years ago; Docker et al. 1999; Li 2014).

Morphological and molecular data clearly show that L. aepyptera is an old non-parasitic species (Docker 2009; Potter et al. 2015). The absence of the SW-MRC in metamorphosed but still immature individuals of this relict species L. aepyptera demonstrates that this cell type is eventually lost following long separation from the anadromous ancestor (Bartels et al. 2015). On the basis of mitochondrial DNA sequence data, L. aepyptera is most closely but still distantly related to the anadromous and parasitic L. fluviatilis (Docker et al. 1999), with molecular clock estimates indicating that this divergence occurred at least 2 million years ago (Bartels et al. 2015). Given the degree to which its dentition is degenerate (Potter et al. 2015) and the increasing appreciation of the “non-clock-like” nature of mitochondrial DNA (see Galtier et al. 2009), 2 million years is probably an underestimate. The crucial point is that L. aepyptera does not possess a SW-MRC, which must thus reflect either the loss of that cell during a long period since its evolution from an anadromous parasitic species or less likely from a freshwater ancestor. Two parasitic species of Ichthyomyzon, for which there are no obvious candidates as ancestors, also do not possess the SW-MRC, which is consistent with the hypothesis that this genus has had a long evolutionary history in fresh water (Bartels et al. 2012, 2015). It is thus concluded that E. mariae, at least in the Vistula River, does not represent a recent derivative of a now extinct anadromous lamprey in the Black Sea, as proposed by Holčík and Renaud (1986). Even if there is some variation in the rate at which SW-MRCs are lost following confinement to fresh water, it is clear that SW-MRCs are not lost rapidly in non-anadromous lampreys. Our results thus strongly indicate that either E. mariae or its parasitic ancestor has had a relatively long history in fresh water. Further studies should verify that E. mariae from other locations likewise lack SW-MRCs. After reviewing the records for the extinct migratory Black Sea lamprey, Kottelat et al. (2005), concluded that earlier reports from the Prut (Danube drainage) refer to E. mariae, but were unable to resolve whether the migratory lamprey recorded in the lower Dnieper River migrated to the sea. Verification that E. mariae from the Dnieper River also lack SW-MRCs indicate that this now extinct migratory lamprey was either not anadromous or was not the recent ancestor of E. mariae from the Dnieper River basin.

Molecular studies

The cytochrome b haplotype network analysis sheds additional light on the possible transition from anadromy to freshwater residency in Eudontomyzon. The lack of SW-MRC indicates that E. mariae in the Vistula River did not evolve recently from an anadromous ancestor, which—on its own—could be interpreted to support the alternative hypothesis that E. mariae is a recent derivative of the freshwater parasitic E. danfordi (Docker 2009; Renaud 2011; Potter et al. 2015). The cytochrome b median-joining haplotype network analysis also suggests that E. danfordi is not a very recent ancestor of the Vistula River E. mariae. Although caution needs to be exercised in interpreting results based on mitochondrial DNA sequence data alone (e.g., since genetic drift between isolated populations can obscure inferences regarding divergence times; Galtier et al. 2009), it is relevant that E. mariae and E. danfordi are one of the very few lamprey pairs studied in which the two species did not share cytochrome b haplotypes (e.g., Espanhol et al. 2007; Docker 2009; Lang et al. 2009; Boguski et al. 2012). Eudontomyzon mariae populations in the Danube River drainage differed from E. danfordi by only 0.7–1.1% in cytochrome b sequence, but E. mariae in the Vistula River differed from E. danfordi by 2.0–2.2%. In contrast, the Vistula River E. mariae (and one of the individuals from both the Dnieper and Danube rivers) were genetically very similar to the hypothetical Eudontomyzon ancestor, i.e., the ancestor to all E. mariae and E. danfordi. The network analysis estimated that there were only five nucleotide substitutions between the hypothetical Eudontomyzon ancestor and E. mariae from the Vistula River. This small genetic distance (0.6%) strongly suggests that E. mariae in the Vistula River would possess SW-MRCs had its ancestor been anadromous. In comparison, the non-parasitic L. appendix still develops the SW-MRC at metamorphosis, even though differences in cytochrome b sequence between this species and its presumed anadromous ancestor range from 0.18 to 1.09% (Li 2014).

This network analysis provides further indications that of the E. mariae in the Black, Baltic, and Caspian Sea basins each evolved indepndently. This is consistent with previous suggestions, based on the wide variation of its taxonomic characters and broad geographical distribution, that E. mariae has evolved independently in different locations from a parasitic ancestor and, as suggested by Renaud (1982, 2011), may even consist of a number of subspecies. If E. mariae did evolve independently in multiple basins, it could be argued that E. mariae in the Baltic Sea basin evolved not from a recent but extinct anadromous ancestor in the Vistula River, but from one in the Black Sea basin. We have not yet tested whether the SW-MRC is also absent in E. mariae from other basins. However, as mentioned above, the cytochrome b network analysis suggests that at least some of the Black Sea E. mariae may be derived from the exclusively freshwater E. danfordi, and that the Danube River E. mariae and E. danfordi have diverged even more from the non-anadromous Eudontomyzon ancestor than has E. mariae in the Vistula River. Thus, although further studies should examine whether E. danfordi and other populations of E. mariae similarly lack SW-MRCs, the results of the cytochrome b network analysis predict that they will lack these cells.

The network analysis showed 25–27 nucleotide substitutions between anadromous L. fluviatilis and the Eudontomyzon ancestor, with freshwater residency presumably arising somewhere along this pathway. Despite this implication, however, the question of how long Eudontomyzon has resided in fresh water remains unresolved. Additional studies are thus needed, using E. mariae from various geographical sources and employing other DNA markers and analyses, to obtain a better understanding of the evolutionary history of these lamprey taxa. Nevertheless, our demonstration that fully metamorphosed E. mariae do not possess SW-MRC provides strong circumstantial evidence that E. mariae was not derived recently from an anadromous ancestor and, in combination with molecular phylogenetic analyses, have placed these findings in the context of the evolutionary relationship between anadromous L. fluviatilis and freshwater populations of E. mariae and E. danfordi.