Molecular resolution of the family Dreissenidae (Mollusca: Bivalvia) with emphasis on Ponto-Caspian species, including first report of Mytilopsis leucophaeata in the Black Sea basin

https://doi.org/10.1016/S1055-7903(03)00240-9Get rights and content

Abstract

Considerable uncertainty exists in determination of the phylogeny among extant members of the Dreissenidae, especially those inhabiting the Ponto-Caspian basin, as multiple systematic revisions based on morphological characteristics have failed to resolve relationships within this group of bivalves. In this study we use DNA sequence analyses of two mitochondrial gene fragments, 16S rRNA and cytochrome c oxidase subunit I (COI), to determine phylogenetic relationships among Dreissena rostriformis, D. bugensis, D. polymorpha, D. stankovici, Congeria kusceri, and Mytilopsis leucophaeata. Dreissena stankovici was determined to represent a sister taxa to D. polymorpha and both are more closely related to other extant Dreissena species than Congeria or Mytilopsis. Sequence divergence between D. rostriformis and D. bugensis was relatively low (0.3–0.4%), suggesting that these two taxa constitute a single species. However, environmental differences suggest two races of D. rostriformis, a brackish water race (rostriformis) and a freshwater race (bugensis). Spread of bugensis-type individuals into habitats in the Caspian Sea that are occupied by rostriformis-type individuals may create novel hybridization opportunities. Species-specific molecular markers also were developed in this study since significant intraspecific variation in morphological features complicates dreissenid identification. Using two gene fragments (nuclear 28S and 16S), we identified restriction fragment length polymorphisms (RFLPs) that distinguish among D. rostriformis/bugensis, D. polymorpha, and D. stankovici and revealed the presence of a cryptic invader to the Black Sea basin, Mytilopsis leucophaeata. This is the first report of this North American native in southern Europe.

Introduction

Dreissenid molluscs are an important group of biofouling bivalves that are rapidly invading habitats around the world (e.g., Hebert et al., 1989; May and Marsden, 1992; Nuttall, 1990). Dreissenids have been reclassified many times, at many levels (i.e., genus, subgenus, species, subspecies, and variety). These analyses have resulted in confusion regarding phylogenetic relationships within the family. For example, Russian systematists have been unable to develop a uniform taxonomic history for the genus Dreissena. Andrusov (1897) outlined the first major taxonomic classification scheme for dreissenids and included Dreissena polymorpha (Pallas), D. rostriformis (Deshayes), and D. bugensis (Andrusov), amongst others, as legitimate species. Zhadin (1952) did not recognize D. rostriformis as a species but subsequent reclassifications by Logvinenko (1965), Logvinenko and Starobogatov (1968), and Starobogatov (1994) did. Zhadin (1952) recognized D. bugensis as a species, while Mordukhai-Boltovskoi (1960) classified D. bugensis as a subspecies of D. rostriformis. Starobogatov (1994) re-elevated D. bugensis to species level within the subgenus Pontodreissena and maintained species level classification for both D. polymorpha (subgenus Dreissena) and D. rostriformis (subgenus Pontodreissena) and included the newly identified D. stankovici (subgenus Carinodreissena). Rosenberg and Ludyanskiy’s (1994) comparative review of dreissenid taxonomy included additional species, subspecies, and varieties based primarily on Russian accounts (e.g., Andrusov, 1897; Babak, 1983; Logvinenko and Starobogatov, 1968; Nevesskaya, 1963; Starobogatov, 1970; Starobogatov, 1994; Taktakishvili, 1973).

Some workers have suggested that Dreissena and Mytilopsis evolved from extinct branches of the genus Congeria (Andrusov, 1897; Babak, 1983; Mackie et al., 1989; Starobogatov, 1994), while others maintain that Dreissena and Congeria arose from Mytilopsis (Marelli, 1994). Mytilopsis was considered a subgenus of Congeria by Russian taxonomists (Andrusov, 1897; Babak, 1983; Starobogatov, 1970), but was elevated to genus level by Nuttall (1990), a classification scheme later supported by Rosenberg and Ludyanskiy (1994). Marelli and Gray (1985) suggested that there exist only five extant species of Mytilopsis, including M. leucophaeata. Unfortunately, as with Dreissena, traditional taxonomic classification of the genus Mytilopsis is complex, discordant and variable through time. We argue that the use of molecular techniques can help clarify the phylogenetics of this group.

Two of four Dreissena species used herein, D. rostriformis and D. stankovici, have never been reported outside their historical ranges (Table 1). Dreissena rostriformis occurs in the Middle and South Caspian Sea at salinities between 12 and 13.5‰, while the closely related D. bugensis is typical of freshwater or oligohaline habitats both within its historical range in the Black Sea basin, and in its introduced range in the Volga River. D. polymorpha occurs in similar habitats as those reported for D. bugensis, but is capable of inhabiting mesohaline waters typical of the northern Caspian Sea (Table 1).

Human activities are rapidly changing aquatic ecosystems. Most notable are activities related to transoceanic shipping and canal creation, both of which link water bodies and allow transfer of nonindigenous species between previously isolated aquatic ecosystems. The Dreissenidae have undergone considerable global redistribution as a result of shipping activities (Nuttall, 1990). Typically considered Ponto-Caspian “endemics” (Geary et al., 2000), two dreissenids have recently invaded the Laurentian Great Lakes. Dreissena polymorpha, the zebra mussel, was first discovered in Lake St. Clair in 1988 (Hebert et al., 1989), while D. bugensis, the quagga mussel, was first reported from Lake Ontario in 1991 (May and Marsden, 1992). A “profundal” variety was reported from deep-water habitats in Lake Erie in 1992 (Dermott and Munawar, 1993) and later identified as D. bugensis using allozymes (Marsden et al., 1996; Spidle et al., 1994). Another Dreissenidae, the dark false mussel Mytilopsis leucophaeata, is native to the Gulf of Mexico, but invaded the Hudson River, New York in the 1930s. The species also has recently been identified in the Upper Mississippi River (Koch, 1989) and at several locations in southern New England (Smith and Boss, 1996). Mytilopsis leucophaeata also has been reported from European waters as early as 1835 (Wolff, 1999) and is found along North Sea coasts from Germany to France (Marelli and Gray, 1983; Oliver et al., 1998) and the River Thames estuary, England (Bamber and Taylor, 2002). European populations occupy both freshwater and brackish estuary habitats (Reise et al., 1999).

Dreissena polymorpha has an extensive distribution in both European and North American freshwaters (Nalepa and Schloesser, 1993). In contrast, D. bugensis has a more restricted distribution in European and North American freshwaters, but is currently undergoing range expansion in the Volga River, Russia, and is replacing D. polymorpha in the lower Great Lakes (Berkman et al., 2000; Mills et al., 1999). Dreissenids are nuisance species in many invaded habitats owing to biofouling (Kharchenko, 1995; Marelli and Gray, 1983), but are considered beneficial in some habitats where they improve water quality (Reeders et al., 1993). Owing largely to human-induced range expansion, co-occurrence of dreissenid species is increasing globally and the ability to discern morphologically similar species has become increasingly important. Dreissena polymorpha, D. bugensis, D. rostriformis, D. stankovici, and M. leucophaeata share many life-history characteristics (e.g., use of byssal threads for attachment, and possession of a free-swimming veliger larva) and exhibit strong morphological and shell colour similarities (Biochino, 1994; Lukashev, 2000; May and Marsden, 1992; O’Neill, 1990; Pathy and Mackie, 1993; Protasov and Gorpinchuk, 2000). Moreover, each species may exhibit pronounced intraspecific variability. Genetic markers may prove particularly useful for discrimination of species, such as dreissenids, with high intraspecific variability or small larval or juvenile size (Claxton et al., 1997, Claxton et al., 1998; Skurikhina et al., 2001). Identification of a few individuals early on in an incipient invasion may allow for implementation of rapid control measures. For example, shortly after M. sallei arrived in Darwin, Australia, a comprehensive eradication campaign was undertaken before the species could become established (Pyne, 1999).

In this study, we use mitochondrial gene sequencing to assess the phylogenetic relationships among members of the family Dreissenidae identified from Ponto-Caspian and Mediterranean regions including the genera Dreissena, Mytilopsis, and Congeria. This is the first study to use molecular techniques to resolve the placement of D. stankovici and D. rostriformis within the family Dreissenidae. In addition, we attempt to resolve the relationship between D. rostriformis and D. bugensis using sequence data. The relationship between these taxa has been highly discordant over time based on traditional taxonomical accounts as some authors consider each a species while others consider D. bugensis a subspecies of D. rostriformis (e.g., Andrusov, 1897; Mordukhai-Boltovskoi, 1960; Starobogatov, 1994; Zhadin, 1952). Furthermore, we use nuclear and mitochondrial DNA restriction digests to identify species that are difficult to distinguish based on morphological characteristics alone.

Section snippets

DNA isolation and PCR amplification

Specimens used in sequencing were collected from their current European ranges (Table 2). Two cryptic dreissenid specimens were collected from the Dniester Liman, Black Sea, and included in our analyses. External shell morphology of these individuals was similar but not identical to those of D. bugensis and D. polymorpha. Total DNA was extracted from mantle muscle tissues of specimens preserved in 95% ethanol or frozen using either a standard phenol–chloroform method or a DNA purification kit

Phylogenetic analysis

The NJ tree based on 16S showed that D. rostriformis and D. bugensis differed by a single nucleotide (Fig. 1). Bootstrap support for distinct nodes in this part of the tree was weak, providing the first molecular evidence that these individuals might represent a common species. However, both NJ and maximum parsimony analyses maintained each taxon as monophyletic. Considering D. rostriformis and D. bugensis as a single species with two possible races (see below), intraspecific differences ranged

Dreissenid phylogenetics

In contrast to traditional dreissenid taxonomy based principally on morphological attributes, our molecular analyses, in combination with environmental tolerances, suggest that D. bugensis and D. rostriformis may represent a single species with two distinct races. This view is supported by only a single base pair difference (0.23%) between D. bugensis and D. rostriformis in the 16S gene, and by 2–3 bp differences (0.36–0.54%) in the COI gene. Consequently, we suggest the ancestral name of D.

Acknowledgements

Drs. I. Grigorovich and C. Lee collected samples of D. bugensis and D. stankovici, without which this work would not have been possible. Financial support was provided by NSERC (T.W.T.) and GLIER (M.F.D.) postdoctoral fellowships, Canada Research Chair (DDH), Premier’s Research Excellence Award (H.J.M.), and by NSERC research grants to D.D.H. and H.J.M.

References (85)

  • B.S. Baldwin et al.

    A diagnostic molecular marker for zebra mussels (Dreissena polymorpha) and potentially co-occurring bivalves: mitochondrial COI

    Mol. Mar. Biol. Biotechnol.

    (1996)
  • R.N. Bamber et al.

    The brackish water mussel Mytilopsis leucophaeata (Conrad, 1831) (Bivalvia: Dreissenidae) in the River Thames

    J. Conchol.

    (2002)
  • P.A. Berkman et al.

    Habitat shift in invading species: zebra and quagga mussel population characteristics on shallow soft substrates

    Biol. Invas.

    (2000)
  • G.I. Biochino

    Polymorphism and geographical variability

  • K.H. Chu et al.

    A biological survey of ballast water in container ships entering Hong Kong

    Hydrobiologia

    (1997)
  • W.T. Claxton et al.

    Discrimination of field-collected juveniles of two introduced dreissenids (Dreissena polymorpha and Dreissena bugensis) using mitochondrial DNA and shell morphology

    Can. J. Fish. Aquat. Sci.

    (1997)
  • W.T. Claxton et al.

    A genetic and morphological comparison of shallow- and deep-water populations of the introduced dreissenid bivalve Dreissena bugensis

    Can. J. Zool.

    (1998)
  • M.E.A. Cristescu et al.

    An invasion history for Cercopagis pengoi based on mitochondrial gene sequences

    Limnol. Oceanogr.

    (2001)
  • R. Dermott et al.

    Invasion of Lake Erie offshore sediments by Dreissena, and its ecological implications

    Can. J. Fish. Aquat. Sci.

    (1993)
  • H.J. Dumont

    The Caspian lake: history, biota, structure, and function

    Limnol. Oceanogr.

    (1998)
  • O.M. Folmer et al.

    DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates

    Mol. Mar. Biol. Biotechnol.

    (1994)
  • J.P.A. Gardner et al.

    Size-dependent, spatial and temporal genetic variation at a leucine aminopeptidase (LAP) locus among blue mussel (Mytilus galloprovincialis) populations along a salinity gradient

    Mar. Biol.

    (1998)
  • P.D.N. Hebert et al.

    Ecological and genetic studies on Dreissena polymorpha (Pallas): a new mollusc in the Great Lakes

    Can. J. Fish. Aquat. Sci.

    (1989)
  • T.J. Hilbish et al.

    Genetics of physiological differentiation within the marine mussel genus Mytilus

    Evolution

    (1994)
  • T.J. Hilbish et al.

    Distribution of Mytilus edulis, M. galloprovincialis, and their hybrids in open-coast populations of mussels in southwestern England

    Mar. Biol.

    (2002)
  • W.R. Hoeh et al.

    Phylogenetic evidence for role-reversals of gender-associated mitochondrial DNA in Mytilus (Bivalvia: Mytilidae)

    Mol. Biol. Evol.

    (1997)
  • M.K. Jacobson

    Congeria leucophaeata (Conrad) in the Hudson River

    Nautilus

    (1953)
  • A.Y. Karataev et al.

    Physical factors that limit the distribution and abundance of Dreissena polymorpha (Pall.)

    J. Shellfish Res.

    (1998)
  • A.F. Karpevich

    Some data on morphological evolution in bivalve molluscs

    Zool. Z.

    (1955)
  • T.A. Kharchenko

    Dreissena: range, ecology, biofouling

    Gidrobiol. Z.

    (1995)
  • L.M. Koch

    Mytilopsis leucophaeata (Conrad, 1831) from the upper Mississippi River (Bivalvia: Dreissenidae)

    Malacol. Data Net

    (1989)
  • R.K. Koehn et al.

    Maintenance of an aminopeptidase allele frequency cline by natural selection

    Proc. Natl. Acad. Sci. USA

    (1980)
  • S. Kumar et al.

    MEGA: Molecular Evolutionary Genetics Analysis, version 1.01

    (1993)
  • B.M. Logvinenko

    Changes in the fauna of Caspian molluscs Dreissena after intrusion of Mytilaster lineatus (Gmelin)

    Nauchn. Dokl. Vyssh. Shk. Biol. Nauki

    (1965)
  • B.M. Logvinenko et al.

    Phillum Mollusca

  • D.V. Lukashev

    Morphological variability of Dreissena bugensis Andrusov under conditions of the regulated Dnieper outflow

    Hydrobiol. J.

    (2000)
  • C. Lydeard et al.

    Molecular systematics and evolution of reproductive traits of North American freshwater unionacean mussels (Mollusca: Bivalvia) as inferred from 16S rRNA gene sequences

    Philos. Trans. R. Soc. Lond. B

    (1996)
  • Mackie, G.L., Gibbons, W.N., Muncaster, B.W., Gray, I.M., 1989. The zebra mussel, Dreissena polymorpha: a synthesis of...
  • MacNeill, D.B., 1991. Potential overlap of Dreissena and Mytilopsis in the Hudson River. In: Moore, S.G. (Ed.),...
  • Markovskii, Yu.M., 1954. Invertebrate fauna from the lower reaches of Ukraine, conditions of its existence and ways for...
  • Marelli, D.C., 1994. The phylogeny of the Dreissenidae: a phylogenetic reconstruction of the zebra mussel tree....
  • D.C. Marelli et al.

    Comments on the status of recent members of the genus Mytilopsis (Bivalvia: Dreissenidae)

    Malacol. Rev.

    (1985)
  • Cited by (99)

    • An integrated reconstruction of the early Pleistocene palaeoenvironment of Homo erectus in the Denizli Basin (SW Turkey)

      2019, Geobios
      Citation Excerpt :

      The species was found in Quaternary deposits of the Konya Basin, located east of the lake (Schütt, 1991). The bivalve Dreissena is an epifaunal filter feeder dwelling on hard substrates in rivers, estuaries and lakes from freshwater to mesohaline conditions down to depths of over 130 m (Therriault et al., 2004; Orlova et al., 2005; Welter-Schultes, 2012; Cummings and Graf, 2015). Lymnocardiinae occur in a wide range of well-oxygenated habitats in lagoons, coastal lakes or river mouths, and are typical of oligohaline to mesohaline conditions; some species occasionally also extend into freshwater (Kijashko in Bogutskaya et al., 2013; Albrecht et al., 2014).

    • Alien Species Invasion: Case Study of the Black Sea

      2019, Coasts and Estuaries: The Future
    View all citing articles on Scopus
    View full text