Laboratory hybridizations between Mytilus species and performance of pure species and hybrid veliger larvae at lowered salinity

The Mytilus species complex consists of three closely related species, Mytilus edulis Linnaeus, 1758, M. galloprovincialis Lamark, 1819 and M. trossulus Gould, 1850, and where the distributions of these species overlap hybridization occurs. In the northern hemisphere M. edulis is the predominant mussel on the eastern and western shores of the Atlantic, while M. trossulus occurs in the northern Pacific Ocean, the northwestern Atlantic Ocean and the Baltic Sea.Mytilus galloprovincialis originates from the Mediterranean Sea and extends out onto the Atlantic coasts of Africa and Europe. It also occurs in Japan and on the Pacific coast of North America. The hybrid zones of these mussel species are often extensive and complex with a mosaic of pure species in sympatry or allopatry, and mixtures of hybrids and pure species. The salinity differences between the North Sea and the Baltic Sea immediately suggest an environmental factor that could be acting in thisM. edulis–M. trossulus zone. Distinct clines in allele frequency at several loci coincide with a cline in salinity (from 32‰ down to 10‰) at the entrance to the Baltic. Laboratory hybridizations have been carried out between M. edulis andM. galloprovincialis and between Atlantic Canadian M. edulis and M. trossulus. Here we report on preliminary laboratory hybridization trials involving Baltic M. trossulus, Irish Sea M. edulis and Mediterranean M. galloprovincialis. Data on larval yield and normality of veliger larva morphology at 72 h, and comparative larval growth data for hybrids and pure species at salinities of 32‰ and 20‰ are presented. Between 1999 and 2001, BalticM. trossuluswere obtained from the Gulf of Gdansk, Mediterranean M. galloprovincialis were collected from the Golfe du Lion, France and Irish Sea M. edulis were collected from Conwy, North Wales, UK. Mussels were scraped clean of epifauna and held in the laboratory in flowthrough tanks at 6+ 18C with a drip feed supply of mixed micro-algae (Pavlova lutheri,Tetraselmis suecica and Rhinomonas reticulata ) until spawned. Mediterranean M. galloprovincialis and Baltic M. trossulus were held under quarantine conditions as required by UK regulations. The Baltic M. trossulus were collected from a salinity of around 10‰ and were held initially in the laboratory at 15‰ salinity. Prior to spawning trials they were gradually acclimated over a 3–4 week period to 25‰ salinity and broodstocks of M. edulis and M. galloprovincialis were gradually acclimated over 3–4 weeks from 32‰ down to 25‰ before spawning. All spawning trials were carried out at 25‰ and all salinity adjustments were made using distilled water. Several spawning trials were attempted of which three produced sufficient numbers of gametes to warrant fertilization. Between 40 and 80 individuals from each species were used in each trial. Spawning was induced, crosses were made and trials were set up following the method described by Beaumont et al. Full reciprocal hybridizations were seldom achieved due to insufficient quality or quantity of M. trossulus eggs. In two of the trials more than two individuals from each species were involved in hybridizations. Most mussels involved in the crosses were subsequently scored at the Me15/16 locus to confirm their species identity. After fertilization, eggs from each cross were distributed in three 1-l glass crystallizing dishes, each with a water volume of 700 ml, at a density of 100,000 eggs per dish. Developing eggs were held undisturbed at 148C and 25‰ salinity to allow development through the trochophore to the early veliger (‘D’ larva) stage over a 72-h period. Larvae were then assessed for (a) the percentage of the eggs that had developed into larvae (5 aliquots/dish 1⁄4% yield) and (b) the percentage of the larvae that exhibited a normal morphology (5 aliquots/dish 1⁄4% normality). Two of the spawning trials (2 and 3) produced a sufficient number and quality of veliger larvae to enable experiments on larval growth at different salinities. The three replicate dishes of veliger larvae from selected crosses (within trials 2 and 3) were pooled and redistributed into four 2-l beakers at a density of 10 larvae/ml. Larvae were grown up to metamorphosis (3–5 weeks) at two salinities – two beakers at 32‰ and two beakers at 20‰. Larvae were reared at 148C according to Beaumont et al. and trials were ended when 50% of the larvae were ‘eyed’ in any one of the beakers. The shell lengths of a random sample of 30 larvae were measured from each beaker using a photographic method. Observations made during water changes indicated that mortalities during the veliger larval stage were negligible. Larval percentage yield, percentage normality and shelllength data were tested for normality and homogeneity of variance before comparing means or medians using ANOVA (parametric data) or Kruskall–Wallis (non-parametric data) tests. Non-normal percentage data were arc-sine transformed and re-tested for normality. Initial tests between replicate dishes within families (72 h data) or between beakers within salinity treatments generally showed no significant differences and the data were therefore pooled within families or within treatments before analysis. Using theMe15/16 locus for species identification, uncertainty was only evident for the BalticM. trossulus, many of which scored as M. edulis or M. edulis hybrids at this locus, a feature already demonstrated by Wood et al. However, these mussels were collected from well inside the Baltic Sea and can safely be regarded as the M. trossulus ‘type’ identified and named by McDonald, Seed & Koehn. We were unable to produce successfully either pure M. trossulus larvae or hybrid larvae from M. trossulus eggs. This was usually due to a consistent reluctance of female Baltic mussels to spawn or possibly simply a lack of females among our broodstock. However, in trial 3, M. trossulus eggs were obtained and were crossed with M. trossulus spermatozoa and heterospecific spermatozoa from both M. galloprovincialis and M. edulis. Very few larvae were produced from any of these crosses (Table 3), and all were abnormal, but this was most probably due to poor quality of the eggs from this particular female, rather than to hybridization, because neither conspecific nor heterospecific spermatozoa produced viable larvae. Correspondence: e-mail: a.r.beaumont@bangor.ac.uk