The Crown Pearl: a draft genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758)

Since historical times, the inherent human fascination with pearls turned the freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758) into a highly valuable cultural and economic resource. Although pearl harvesting in M. margaritifera is nowadays residual, other human threats have aggravated the species conservation status, especially in Europe. This mussel presents a myriad of rare biological features, e.g. high longevity coupled with low senescence and Doubly Uniparental Inheritance of mitochondrial DNA, for which the underlying molecular mechanisms are poorly known. Here, the first draft genome assembly of M. margaritifera was produced using a combination of Illumina Paired-end and Mate-pair approaches. The genome assembly was 2.4 Gb long, possessing 105,185 scaffolds and a scaffold N50 length of 288,726 bp. The ab initio gene prediction allowed the identification of 35,119 protein-coding genes. This genome represents an essential resource for studying this species’ unique biological and evolutionary features and ultimately will help to develop new tools to promote its conservation.     

Development and characterization of microsatellite markers for the endangered northern riffleshell mussel Epioblasma torulosa rangiana (Bivalvia: Unionidae)

Primers for six polymorphic microsatellite loci were developed for the endangered northern riffleshell Epioblasma torulosa rangiana from the Sydenham River, Ontario, Canada. These loci along with an additional nine microsatellite loci, developed from other unionoid species, available in the literature, were tested and characterized on individuals from two populations in the Allegheny River; one population in French Creek, PA, USA, and one population from the Sydenham River. Allelic diversity ranged from two to 28 alleles per locus and averaged 13.7 per locus. These primers are being used in a rangewide study on the conservation genetics of remaining extant populations of the northern riffleshell.     

Population density and age structure of the freshwater pearl mussel, Margaritifera margaritifera, in two Iberian rivers

1. The European freshwater pearl mussel, Margaritifera margaritifera (Bivalvia: Unionoida), is one of the most threatened mussels. The Iberian populations of this species are considered peripheral because their distinct characters such as growth rate and longevity, and require development of effective conservation strategies. 2. We assessed population density and age structure of pearl mussels in two Galician rivers (Eo and Masma in north‐west Spain). Four sampling sites were selected in each river to cover stretches of 100 m. The mean density of mussels in each of these sampling transects was estimated using the adaptive sampling technique, given that mussels occur at low densities and are highly aggregated in these rivers. 3. Age structure was inferred for each population using length–age keys. The empty shells encountered during sampling were used to determine the length of the specimens at different ages (years), together with length‐at‐age data from shells previously analysed for computing growth rates from the same rivers. Water samples from both rivers were analysed for typical physicochemical parameters. 4. Mean densities were very variable, even within the same river (from 0.27 to 6.55 m^?2 in the River Eo and from 0.98 to 2.38 m^?2 in the River Masma). Individuals in the 0‐ to 5‐year age class were scarce in both rivers. 5. Margaritifera margaritifera showed a preference for the strip of river bed within 1.5 m from the river bank and avoided sites at greater distances. The species also showed a preference for sites with more than 80% tree cover and avoided sites with <50% cover. 6. Iberian populations exhibit the highest growth rate, together with the lowest maximum age and maximum length known for M. margaritifera. Detailed knowledge about these peripheral Iberian populations will contribute to developing strategies for conservation and management of this endangered species.     

Duration of the parasitic phase determines subsequent performance in juvenile freshwater pearl mussels (Margaritifera margaritifera)

Host–parasite systems have been useful in understanding coevolutionary patterns in sympatric species. Based on the exceptional interaction of the long‐lived and highly host‐specific freshwater pearl mussel (FPM; Margaritifera margaritifera) with its much shorter‐lived host fish (Salmo trutta or Salmo salar), we tested the hypotheses that a longer duration of the parasitic phase increases fitness‐related performance of mussels in their subsequent post parasitic phase, and that temperature is the main factor governing the duration of the parasitic phase. We collected juvenile mussels from naturally and artificially infested fish from eight rivers in Norway. Excysted juvenile mussels were maintained separately for each collection day, under similar temperature and food regimes, for up to 56 days. We recorded size at excystment, post excystment growth, and survival as indicators of juvenile fitness in relation to the duration of the parasitic phase. We also recorded the daily average temperatures for the entire excystment period. We observed strong positive relationships between the length of the parasitic phase and the post parasitic growth rate, size at excystment and post parasitic survival. Temperature was identified as an important factor governing excystment, with higher temperatures decreasing the duration of the parasitic phase. Our results indicate that juvenile mussels with the longest parasitic phase have better resources (larger size and better growth rate) to start their benthic developmental phase and therefore to survive their first winter. Consequently, the parasitic phase is crucial in determining subsequent survival. The temperature dependence of this interaction suggests that climate change may affect the sensitive relationship between endangered FPMs and their fish hosts.     

Development and characterization of microsatellite loci in the endangered oyster mussel Epioblasma capsaeformis (Bivalvia: Unionidae)

Primers for 10 polymorphic microsatellite loci were developed and characterized for the endangered oyster mussel Epioblasma capsaeformis from the Clinch River, Tennessee. Microsatellite loci also were tested in four other populations or species. Amplification was successful for most loci in these closely related endangered species or populations; therefore, a high level of flanking sequence similarity was inferred for this group of species and populations. Allelic diversity ranged from nine to 20 alleles/locus, and averaged 13.6/locus. This study demonstrated the feasibility of using polymerase chain reaction (PCR) primers to amplify microsatellite loci across freshwater mussel species to conduct population genetics studies.     

Glochidium metamorphosis in the endangered freshwater mussel Margaritifera auricularia (Spengler, 1793): a histological and scanning electron microscopy study

The metamorphosis of the glochidium of the critically endangered Margaritifera auricularia in the gills of a host is studied here for the first time. Siberian sturgeon, Acipenser baeri, were infected with glochidia and regularly inspected using scanning and optical microscopy. The mature glochidia immediately attach to the epithelium of the sturgeon gill filaments, piercing the secondary lamellae and the connective tissues, blood cells, and vessels within the lamellae. Once the epithelium is pierced, overlapping host lamellae cover the glochidium and form a cyst. Metamorphosis takes place inside the cyst. Sixteen days after infection the glochidium becomes spherical in shape and the larval muscle is reabsorbed. The two adductor muscles of the juvenile are observed 34 days after infection at 16-20 degrees C. Metamorphosis is complete in approximately 51 days at 18-22 degrees C and in 65 days at 16-17 degrees C. Released juveniles have a spherical shell with a thin rim of new shell material and a finely ciliated foot. Juvenile mean measurements are: length = 190 microm, width = 193 microm, and height = 210 microm.     

Timing of spawning and glochidial release in Scottish freshwater pearl mussel (Margaritifera margaritifera) populations

1. The timing of reproduction was investigated in six Scottish freshwater pearl mussel populations from 1993 to 2002. Gravid females were examined and the release of mussel larvae (glochidia) was monitored. 2. Annual spawning (oviposition) and spat (glochidial release) events occurred during June to July and June to September, respectively. 3. Between‐river differences in timing seem to be related to water temperature. Mussels in the warmest rivers tend to spawn and spat first, and vice‐versa. 4. Thermal variations also seem to influence the timing of reproduction within rivers, which can be delayed by several weeks during cold years. At least 3000°‐days occur between annual episodes of glochidial release. 5. The timing of spawning is determined gradually, probably by a thermal summation effect. 6. The release stage occurs as a sudden, synchronised event, with most of the glochidia spat over 1–2 days, indicating that it is triggered by an environmental cue. Sudden changes in water temperature and/or river level often result in spats, and the underlying mechanism may be respiratory.     

Physical microhabitat requirements of freshwater pearl mussels,Margaritifera margaritifera (L.)

Surface sediment diatoms from the east coast of Lake Tanganyika were analysed using ordination and classification techniques, and compared with assemblages previously described from the northern part of the lake. Grain-size analyses were performed on subsamples. Four groups of diatom assemblages were recognised. The first group clusters samples taken in the north, far from the Rusizi river mouth. The second group comprises samples taken on silty sediment along the Tanzanian coast, including one sample taken near the mouth of the Malagarazi river and those from the northernmost part of the lake. The third group comprises surface sediments along the Burundian coast (near Ramba and Magara), and the fourth is characterised by epipsammic taxa. A sample taken near the central arm of the Malagarazi river is included in the latter group. The impact of small rivers on the diatom assemblages in the surface sediments is restricted to the mouth area.     

Growth characteristics of freshwater pearl mussels,Margaritifera margaritifera (L.)

• 1 Shell growth in the freshwater pearl mussel, Margaritifera margaritifera, was investigated. Three non‐linear growth models (i.e. power, logistic and von Bertalanffy) were fitted to Scottish length‐at‐age data sets and compared.
• 2 Overall, the von Bertalanffy model outperformed the other two approaches, generating the smallest residuals in eight out of 11 samples (the logistic model provided slightly better fits to the other three). It was concluded that individual M. margaritifera appear to grow in an approximately asymptotic fashion and that the von Bertalanffy equation is an appropriate growth model to fit to freshwater pearl mussel length‐at‐age data.
• 3 The ranges in von Bertalanffy parameter estimates observed (k = 0.023–0.075 year^‐1, L_∞ = 77–158 mm, t_o = ‐3.93–4.33 years) are typical of those reported in northern European populations.
• 4 Most of the populations investigated had relatively low k‐values and high maximum age (A_max) estimates. This feature, which suggests high long‐term productivity and less vulnerability to decline (i.e. larger, longer‐living mussels produce more offspring), may be a reason why these populations have survived until now. The population which appears to be the most vulnerable (i.e. which has the highest k and lowest A_max) is probably not recruiting adequately at present.
• 5 An index of absolute growth (mean shell length‐at‐age) was also used for comparing different populations. Observed between‐ and within‐river differences in mussel growth patterns may be associated with a number of environmental factors, particularly water temperature and productivity.
• 6 A significant positive relationship between river length and mean mussel length‐at‐age was observed. In general, mussels grow large in large, cold rivers and vice versa, although there are exceptions which suggest that additional factors may be involved.

     

Phylogeography and conservation genetics of endangered European Margaritiferidae (Bivalvia: Unionoidea)

Margaritifera margaritifera and M. auricularia are among the most endangered freshwater mussels in the world, and the only species of the genus found in Europe. Our genetic study explores allozymic variability (27 loci) and differentiation at the mitochondrial sequence level (partial COI and 16S rRNA gene sequences). The Spanish M. auricularia population showed genetic parameters of variation that were of the same order as those of other freshwater molluscs (though at the lower end of the range), probably permitting its potential recovery. The difference between this species and M. margaritifera was clearly established (ten diagnostic allozymic loci, Nei = 0.462, and mean nucleotide divergence around 9.4%). The M. margaritifera populations analysed showed a certain degree of population genetic structure (according to allozyme data) that was not, however, related to a geographical cline. Nevertheless, two mitochondrial lineages (albeit very closely related) were identified: a northern lineage extending from Ireland to the Kola Peninsula including the western Atlantic coast, and a second cluster distributed from Ireland to the Iberian Peninsula. The phylogenetic relationships between these two species and other related taxa were established. The putative M. m. durrovensis could be considered an 'ecophenotype'. Palaeobiogeographical scenarios are presented and indicate unexpected 'recent' gene flow between M. margaritifera populations that were theoretically isolated in the early Tertiary.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society 2003, 78, 235–252.     

Threatened freshwater pearl mussel <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> L. in NW Spain: low and very structured genetic variation in southern peripheral populations assessed using microsatellite markers

A genetic analysis of freshwater pearl mussel Margaritifera margaritifera populations from NW Spain, a peripheral area of its European distribution, was carried out using microsatellite markers. These populations were formerly reported as genetically differentiated on the basis of growth and longevity studies. Ten loci previously characterized in populations from central Europe were used to comparatively analyze the genetic variability at the southern edge of the species’ range. Iberian pearl mussel populations showed very low genetic variability and significant high genetic differentiation. Half of the total genetic diversity observed appeared to be distributed between populations, which suggested a highly structured adaptive potential in pearl mussel at the southern peripheral distribution of the species. Population distinctiveness was evidenced by assignment tests, which revealed a high accuracy of individual assignments to their population of origin. All data suggested low effective population size and major effects of genetic drift on population genetic structure. In order to avoid further loss of genetic variation in biologically distinctive populations from NW Spain, prioritization of genetic resources of this species is required for conservation and management.     

Isolation and characterization of microsatellite loci in the western pearlshell mussel, Margaritifera falcata (Gould)

Ten microsatellite loci were isolated from the western pearlshell, Margaritifera falcata (Gould, 1850) and characterized in populations from Washington and Montana, USA. We also assessed eight microsatellite loci developed in M. margaritifera, two of which showed utility. Both of our test populations showed significant heterozygote deficiencies at most loci, consistent with a hermaphroditic life history. Populations differed markedly with respect to allelic richness, allele frequencies and numbers of identical multilocus genotypes. This panel of loci should prove useful in describing gene flow and genetic diversity patterns among M. falcata populations, information that should aid future conservation efforts.     

The revenant: rediscovery of Margaritifera homsensis from Orontes drainage with remarks on its taxonomic status and conservation (Bivalvia: Margaritiferidae)

Since Margaritifera marocana (Pallary, 1918) and M. laosensis (Lea, 1863) were rediscovered, M. homsensis (Lea, 1865) remains the only pearl mussel species known solely based on old shell samples from natural history museums. This is also the last pearl mussel species, which is absent in a phylogeny of the family. Here, we aimed to provide an integrative revision of the taxonomic status of M. homsensis from the Orontes Basin. Using a newly collected specimen from the River Karasu, Hatay Province, southern Turkey, five gene partitions were sequenced, the cytochrome c oxidase subunit I (COI), large ribosomal subunit rRNA (16S), large ribosomal subunit rDNA (28S) and its D3 expansion segment (D3), and small ribosomal subunit rDNA (18S). The multi-gene phylogeny indicates that M. homsensis is a sister taxon of M. auricularia, but both these species are closely related to M. marocana by nuclear genes. The main conchological features, i.e., the shell shape, teeth morphology, and mantle attachment scars, as well as Fourier shell shape analysis have not shown principal differences between M. homsensis and M. auricularia. Based on these data, we concluded that M. homsensis is a valid species that is most closely related to M. auricularia. Special conservation efforts for a population of M. homsensis discovered in Turkey, including the formation of a nature reserve, might contribute to the conservation of the species. Finally, an extensive search for surviving populations in Orontes drainage (southern Turkey, Lebanon, and Syria) and the Nahr-el-Kabir River (Lebanon and Syria) remains necessary to develop a transboundary conservation strategy for this unique taxon.     

Physicochemical stream bed characteristics and recruitment of the freshwater pearl mussel (Margaritifera margaritifera)

1. The freshwater pearl mussel (Margaritifera margaritifera) is endangered and of conservation importance. We used its survival/mortality during the critical post‐parasitic phase as a biological indicator for the habitat quality of the stream substratum. 2. We established and tested biological, physical and chemical methods of assessing the stream bed in 26 streams from seven European countries. We analysed penetration resistance, texture, the concentrations and ratios of C, N, S, P, Fe, Mn in fine material <100 μm, and redox, pH and electric conductivity at the surface and at 5 and 10 cm into the substratum. 3. Sites with high stream bed quality (promoting pearl mussel populations with good juvenile recruitment) had coarser and better sorted substrata with significantly lower quantities of fines, and a higher Mn concentration in the fines, than poor quality sites. Redox potential (Eh) at sites without recruitment differed markedly between the free‐flowing water at the surface and at 5 and 10 cm in the bed, whereas no differences were detectable at good quality sites. This was also true of electric conductivity and, to a lesser extent, pH. The stream bed at sites lacking pearl mussel recruitment had a more variable and higher penetration resistance, indicating clogging of the interstitial macropore system by the deposition of mud and compaction of the stream bed. 4. Our results show that habitat quality for pearl mussels depends strongly on the exchange between the surface and the interstices, which is governed by physicochemical characteristics of the stream substratum. Combined measurements of penetration resistance, depth gradients of Eh and texture were most suitable for assessing stream bed quality, while water chemistry was insufficient because of the decoupling of interstitial and free‐flowing water at poor quality sites.     

Development and characterization of novel tetra‐ and dinucleotide microsatellite markers for the French Polynesia black‐lipped pearl oyster,Pinctada margaritifera

Ten microsatellite loci were developed for the black‐lipped pearl oyster Pinctada margaritifera with a magnetic bead enrichment protocol. These tetra‐ and dinucleotide markers were polymorphic, with 10 to 43 alleles observed in 97 individuals from two Tuamotu atoll populations. Most loci revealed significant genic differentiation between the two populations and also exhibited some degree of heterozygote deficiencies, probably due to the presence of null alleles. These loci should be very useful to describe genetic structure, genetic variability and reproductive success in the various aquaculture and wild populations of pearl oyster in French Polynesia.     

Genetic diversity and differentiation of central European freshwater pearl mussel (Margaritifera margaritifera L.) populations: implications for conservation and management

Despite the fact that mollusc species play an important role in many aquatic ecosystems, little is known about their biodiversity and conservation genetics. Freshwater pearl mussel (Margaritifera margaritifera L.) populations are seriously declining all over Europe and a variety of conservation programs are being established to support the remaining endangered central European populations. In order to provide guidelines for conservation strategies and management programs, we investigated the genetic structure of 24 freshwater pearl mussel populations originating from five major central European drainages including Elbe, Danube, Rhine, Maas and Weser, representing the last and most important populations in this area. We present a nondestructive sampling method of haemolymph for DNA analyses, which is applicable for endangered bivalves. The analyses of nine microsatellite loci with different levels of polymorphism revealed a high degree of fragmented population structure and very different levels of genetic diversity within populations. These patterns can be explained by historical and demographic effects and have been enforced by anthropogenic activities. Even within drainages, distinct conservation units were detected, as revealed from high F(ST) values, private alleles and genetic distance measures. Populations sampled close to contact zones between main drainage systems showed lowest levels of correct assignment to present-day drainage systems. Populations with high priority for conservation should not only be selected by means of census population size and geographical distance to other populations. Instead, detailed genetic analyses are mandatory for revealing differentiation and diversity parameters, which should be combined with ecological criteria for sustainable conservation and recovery programs.     

Parasitic freshwater pearl mussel larvae (Margaritifera margaritifera L.) reduce the drift-feeding rate of juvenile brown trout (Salmo trutta L.)

In this paper we describe, for the first time, the effects of freshwater pearl mussel (Margaritifera margaritifera L.) encystment on the drift-feeding behavior of juvenile brown trout (Salmo trutta L.). Because both mussel and salmonid populations are often threatened, this study not only adds knowledge to the understanding of host-parasite systems, but it is also of conservation value. Individual trout, mussel-encysted (25.1?±?5.7 larvae?·?g^?1 body weight, n?=?5) or non-encysted (n?=?5), were fed with chironomid larvae in a flow-through stream aquarium. Feeding trials were filmed and analyzed by counting the numbers of chironomid larvae each individual ate, and by estimating the prey-capture distance. Non-encysted trout had a significantly higher drift-foraging rate than did encysted trout, and they captured significantly more prey further away from their focal point. The reduced foraging success of encysted trout was mainly due to their failure to catch prey relatively further from their focal point. This suggests that reduced foraging success of encysted trout may be due to poorer energetic status, but the physical effects of mussel larvae on prey handling time cannot be ruled out. Encysted trout caught approximately 20 % fewer prey, which would result in a reduction in growth potential during the period of mussel encystment. Reduced energetic status might also result in reduced competitive ability or in increased exposure to predation risk.     

Characterization of microsatellite DNA loci for the southern flying squirrel (Glaucomys volans)

Polymerase chain reaction primers for microsatellite DNA loci (one dinucleotide, four tetranucleotide and two compound) and the conditions necessary to amplify each are described for the southern flying squirrel (Glaucomys volans). These primers were tested on 22 or more individuals from a population at the Savannah River Site in South Carolina. These microsatellite primers yielded a high allelic diversity (6–22 alleles/locus), and moderate to high observed heterozygosities (0.318–0.826). Primers developed for the northern flying squirrel (Glaucomys sabrinus) were also tested for use on G. volans, with only two successful cross amplifications from the seven loci.     

Optimization of diet and culture environment for larvae and juvenile freshwater pearl mussels,Hyriopsis (Limnoscapha) myersiana Lea, 1856

Summary

Culture of the freshwater pearl mussel, Hyriopsis (Limnoscapha) myersiana, was carried out in three consecutive steps: (1) culture of glochidia larvae in artificial media, (2) rearing the early juveniles (0–120 days old) in a nursery, and (3) rearing the juveniles (120–360 days old) in an earthen pond. The percentage survival of glochidia in standard tissue culture medium (M199) supplemented with common carp plasma was 95±2.5. All surviving larvae (100%) transformed to juveniles, the duration of transformation being 8 days. The early juveniles (0–60 days old) were fed with a mixture of four selected phytoplankton species (Chlorella sp., Kirchneriella incurvata, Navicula sp. and Coccomyxa sp.). The survival rate of juveniles was 8±0.2%. The average length of these juveniles increased from 0.13±0.01 mm to 1.41±0.16 mm and the average height from 0.16±0.01 mm to 0.98±0.09 mm. Subsequently, 60–120-day juveniles were fed with one of the same four phytoplankton species or a combination of the four. Feeding the juveniles with K. incurvata resulted in the highest survival rate (65±8.32%), with an average length of 3.46±0.04 mm and an average height of 1.94±0.04 mm. Finally, the 120–360-day juveniles were cultured in an earthen pond. There were progressive changes in average weight (0.0037±0.002 g to 11.24±5.02 g), length (3.48±0.39 mm to 54.08±6.21 mm), height (1.97±0.24 mm to 25.09±2.48 mm) and width (0.98±0.06 mm to 12.28±3.21 mm) from 120 to 360 days. The average growth rates per day of these parameters were 0.0497±0.01 g, 0.2414±0.15 mm, 0.0975±0.08 mm and 0.0493±0.03 mm, respectively. H. (L.) myersiana juveniles developed the complete structural composition of the adult by 160 days, and at 360 days, gametogenesis was complete.