Strong genetic population structure in the boring giant clam, Tridacna crocea, across the Indo-Malay Archipelago: implications related to evolutionary processes and connectivity

Even though the Indo-Malay Archipelago hosts the world's greatest diversity of marine species, studies on the genetic population structure and gene flow of marine organisms within this area are rather rare. Consequently, not much is known about connectivity of marine populations in the Indo-Malay Archipelago, despite the fact that such information is important to understand evolutionary and ecological processes in the centre of marine biodiversity. This study aims to investigate the genetic population structure of the boring giant clam, Tridacna crocea . The analysis is based on a 456-bp fragment of the cytochrome oxidase I gene from 300 individuals collected from 15 localities across the Indo-Malay Archipelago. Tridacna crocea shows a very strong genetic population structure and isolation by distance, indicating restricted gene flow between almost all sample sites. The observed Φ_ST-value of 0.28 is very high compared to other studies on giant clams. According to the pronounced genetic differences, the sample sites can be divided into four groups from West to East: (i) Eastern Indian Ocean, (ii) Java Sea, (iii) South China Sea, Indonesian throughflow, as well as seas in the East of Sulawesi, and (iv) Western Pacific. This complex genetic population structure and pattern of connectivity, characterised by restricted gene flow between some sites and panmixing between others can be attributed to the geological history and prevailing current regimes in the Indo-Malay Archipelago.     

Distribution,burrowing, and growth rates of the clam Tridacna crocea on interior reef flats

Summary Larvae of the burrowing clam Tridacna crocea (Tridacnidae) settle preferentially on top of detached coral heads lying on the surface of the interior reef flat in the Great Barrier Reef province. This species burrows as it grows, eroding the central top surfaces of coral boulders, producing structures that superficially resemble micro-atolls. Storm surges roll these coral heads onto the now flattened surface, killing the live population of clams, and exposing the fresh underside for unimpeded larval settlement. As these clams grow and burrow into the substratum, the coral head becomes progressively flattened and finally breaks apart. Field observations and growthring data documented growth rate; growth rates plus burrow volumes were converted to annual sediment production. At average population densities approximately 140 gm/m²/yr of coral are eroded. Concomitant with erosion is a calcium carbonate increase in the shell of these clams amounting to 60gm/m²/yr. Assuming a stable population structure, with annual mortality equal to annual estimated growth, total sediment production is 200 gm/m²/yr. Clams are usually aggregated at higher densities, however, with numbers regularly exceeding 100 clams/m². Consequently maximum sediment production rate locally is often 4,500 gm/m²/yr.     

Two kinds of thick filament in smooth muscle cells in the adductor of a clam, Chlamys nobilis

The ultrastructure of the opaque portion of the adductor muscle in the pecten Chlamys nobilis was investigated. The opaque portion was composed of smooth muscle cells that contained thin and thick filaments. The thick filaments were classified into two kinds, thinner and thicker, according to the statistical analysis of diameters. They were also classified as being shorter and longer, when isolated native filaments were examined. The thick filaments may consequently be classified into two kinds: thinner and shorter filaments, and thicker and longer ones. The thinner and shorter filaments were about 26.5 nm in diameter and 7.5 mum in length, and the thicker and longer ones were about 42.0 nm in diameter and 13.0 mum in length, respectively. A regular periodicity was apparent on the surface of the core after removal of myosin molecules from its surface. The periodicity seemed similar for the two kinds of thick filament.     

Intraspecific competition in Tridacna crocea,a burrowing bivalve

Summary Intraspecific competition for space and light occurred when Tridacna crocea burrowed into coralline substratum of boulders on leeward coral reefs in the central Great Barrier Reef near Townsville, Australia. Intensity of competition was linearly related to clam density. Above about 200 clams/m², all clams physically contacted one another and all shells sustained damage. Mortality in isolated populations due to intraspecific competition was estimated at 40%. Principles of intraspecific competition in plants were tested for applicability to T. crocea populations. Juvenile mortality due to competitive stress was density dependent. Aggregated distributions of one year old clams changed to random or regular distribution of adults. Normal size-frequency distribution for juveniles became skewed for older groups. A bimodal size-frequency distribution of the population was related to selective mortality in 1–3 year old clams. Adult mortality due to crowding was less severe but significant. Growth rates were inhibited by competition. Deformations in morphology resulted from crowding. Intraspecific competition for space and light by adults inhibited recruitment of young. Animal adaptations to reduce mortality under crowded conditions were also important. Larvae aggregated on settling and oriented with posterior ends pointed away from nearest neighbors. Positional alignment within the substratum was selectively advantageous. Burrowing posteriorly was preferential, but anterior and sideways burrowing as well as twisting within the burrow were also observed. Movement within substratum served to reduce local damage to the shell. Proteinaceous deposits secreted through perforations in the shell reduced subsequent damage. T. crocea populations exhibited many animal adaptations that reduced mortality during the first years of life, but as cohorts matured, plant-like patterns of competitive interaction became more significant.     

Sequence Variation in the Ribosomal DNA Internal Transcribed Spacer of Tridacna crocea

DNA-based genetic markers are needed to augment existing allozyme markers in the assessment of genetic diversity of wild giant clam populations. The dearth of polymorphic mitochondrial DNA regions amplified from known universal polymerase chain reaction (PCR) primers has led us to search other regions of the genome for viable sources of DNA polymorphism. We have designed tridacnid-specific PCR primers for the amplification of internal transcribed spacer regions. Sequences of the first internal transcribed spacer segment (ITS-1) revealed very high polymorphism, showing 29% variation arising from base substitutions alone. Preliminary restriction analysis of the ITS regions using 8 restriction enzymes revealed cryptic changes in the DNA sequence. These mutations are promising as marker tools for differentiating geographically separated populations. Such variation in the ITS region can possibly be used for population genetic analysis. Received February 1, 2000; accepted May 8, 2000.     

Phagocytosis in invertebrates: Studies on the hemocytes of the clam Tridacna maxima

The two types of cells found in the hemolymph of the clam Tridacna maxima have been examined in vitro by light microscopy, and their morphological charcteristics described and illustrated. It was shown that the hemocytes, which exhibited rapidly spreading cytoplasm and extensive ruffled membrane activity, were able to phagocytose carbon particles experimentally introduced into the animals. Attention is drawn to the part that the phagocytic hemocytes of invertebrates may have to play in putative host defense mechanisms and physiological waste removal processes.     

Four-stranded coiled-coil elastic protein in the byssus of the giant clam, Tridacna maxima

An elastic protein with a secondary structure distinct from all well-known load-bearing proteins is found in the byssus of the giant clam, Tridacna maxima . The byssus consists of a bundle of hundreds of individual threads, each measuring about about 100 μm in diameter, which exhibit a tendon-like mechanical response. The amino acid composition of Tridacna byssus, however, is unlike tendon collagen, lacking high glycine, proline, and hydroxyproline. Wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) measurements suggest that the constituent nanofibrils of the byssal threads are distinct from known secondary structure motifs previously reported for elastic proteins including the collagen triple-helix, the β-sheet nanocrystalline domains of silks, or the double-stranded coiled-coil regions of intermediate filaments. Instead, X-ray diffraction data indicate a structural organization in which four coiled-coil α-helices form a stable rope-like structure, which then further pack in a pseudohexagonal lattice to form nanofibrils. Amino acid composition analysis shows unusually high concentrations of acidic as well as basic residues, suggesting that the four-helix structure is stabilized by strong ionic interactions between oppositely charged residues in neighboring strands. The composition also suggests additional stabilization by disulfide cross-linking. On a larger scale, scanning and conventional transmission electron microscope (STEM and TEM) observations indicate that the nanofibrils exhibit an alternating periodicity of about 500 nm along the axial direction. A molecular model that combines the mechanical properties with the structural characteristics of the Tridacna byssal threads is proposed.     

Ultrastructure of muscle satellite cells in hypersomatotropic rats

Female Wistar-Furth rats were injected at one week of age with cells from either the GH1 or GH3 rat pituitary cell lines. Controls were injected with vehicle. Rats were killed at 11 weeks of age and satellite cells in the soleus and extensor digitorum longus (EDL) muscles were examined using transmission electron microscopy. Satellite cells in both the soleus and EDL muscles of rats with tumours which secreted growth hormone generally appeared to be metabolically more active than those cells seen in the muscles of control rats. The source of pituitary cell line did not appear to influence satellite cell ultrastructure. In rare instances, myofibers of tumor-bearing rats appeared to extend cytoplasmic projections around satellite cells as if to engulf the latter. There was no evidence of a pathological condition. Since only one time frame was observed, the effects of prolonged exposure to elevated blood growth hormone levels on satellite cells are not known.     

Electrophoretic variation of adductor muscle protein and tetrazolium oxidase in the smooth Washington clam, Saxidomus giganteus (Deshayes 1839)

Two electrophoretic polymorphisms (muscle protein 2 alleles and tetrazolium oxidase 3 alleles) were found in Saxidomus giganteus. The muscle protein system showed significant differences between collections from different locations, while the tetrazolium oxidase system showed variation in allele frequency suggestive of a north-south cline.     

Genetic structure of giant clam (Tridacna maxima) populations from reefs in the Western Coral Sea

Allozyme variation at six polymorphic loci was examined in 10 populations of Tridacna maxima from reefs in the Western Coral Sea, to test whether patterns of relatedness previously reported for foraminiferan populations reflected a fundamental structuring of the fauna in the region. Genetic distances (Nei's D) among populations of T. maxima ranged from 0–0.065 and increased with increasing geographical separation. No significant differences in gene frequencies were observed among populations within two groups of reefs identified by cluster analysis: the Great Barrier Reef (GBR), and among the offshore reefs excluding Lihou and Osprey. Significant genetic differences among these groups and the outliers Lihou and Osprey were consistent with the greater geographical separation of populations between areas than within areas. There was no evidence of differentiation along a north-south axis as reported for the foraminiferan Marginopora vertebralis, nor did populations from offshore reefs on the Queensland Plateau form a well-defined group that was genetically distinct from the GBR. The patterns observed for M. vertebralis do not appear to reflect a fundamental structuring of biota in the region. The differences in the pattern of genetic variation for M. vertebralis as compared with those for T. maxima may be due to several differences in the biological characteristics of the two species. The time of breeding in particular may influence the extent to which the divergence of the East Australian Current restricts larval dispersal among reefs in the central Queensland Plateau.     

Responses to temperature,heavy metal and sediment stress by the giant clam Tridacna squamosa

Increasing seawater temperature, heavy metal concentration and sediment load, all represent environmental stress factors found in the Gulf of Thailand. In the present study we investigate and compare the physiological responses of exposure to increased water temperature (+ 3°C) and copper (50 μg Cu/l) by the giant clam Tridacna squamosa, and the behavioural response to sedimentation.

Both temperature and copper caused significant decrease in P_g: R ratios, although by different physiological responses. In clams exposed to copper, the decrease in P_g: R is caused by reduced photosynthesis, whereas the clams exposed to increased temperature maintained a high photosynthetic rate, but significantly increased their respiration.

Clams responded to additional sediment with increased activity. This augmented activity was further increased for clams previously stressed by copper.     

A novel carbonic anhydrase from the giant clam Tridacna gigas contains two carbonic anhydrase domains

This report describes the presence of a unique dual domain carbonic anhydrase (CA) in the giant clam, Tridacna gigas. CA plays an important role in the movement of inorganic carbon (Ci) from the surrounding seawater to the symbiotic algae that are found within the clam's tissue. One of these isoforms is a glycoprotein which is significantly larger (70 kDa) than any previously reported from animals (generally between 28 and 52 kDa). This alpha-family CA contains two complete carbonic anhydrase domains within the one protein, accounting for its large size; dual domain CAs have previously only been reported from two algal species. The protein contains a leader sequence, an N-terminal CA domain and a C-terminal CA domain. The two CA domains have relatively little identity at the amino acid level (29%). The genomic sequence spans in excess of 17 kb and contains at least 12 introns and 13 exons. A number of these introns are in positions that are only found in the membrane attached/secreted CAs. This fact, along with phylogenetic analysis, suggests that this protein represents the second example of a membrane attached invertebrate CA and it contains a dual domain structure unique amongst all animal CAs characterized to date.     

Effects of low light and high temperature on pediveligers of the fluted giant clam Tridacna squamosa

ABSTRACT

This paper examines the responses of the fluted giant clam Tridacna squamosa pediveligers to elevated temperature and reduced light levels. In a light reduction experiment, a total of 104,000 T. squamosa pediveligers were exposed to four different levels of shading for approximately one month. The most heavily shaded treatment, at 0.4% of ambient light, had significantly lower survival than the other groups, which all received 1% or more of ambient light. In a second experiment, for approximately two weeks 13,000 T. squamosa pediveligers were divided among three treatments: one at ambient temperature averaging 29.5 °C, and two with elevated temperatures averaging 32.2 °C and 34.8 °C. The elevated temperature treatments resulted in near total mortality. The highest temperature survived by any pediveliger was 32.8 °C. Our results indicate a potential synergetic effect, with turbidity causing giant clam pediveligers to settle in shallower water―where they will likely be exposed to higher temperatures.     

Polymorphism and crypsis in the boring giant clam (<Emphasis Type="Italic">Tridacna crocea</Emphasis>): potential strategies against visual predators

The giant clam, Tridacna crocea, is a sessile bivalve that bores into rocks and dead corals found on shallow Indo-Pacific coral reefs. Clams are a valuable prey item and T. crocea have developed at least two effective strategies to avoid predation: borrowing, and simultaneous rapid mantle withdrawal and valve contraction. Tridacna crocea obtain their food via photosynthetic products of symbiotic zooxanthellae and from filter-feeding. When they retract their mantles in response to predators, both photosynthesis and feeding are disrupted. Visible polymorphism and crypsis should reduce the frequency of attack and corresponding mantle retraction. In order to determine whether T. crocea is polymorphic and/or cryptic, samples (n = 573) from Tioman Island, Malaysia, were surveyed and photographed. Classification of images into categories of size and mantle colour/pattern demonstrates that T. crocea is colour/pattern polymorphic and morph frequency is size-dependent. Eight morphs were identified, but there also existed considerable intra-morph variation. Morphs with brown and/or and green pigments dominated the larger clam size category (>8 cm). Furthermore, red/blue/green (RGB) values extracted from a subset (n = 93) of digital images of T. crocea showed significant positive correlations between mantle and substrate colours (blue, r = 0.641; green, r = 0.540; red, r = 0.528), indicating background matching (crypsis).     

High variability of genetic pattern in giant clam (Tridacna maxima) populations within French Polynesia

Nine populations of giant clams, Tridacna maxima, from six islands of French Polynesia were screened for allozyme variation at ten polymorphic loci. The genetic structure of populations of T. maxima were studied at different spatial scales: within an island, between islands of the same archipelago and between archipelagos. Significant genetic differences were observed only between populations from different archipelagos, and genetic differentiation was correlated with geographical separation. However, these results were only supported by a single locus, PEP * and all other loci were homogeneous between studied populations. According to Lewontin & Krakauer's model, the genetic structure can be explained by selection. The selective factors most likely depend on the respective habitat of each archipelago. We also studied genotype–phenotype correlation using the colour of the clam mantle, and did not find any relationship between the mantle colour and the genetic structure of the individuals.  © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 77 , 221–231.     

Ultrastructure of muscle cells in Siboglinum fiordicum (Pogonophora)

Two different muscle types are found in the body of Siboglinum fiordicum: body wall muscle and blood vessel muscle. Both are of a myomesothelial type. The myofibrils of the body wall muscle are non-striated and consist of thick and thin myofilaments. Scattered dense bodies and attachment plaques are described. The sarcoplasmic reticulum forms a three-dimensional network in the myofibrils and only peripheral couplings are observed. The thick filaments are of a paramyosin type and have a diameter ranging from 400-1500 A. The blood vessels muscle is non-striated, but sometimes a sarcomere-like organization has been observed. Both thick and thin filaments are present. The thick filaments have a diameter of 250-400 A and lack transverse striations. Dense bodies and attachment of plaques are few. The sparse sarcoplasmic reticulum is restricted to the myofibril periphery where it makes peripheral couplings with sarcolemma. The luminal surface of the vessels is lined by a basal lamina with collagen-like inclusions. No endothelium is found. The body wall muscle and the blood vessel muscle are compared with other muscle types described in invertebrates.