Ricardo Beldade

ALLOPATRIC DIVERGENCE AND SPECIATION IN CORAL REEF FISH: THE THREE‐SPOT DASCYLLUS, DASCYLLUS TRIMACULATUS, SPECIES COMPLEX

Long pelagic larval phases and the absence of physical barriers impede rapid speciation and contrast the high diversity observed in marine ecosystems such as coral reefs. In this study, we used the three‐spot dascyllus (Dascyllus trimaculatus) species complex to evaluate speciation modes at the spatial scale of the Indo‐Pacific. The complex includes four recognized species and four main color morphs that differ in distribution. Previous studies of the group using mitochondrial DNA revealed a noncongruence between color morphs and genetic groupings; with two of the color morphs grouped together and one color morph separated into three clades. Using extensive geographic sampling of 563 individuals and a combination of mitochondrial DNA sequences and 13 nuclear microsatellites, we defined population/species boundaries and inferred different speciation modes. The complex is composed of seven genetically distinct entities, some of which are distinct morphologically. Despite extensive dispersal abilities and an apparent lack of barriers, observed genetic partitions are consistent with allopatric speciation. However, ecological pressure, assortative mating, and sexual selection, were likely important during periods of geographical isolation. This study therefore suggests that primarily historical factors later followed by ecological factors caused divergence and speciation in this group of coral reef fish.

Larger female fish contribute disproportionately more to self-replenishment

While chance events, oceanography and selective pressures inject stochasticity into the replenishment of marine populations with dispersing life stages, some determinism may arise as a result of characteristics of breeding individuals. It is well known that larger females have higher fecundity, and recent laboratory studies have shown that maternal traits such as age and size can be positively associated with offspring growth, size and survival. Whether such fecundity and maternal effects translate into higher recruitment in marine populations remains largely unanswered. We studied a population of Amphiprion chrysopterus (orange-fin anemonefish) in Moorea, French Polynesia, to test whether maternal size influenced the degree of self-recruitment on the island through body size–fecundity and/or additional size-related maternal effects of offspring. We non-lethally sampled 378 adult and young juveniles at Moorea, and, through parentage analysis, identified the mothers of 27 self-recruits (SRs) out of 101 recruits sampled. We also identified the sites occupied by each mother of an SR and, taking into account variation in maternal size among sites, we found that females that produced SRs were significantly larger than those that did not (approx. 7% greater total length, approx. 20% greater biomass). Our analyses further reveal that the contribution of larger females to self-recruitment was significantly greater than expected on the basis of the relationship between body size and fecundity, indicating that there were important maternal effects of female size on traits of their offspring. These results show, for the first time in a natural population, that larger female fish contribute more to local replenishment (self-recruitment) and, more importantly, that size-specific fecundity alone could not explain the disparity.

Full-Sibs in Cohorts of Newly Settled Coral Reef Fishes

Reef fishes exhibit a bipartite life cycle where a benthic adult stage is preceded by a pelagic dispersal phase during which larvae are presumed to be mixed and transported by oceanic currents. Genetic analyses based on twelve microsatellite loci of 181 three-spot dascyllus (Dascyllus trimaculatus) that settled concurrently on a small reef in French Polynesia revealed 11 groups of siblings (1 full sibs and 10 half-sibs). This is the first evidence that fish siblings can journey together throughout their entire planktonic dispersal phase (nearly a month long for three-spot dascyllus). Our findings have critical implications for the dynamics and genetic structure of fish populations, as well as for the design of marine protected areas and management of fisheries.

Evidence for Cohesive Dispersal in the Sea

As with many marine species, the vast majority of coral-reef fishes have a bipartite life cycle consisting of a dispersive larval stage and a benthic adult stage. While the potentially far-reaching demographic and ecological consequences of marine dispersal are widely appreciated, little is known of the structure of the larval pool and of the dispersive process itself. Utilizing Palindrome Sequence Analysis of otolith micro-chemistry (PaSA;) we show that larvae of Neopomacentrus miryae (Pomacentridae) appear to remain in cohesive cohorts throughout their entire pelagic larval duration (PLD; ∼28 days). Genetically, we found cohort members to be maternally (mtDNA) unrelated. While physical forcing cannot be negated as contributing to initial cohort formation, the small scale of the observed spatial structure suggests that some behavioral modification may be involved from a very early age. This study contributes to our ongoing re-evaluation of the processes that structure marine populations and communities and the spatial scales at which they operate.

Multiple paternity and competition in sympatric congeneric reef fishes, Embiotoca jacksoni and E. lateralis

The black surfperch Embiotoca jacksoni and the striped surfperch E. lateralis (Embiotocidae, Perciformes) are livebearing temperate reef fishes that live sympatrically over a large portion of their distribution range, where they exhibit strong ecological competition. In order to assess whether mating strategies reflect competition, we investigated multiple paternity in these two species in an area of sympatry. We sampled 24 pregnant females (12 for each species) in Monterey Bay, California, used microsatellite analysis and assessed paternity with the COLONY software. While broods are relatively small (12 to 36 offspring), they were always sired by multiple fathers (2 to 9), with no correlation between the size of a brood and the number of fathers. The number of sires for each brood was not significantly different between the two species (approximately 3.5 sires per brood). We tested the deviation from stochasticity of fathered offspring for each father in one brood. Results showed a significant deviation for both E. jacksoni and E. lateralis. However, this deviation was not found to be significant between species. The striking similarity in the dynamics of multiple paternity in these species, when sampled in sympatry, may result from several alternative scenarios, including phylogenetic inertia, reproductive behaviour, and ecological competition.

Composition and temporal dynamics of a temperate rocky cryptobenthic fish assemblage

Temporal variation in a temperate cryptobenthic fish assemblage at the Arrabida Marine Park (Portugal) was assessed by visual surveys during 2002 and 2003. A total of 9596 fish from 11 families and 30 species was recorded. There were no changes in structure or density at the assemblage level between years, whereas diversity changed significantly due to a higher number of abundant species in the second year. A similar seasonal trend was found between years, with a significant overall density increase in autumn. This is partially explained by the arrival of new recruits of some of the most abundant species in the assemblage. Assemblage diversity and structure also changed across seasons. A group of species encompassing Gobius xanthocephalus , Tripterygion delaisi , Parablennius pilicornis , Gobius paganellus , Lepadogaster candollii and Lepadogaster spp. were analysed in detail. The temporal patterns of two of the most abundant species, G. xanthocephalus and T. delaisi , mimicked the overall temporal patterns of the assemblage. We suggest that the inter-annual stability in density of this subtidal fish assemblage may be similar to what has been reported for the intertidal and that strong post-settlement processes are probably shaping this assemblage.

Isolation and characterization of eight polymorphic microsatellite markers from the orange-fin anemonefish, Amphiprion chrysopterus

Eight polymorphic microsatellite loci were isolated and characterized from the orange-fin anemonefish (Amphiprion chrysopterus). These loci provided markers with polymorphisms of 4–14 alleles per locus within 151 individuals from Moorea, French Polynesia. The expected heterozygosities ranged from 0.53 to 0.88. High variability suggests that these markers should be useful to study paternity, population structure, and connectivity in this species.

Lime Juice and Vinegar Injections as a Cheap and Natural Alternative to Control COTS Outbreaks.

Outbreaks of the corallivorous crown-of-thorns seastar Acanthaster planci (COTS) represent one of the greatest disturbances to coral reef ecosystems in the Indo-Pacific, affecting not only coral reefs but also the coastal communities which rely on their resources. While injection approaches are increasingly used in an attempt to control COTS densities, most of them display severe drawbacks including logistical challenges, high residual environmental impacts or low cost-effectiveness. We tested a new alternative control method based upon acidic injections of cheap, 100% natural products. We investigated the lethal doses, intra- and inter-specific disease transmission and immune responses of COTS when injected with fresh lime juice (extracted from local Citrus arantifolia) and white spirit vinegar. High COTS mortality was achieved with small volumes: 10–20 ml per seastar induced death in 89%/97% of injected specimens after an average 34.3 h/29.8 h for lime juice and vinegar respectively. Highest efficiency was reached for both solutions with double shots of (2 × 10 ml) in two different areas on the body: 100% mortality occurred within 12–24 h, which is similar or faster compared with other current injection methods. Multiple immune measures suggested that death was very likely caused by pH stress from the acidic solutions rather than a bacterial infection. Contagion to either conspecifics or a variety of other reef species was not observed, even at COTS densities 15 times higher than the highest naturally reported. 10 to 20 l lime juice/vinegar could kill up to a thousand COTS at a cost of less than 0.05 USD per specimen; no permits or special handling procedures are required. We conclude that injections of lime juice and vinegar offer great advantages when compared to current best practises and constitute a cheap and natural option for all reefs affected by COTS.

Incorporating historical and ecological genetic data for leopard grouper (Mycteroperca rosacea) into marine reserve design in the Gulf of California

Effective design of marine reserves for use in fisheries management and conservation requires a clear understanding of patterns of larval transport and sink-source dynamics between populations, as well as a clear understanding of population demography. Mitochondrial and nuclear markers were analyzed to investigate potential mechanisms impacting connectivity among and the demographic history of subpopulations of a commercially important species in the Gulf of California, the leopard grouper (Mycteroperca rosacea). Demographic history and connectivity analyses included a coalescent analysis, estimating neutrality indices, estimating global and pairwise F’ST, ΦST, or G’’ST, and a priori methodologies to identify genetically distinct units and barriers to dispersal. Average, long-term connectivity between geographic regions in the Gulf was also estimated. Divergence of mitochondrial lineages of leopard grouper dated to the late Pleistocene, with deep-water islands serving as demographically stable populations that may have acted as sources for new populations during periods of climate variability. Additionally, we observed genetically distinct units of leopard grouper in the Gulf, particularly between peninsular and mainland sites, as well as asymmetrical migration between the northern and central Gulf. Observed patterns of genetic differentiation are likely attributed to complex asymmetrical oceanographic currents and local larval retention. Based on our genetic findings and current fishing pressure in certain regions, we recommend implementing small, upstream no-take zones in the areas east of Isla Ángel de la Guarda, around Isla San Lorenzo and Isla San Esteban, and north of Isla Tiburón, that would enhance connectivity among subpopulations, preserve sites with high genetic diversity, and benefit fisheries downstream of these sites.

Isolation and characterization of 13 polymorphic nuclear microsatellite primers for the widespread Indo‐Pacific three‐spot damselfish, Dascyllus trimaculatus, and closely related D. auripinnis

A set of 13 simple sequence repeat markers was developed from D. trimaculatus genomic DNA, tested for D. auripinnis and characterized using 40 individuals per species. All the loci were polymorphic with a number of alleles ranging from three to 30. Observed heterozygosities varied from 0.23 to 0.89 for D. trimaculatus and from 0.11 to 0.85 for D. auripinnis. Early results show that these will be powerful markers for the study of ecological and evolutionary mechanism in this coral reef fish species complex.