Mechanisms of reproductive isolation among sympatric broadcast-spawning corals of the Montastraea annularis species complex

Many coral species spawn simultaneously and have compatible gametes, leading to controversy over the nature of species boundaries and the frequency with which hybridization occurs. Three western Atlantic corals, Montastraea annularis, M. faveolata, and M. franksi, typify this controversy; they all spawn sympatrically on the same evenings after the fall full moons. Here we show, in both Panama and the Bahamas for multiple years, how a variety of mechanisms may act in concert to reproductively isolate all three species. Field studies indicate that M. franksi spawns two hours earlier than the other two species, and the eggs released during this earlier period disperse an average of 500 m by the time the other species spawn. Field measures of fertilization indicate that peak fertilization occurs when spawning synchrony is high and that corals that spawn at the tails of the spawning distributions have greatly reduced fertilization success. Laboratory studies indicate that there is a gametic incompatibility between M. faveolata and the other two species. There are regional differences in the gametic compatibility of M. franksi and M. annularis. In Panama, the two species are completely compatible, whereas in the Bahamas, M. franksi sperm can fertilize M. annularis eggs but the reciprocal cross often fails. Gamete age influences patterns of fertilization, such that very young eggs seem resistant to fertilization and old sperm lose viability after two hours. In sum, the combination of temporal differences in spawning, sperm aging, gamete dispersal and dilution, and gametic incompatibility act in various combinations among the three species, making it unlikely that hybrid fertilization would occur.     

Quantifying the colony shape of the Montastraea annularis species complex

The reef coral Montastraea annularis has been used in a wide range of investigations. Recently, it has been recognized as a complex of three species based on field observations of the variation in colony shape. These observations have also been confirmed by molecular methods as well as morphometrics on individual corallites in the colonies. This paper presents a new quantitative method for measuring overall colony shape based on geostatistics. Seventeen colonies collected from San Blas, Panama in 1995 and 1996 were examined using the Polhemus 3SPACE FASTRAK system to construct three-dimensional coordinates of the center of several hundred corallites in each colony. This method measures the larger bumps or “ridges” as well as the smaller bumps or “lumps” of the colonies. Variograms were then calculated for all the specimens and the lag distances and the values of the variogram were used in a multivariate statistical analysis. Overall, this method indicated that M. annularis and Montastaea faveolata overlap in their relative amount of bumpiness in colony shape while Montastraea franksi is distinct from the other two species. This method has implications for both the modern and fossil record of Montastraea as well as other organisms with similar shapes.     

Genetic, spatial, and temporal components of precise spawning synchrony in reef building corals of the Montastraea annularis species complex

When organisms release gametes into the sea, synchrony must be precise to increase fertilization and decrease hybridization. We tagged and genotyped over 400 spawning corals from the three species in the Montastraea annularis species complex. We report on the influence of species, individuals, and genotypes on timing of spawning from 2002 through 2009. During their annual spawning event M. franksi spawns on average 2 h after sunset, whereas M. annularis and M. faveolata spawn 3.5 h after sunset. Only M. franksi and M. annularis have compatible gametes. Individual colonies of the same genotype spawn at approximately the same time after sunset within and across years (within minutes), but different genotypes have significantly different spawning times. Neighboring colonies, regardless of genotype, spawn more synchronously than individuals spaced further apart. At a given distance, clone-mates spawn more synchronously than nonclone-mates. A transplant experiment indicates a genetic and environmental influence on spawn time. There is strong, but not absolute, concordance between spawn time, morphology, and genetics. Tight precision in spawning is achieved via a combination of external cues, genetic precision, and perhaps conspecific signaling. These mechanisms are likely to influence reproductive success and reproductive isolation in a density-dependent manner.     

Molecular determination of species boundaries in corals: genetic analysis of the Montastraea annularis complex using amplified fragment length polymorphisms and a microsatellite marker

Analyses of DNA have not been widely used to distinguish coral sibling species. The three members of the Montastraea annularis complex represent an important test case: they are widely studied and dominate Caribbean reefs, yet their taxonomic status remains unclear. Analysis of amplified fragment length polymorphisms (AFLPs) and a microsatellite locus, using DNA from sperm, showed that Montastraea faveolata is genetically distinct. One AFLP primer yielded a diagnostic product (880 bp in M. faveolata 920 bp in M. franksi and M. annularis) whose homology was established by DNA sequencing. A second primer revealed a 630 bp band that was fixed in M. faveolata, and rare in M. franksi and M. annularis; in this case homologies were confirmed by Southern hybridizations. A tetranucleotide microsatellite locus with several alleles exhibited strong frequency differences between M. faveolata and the other two taxa. We did not detect comparable differences between M. annularis and M. franksi with either AFLPs (12 primers screened) or the microsatellite locus. Comparisons of AFLP patterns obtained from DNA from sperm, somatic tissues, and zooxanthellae suggest that the technique routinely amplifies coral (animal) DNA. Thus analyses based on somatic tissues may be feasible, particularly after diagnostic differences have been established using sperm DNA.     

Geographic differences in species boundaries among members of the Montastraea annularis complex based on molecular and morphological markers

The three members of the Montastraea annularis complex (M. annularis, M. franksi, and M. faveolata) are dominant reef builders in the western Atlantic whose species status has been controversial for over a decade. Although differences in colony morphology and reproductive characteristics exist, interspecific fertilizations are possible in the laboratory and genetic differentiation is slight. Here we compare the three taxa genetically and morphologically in Panama and the Bahamas, widely separated locations spanning most of their geographic ranges. In Panama, analyses of three AFLP loci, a noncoding region of the mitochondrial genome, and ITS sequences reveal that M. faveolata is strongly differentiated genetically. Discriminant function analysis also indicates no overlap with the other two species in the fine structure of the corallites that comprise the colony. Genetic analyses of larvae from interspecific crosses between M. faveolata and the other two taxa confirmed the hybrid status of the larvae, but no examples of the most probable F1 genotype were observed in the field. Although M. annularis and M. franksi were more similar, they also exhibited strong frequency differences at two AFLP loci and in the mitochondrial noncoding region, as well as distinct corallite structure. In the Bahamas, in contrast, the three taxa exhibited overlapping morphologies. Montastraeafranksi and M. annularis were indistinguishable genetically, and M. faveolata was distinct at fewer genetic loci. Once again, however, the most probable F1 genotype involving M. faveolata was not observed. Geographic differences between Panama and the Bahamas explain why past studies have come to different conclusions concerning the status of the three species. In general, the genetic and morphological data suggest a north to south hybridization gradient, with evidence for introgression strongest in the north. However, reproductive data show no such trend, with intrinsic barriers to gene flow comparable or stronger in the north.     

Asymmetric conspecific sperm precedence in relation to spawning times in the Montastraea annularis species complex (Cnidaria: Scleractinia)

In broadcast spawners, prezygotic reproductive isolation depends on differences in the spatial and temporal patterns of gamete release and gametic incompatibility. Typically, gametic incompatibility is measured in no‐choice crosses, but conspecific sperm precedence (CSP) can prevent hybridization in gametes that are compatible in the absence of sperm competition. Broadcast spawning corals in the Montastraea annularis species complex spawn annually on the same few evenings. Montastraea franksi spawns an average of 110 min before M. annularis, with a minimum gap of approximately 40 min. Gametes are compatible in no‐choice heterospecific assays, but it is unknown whether eggs exhibit choice when in competition. Hybridization depends on either M. franksi eggs remaining unfertilized and in proximity to M. annularis when the latter species spawns or M. franksi sperm remaining in sufficient viable concentrations when M. annularis spawns. We found that the eggs of the early spawning M. franksi demonstrate strong CSP, whereas CSP appears to be lacking for M. annularis eggs. This study provides evidence of diverging gamete affinities between these recently separated species and suggests for the first time that selection may favour CSP in earlier spawning species when conspecific sperm is diluted and aged and is otherwise at a numeric and viability disadvantage with heterospecific sperm.     

Patterns of association between Symbiodinium and members of the Montastraea annularis species complex on spatial scales ranging from within colonies to between geographic regions

Patterns of associations between coral colonies and the major clades of zooxanthellae can vary across scales ranging from individual colonies to widely separated geographic regions. This is exemplified in this study of the Montastraea annularis species complex from six sites on the Mesoamerican Reef, Belize and nine sites in the Bocas del Toro archipelago, Panama. Restriction fragment length polymorphism (RFLP) analysis of small subunit ribosomal DNA (SSU rDNA) was used to identify the zooxanthellae. In Belize (M. annularis), Symbiodinium B (79% of the colonies), Symbiodinium A, and Symbiodinium C were observed. In Panama (primarily M. franksi, but also M. annularis and M. faveolata), there was greater diversity and evenness with Symbiodinium A, B, C, C′ (a new symbiont) and D all being common in at least some host/habitat combinations. Non-metric multidimensional scaling ordinations showed that distribution patterns of symbionts across sites are best explained by enclosure (relative influence of open ocean vs. coastal water) and total suspended solids. Because members of clade D are known to be temperature resistant and Symbiodinium C′ was found in environments characterized by high sedimentation, these Panamanian reefs may have importance from a management perspective as reservoirs of corals better able to tolerate human impacts.     

Repopulation of Zooxanthellae in the Caribbean corals Montastraea annularis and M. faveolata following experimental and disease-associated bleaching.

Caribbean corals of the Montastraea annularis species complex associate with four taxa of symbiotic dinoflagellates (zooxanthellae; genus Symbiodinium) in ecologically predictable patterns. To investigate the resilience of these host-zooxanthella associations, we conducted field experiments in which we experimentally reduced the numbers of zooxanthellae (by transplanting to shallow water or by shading) and then allowed treated corals to recover. When depletion was not extreme, recovering corals generally contained the same types of zooxanthellae as they did prior to treatment. After severe depletion, however, recovering corals were always repopulated by zooxanthellae atypical for their habitat (and in some cases atypical for the coral species). These unusual zooxanthellar associations were often (but not always) established in experimentally bleached tissues even when adjacent tissues were untreated. Atypical zooxanthellae were also observed in bleached tissues of unmanipulated Montastraea with yellow-blotch disease. In colonies where unusual associations were established, the original taxa of zooxanthellae were not detected even 9 months after the end of treatment. These observations suggest that zooxanthellae in Montastraea range from fugitive opportunists and stress-tolerant generalists (Symbiodinium A and E) to narrowly adapted specialists (Symbiodinium B and C), and may undergo succession.     

Zooxanthellae of the Montastraea annularis species complex: patterns of distribution of four taxa of Symbiodinium on different reefs and across depths.

Corals of the Montastraea annularis complex host several different dinoflagellates in the genus Symbiodinium. Here we address two questions arising from our previous studies of these associations on an offshore reef. First, do the same taxa and patterns of association (Symbiodinium A and B found in higher irradiance habitats than Symbiodinium C) occur on an inshore reef? Second, does M. franksi at the limits of its depth range host only Symbiodinium C, as it does at intermediate depths? In both surveys, a new Symbiodinium taxon and different patterns of distribution (assayed by analyses of small ribosomal subunit RNA genes [srDNA]) were observed. Inshore, a taxon we name Symbiodinium E predominated in higher irradiance habitats in M. franksi and its two sibling species; the only other zooxanthella observed was Symbiodinium C. Offshore, M. franksi mainly hosted Symbiodinium C, but hosted Symbiodinium A, B, C, and E in shallow water and Symbiodinium E and C in very deep water. Symbiodinium E may be stress-tolerant. Observed srDNA heterogeneity within samples of Symbiodinium B, C, and E is interpreted as variation across copies within this multigene family. Experimental bleaching of Symbiodinium C supported this interpretation. Thus sequences from natural samples should be interpreted cautiously.     

Development of bacterial biofilms on artificial corals in comparison to surface-associated microbes of hard corals

Numerous studies have demonstrated the differences in bacterial communities associated with corals versus those in their surrounding environment. However, these environmental samples often represent vastly different microbial micro-environments with few studies having looked at the settlement and growth of bacteria on surfaces similar to corals. As a result, it is difficult to determine which bacteria are associated specifically with coral tissue surfaces. In this study, early stages of passive settlement from the water column to artificial coral surfaces (formation of a biofilm) were assessed. Changes in bacterial diversity (16S rRNA gene), were studied on artificially created resin nubbins that were modelled from the skeleton of the reef building coral Acropora muricata. These models were dip-coated in sterile agar, mounted in situ on the reef and followed over time to monitor bacterial community succession. The bacterial community forming the biofilms remained significantly different (R = 0.864 p<0.05) from that of the water column and from the surface mucus layer (SML) of the coral at all times from 30 min to 96 h. The water column was dominated by members of the α-proteobacteria, the developed community on the biofilms dominated by γ-proteobacteria, whereas that within the SML was composed of a more diverse array of groups. Bacterial communities present within the SML do not appear to arise from passive settlement from the water column, but instead appear to have become established through a selection process. This selection process was shown to be dependent on some aspects of the physico-chemical structure of the settlement surface, since agar-coated slides showed distinct communities to coral-shaped surfaces. However, no significant differences were found between different surface coatings, including plain agar and agar enhanced with coral mucus exudates. Therefore future work should consider physico-chemical surface properties as factors governing change in microbial diversity.     

Diseases and partial mortality in Montastraea annularis species complex in reefs with differing environmental conditions (NW Caribbean and Gulf of Mexico)

We documented the prevalence of diseases, syndromes and partial mortality in colonies of the Montastraea annularis species complex on 3 reefs, and tested the assumption that a higher prevalence of these parameters occurs when reefs are closer to point-sources of pollution. One reef was isolated from the impact of local factors with the exception of fishing, 1 potentially influenced by local industrial pollutants, and 1 influenced by local urban pollution. Two reefs were surveyed in 1996 and again in 2001 and 1 in 1998 and again in 2001. In 2001, colonies on all reefs had a high prevalence of the yellow-band syndrome and a relatively high degree of recent partial mortality, while the prevalence of black-band and white-plague diseases was low although a new sign, that we named the thin dark line, had relatively high prevalence in all reefs. As no direct relationship was found between disease prevalence and local environmental quality, our results open the possibility that regional and/or global factors may already be playing an important role in the prevalence of coral disease in the Caribbean, and contradict the theory that coral disease prevalence is primarily related to local environmental degradation. Reasons that may partially explain these findings are the high level of potential pathogen connectivity within the Caribbean as a result of its circulation patterns coupled to the large land-derived pollutants and pathogens input into this Mediterranean sea, together with the surface water warming effects which stress corals and enhance pathogen activity.     

Analysis of complete mitochondrial DNA sequences of three members of the Montastraea annularis coral species complex (Cnidaria, Anthozoa, Scleractinia)

Complete mitochondrial nucleotide sequences of two individuals each of Montastraea annularis, Montastraea faveolata, and Montastraea franksi were determined. Gene composition and order differed substantially from the sea anemone Metridium senile, but were identical to that of the phylogenetically distant coral genus Acropora. However, characteristics of the non-coding regions differed between the two scleractinian genera. Among members of the M. annularis complex, only 25 of 16,134 base pair positions were variable. Sixteen of these occurred in one colony of M. franksi, which (together with additional data) indicates the existence of multiple divergent mitochondrial lineages in this species. Overall, rates of evolution for these mitochondrial genomes were extremely slow (0.03–0.04% per million years based on the fossil record of the M. annularis complex). At higher taxonomic levels, patterns of genetic divergence and synonymous/nonsynonymous substitutions suggest non-neutral and unequal rates of evolution between the two lineages to which Montastraea and Acropora belong.     

Consequences of yellow band disease (YBD) on Montastraea annularis (species complex) populations on remote reefs off Mona Island, Puerto Rico

The rate and extent of mortality from yellow band disease (YBD) to Montastraea annularis (species complex) on reefs off Mona Island, Puerto Rico, was evaluated over 8 yr. Isolated YBD infections were first observed in 1996. Prevalence of YBD increased dramatically in 1999, with a maximum of 52 % of all M. annularis colonies infected in 1 shallow site. YBD continued to spread among adjacent, previously uninfected corals over the next 4 yr, and disease prevalence progressively increased in deeper sites. Linear rates of disease advance and tissue mortality have been slow (5 to 15 cm yr(-1)), although colonies with single YBD lesions have become infected in multiple locations. Most (85%) colonies identified with YBD in 1999 and 2000 were still affected in 2003, and these corals have lost a mean of 60% of their living tissue. Mortality from YBD is being compounded by black band disease, white plague and other syndromes; bioeroding sponges, macroalgae, cyanobacteria and other competitors have colonized tissue-denuded skeleton, minimizing the likelihood of resheeting. The deteriorating health of M. annularis is of particular concern, as these are the dominant corals on these reefs, the largest (2 to 3 m diameter and height) and presumably oldest colonies were infected with YBD more frequently than small colonies, and no recruitment has been observed. YBD is causing extensive mortality to key reef-building taxa in a remote location where anthropogenic stressors are minimal. Additional research on causes of YBD, mechanisms of infection, and strategies to mitigate YBD is needed; otherwise, M. annularis may suffer a fate similar to that of the Atlantic acroporids.     

Combination of biomolecular and stable isotope techniques to determine the origin of organic matter used by bacterial communities: application to sediment.

Natural isotopic composition is a good tool to trace organic matter in ecosystems. Recent studies used a combination of molecular and stable isotope techniques to determine the origin of the organic carbon used by bacteria in the water column. In our study, we show that this procedure can be used for analysis of sediment bacterial communities with few modifications. In the water column, bacterial recovery is done before DNA extraction. In the sediment, we tested qualitatively and quantitatively a direct and indirect extraction of DNA. The direct extraction was the most efficient. It recovered between 3.1 and 15.8 microg DNA g(-1) dry sediment and the contamination of field samples by eucaryotic DNA was less than 13%. In this preliminary study of the salt marsh ecosystem, the delta(13)C values of DNA (-26 to - 24%) recovered from the sediment were close to the delta(13)C values of halophytic plants (-26.4 and - 25.3%) showing a relationship between plants and microorganisms. Thus, this procedure can be used to trace the flow of carbon through the sediment microbial biomass and to understand the variation of bacterial activity according to the inputs of allocthonous and autochtonous organic matter.     

A comparative study of bacterial diversity based on culturable and culture-independent techniques in the rhizosphere of maize (Zea mays L.)

ObjectiveMaize is an important crop for fodder, food and feed industry. The present study explores the plant-microbe interactions as alternative eco-friendly sustainable strategies to enhance the crop yield.MethodologyBacterial diversity was studied in the rhizosphere of maize by culture-dependent and culture-independent techniques by soil sampling, extraction of DNA, amplification of gene of interest, cloning of desired fragment and library construction.ResultsCulturable bacteria were identified as Achromobacter, Agrobacterium, Azospirillum, Bacillus, Brevibacillus, Bosea, Enterobacter, Microbacterium, Pseudomonas, Rhodococcus, Stenotrophomonas and Xanthomonas genera. For culture-independent approach, clone library of 16S ribosomal RNA gene was assembled and 100 randomly selected clones were sequenced. Majority of the sequences were related to Firmicutes (17%), Acidobacteria (16%), Actinobacteria (17%), Alpha-Proteobacteria (7%), Delta-proteobacteria (4.2%) and Gemmatimonadetes (4.2%) However, some of the sequences (30%) were novel that showed no homologies to phyla of cultured bacteria in the database. Diversity of diazotrophic bacteria in the rhizosphere investigated by analysis of PCR-amplified nifH gene sequence that revealed abundance of sequences belonging to genera Azoarcus (25%), Aeromonas (10%), Pseudomonas (10%). The diazotrophic genera Azotobacter, Agrobacterium and Zoogloea related nifH sequences were also detected but no sequence related to Azospirillum was found showing biasness of the growth medium rather than relative abundance of diazotrophs in the rhizosphere.ConclusionThe study provides a foundation for future research on focussed isolation of the Azoarcus and other diazotrophs found in higher abundance in the rhizosphere.     

Bacterial communities inhabiting the healthy tissues of two Caribbean reef corals: interspecific and spatial variation

Bacterial communities inhabiting healthy tissues of the reef-building corals Diploria strigosa and Montastraea annularis were evaluated across a human-induced environmental gradient along the southern coast of Curaçao, Netherlands Antilles. Variations in bacterial communities inhabiting coral tissues were determined using terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes, and the ¹⁵N value of coral tissue was used to assess the relative amount of human contaminants at each reef locality. Bacterial communities of D. strigosa were more variable than M. annularis, but there were no systematic differences in the populations of healthy M. annularis and D. strigosa. The ¹⁵N value of coral tissues showed as much as a 1.5 increase in the impacted versus the non-impacted localities. While M. annularis showed no significant variation in bacterial community structure due to local reef conditions, the bacterial communities of D. strigosa showed dramatic shifts in community structure. The most abundant bacterial taxa inhabiting D. strigosa display increased dominance at impacted localities. By linking variations in microbial communities with an understanding of variations in local environmental conditions, this study provides a means of assessing potential factors that may impact the microbial habitat of coral tissues as well as overall reef health.     

The roles of age,parentage and environment on bacterial and algal endosymbiont communities in Acropora corals

The bacterial and microalgal endosymbiont (Symbiodiniaceae spp.) communities associated with corals have important roles in their health and resilience, yet little is known about the factors driving their succession during early coral life stages. Using 16S rRNA gene and ITS2 metabarcoding, we compared these communities in four Acropora coral species and their hybrids obtained from two laboratory crosses (Acropora tenuis × Acropora loripes and Acropora sarmentosa × Acropora florida) across the parental, recruit (7 months old) and juvenile (2 years old) life stages. We tested whether microbiomes differed between (a) life stages, (b) hybrids and purebreds, and (c) treatment conditions (ambient/elevated temperature and pCO₂). Microbial communities of early life stage corals were highly diverse, lacked host specificity and were primarily determined by treatment conditions. Over time, a winnowing process occurred, and distinct microbial communities developed between the two species pair crosses by 2 years of age, irrespective of hybrid or purebred status. These findings suggest that the microbial communities of corals have a period of flexibility prior to adulthood, which can be valuable to future research aimed at the manipulation of coral microbial communities.     

水产养殖不同物种对水体和沉积物中细菌群落的影响

为探究水产养殖中养殖不同物种对水体和沉积物中细菌群落的影响,以养殖克氏原螯虾(Procambarus clarkii, PC)和中华鳖(Pelodiscus sinensis, PS)的水体和沉积物样品为研究对象,利用基于16S rRNA基因的高通量测序技术,对细菌群落多样性和群落组成进行分析,并结合环境因子,探究水产养殖对细菌群落的影响。结果显示,水体和沉积物中细菌群落的α多样性均呈现PS>PC的显著差异(P<0.05)。非度量多维尺度分析的结果显示,PC和PS区的水体和沉积物细菌群落结构均呈现明显差异。冗余分析(RDA)的结果表明,水体氨氮(NH~+₄-N)和硝酸盐氮(NO~-₃-N)是影响水体细菌群落结构的最主要环境因子,沉积物总氮(TN)、总磷(TP)和有机碳(OC)均对沉积物细菌群落结构有显著影响(P<0.05)。PC和PS区中的细菌隶属于34门、114纲、258目、504科和955属,水体中共筛选出了11个优势菌门(相对丰度>0.5%),沉积物中筛选出了13个。2个养殖区域的水体样品中共筛选出了15个优势(...