Divergence through species interactions: The role of the
sponge
Clathria oxeota
in ecological speciation of the
octocoral
Briareum asbestinum
1Lina M. Gutiérrez-‐Cala.
Laboratorio de Biología Molecular Marina BIOMMAR. Universidad de los Andes. Bogotá, Colombia.
Summary
Organisms often cope with environmental variation by altering their
phenotypes. Such responses may be driven by phenotypic plasticity, which may even precede genetic differences between populations. Morphological variation across environments if reinforce by divergent selection may reduce gene flow and eventually lead fixed differences in ecologically-‐segregated populations and to ecological speciation. Here we studied the ecological, morphological and genetic divergence between encrusting and erect morphotypes of the Caribbean octocoral Briareum asbestinum. We used an ecological approach to test for the association of the encrusting morphs with a symbiotic sponge and to determine its role as a driving factor of ecomorphological differentiation. We studied morphological differentiation induced by the symbiotic sponge Clathria oxeota. We also measured the degree of genetic differentiation between the growth forms. We found ecological differences between growth forms mediated by the symbiotic sponge and intermediate levels of genetic differences that suggest an early genetic divergence between morphs inhabiting contrasting environments.
Key Words
Ecological speciation, symbiotic species interactions, reproductive isolation, coral reefs
Introduction
The origin of biodiversity is a challenging topic that has intrigued scientists since Darwin [1] and today we have clearer ideas about species declines than how species form [2-‐4]. Traditional speciation models seem insufficient to explain the vast biodiversity in coral reefs [5] and this failure is due, to some degree, to a differential understanding between processes operating in model species [6] and those rampant in natural environments.
To understand early divergence through integrative approaches, we propose the octocoral Briareum asbestinum with its ecotypic variation as a model to
understand the process of ecological speciation. The association of the
encrusting ecomorph with the sponge Clathria oxeota promotes morphological differentiation and unequal survival in different habitats, compared to the erect form, thus generating divergence between morphotypes that can reduce gene flow and lead to evolutionary divergence.
The formation of species has fascinated biologist for many years and particularly during the neo-‐Darwinian synthesis [7, 8], with an emphasis on defining the biological species concept and the preponderant role of geography in driving speciation. Research in the last two decades has moved from the geography of speciation to the understanding on the causes of reproductive isolation , niche divergence and considering ecological variables as potential drivers of speciation [9].
Ecological speciation causes reproductive isolation via divergent selection acting on phenotypic variation across environments [10]. Ecologically-‐based selection acts on existing variation to generate divergent populations on their quest for differential use of resources and survival [9]. Ecological speciation can occur either sympatrically or allopatrically and can arise as an indirect consequence of natural divergent selection between environments on morphological,
physiological or behavioral traits which, in turn, lead to reproductive isolation [11]. For example the system of the Threespine sticklebacks Gasterosteus aculeatus for which 5 independent parallel transitions originated new species, developing “limnetic” and “benthic” ecotypes in response to differences in the
foraging niche [12-‐17]. While these examples show ecological speciation in wild populations, few have studied the role of symbiotic partners during ecological speciation, but a remarkable example is Prada and Hellberg 2014 [18] in which the octocoral Eunicea flexuosa shows depth segregated populations with
coincident clines in morphology, genetics, and symbiont association. Despite the study of ecological speciation has improved considerably, strong evidence is missing on how to identify the exact evolutionary forces acting on each case of speciation as well as the genetic mechanism underlying reproductive isolation [9].
In corals much of the work has shown high levels of phenotypic plasticity across scleractinians and octocorals related to environmental factors, such depth [19-‐ 22]. We however lack an understanding of the evolutionary basis underlying ecologically relevant traits and how such phenotypic differences are generated. For example deepening in the evolutionary study of the brooder reproductive strategy of corals is a key aspect because brooder larvae are incubated over the colony and are highly phylopatric, showing reduced dispersion but high
potential for local adaptation. Likewise phylogeographic insights could also be explored given the limited gene flow between populations [23-‐25].
The octocoral Briareum asbestinum is a brooder coral widely distributed across the Caribbean and the Sargasso Sea [26]. This species shows two ecotypes throughout its distribution; an encrusting form that overgrows substrata including other corals, it is often called B. polyanthes (although the species
designation is still unclear). The other is Briareum asbestinum an erect form with vertical branches that can reach over 1 m length. It has been proposed that morphotypes replace each other over a depth gradient but [27] the work only studied plastic response on the erect form. In this project we introduced a new dimension and studied the evolutionary ecology of the species. The encrusting morphotype [from now on, Briareum polyanthes] bears a sponge living under and within the coral tissue. Such interaction, unnoticed so far, could play a role in defining the ecomorphology of this morphotype and triggering divergence
context of ecological speciation as the two ecotypes are responding differently to contrasting environments and in one case largely mediated by the symbiotic interaction with the sponge.
Divergence triggered by symbiont-‐host association has been studied in insect-‐ bacteria interactions [28] [29]. Speciation induced by symbionts may generate a pattern of coevolution between the two partners. Speciation events driven by interactions does not imply environmental and abiotic differences per se, but could be considered ecological speciation if such interaction affects resource use for either organism [10]. If the symbiosis with the sponge Clathria oxeota drives the divergence between Briareum growth forms, it may suggest symbiont-‐
induced ecological speciation.
The genetic divergence in Briareum is uncertain. Allozyme analyses suggest two lineages with different allelic frequencies, indicating restricted gene flow
between them. However, ITS and msh suggest monotypy for this species as both morphotypes shared some haplotypes.[30] [31] Further studies based on more complete sampling would resolve the monotypy of the group.
The expression of either morphotype may be the result of selection acting on morphological characters due to variations in oceanic currents, feeding habits, competitive interactions and differential predation [27, 32]. My motivation here is to study whether the symbiotic sponge may have promoted the morphological, ecological and genetic divergence in Briareum asbestinum. The main hypothesis states that the presence/absence of the sponge and its variable distribution across habitats generates variable selection that may lead to morphological differences across environments. This difference in morphology and their distribution in different environments may also lead to reduced gene flow. To understand the role of the sponge on the ecological divergence in Briareum, we tested for an association between B. polyanthes and the sponge, the role of the sponge in determining morphology and the genetic differentiation between growth forms. Morphotypes show differential allele distributions, suggesting reduced gene flow thus some reproductive isolation. Furthermore a significantly
frequent association between Briareum polyanthes and Clathria oxeota was found as well as strong evidence of the role of C. oxeota on determining the coral morphology.
Methods
Currently the valid status of the species recognizes both growth forms as
Briareum asbestinum, as proposed in [33]. However in this project, growth forms were independently: B.polyanhtes for the encrusting growth form and B.
asbestinum for the erect. .
Fieldwork and sampling.
Fieldwork was conducted between 2013 and 2015 in nine Caribbean fringing reefs: Santo Domingo-‐Barú, Bajo San Felipe [BSF], Contour and Espiral-‐ Providencia, Colombia; Pinnacles, Turromote and Mario reefs in La Parguera, southwest Puerto Rico.
To determine the relative abundance of each growth form, we performed ecological surveys in Providencia and Puerto Rico using 30 m line transects. We recorded the frequency of the association between the sponge and the two Briareum forms and the frequency of overgrowing interactions between one of the growth forms, B. polyanthes, and other coral species. Four depths were established in each reef, ranging from 5-‐22 m according to the conditions of the site. The transect had a width of 2 meters, so each transect represented a sampled area of 120 m2 , in four transects for a total area of 480m2 per site. I
checked for the presence of the sponge towards the base of the colonies, specially in B. polyanthes, where it is common to find the sponge intertwined with the coral tissue.
To test for differences in the frequencies of association between the sponge and the growth forms between and within localities, Pearson’s correlations andchi-‐ square tests with Yate’s correction were performed [34] . I tested for normality
using Shapiro-‐wilk test with a Wilcoxon signed-‐rank test. All ecological analyses and figures were done in R software (CRAN).
To test for genetic differences, I collected pieces of 2-‐5cm2 and preserved them
in 96% ethanol or liquid nitrogen for DNA analysis. When B. polyanthes samples were attached to sponge tissue, they were manually separated and the sponge was also preserved in ethanol 96% for DNA analysis. Tissue samples were collected in the same transects in which we recorded ecological data. To avoid sampling clones, samples were taken with the minimum distance of 5m between colonies.
Experimental setting of sponge graft
To explore the potential of the sponge to modify coral morphology, we set sponge grafts in the erect B. asbestinum often asymbiotic and monitored the responses of the coral-‐sponge interaction [Fig.1]. We collected sponge tissue from B. polyanthes colonies and then attached them to 35 B. asbestinum colonies [Fig1]. They were randomly distributed on the reef of Bajo San Felipe,
Providencia [10-‐13m deep]; identified with metallic tags and attached to colonies with plastic cable ties. We chose colonies that have at least 5 branches so all the treatments were set in the same individual and separated by 6 m or more in order to avoid clonality.
To attach the grafts an 8 cm longitudinal cut was performed towards the base of one of the colony and introduced one a piece of sponge tissue. The sponge was completely surrounded by the coral’s tissue. To increase the graft’s success, a thin cotton stripe was tied up around de branch to hold the coral tissue and keep the sponge inside the branch. I followed the same procedure but without the sponge tissue to test for any artifact of the cut itself. I also left uncut colonies to control for any other environmental variation.
If the sponge were to make any change on the basal morphology of the colony we should expect an increase of circumference or a morphological change
resembling the encrusting form. In April 2015, we revisited the reef and check the experimental colonies and the effect of the sponge on the treatments.
Molecular approaches
Preserved samples were taken to the laboratory and genomic DNA was extracted by CTAB and CIA/FCIA following [35]. DNA concentrations were measured in a NanoDrop 2000 UV-‐Vis Spectrophotometer [Thermo Scientific].
Sponge identification and barcoding
Sponge samples were randomly chosen from field collections [n=10]. Sponge DNA was extracted as for Briareum. To barcode the samples, I used universal primers for the mitochondrial cytochrome c oxidase subunit I [COI] previously used for sponges [36].
Sequences were cleaned and edited in Sequencher 4.1.4, and then BLAST to identify the closest matched for the sponge tissue. Highest match species were recorded and checked their reported distribution on [37]. Neotropical sponge expert Sven Zea furthered confirmed species assignments based on morphology.
Molecular approach-‐Briareum.
To genotype Briareum colonies, we used the ribosomal ITS-‐2 [Internal
Transcribed Spacer 2; 230 bp] that is located in the region between the 5’ end of the 5.8s ribosomal gene and the 3’ end of the 28s. Amplifications were
performed using the octocoral universal primers [5.8S-‐436: 5’-‐ AGCATGTCTGTCCTGAGTGTTGG-‐3’ and 28S-‐663: 5’-‐
GGGTAATCTTGCCTGATCTGAG-‐3’] designed by [38].
For successful amplification of ITS-‐2, DNA extractions were diluted 1:1000 or 1:500 given the high concentration of DNA and phenols in extracts. PCR volume was 15 μl with 120 s at 94°C followed by 35 cycles of 30 s at 94°C, 45 s at 58°C, 60 s at 72°C and a final extension step for 5 min at 72°C. Successfully amplified samples were sequenced using a Sanger ABI 3730 sequencer.
Determination of ITS2 nuclear genotypes
Sequences for 67 individuals were cleaned and edited in Sequencher 4.1.4. Double peaks were present in some chromatograms suggesting multiple copies per individuals as often found for ITS2. Procedures for genotype determination followed [39]. Identifying the sequence for homozygous was straightforward because no double peaks were observed. Heterozygous displaying only one heterozygous site were disentangled by placing each variant in each haplotype. For heterozygous with several double peaks, each doubtful position was
replaced with IUPAC codes; the main criteria for accounting a double peak as true heterozygote, was the presence of such double peak in both forward and reverse contigs. In more doubtful situations, we analyzed the height difference between both peaks. If they were nearly of same height, it was counted as a true heterozygote and an IUPAC code was used. If neither criterion were met, we would call a false double peak and assume it was sequencing noise.
Sequences were aligned in AliView v 1.11 [40] using MUSCLE algorithm. To reconstruct the most probable alleles for each of the ambiguous individuals, we generated an input file in SeqPHASE [41] and then use it in PHASE2.1.1 [42]. Prior to running PHASE2.1.1, we recovered 25 out of 67 pairs of alleles by following [43]. Afterwards PHASE2.1.1 was run for the remaining individuals using information from all known alleles. To detect an association between haplotypes and growth form, a Fisher Exact Test was ran as implemented in PHASE2.1.1 a [α = 0.05].
Once the alleles were determined, a median joining haplotype network [44] was inferred using the program PopART [available at [45]]. Using the enhanced metafile [emf] format of the image, it was imported in Microsoft Powerpoint and connections between co-‐occurring alleles in an individual were established by drawing lines with their thickness proportional to the number of individuals harboring such configuration. By establishing such connections I turned the
haplonet into a haploweb showing the distribution of alleles in the population [39]
To visualize the phylogenetic relationships among alleles, we first used jModelTest 2.1.4 [46] to determine the best model of nucleotide evolution and then a Maximum Likelihood analysis using RAxML [8.1.11] [47].
The network was studied using graph theory concepts in order to identify hidden patterns of allele distribution. The node degree was calculated for all the alleles [48]
Results
Ecological information
Briareum polyanthes colonies often associated with the sponge throughout the surveyed reefs in Puerto Rico and Providencia. In the former, the total number of B. polyanthes colonies associated with the sponge was 193 , [V=6105, p= 0.05].
Likewise in Providencia we found 199 colonies of B. polyanthes associated with the sponge [V=6105, p= 0.05]. It is evident that in both places the number B. polyanthes colonies associated with the sponge was significantly higher to those asymbiotic [Fig.3]. This is the first time such a consistent interaction is reported for B. polyanthes. The pattern of differential association holds within each reef of both localities.
Regarding the general abundances of both growth forms registered along the surveys in Puerto Rico and Providencia, we found the data to be non-‐parametric [W=0.757, W= W=0.7412 respectively, p < 0.05] and significantly higher abundance of B. asbestinum than B. polyanthes [V= 23220, V=34453, p < 0.05]. In both
localities B. asbestinum colonies were more abundant than B. polyanthes and the pattern holds for all the surveyed reefs within each locality [Fig.2]
The overgrowing behavior of B. polyanthes was present in some of the surveyed colonies. However 100% of B. polyanthes colonies that showed overgrow
harbored sponge tissue intertwined within. This suggests an interesting role of the sponge on helping the coral outcompete other gorgonians in the reef. The
overgrowth data for Puerto Rico and Providencia were no parametric either [W= 0.7801, p = 3.28 x 10-‐10,, W= 0.7589, p = 3.76*e^-‐12; respectively ]. Furthermore there
were significant differences on the coral genera affected by this overgrowth interaction in both Puerto Rico and Providencia [ χ2[k-‐w] = 21.5, p= 0.005931; χ2[k-‐
w] = 15.6659, p= 0.01566; respectively]. In the surveyed reefs of Puerto Rico and
Providencia the most frequently overgrown genus was Gorgonia sp. followed by Millepora sp [Fig.4]. A remarkable observation was a overgrowing behavior of an encrusting colony of B. polyanthes over an erect Briareum asbestinum, for this suggests the existence of spatial competence and a possible advantage of having an associated sponge.
Experimental setting of sponge graft
From the initial 35-‐tagged colonies, only 12 colonies were recovered. In 11 out of the 12 colonies, no signs of the sponge graft were found, the cut healed completely and no increase of horizontal growth or deformation was observed. The cotton stripes remained tightened around the branch. The branch
surrounded by the plastic cable tie showed some physical damage around, characterized by tissue loss and some tissue molting.
However, in one experimental colony, identified with the tag 734, an important deformation was observed towards the base of the colony [Fig.5], also the sponge was visibly intertwined within the coral tissue. One of the most
remarkable result was the evident deformation of the colony where the sponge was embedded, resulting in a bulky, amorphous phenotype [Fig.5]. In the control branches no significant change was observed. This observation can be
considered as preliminary, but encouraging to expand the experiment and fully test the Briareum-‐sponge interaction.
Molecular Results.
Barcoding identification of the sponge
Our mitochondrial COI survey of 10 sponge samples matched Clathria [Thalysias] reinwardti [Duchassaing & Micheloti, 1864] with a 97% identity score and E-‐ value= 0 in BLAST searches. However records for that species in WoRMS [World
Register of Marine Species, at www.marinespecies.org] and in the World Porifera Database [37] showed the species distribution spans the Indo and Eastern Pacific ocean. I decided to take into account the match for Clathria [Thalysias] schoenus [Laubenfels, 1936] which was the third highest Max Score [996] that also
showed 97% of identity and E-‐value= 0. It is worth noting that Clathria oxeota showed exactly the same values for BLAST species match. Records suggest Clathria oxeota spans throughout the Caribbean, including Panama, Southern Caribbean and Belize, all of which are nearby Providencia Island, hence is more plausible assuming those are the actual match for my sample sequences.
Furthermore, morphological examination by sponge expert Sven Zea, supported Clathria oxeota as the symbiotic sponge.
Molecular approach – Briareum
Successful amplification of ITS-‐2 was achieved for 67 individuals, 37 from Cartagena and 30 from Providencia. Among the sequenced individuals, 6 were homozygous and 61 were heterozygous. As mentioned before, 25 individuals were manually phased. For all other samples after running PHASE2.1.1, 33 individuals were phased with posterior probabilities ≥ 0.9; whereas 34 presented different values for posterior probability. After choosing the most probable phases for each individual, they were realigned.
Haploweb analysis [Fig.6] suggests high levels of allele co-‐occurrence among growth forms. However, I found a non-‐random distribution of the 134 alleles across morphs (p = 0.01), suggesting that despite some sharing, genetic variation is partition across sympatrically distributed growth forms. Most of the sharing is due to the most common haplotypes 2, 3, 4, 9. The next most common allele was number 1, harboring 9 individuals but in this case the allele is slightly more common in B.asbestinum rather than B. polyanthes. Allele number 5 was present in 7 individuals but it was predominantly frequent in B. polyanthes. Additionally, allele 6 is unique to B. asbestinum and it is present in 6 individuals.
In addition to the assortative distribution of common alleles, we found 2 unique allelic combinations present in 2 distinct individuals, these are known as
“singletons” [39] and are represented by a dashed oval. The haploweb shows an interesting link between alleles 3, 4, 6 as many individuals co-‐occur with any of these three variants [Fig.7].
The histogram of node degree [Fig.8] shows that most of the alleles present degree 1, which means there is a high density of low-‐degree nodes, thus there is a high exclusivity of alleles and only few of them are shared. As stated by the above-‐mentioned Fisher exact test, we can see non-‐random distributions of alleles among ectypes, [Fig.9] shows 14 exclusive alleles for B. polyanthes,
whereas just 9 for Briareum asbestinum, and only 6 alleles shared between them. Finally we can see a larger amount of heterozygote individuals than homozygote, which suggests a high genetic variation in both ecotype populations [Fig.10]
Discussion
This study increased the ecological and genetic knowledge for Briareum asbestinum. Not only obtained baseline ecological information on the species abundance and distribution, but also the first report of a common interaction between the coral encrusting growth form [B. polyanthes] and the sponge Clathria oxeota. Furthermore important insights were obtained regarding the role of C. oxeota on driving the coral morphology. Despite the need for additional testing, this result is encouraging considering how difficult is to test the impact of an interspecific interaction on the phenotypes of the partners. The subtle genetic divergence in sympatrically living morphs suggests non-‐random mating and an incipient divergence process.
An important consideration is the possibility of phenotypic plasticity might be playing an important role in driving morphology and would need further studies to confirm the exact trigger of morphological change. The preliminary results on the graft experiment suggest a strong role of plasticity induced by the sponge contact. Given that phenotypic plasticity is defined as the ability of a single genotype of producing a range of phenotypes in response to different environmental conditions [49, 50]. However this would not preclude the possibility of a speciation event, because plasticity is an emergent property of
the genotype plausible to evolve. Thus through adaptive plasticity some
genotpyes would respond to environment even in future generations, not just its lifespan [51].
Association between B. polyanthes and Clathria oxeota.
Our report of a close association between Briareum polyanthes and the sponge Clathria oxeota is described for the first time. The data gathered in field surveys showed high frequency of the interaction related to the abundance of Briareum in the reef. Despite being abundant organisms in coral reefs worldwide,
associations between corals/octocorals with sponges are rarely reported in the scientific literature. Most of the examples are related to spatial competition between them [52-‐54]. However one of the first associations ever described [55] reported the interaction of the coral Tubipora musica and a sponges of the genus Mycale. As in the association studied here, the tissues of the sponge grow
intertwined and no signs of negative consequences were observed for either partner. It suggests the association could be a commensalism or even
mutualistic. Another characteristic that would be useful for future work is to measure the number of discharged cnidae in tissues of the interacting sponge. This measure would be a direct proxy to test whether the association is
mutualistic or not.
Another reported association between an octocoral and a sponge is between Carijoa riseii and Desmapsammma acnhorata [56]. This study shows how C. riseii responds to the sponge by changing its morphology, nematocysts concentration, and redistribution of its polyps. This could be the case for B. asbestinum and B.polyanthes as shown by the morphological change in colony 734 [Fig.5]. A new experimental set up should be designed contemplating the effect of
removing the sponge from B. polyanthes, as well as making the grafts in branches of different lengths in order to study the degree of plasticity in different stages of the animal.
A recent study [57] of reefs around La Parguera, Puerto Rico focused on the interactions of several gorgonian species with different sponge species. The
general conclusion of this survey is that gorgonian-‐sponge interactions are context dependent and are strongly related to the substrate abundance of the reef. If gorgonians are the dominant component in the reef, the probability of finding such associations increase. Furthermore they remarked that most of the gorgonians interacting with the sponge, presented negative consequences like overgrowth and smothering. However they state “A notable exception to this overall pattern was Briareum, which was found highly associated with sponges relative to its low abundance” […] “A notable exception to the negative
interactions with gorgonians was observed between Briareum and
Desmapsamma anchorata, Amphimedon compressa and Mycale carmigropila”. The first statement suggests that Briareum has a non-‐random association
triggered by other factor rather than distribution, and this non-‐randomness was statistically supported by my field data. Actually, the non-‐random pattern is so strong that I did not find colonies of Briareum asbestinum per se [erect colonies] interacting with the sponges, rather only B. polyanthes. Given the various
ecological differences between growth forms in Briareum, it would be very useful to report exactly the specific growth form participating in the interaction. That way would be clearer if the species establishes interaction networks at the multispecies level, or if it is somehow species-‐specific.
The second statement, which says that no negative sign is observed in the coral tissue, as it is associated with the sponge. I observed the same pattern and a clear sign of non-‐negative interaction is that coral polyps are never smothered or covered by the sponge, which means that vital activities such as nutrition and photosynthesis are not prevented by the sponge.
Finally one important difference between this work and [57] is on the species of sponge participating on the association. They found D. anchorata just as in [56], whereas I found Clathria oxeota. However, this suggests that further work needs to be done regarding more replicate surveys in different parts of the Caribbean in order to record more species of sponges species interacting or determining if this is a species specific association. Additionally at least 2 identification sources need to be included, namely genetic, morphological, histological, spicule, etc., in order to get confident identification results.
To understand the possible reasons that might explain these interactions we have to analyze some characteristics especially important in Porifera: Sponges are responsible for the highest rates of Dissolved Organic Matter [DOM] uptake, exceeding even bacterial rates for the same process. Furthermore, they recycle elements like N, which means they are keystones for reef functioning: organically and inorganically [58, 59]. From a coral’s perspective having a constant source of food and nutrient could represent various benefits for thriving in a highly
competitive, resource-‐scarce ecosystem like coral reefs. For future addresses, using stable isotope approaches would be useful to test whether a nutrition-‐ driven interaction takes place between Briareum polyanthes and Clathria oxeota.
Molecular Results
Haploweb as an approach for allele delimitation.
Even though the reconstructed haploweb did not separate major alleles by morphotypes, it was useful when working with “problematic” markers such as rDNA internal transcriber 2 [ITS-‐2]. Despite being the most variable marker in Octocorallia and the most suited for intraspecific resolution [60], it poses
problems regarding high rates of intragenomic variation including length variant and multiple copies [61, 62].
A notable result was the relatively good quality of the sequences right after direct sequencing. Unlike stated by [30], ITS-‐2 sequences did not result in truncated chromatograms after direct sequencing. Only 1 out of 68 sequences presented such low quality that couldn’t be used at all. For the remaining
sequences, although some noisy, haplotypes were reconstructed either by Clark’s method, [43] or by PHASE2.1.1. Since Doyle’s Field for Recombination [FFR’s] approach is based on presence/absence of haplotypes [63] and not the exact definition of the genotype, dealing with copy-‐number and intragenomic variation becomes less problematic.
Genetic data showed extensive allele sharing but a non-‐random distribution of alleles with morphology, suggesting early divergence of sympatrically living and ecologically segregated ecotypes of Briareum.
The pattern allele sharing and private alleles, is one of the signals of intermediate divergence known as “Neotypy” [64]. It is important to consider all possible intermediate scenarios of divergence. Reciprocal monophyly should not be the definitive pattern to look for in a incipient/recently diverged system, rather this is the pattern we expect at the “end” of the divergence process or if looking at a pair of lineages split long time ago.
We found an intermediate stage through genetic data, but ecologically we were able to find strong differentiation that can promote ecologically based
divergence, which is a key step into an ecological speciation process because it promotes ecological-‐based selection [10]. This findings are strongly related with the isolation-‐by-‐ecology approach (IBE)[65] which is based on differential resource exploitation between populations albeit in the face of gene flow, which means that gene responsible for IBE are different form genes responsible for reproductive isolation, so further genomic studies would complete the knowledge gap in such eco-‐evolutionary processes. Additionally we are
reporting a new suitable non-‐model system in which to make future experiments aimed to deepen on the knowledge of symbiont-‐induced speciation [66].
Conclusions
Our results confirm the suitability of Briareum to study the degree of divergence between growth forms and the role of ecology during early divergence. We found differential associations with the sponge Clathria oxeota and differential
competition for substrate between growth forms. Despite such extensive morphological and ecological divergence,,some major alleles were shared between growth forms, but there was a substantial presence of exclusive alleles in sympatrically segregated forms, which suggests not-‐random mating between ecotypes. While some work is needed, my results highlight the importance of species interactions during early stage on the formation of species and its role during ecological speciation in hyper-‐diverse ecosystems such coral reefs.
Figures and graphs.
Figure. 1
Fig1. Initial setup of the graft experiment. Branches 1,2,4 control the effect of the lace and the wound. Branch 3 holds the experimental graft.
Figure 2.
Fig2. Abundance of Briareum asbestinum [black] and Briareum polyanthes [grey] 1 2 3 4 0.0 0.2 0.4 0.6 0.8 1.0 1.2
Puerto Rico Providencia
Ab
un
da
nc
e
(c ol on ia s* m -‐2 )
Figure 3.
Fig3. Colonies of Briareum polyanthes associated [black] with Clathria oxeota, versus non-‐associated [gray].
Figure 4.
Fig4: Overgrown species in Providencia [grey] and Puerto Rico [black] 0.0 0.1 0.2 0.3 0.4
Puerto Rico Providencia
Ab
un
da
nc
e
(c ol on ia s* m -‐2 ) 0.000# 0.010# 0.020# 0.030# 0.040# 0.050# 0.060# 0.070# 0.080# An.ll ogorg ia.sp # B.#asb es.n um# D.cylindr us# Gorgo nia.sp # Mille pora. sp# Muric eops is.sp# Eunic ea.sp # Muric ea.sp # Plexau ra.sp # Pseu dople xaura. sp# A.cervi corni s.sp# Providencia# Puerto#Rico# Antill ogorgi
a sp. B.
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rus
Gorgo
nia sp
.
Millep
ora sp
.
Murice
opsis sp.
Eunic ea sp.
Plexau
ra sp.
Pseud
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ura sp.
Murice
a sp.
A. cer
vicorn is
Ab
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-‐
Figure 5.
Fig5: Picture of the graft experiment 8 months after. The arrow points the deformation at the base of the colony. The orange tissue is the living sponge within the coral..
Figure 6.
Fig6: Allele network. Red: Briareum asbestinum, Yellow: Briareum polyanthes. P4b P4a BP9b BP9a BP27b BA28b BA20b BA17b BA17a P15b P15a BP8b BP7b BP6b BP4a BP3b BP29 BP24b BP19a BP17b BP11b BP11a BP10b BA5b A14b A12b A12a 767Ab 767Aa 10 samples 1 sample B_asbestinum B_polyanhtes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Figure 7.
Fig7: Haploweb with co-‐ocurring alleles connected. Dashed lines: singletons. Red: Briareum asbestinum, Yellow: Briareum polyanthes.
Figure 8.
Fig8: Shows the node degree: Frequency of nodes connected to a focal node.
Histogram of nod$grado
Grado de nodos
F
re
qu
en
cy
1 2 3 4 5 6
0
5
10
15
Figure 9.
Fig9: Number of exclusive and shared alleles in each species.
Figure 10.
Fig 10: Number of heterozygote and homozygote individuals of each species. Red: Briareum asbestinum, Yellow: Briareum polyanthes
0
5
10
15
Bp Ba Compartidos
Briareum polyanthes Briareum asbestinum Comparitidos
#
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40
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Heterocigoto Homocigoto # In di vi du os
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