Microbial community structure and settlement induction capacity of marine biofilms developed under varied reef conditions.

Coral larval settlement relies on biogenic cues such as those elicited by microbial biofilm communities, a crucial element of coral recruitment. Eutrophication can modify these biofilm-associated communities, but studies on how this affects coral larval settlement are limited. In this study, we developed biofilm communities on glass slides at four sites with increasing distance from a mariculture zone. Biofilms farthest from the mariculture area were more effective at inducing the settlement of Acropora tenuis larvae. These biofilms were characterized by a greater proportion of crustose coralline algae (CCA) and gammaproteobacterial taxa compared to biofilms from sites closer to the mariculture zone, which had a greater proportion of cyanobacteria and no CCA. These findings suggest that nutrient enrichment due to mariculture activities alters the composition of biofilm-associated microbiome at nearby reef sites and indirectly causes poor coral larval settlement.

Specific bacterial microbiome enhances the sexual reproduction and auxospore production of the marine diatom, Odontella.

Auxospore production is a sexual reproductive strategy by diatoms to re-attain normal size after the size-reducing effect of clonal reproduction. Aside from the minimum size threshold used as a sex clock by diatoms, the environmental or chemical triggers that can induce sex in diatoms are still not well understood. Here we investigated the influence of six marine bacteria from five families on the production of sexual cells and auxospores of the ubiquitous marine polar centric diatom, Odontella sp. Microbiome association and co-occurrence with the diatom in culture and in nature were investigated using 16S rRNA amplicon sequencing. Indole acetic acid (IAA) secretion, a phytohormone that regulates plants' growth and sexual development, was explored as a potential inducer of sexual reproduction in Odontella and compared between bacterial associates. We found that Odontella co-cultured with Flavobacteriaceae (Polaribacter and Cellulophaga) have significantly more sexual cells and auxospores than bacteria-free Odontella and Odontella co-cultured with other bacteria from Vibrionaceae (Vibrio), Pseudoalteromonadaceae (Pseudoalteromonas), Rhodobacteraceae (Sulfitobacter), or Planococcaceae (Planococcus) family. Differences in IAA secretion were observed between bacterial isolates, but this did not correspond consistently with the diatom's clonal growth or production of sexual cells and auxospores. Microbiome composition survey of Odontella cultures showed that the diatom harbors homologous sequences of the four bacterial isolates at varying proportions, with Sulfitobacter and Polaribacter at high abundances. Microbiome surveys at Santa Cruz Wharf, Monterey Bay, from 2014-2015 showed that Odontella abundance is positively correlated with Flavobacteriaceae and Rhodobacteraceae abundances. Our study demonstrates that specific members of the diatom microbiome can enhance the host's sexual reproduction, with the interkingdom interaction driven by partner compatibility and long-term association. Sex-enhancing bacteria may even be needed by the diatom host to carry out the optimal inducement of sex under normal conditions, allowing for size restitution and maintaining genetic diversity in culture and in nature.

A novel random forest approach to revealing interactions and controls on chlorophyll concentration and bacterial communities during coastal phytoplankton blooms.

Increasing occurrence of harmful algal blooms across the land-water interface poses significant risks to coastal ecosystem structure and human health. Defining significant drivers and their interactive impacts on blooms allows for more effective analysis and identification of specific conditions supporting phytoplankton growth. A novel iterative Random Forests (iRF) machine-learning model was developed and applied to two example cases along the California coast to identify key stable interactions: (1) phytoplankton abundance in response to various drivers due to coastal conditions and land-sea nutrient fluxes, (2) microbial community structure during algal blooms. In Example 1, watershed derived nutrients were identified as the least significant interacting variable associated with Monterey Bay phytoplankton abundance. In Example 2, through iRF analysis of field-based 16S OTU bacterial community and algae datasets, we independently found stable interactions of prokaryote abundance patterns associated with phytoplankton abundance that have been previously identified in laboratory-based studies. Our study represents the first iRF application to marine algal blooms that helps to identify ocean, microbial, and terrestrial conditions that are considered dominant causal factors on bloom dynamics.

Presence and abundance of bacteria with the Type VI secretion system in a coastal environment and in the global oceans.

Marine bacteria employ various strategies to maintain their competitive advantage over others in a mixed community. The use of Type VI Secretion Systems (T6SS), a protein secretion apparatus used as a molecular weapon for interbacterial competition and eukaryotic interactions, is one of the competitive strategies that is least studied among heterotrophic bacteria living in the water column. To get an insight into the temporal and spatial distribution of bacteria with T6SS in this portion of the marine environment, we examine the presence and abundance of T6SS-bearing bacteria at both local and global scales through the use of metagenome data from water samples obtained from the coast of Monterey Bay and the TARA Oceans project. We also track the abundance of T6SS-harboring bacteria through a two-year time series of weekly water samples in the same coastal site to examine the environmental factors that may drive their presence and abundance. Among the twenty-one T6SS-bearing bacterial genera examined, we found several genera assume a particle-attached lifestyle, with only a few genera having a free-living lifestyle. The abundance of T6SS-harboring bacteria in both niches negatively correlates with the abundance of autotrophs. Globally, we found that T6SS genes are much more abundant in areas with low biological productivity. Our data suggest that T6SS-harboring bacteria tend to be abundant spatially and temporally when organic resources are limited. This ecological study agrees with the patterns observed from several in vitro studies; that T6SS could be an adaptive strategy employed by heterotrophic bacteria to obtain nutrients or reduce competition when resources are in limited quantity.

The Mercury-Tolerant Microbiota of the Zooplankton Daphnia Aids in Host Survival and Maintains Fecundity under Mercury Stress.

Many aquatic organisms can thrive in polluted environments by having the genetic capability to withstand suboptimal conditions. However, the contributions of microbiomes under these stressful environments are poorly understood. We investigated whether a mercury-tolerant microbiota can extend its phenotype to its host by ameliorating host survival and fecundity under mercury-stress. We isolated microbiota members from various clones of Daphnia magna, screened for the mercury-biotransforming merA gene, and determined their mercury tolerance levels. We then introduced the mercury-tolerant microbiota, Pseudomonas-10, to axenic D. magna and quantified its merA gene expression, mercury reduction capability, and measured its impact on host survival and fecundity. The expression of the merA gene was up-regulated in Pseudomonas-10, both in isolation and in host-association with mercury exposure. Pseudomonas-10 is also capable of significantly reducing mercury concentration in the medium. Notably, mercury-exposed daphnids containing only Pseudomonas-10 exhibited higher survival and fecundity than mercury-exposed daphnids supplemented with parental microbiome. Our study showed that zooplankton, such as Daphnia, naturally harbor microbiome members that are eco-responsive and tolerant to mercury exposure and can aid in host survival and maintain host fecundity in a mercury-contaminated environment. This study further demonstrates that under stressful environmental conditions, the fitness of the host can depend on the genotype and the phenotype of its microbiome.

Host genotype-specific microbiota do not influence the susceptibility of D. magna to a bacterial pathogen.

Host-associated microbiota have been claimed to play a role in hosts' responses to parasitic infections, often protecting the hosts from infection. We tested for such a role in the crustacean Daphnia and the parasitic bacterium Pasteuria ramosa, a widely used model system for host-parasite interactions. We first determined the infection phenotype (i.e., resistotype) of eight clonal D. magna genotypes against four strains of P. ramosa by attachment test, followed by 16 S rDNA amplicon sequencing to determine if their genotype or their parasite resistotype influences the composition of their microbiome. We then reciprocally transplanted the microbiota of two host genotypes with opposite resistotypes to four P. ramosa isolates, followed by a reassessment of their resistotype after transplantation. We found significant differences in microbiome composition and structure between Daphnia genotypes and between Daphnia resistotypes to specific P. ramosa strains. Reciprocal microbiota exchange or making the Daphnia hosts bacteria-free, however, did not influence the resistotypes of the hosts. Thus, in contrary to what has been observed in some taxa, our results suggest that D. magna susceptibility to P. ramosa is strongly dictated by the genetic differences of the hosts and is still dependent on Daphnia's first line of immune defense against the esophageal attachment of P. ramosa, which appears to be uninfluenced by the host's microbiota.

Phytoplankton-Associated Bacterial Community Composition and Succession during Toxic Diatom Bloom and Non-Bloom Events.

Pseudo-nitzschia blooms often occur in coastal and open ocean environments, sometimes leading to the production of the neurotoxin domoic acid that can cause severe negative impacts to higher trophic levels. Increasing evidence suggests a close relationship between phytoplankton bloom and bacterial assemblages, however, the microbial composition and succession during a bloom process is unknown. Here, we investigate the bacterial assemblages before, during and after toxic and non-toxic Pseudo-nitzschia blooms to determine the patterns of bacterial succession in a natural bloom setting. Opportunistic sampling of bacterial community profiles were determined weekly at Santa Cruz Municipal Wharf by 454 pyrosequencing and analyzed together with domoic acid levels, phytoplankton community and biomass, nutrients and temperature. We asked if the bacterial communities are similar between bloom and non-bloom events and if domoic acid or the presence of toxic algal species acts as a driving force that can significantly structure phytoplankton-associated bacterial communities. We found that bacterial diversity generally increases when Pseudo-nitzschia numbers decline. Furthermore, bacterial diversity is higher when the low-DA producing P. fraudulenta dominates the algal bloom while bacterial diversity is lower when high-DA producing P. australis dominates the algal bloom, suggesting that the presence of algal toxin can structure bacterial community. We also found bloom-related succession patterns among associated bacterial groups; Gamma-proteobacteria, were dominant during low toxic P. fraudulenta blooms comprising mostly of Vibrio spp., which increased in relative abundance (6-65%) as the bloom progresses. On the other hand, Firmicutes bacteria comprising mostly of Planococcus spp. (12-86%) dominate during high toxic P. australis blooms, with the bacterial assemblage showing the same bloom-related successional patterns in three independent bloom events. Other environmental variables such as nitrate and phosphate and temperature appear to influence some low abundant bacterial groups as well. Our results suggest that phytoplankton-associated bacterial communities are strongly affected not just by phytoplankton bloom in general, but also by the type of algal species that dominates in the natural bloom.

Response of Vibrio cholerae to Low-Temperature Shifts: CspV Regulation of Type VI Secretion, Biofilm Formation, and Association with Zooplankton.

UNLABELLED: The ability to sense and adapt to temperature fluctuation is critical to the aquatic survival, transmission, and infectivity of Vibrio cholerae, the causative agent of the disease cholera. Little information is available on the physiological changes that occur when V. cholerae experiences temperature shifts. The genome-wide transcriptional profile of V. cholerae upon a shift in human body temperature (37°C) to lower temperatures, 15°C and 25°C, which mimic those found in the aquatic environment, was determined. Differentially expressed genes included those involved in the cold shock response, biofilm formation, type VI secretion, and virulence. Analysis of a mutant lacking the cold shock gene cspV, which was upregulated >50-fold upon a low-temperature shift, revealed that it regulates genes involved in biofilm formation and type VI secretion. CspV controls biofilm formation through modulation of the second messenger cyclic diguanylate and regulates type VI-mediated interspecies killing in a temperature-dependent manner. Furthermore, a strain lacking cspV had significant defects for attachment and type VI-mediated killing on the surface of the aquatic crustacean Daphnia magna Collectively, these studies reveal that cspV is a major regulator of the temperature downshift response and plays an important role in controlling cellular processes crucial to the infectious cycle of V. cholerae IMPORTANCE: Little is known about how human pathogens respond and adapt to ever-changing parameters of natural habitats outside the human host and how environmental adaptation alters dissemination. Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, experiences fluctuations in temperature in its natural aquatic habitats and during the infection process. Furthermore, temperature is a critical environmental signal governing the occurrence of V. cholerae and cholera outbreaks. In this study, we showed that V. cholerae reprograms its transcriptome in response to fluctuations in temperature, which results in changes to biofilm formation and type VI secretion system activation. These processes in turn impact environmental survival and the virulence potential of this pathogen.

Water fleas require microbiota for survival, growth and reproduction.

Microbiota have diverse roles in the functioning of their hosts; experiments using model organisms have enabled investigations into these functions. In the model crustacean Daphnia, little knowledge exists about the effect of microbiota on host well being. We assessed the effect of microbiota on Daphnia magna by experimentally depriving animals of their microbiota and comparing their growth, survival and fecundity to that of their bacteria-bearing counterparts. We tested Daphnia coming from both lab-reared parthenogenetic eggs of a single genotype and from genetically diverse field-collected resting eggs. We showed that bacteria-free hosts are smaller, less fecund and have higher mortality than those with microbiota. We also manipulated the association by exposing bacteria-free Daphnia to a single bacterial strain of Aeromonas sp., and to laboratory environmental bacteria. These experiments further demonstrated that the Daphnia-microbiota system is amenable to manipulation under various experimental conditions. The results of this study have implications for studies of D. magna in ecotoxicology, ecology and environmental genomics.

Host-specific adaptation governs the interaction of the marine diatom, Pseudo-nitzschia and their microbiota.

The association of phytoplankton with bacteria is ubiquitous in nature and the bacteria that associate with different phytoplankton species are very diverse. The influence of these bacteria in the physiology and ecology of the host and the evolutionary forces that shape the relationship are still not understood. In this study, we used the Pseudo-nitzschia-microbiota association to determine (1) if algal species with distinct domoic acid (DA) production are selection factors that structures the bacterial community, (2) if host-specificity and co-adaptation govern the association, (3) the functional roles of isolated member of microbiota on diatom-hosts fitness and (4) the influence of microbiota in changing the phenotype of the diatom hosts with regards to toxin production. Analysis of the pyrosequencing-derived 16S rDNA data suggests that the three tested species of Pseudo-nitzschia, which vary in toxin production, have phylogenetically distinct bacterial communities, and toxic Pseudo-nitzschia have lower microbial diversity than non-toxic Pseudo-nitzschia. Transplant experiments showed that isolated members of the microbiota are mutualistic to their native hosts but some are commensal or parasitic to foreign hosts, hinting at co-evolution between partners. Moreover, Pseudo-nitzschia host can gain protection from algalytic bacteria by maintaining association with its microbiota. Pseudo-nitzschia also exhibit different phenotypic expression with regards to DA production, and this depends on the bacterial species with which the host associates. Hence, the influences of the microbiota on diatom host physiology should be considered when studying the biology and ecology of marine diatoms.

Positive selection of a duplicated UV-sensitive visual pigment coincides with wing pigment evolution in Heliconius butterflies.

The butterfly Heliconius erato can see from the UV to the red part of the light spectrum with color vision proven from 440 to 640 nm. Its eye is known to contain three visual pigments, rhodopsins, produced by an 11-cis-3-hydroxyretinal chromophore together with long wavelength (LWRh), blue (BRh) and UV (UVRh1) opsins. We now find that H. erato has a second UV opsin mRNA (UVRh2)-a previously undescribed duplication of this gene among Lepidoptera. To investigate its evolutionary origin, we screened eye cDNAs from 14 butterfly species in the subfamily Heliconiinae and found both copies only among Heliconius. Phylogeny-based tests of selection indicate positive selection of UVRh2 following duplication, and some of the positively selected sites correspond to vertebrate visual pigment spectral tuning residues. Epi-microspectrophotometry reveals two UV-absorbing rhodopsins in the H. erato eye with lambda(max) = 355 nm and 398 nm. Along with the additional UV opsin, Heliconius have also evolved 3-hydroxy-DL-kynurenine (3-OHK)-based yellow wing pigments not found in close relatives. Visual models of how butterflies perceive wing color variation indicate this has resulted in an expansion of the number of distinguishable yellow colors on Heliconius wings. Functional diversification of the UV-sensitive visual pigments may help explain why the yellow wing pigments of Heliconius are so colorful in the UV range compared to the yellow pigments of close relatives lacking the UV opsin duplicate.

Impact of duplicate gene copies on phylogenetic analysis and divergence time estimates in butterflies.

BACKGROUND: The increase in availability of genomic sequences for a wide range of organisms has revealed gene duplication to be a relatively common event. Encounters with duplicate gene copies have consequently become almost inevitable in the context of collecting gene sequences for inferring species trees. Here we examine the effect of incorporating duplicate gene copies evolving at different rates on tree reconstruction and time estimation of recent and deep divergences in butterflies. RESULTS: Sequences from ultraviolet-sensitive (UVRh), blue-sensitive (BRh), and long-wavelength sensitive (LWRh) opsins,EF-1 and COI were obtained from 27 taxa representing the five major butterfly families (5535 bp total). Both BRh and LWRh are present in multiple copies in some butterfly lineages and the different copies evolve at different rates. Regardless of the phylogenetic reconstruction method used, we found that analyses of combined data sets using either slower or faster evolving copies of duplicate genes resulted in a single topology in agreement with our current understanding of butterfly family relationships based on morphology and molecules. Interestingly, individual analyses of BRh and LWRh sequences also recovered these family-level relationships. Two different relaxed clock methods resulted in similar divergence time estimates at the shallower nodes in the tree, regardless of whether faster or slower evolving copies were used, with larger discrepancies observed at deeper nodes in the phylogeny. The time of divergence between the monarch butterfly Danaus plexippus and the queen D. gilippus (15.3-35.6 Mya) was found to be much older than the time of divergence between monarch co-mimic Limenitis archippus and red-spotted purple L. arthemis (4.7-13.6 Mya), and overlapping with the time of divergence of the co-mimetic passionflower butterflies Heliconius erato and H. melpomene (13.5-26.1 Mya). Our family-level results are congruent with recent estimates found in the literature and indicate an age of 84-113 million years for the divergence of all butterfly families. CONCLUSION: These results are consistent with diversification of the butterfly families following the radiation of angiosperms and suggest that some classes of opsin genes may be usefully employed for both phylogenetic reconstruction and divergence time estimation.

The lycaenid butterfly Polyommatus icarus uses a duplicated blue opsin to see green.

The functional significance of gene duplication is rarely addressed at the level of animal behavior. Butterflies are excellent models in this regard because they can be trained and the use of their opsin-based visual pigments in color vision can be assessed. In the present study, we demonstrate that the lycaenid Polyommatus icarus uses its duplicate blue (B2) opsin, BRh2, in conjunction with its long-wavelength (LW) opsin, LWRh, to see color in the green part of the light spectrum extending up to 560 nm. This is in contrast to butterflies in the genus Papilio, which use duplicate LW opsins to discriminate colors in the long-wavelength range. We also found that P. icarus has a heterogeneously expressed red filtering pigment and red-reflecting ommatidia in the ventral eye region. In behavioural tests, the butterflies could not discriminate colors in the red range (570-640 nm). This finding is significant because we have previously found that the nymphalid butterfly Heliconius erato has filter-pigment mediated color vision in the long wavelength range. Our results suggest that lateral filtering pigments may not always influence color vision in insects.

Gene duplication is an evolutionary mechanism for expanding spectral diversity in the long-wavelength photopigments of butterflies.

Butterfly long-wavelength (L) photopigments are interesting for comparative studies of adaptive evolution because of the tremendous phenotypic variation that exists in their wavelength of peak absorbance (lambda(max) value). Here we present a comprehensive survey of L photopigment variation by measuring lambda(max) in 12 nymphalid and 1 riodinid species using epi-microspectrophotometry. Together with previous data, we find that L photopigment lambda(max) varies from 510-565 nm in 22 nymphalids, with an even broader 505- to 600-nm range in riodinids. We then surveyed the L opsin genes for which lambda(max) values are available as well as from related taxa and found 2 instances of L opsin gene duplication within nymphalids, in Hermeuptychia hermes and Amathusia phidippus, and 1 instance within riodinids, in the metalmark butterfly Apodemia mormo. Using maximum parsimony and maximum likelihood ancestral state reconstructions to map the evolution of spectral shifts within the L photopigments of nymphalids, we estimate the ancestral pigment had a lambda(max) = 540 nm +/- 10 nm standard error and that blueshifts in wavelength have occurred at least 4 times within the family. We used ancestral state reconstructions to investigate the importance of several amino acid substitutions (Ile17Met, Ala64Ser, Asn70Ser, and Ser137Ala) previously shown to have evolved under positive selection that are correlated with blue spectral shifts. These reconstructions suggest that the Ala64Ser substitution has indeed occurred along the newly identified blueshifted L photopigment lineages. Substitutions at the other 3 sites may also be involved in the functional diversification of L photopigments. Our data strongly suggest that there are limits to the evolution of L photopigment spectral shifts among species with only one L opsin gene and that opsin gene duplication broadens the potential range of lambda(max) values.

Adaptive evolution of color vision as seen through the eyes of butterflies.

Butterflies and primates are interesting for comparative color vision studies, because both have evolved middle- (M) and long-wavelength- (L) sensitive photopigments with overlapping absorbance spectrum maxima (lambda(max) values). Although positive selection is important for the maintenance of spectral variation within the primate pigments, it remains an open question whether it contributes similarly to the diversification of butterfly pigments. To examine this issue, we performed epimicrospectrophotometry on the eyes of five Limenitis butterfly species and found a 31-nm range of variation in the lambda(max) values of the L-sensitive photopigments (514-545 nm). We cloned partial Limenitis L opsin gene sequences and found a significant excess of replacement substitutions relative to polymorphisms among species. Mapping of these L photopigment lambda(max) values onto a phylogeny revealed two instances within Lepidoptera of convergently evolved L photopigment lineages whose lambda(max) values were blue-shifted. A codon-based maximum-likelihood analysis indicated that, associated with the two blue spectral shifts, four amino acid sites (Ile17Met, Ala64Ser, Asn70Ser, and Ser137Ala) have evolved substitutions in parallel and exhibit significant d(N)/d(S) >1. Homology modeling of the full-length Limenitis arthemis astyanax L opsin placed all four substitutions within the chromophore-binding pocket. Strikingly, the Ser137Ala substitution is in the same position as a site that in primates is responsible for a 5- to 7-nm blue spectral shift. Our data show that some of the same amino acid sites are under positive selection in the photopigments of both butterflies and primates, spanning an evolutionary distance >500 million years.

Beauty in the eye of the beholder: the two blue opsins of lycaenid butterflies and the opsin gene-driven evolution of sexually dimorphic eyes.

Although previous investigations have shown that wing coloration is an important component of social signaling in butterflies, the contribution of opsin evolution to sexual wing color dichromatism and interspecific divergence remains largely unexplored. Here we report that the butterfly Lycaena rubidus has evolved sexually dimorphic eyes due to changes in the regulation of opsin expression patterns to match the contrasting life histories of males and females. The L. rubidus eye contains four visual pigments with peak sensitivities in the ultraviolet (UV; lambdamax=360 nm), blue (B; lambdamax=437 nm and 500 nm, respectively) and long (LW; lambdamax=568 nm) wavelength range. By combining in situ hybridization of cloned opsin-encoding cDNAs with epi-microspectrophotometry, we found that all four opsin mRNAs and visual pigments are expressed in the eyes in a sex-specific manner. The male dorsal eye, which contains only UV and B (lambdamax=437 nm) visual pigments, indeed expresses two short wavelength opsin mRNAs, UVRh and BRh1. The female dorsal eye, which also has the UV and B (lambdamax=437 nm) visual pigments, also contains the LW visual pigment, and likewise expresses UVRh, BRh1 and LWRh mRNAs. Unexpectedly, in the female dorsal eye, we also found BRh1 co-expressed with LWRh in the R3-8 photoreceptor cells. The ventral eye of both sexes, on the other hand, contains all four visual pigments and expresses all four opsin mRNAs in a non-overlapping fashion. Surprisingly, we found that the 500 nm visual pigment is encoded by a duplicate blue opsin gene, BRh2. Further, using molecular phylogenetic methods we trace this novel blue opsin gene to a duplication event at the base of the Polyommatine+Thecline+Lycaenine radiation. The blue opsin gene duplication may help explain the blueness of blue lycaenid butterflies.

Color discrimination in the red range with only one long-wavelength sensitive opsin.

The basic precondition for color vision is the presence of at least two receptor types with different spectral sensitivities. The sensitivity of a receptor is mostly defined by the opsin-based visual pigment expressed in it. We show here, through behavioral experiments, that the nymphalid butterfly Heliconius erato, although it expresses short and medium wavelength opsins and only one long wavelength opsin, discriminates colors in the long-wavelength range (590 nm, 620 nm and 640 nm), whereas another nymphalid, Vanessa atalanta, despite having color vision, is unable to do so. In the eyes of H. erato we identified filtering pigments very close to the rhabdom which differ between ommatidia and produce the yellow and red ommatidial reflection seen under orthodromic illumination. The eyes of V. atalanta lack the filtering pigments, and reflect a homogeneous orange. We hypothesize that the filtering pigments found in the eyes of H. erato may shift the spectral sensitivity peak of the long wavelength receptors in some ommatidia towards longer wavelengths. The comparison of the signals between the two new receptor types makes color discrimination in the red range possible. To our knowledge, this is the first behavioral proof of color vision based on receptors expressing the same opsin.