Feasible mechanisms for algal digestion in the king angelfish

To determine the ability of the king angelfish Holacanthus passer to digest algae, three algal species were immersed in acidic conditions similar to that found in the stomach of fish. Only one of them was not susceptible to acidic lysis; two were affected after 40 and 60 min at pH 2·0. King angelfish have an expanded region of the intestine called here the hindgut chamber (HC) containing populations of micro-organisms. Some of these micro-organisms have the capacity to grow in cellulose, agar, and alginic acid; the main components of algal cell walls. Micro-organisms grew in carboxymethylcellulose cultures under aerobic and micro-aerobic conditions. The HC is highly vascularized, which could increase absorptive efficiency of material digested in it.     

Differences in the pattern of antipredator behaviour between hatchery‐reared and wild European sea bass juveniles

Shoals of hatchery‐reared and wild sea bass juveniles Dicentrarchus labrax were tested for differences in their antipredator responses towards a potential live predator, the eel Anguilla anguilla . Eight experimental shoals ( i.e . replicates), each composed of 15 individuals from the same stock of juveniles ( i.e . wild or hatchery), were video recorded for 5 min before and after predator exposure. A set of behavioural variables were measured during the pre‐stimulus and stimulus phases of each test and compared between the two groups of replicates. Results showed that in both hatchery‐reared and wild juveniles predator exposure elicited a significant increase in the mean level of shoal cohesiveness and mean shoal distance from the predator, and a significant decrease in the mean shoal distance from the bottom. Shoals of wild juveniles, however, aggregated more quickly and reached higher shoal cohesiveness within the first 20 s of the stimulus period than shoals of hatchery‐reared fish. During this period, the wild fish also reached the highest peak in shoal cohesiveness, which then decreased gradually towards the levels observed before predator exposure. Another component of the antipredator response, the predator inspection behaviour, was fully developed in both wild and hatchery fish. Wild fish, however, tended to inspect the predator at a closer distance than hatchery fish.     

Effects of life phase and schooling patterns on the foraging behaviour of coral‐reef fishes from the genus Haemulon

During this study (December 2009 to December 2010), underwater visual surveys using the focal animal method were performed in the coastal reefs of Tamandaré, north‐eastern Brazil. The aim was to analyse the effects of the life phase (juvenile and adult) and schooling patterns (school and solitary) on the feeding behaviour (foraging rates and substratum preferences) of four species of the genus Haemulon (Haemulon aurolineatum, Haemulon parra, Haemulon plumieri and Haemulon squamipinna). PERMANOVA analysis (P < 0·05) indicated that ontogenetic changes and schooling patterns directly influence foraging behaviour. Schooling individuals had low foraging rates (mean ± s.d . = 2·3 ± 2·1 bites 10 min^?1) and mobility, usually remaining near the bottom; however, solitary fishes had high foraging rates (mean ± s.d . = 12·5 ± 4·6 bites 10 min^?1). Juveniles preferred feeding in the water column (75% of the total number of bites), whereas adults foraged mainly in sand (80%) and bare rock (20%). All four Haemulon species displayed similar patterns of feeding behaviour as well as preferences for foraging sites and display competition for food resources. In contrast, little is known about their habitat use and foraging behaviour over the diel cycle, particularly the newly settled and early juvenile stages.     

Phylogenetic relationships of Combretaceae inferred from nuclear and plastid DNA sequence data: implications for generic classification

The putative complexity of Combretaceae and lack of information on phylogenetic relationships within the family led us to explore relationships between genera of Combretaceae by means of combined analyses of plastid and nuclear sequences. We collected DNA sequence data from the nuclear ribosomal internal transcribed spacer region and plastid rbcL, psaA‐ycf3 spacer and psbA‐trnH spacer for 14 of the 17 genera of Combretaceae. The current classification of the family into two subfamilies, Strephonematoideae and Combretoideae, is corroborated. Within Combretoideae, division into two tribes, Laguncularieae and Combreteae, is strongly supported. Within Combreteae subtribe Terminaliinae, relationships between genera are largely unresolved. Terminalia is not supported as monophyletic and two groups were identified, one containing mainly African species and another of mostly Asian species. Pteleopsis, Buchenavia and Anogeissus are embedded within Terminalia, and we suggest that all genera of Terminaliinae, with the exception of Conocarpus, should be included in an expanded circumscrition of Terminalia. Within subtribe Combretinae, a clade formed by the two monotypic genera Guiera and Calycopteris is sister to the rest of the subtribe. Groupings in Combretinae are consistent with recent results based on morphological data. Combretum is currently divided into three subgenera: Apethalanthum, Cacoucia and Combretum. The last two were included in this study and supported as monophyletic if Quisqualis is included within subgenus Cacoucia. Meiostemon is sister to subgenus Combretum. We recommend that subgenus Combretum should be expanded to include Meiostemon and subgenus Cacoucia to include Quisqualis. The sectional classification within Combretum proposed in earlier morphological studies is confirmed except for the exclusion of C. imberbe from section Hypocrateropsis in a separate and monotypic section and the inclusion of C. zeyheri (section Spathulipetala) in section Macrostigmatea. In order to accommodate C. imberbe, a new section is suggested. The reinstatement of previously recognized sections Grandiflora and Trichopetala, both of which had been sunk into subgenus Cacoucia section Poivrea, is proposed. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 162 , 453–476.     

Microsatellite loci for studies on population differentiation and connectivity of the red‐bellied yellow tail fusilier,Caesio cuning (Caesionidae)

Ten polymorphic microsatellites were isolated from the red‐bellied yellow tail fusilier Caesio cuning, a reef‐associated fish which occurs widely in the Indo‐Pacific region. The species is exploited by both small‐scale and commercial fisheries. Fifty individuals from six populations were genotyped using primers that reliably amplified 10 polymorphic loci. The number of alleles per locus ranged from three to 11. Observed heterozygosity (H_O) and expected heterozygosity (H_E) ranged between 0.400 to 0.880 and 0.337 to 0.843, respectively. These microsatellite loci may be used to study population structure, genetic diversity and connectivity of C. cuning in the range of its distribution.     

Seasonal stability and spatial variability of parasites in Brazilian sandperch Pinguipes brasilianus from the Northern Argentine Sea: evidence for stock discrimination

The use of parasites as biological tags allowed the identification of two stocks of Brazilian sandperch Pinguipes brasilianus , in the Bonaerense region of the Argentine Sea. A total of 156 adult specimens of P. brasilianus were examined for parasites. Temporal variability in parasite burdens was assessed from fish caught seasonally in coastal waters of two zones, south Bonaerense (during autumn and winter) and north Bonaerense (during summer). Additional data from a previous study, comprising P. brasilianus caught during spring in these two zones, as well as in two populations from north Patagonian gulfs were used for comparative analyses of spatial variability. A total of 14 193 metazoan parasites belonging to 19 species were found. Comparisons of seasonal variability in pooled samples and within each locality showed that locality effects exceeded seasonal ones, suggesting the possible existence of two discrete stocks in the Bonaerense region. These findings were strongly supported by discriminant analyses and comparisons of prevalence and abundance between zones, after pooling seasonal samples within each zone. Further evidence of the discreteness of both stocks was assessed by inclusion of samples from Patagonian gulfs in the discriminant analysis, confirming that their differences were at a inter-population level. The parasite species that contributed most to the separation of the samples were generally those identified as biological markers in previous studies. Differing oceanographic conditions are discussed as potential causes of inter-population variation of parasite burdens.     

A computational fluid dynamics (CFD) based method for assessing the hydrodynamic impact of animal borne data loggers on host marine mammals

Animal‐borne data loggers (ABDLs) or “tags” are regularly used to elucidate animal ecology and physiology, but current literature highlights the need to assess associated deleterious impacts including increased resistive force to motion. Previous studies have used computational fluid dynamics (CFD) to estimate this impact, but many suffer limitations (e.g., inaccurate turbulence modeling, neglecting boundary layer transition, neglecting added mass effects, and analyzing the ABDL in isolation from the animal). A novel CFD‐based method is presented in which a “tag impact envelope” is defined utilizing simulations with and without transition modeling to define upper and lower drag limits, respectively, and added mass coefficients are found via simulations with sinusoidally varying inlet velocity, with modified Navier‐Stokes conservation of momentum equations enforcing a shift to the animal's noninertial reference frame. The method generates coefficients for calculating total resistive force for any velocity and acceleration combination, and is validated against theory for a prolate spheroid. An example case shows ABDL drag impact on a harp seal of 11.21%–16.24%, with negligible influence on added mass. By considering the effects of added mass and boundary layer transition, the approach presented is an enhancement to the CFD‐based ABDL impact assessment methods previously applied by researchers.     

Genomewide SNP data reveal cryptic phylogeographic structure and microallopatric divergence in a rapids‐adapted clade of cichlids from the Congo River

The lower Congo River is a freshwater biodiversity hot spot in Africa characterized by some of the world's largest rapids. However, little is known about the evolutionary forces shaping this diversity, which include numerous endemic fishes. We investigated phylogeographic relationships in Teleogramma, a small clade of rheophilic cichlids, in the context of regional geography and hydrology. Previous studies have been unable to resolve phylogenetic relationships within Teleogramma due to lack of variation in nuclear genes and discrete morphological characters among putative species. To sample more broadly across the genome, we analysed double‐digest restriction‐associated sequencing (ddRAD) data from 53 individuals across all described species in the genus. We also assessed body shape and mitochondrial variation within and between taxa. Phylogenetic analyses reveal previously unrecognized lineages and instances of microallopatric divergence across as little as ~1.5 km. Species ranges appear to correspond to geographic regions broadly separated by major hydrological and topographic barriers, indicating these features are likely important drivers of diversification. Mitonuclear discordance indicates one or more introgressive hybridization events, but no clear evidence of admixture is present in nuclear genomes, suggesting these events were likely ancient. A survey of female fin patterns hints that previously undetected lineage‐specific patterning may be acting to reinforce species cohesion. These analyses highlight the importance of hydrological complexity in generating diversity in certain freshwater systems, as well as the utility of ddRAD‐Seq data in understanding diversification processes operating both below and above the species level.     

Consequences of marine barriers for genetic diversity of the coral‐specialist yellowbar angelfish from the Northwestern Indian Ocean

Ocean circulation, geological history, geographic distance, and seascape heterogeneity play an important role in phylogeography of coral‐dependent fishes. Here, we investigate potential genetic population structure within the yellowbar angelfish (Pomacanthus maculosus) across the Northwestern Indian Ocean (NIO). We then discuss our results with respect to the above abiotic features in order to understand the contemporary distribution of genetic diversity of the species. To do so, restriction site‐associated DNA sequencing (RAD‐seq) was utilized to carry out population genetic analyses on P. maculosus sampled throughout the species’ distributional range. First, genetic data were correlated to geographic and environmental distances, and tested for isolation‐by‐distance and isolation‐by‐environment, respectively, by applying the Mantel test. Secondly, we used distance‐based and model‐based methods for clustering genetic data. Our results suggest the presence of two putative barriers to dispersal; one off the southern coast of the Arabian Peninsula and the other off northern Somalia, which together create three genetic subdivisions of P. maculosus within the NIO. Around the Arabian Peninsula, one genetic cluster was associated with the Red Sea and the adjacent Gulf of Aden in the west, and another cluster was associated with the Arabian Gulf and the Sea of Oman in the east. Individuals sampled in Kenya represented a third genetic cluster. The geographic locations of genetic discontinuities observed between genetic subdivisions coincide with the presence of substantial upwelling systems, as well as habitat discontinuity. Our findings shed light on the origin and maintenance of genetic patterns in a common coral reef fish inhabiting the NIO, and reinforce the hypothesis that the evolution of marine fish species in this region has likely been shaped by multiple vicariance events.     

Phylogeography of European sea bass in the north‐east Atlantic: a correction and reanalysis of the mitochondrial DNA data from Coscia & Mariani (2011)

The re‐analysis of mtDNA sequence data on sea bass (Dicentrarchus labrax) in the north‐east Atlantic revealed the presence of a ‘slippage error’ in the alignment of the mitochondrial DNA (mtDNA) control region sequences. This induced an overestimation of the genetic variability within this species, and hence the inference of a striking multi‐clade partitioning of D. labrax populations within this area. After correction, the existence of highly distinct D. labrax haplogroups in Atlantic areas does not hold anymore, but the robust dichotomy between a single Mediterranean and a single Atlantic subgroup is confirmed. Here we present the new results related to the amended mtDNA control region alignment, and also summarize the key message in relation to the microsatellite data, which are unaffected by this revision. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 455–458.     

Phylogeography and the origins of range disjunctions in a north temperate fish,the pygmy whitefish (Prosopium coulterii), inferred from mitochondrial and nuclear DNA sequence analysis

Aim To investigate the degree of phylogeographical divergence within pygmy whitefish (Prosopium coulterii) and to test hypotheses concerning the origin of disjunct populations within North America. Location North America from western Alaska to Lake Superior. Methods Mitochondrial (ATPase subunit VI) and nuclear (ITS‐1, ITS‐2) DNA sequence variation was assessed across the species’ North American range to test for the existence of distinct phylogeographical groupings of pygmy whitefish associated with known glacial refugia. Coalescent simulations of the mitochondrial DNA (mtDNA) data were used to test hypotheses of population structure. Results This species is composed of two monophyletic mitochondrial clades across its North American range. The two mtDNA clades differed by an average 3.3% nucleotide sequence divergence. These clades were also distinguished by ITS‐2, but the relationships among lineages were not resolved by the ITS‐1 analysis. Coalescent analyses rejected the null hypothesis of the current disjunct distributions being a result of fragmentation of a single widespread ancestral lineage across a variety of effective population sizes and divergence times. Main conclusions The current range disjunctions of pygmy whitefish in North America probably resulted from isolation, genetic divergence, and selective dispersal from at least two major Pleistocene glacial refugia: Beringia and Cascadia. More recent isolation and dispersal from an upper Mississippi refugium is suggested by relationships within one of the clades and by distributional evidence from co‐distributed species. The Beringian and Cascadian refugia have played major roles in the zoogeography of Nearctic temperate aquatics, but the roles of smaller refugia appear more variable among other species.     

Inferring the geographic origin of a range expansion: Latitudinal and longitudinal coordinates inferred from genomic data in an ABC framework with the program x‐origin

Climatic or environmental change is not only driving distributional shifts in species today, but it has also caused distributions to expand and contract in the past. Inferences about the geographic locations of past populations especially regions that served as refugia (i.e., source populations) and migratory routes are a challenging endeavour. Refugial areas may be evidenced from fossil records or regions of temporal stability inferred from ecological niche models. Genomic data offer an alternative and broadly applicable source of information about the locality of refugial areas, especially relative to fossil data, which are either unavailable or incomplete for most species. Here, we present a pipeline we developed (called x ‐origin ) for statistically inferring the geographic origin of range expansion using a spatially explicit coalescent model and an approximate Bayesian computation testing framework. In addition to assessing the probability of specific latitudinal and longitudinal coordinates of refugial or source populations, such inferences can also be made accounting for the effects of temporal and spatial environmental heterogeneity, which may impact migration routes. We demonstrate x ‐origin with an analysis of genomic data collected in the Collared pika that underwent postglacial expansion across Alaska, as well as present an assessment of its accuracy under a known model of expansion to validate the approach.     

Risk‐taking and the evolution of mechanisms for rapid escape from predators

Flight initiation distance (FID) is the distance at which an individual animal takes flight when approached by a human. This behavioural measure of risk‐taking reflects the risk of being captured by real predators, and it correlates with a range of life history traits, as expected if flight distance optimizes risk of predation. Given that FID provides information on risk of predation, we should expect that physiological and morphological mechanisms that facilitate flight and escape predict interspecific variation in flight distance. Haematocrit is a measure of packed red blood cell volume and as such indicates the oxygen transport ability and hence the flight muscle contracting reaction of an individual. Therefore, we predicted that species with short flight distances, that allow close proximity between a potential prey individual and a predator, would have high haematocrit. Furthermore, we predicted that species with large wing areas and hence relatively low costs of flight and species with large aspect ratios and hence high manoeuvrability would have evolved long flight speed. Consistent with these predictions, we found in a sample of 63 species of birds that species with long flight distances for their body size had low levels of haematocrit and large wing areas and aspect ratios. These findings provide evidence consistent with the evolution of risk‐taking behaviour being underpinned by physiological and morphological mechanisms that facilitate escape from predators and add to our understanding of predator–prey coevolution.     

Phylogeography and phylogeny of the epineritic cosmopolitan bonitos of the genus Sarda (Cuvier): inferred patterns of intra‐ and inter‐oceanic connectivity derived from nuclear and mitochondrial DNA data

Aim To reconstruct the phylogenetic relationships of the four species of the genus Sarda (Sarda sarda, Sarda orientalis, Sarda australis and Sarda chilensis) and their phylogeographic history in the context of historical and ecological biogeography. Also, to reconstruct within‐species phylogenetic relationships to test whether the North Atlantic and Mediterranean populations of Atlantic bonito (S. sarda) warrant subspecies status, and the validity of the allopatric northern and southern populations of eastern Pacific bonito (S. chiliensis), recognized as S. chiliensis lineolata and S. chiliensis chiliensis. Location Representative samples of all four Sarda species collected world‐wide were analysed. Methods Phylogenetic inference was carried out with neighbour‐joining, maximum parsimony and maximum likelihood, employing nucleotide sequences of the mitochondrial DNA (mtDNA) control region I (CR‐I) and of the single‐copy nuclear DNA (nDNA) Tmo‐4c4 gene. Analysis of molecular variance was used on the mtDNA data to estimate the extent of geographic population structuring. Results Gene trees derived from mtDNA and nDNA data yielded concordant phylogenies that support the monophyly of the genus Sarda. The following sibling pairs received strong statistical support: striped bonito (S. orientalis) with Australian bonito (S. australis), and Atlantic bonito (S. sarda) with eastern Pacific bonito (S. chiliensis). Furthermore, the origin of S. sarda mtDNA is paraphyletic with respect to S. chiliensis, and these results are indicative of introgression. The analysis of Tmo‐4c4 sequences corroborates the ancestral hybridization between these allopatric species. Comparisons of north‐west Atlantic and Mediterranean populations of S. sarda using mtDNA CR‐I data revealed substantial genetic differentiation. By contrast, no differences between the putative northern and southern allopatric subspecies of S. chiliensis were detected. Main conclusions The monophyly of the genus Sarda as indicated by morphology is corroborated using both molecular markers. However, molecular phylogenies depicted a paraphyletic relationship between S. sarda and S. chiliensis. This phylogeographical relationship is better explained by an ancestral introgression facilitated by trans‐Arctic contact during the Pleistocene. The pronounced genetic differentiation between S. sarda samples from the north‐west Atlantic and the Mediterranean is consistent with the differentiation of these two regions, but not with the amphi‐Atlantic speciation hypothesis. Finally, the S. chiliensis lineolata and S. chiliensis chiliensis subspecies status is not supported by the molecular data.     

Predictive cross-validation for the choice of linear mixed-effects models with application to data from the Swiss HIV Cohort Study

Model choice in linear mixed-effects models for longitudinal data is a challenging task. Apart from the selection of covariates, also the choice of the random effects and the residual correlation structure should be possible. Application of classical model choice criteria such as Akaike information criterion (AIC) or Bayesian information criterion is not obvious, and many versions do exist. In this article, a predictive cross-validation approach to model choice is proposed based on the logarithmic and the continuous ranked probability score. In contrast to full cross-validation, the model has to be fitted only once, which enables fast computations, even for large data sets. Relationships to the recently proposed conditional AIC are discussed. The methodology is applied to search for the best model to predict the course of CD4+ counts using data obtained from the Swiss HIV Cohort Study.     

Higher phylogeny of zygaenid moths (Insecta: Lepidoptera) inferred from nuclear and mitochondrial sequence data and the evolution of larval cuticular cavities for chemical defence

Zygaenid moths are capable of releasing hydrogen cyanide in their defense by enzymatic break-down of cyanoglucosides, but only larvae of chalcosiine and zygaenine moths store cyanogenic compounds in cuticular cavities and thus are able to discharge defense droplets, which effectively deter potential predators. A previously proposed phylogeny of Zygaenidae hypothesized a sister group relationship of chalcosiine and zygaenine moths because of their similar larval defense system. Not all chalcosiine taxa possess cuticular cavities, however, and a comparable defense mechanism has been reported in larvae of the zygaenoid family Heterogynidae. Considering sequence data of seven molecular loci, the present study estimates the posterior probability of phylogenetic hypotheses explaining the occurrence of larval cuticular cavities. The molecular data confirm the previous exclusion of Himantopteridae from Zygaenidae and suggest their close affinity to Somabrachyidae. The sequence data also corroborate the recently proposed exclusion of the Phaudinae from the Zygaenidae, because this subfamily is recovered in a reasonably well supported species cluster consisting of members of the families Lacturidae, Limacodidae, Himantopteridae, and Somabrachyidae. We consequently agree to raise Phaudinae to family rank. Within Zygaenidae, the subfamilies Callizygaeninae, Chalcosiinae, and Procridinae most likely constitute a monophyletic group, which is sister to the Zygaeninae. Our results imply that cuticular cavities were probably present in the larvae of the most recent common ancestor of Zygaenidae. Heterogynidae cannot be confirmed as sister taxon to this family, but appear at the very first split of the Zygaenoidea, although with poor support. The specific pattern of taxa in the molecular phylogeny showing larval cuticular cavities opens the possibility that these structures could have been already present in the most recent common ancestor of the Zygaenoidea.     

Molecular evidence for adaptive radiation of Micromeria Benth. (Lamiaceae) on the Canary Islands as inferred from chloroplast and nuclear DNA sequences and ISSR fingerprint data

The Canary Islands have been a focus for phylogeographic studies on the colonization and diversification of endemic angiosperm taxa. Based on phylogeographic patterns, both inter island colonization and adaptive radiation seem to be the driving forces for speciation in most taxa. Here, we investigated the diversification of Micromeria on the Canary Islands and Madeira at the inter- and infraspecific level using inter simple sequence repeat PCR (ISSR), the trnK-Intron and the trnT-trnL-spacer of the cpDNA and a low copy nuclear gene. The genus Micromeria (Lamiaceae, Mentheae) includes 16 species and 13 subspecies in Macaronesia. Most taxa are restricted endemics, or grow in similar ecological conditions on two islands. An exception is M. varia, a widespread species inhabits the lowland scrub on each island of the archipelago and could represent an ancestral taxon from which radiation started on the different islands. Our analyses support a split between the "eastern" islands Fuerteventura, Lanzarote and Gran Canaria and the "western" islands Tenerife, La Palma and El Hierro. The colonization of Madeira started from the western Islands, probably from Tenerife as indicated by the sequence data. We identified two lineages of Micromeria on Gomera but all other islands appear to be colonized by a single lineage, supporting adaptive radiation as the major evolutionary force for the diversification of Micromeria. We also discuss the possible role of gene flow between lineages of different Micromeria species on one island after multiple colonizations.     

Genetic support for random mating between left and right‐mouth morphs in the dimorphic scale‐eating cichlid fish Perissodus microlepis from Lake Tanganyika

Population genetic analyses were conducted to investigate whether random mating occurs between left and right‐mouth morphs of the dimorphic scale‐eating cichlid fish Perissodus microlepis from two geographical sites in southern Lake Tanganyika. The mitochondrial and nuclear DNA markers (13 microsatellite loci) revealed no genetic differentiation between left and right morphs (i.e. widespread interbreeding). The observed lack of genetic divergence between the different morphs allowed for the exclusion of the possibility of assortative mating between same morph types. The microsatellite data showed no significant departures of heterozygosity from Hardy–Weinberg equilibrium, suggesting purely random mating between the morphs. Overall, this study indicated no genetic evidence for either assortative or disassortative mating, but it did provide support for the random mating hypothesis. Highly significant, albeit weak, spatial population structure was also found when samples of different morphs were pooled according to geographical sites. An additional analysis of two microsatellite loci that were recently suggested to be putatively linked to the genetic locus that determines the laterality of these mouth morphs did not show any such association.     

Dual‐color‐emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy

We isolated and characterized a green fluorescent protein (GFP) from the sea cactus Cavernularia obesa. This GFP exists as a dimer and has absorption maxima at 388 and 498 nm. Excitation at 388 nm leads to blue fluorescence (456 nm maximum) at pH 5 and below, and green fluorescence (507 nm maximum) at pH 7 and above, and the GFP is remarkably stable at pH 4. Excitation at 498 nm leads to green fluorescence (507 nm maximum) from pH 5 to pH 9. We introduced five amino acid substitutions so that this GFP formed monomers rather than dimers and then used this monomeric form to visualize intracellular pH change during the phagocytosis of living cells by use of fluorescence microscopy. The intracellular pH change is visualized by use of a simple long‐pass emission filter with single‐wavelength excitation, which is technically easier to use than dual‐emission fluorescent proteins that require dual‐wavelength excitation. Copyright © 2013 John Wiley & Sons, Ltd.