Field‐readable alphanumeric flags are valuable markers for shorebirds: use of double‐marking to identify cases of misidentification

Implicit assumptions for most mark‐recapture studies are that individuals do not lose their markers and all observed markers are correctly recorded. If these assumptions are violated, e.g., due to loss or extreme wear of markers, estimates of population size and vital rates will be biased. Double‐marking experiments have been widely used to estimate rates of marker loss and adjust for associated bias, and we extended this approach to estimate rates of recording errors. We double‐marked 309 Piping Plovers (Charadrius melodus) with unique combinations of color bands and alphanumeric flags and used multi‐state mark recapture models to estimate the frequency with which plovers were misidentified. Observers were twice as likely to read and report an invalid color‐band combination (2.4% of the time) as an invalid alphanumeric code (1.0%). Observers failed to read matching band combinations or alphanumeric flag codes 4.5% of the time. Unlike previous band resighting studies, use of two resightable markers allowed us to identify when resighting errors resulted in reports of combinations or codes that were valid, but still incorrect; our results suggest this may be a largely unappreciated problem in mark‐resight studies. Field‐readable alphanumeric flags offer a promising auxiliary marker for identifying and potentially adjusting for false‐positive resighting errors that may otherwise bias demographic estimates.     

An experimental evaluation of the effects of geolocator design and attachment method on between‐year survival on whinchats Saxicola rubetra

Data from location logging tags have revolutionised our understanding of migration ecology, but methods of tagging that do not compromise survival need to be identified. We compared resighting rates for 156 geolocator‐tagged and 316 colour ringed‐only whinchats on their African wintering grounds after migration to and from eastern Europe in two separate years. We experimentally varied both light stalk length (0, 5 and 10 mm) and harness material (elastic or non‐elastic nylon braid tied on, leg‐loop ‘Rappole’ harnesses) in the second year using a reasonably balanced design (all tags in the first year used an elastic harness and 10 mm light stalk). Tags weighed 0.63 g (0.01 SE), representing 4.1% of average body mass. There was no overall significant reduction in between‐year resighting rate (our proxy for survival) comparing tagged and untagged birds in either year. When comparing within tagged birds, however, using a tied harness significantly reduced resighting rate by 53% on average compared to using an elastic harness (in all models), but stalk length effects were not statistically significant in any model considered. There was no strong evidence that the fit (relative tightness) or added tag mass affected survival, although tied tags were fitted more tightly later in the study, and birds fitted with tied tags later may have had lower survival. Overall, on a precautionary principle, deploying tags with non‐elastic tied harnesses should be avoided because the necessary fit, so as not to reduce survival, is time‐consuming to achieve and does not necessarily improve with experience. Geolocator tags of the recommended percentage of body mass fitted with elastic leg‐loop harnesses and with short light stalks can be used without survival effects in small long‐distance migrant birds.     

In situ tagging technique for fishes provides insight into growth and movement of invasive lionfish

Information on fish movement and growth is primarily obtained through the marking and tracking of individuals with external tags, which are usually affixed to anesthetized individuals at the surface. However, the quantity and quality of data obtained by this method is often limited by small sample sizes owing to the time associated with the tagging process, high rates of tagging‐related mortality, and displacement of tagged individuals from the initial capture location. To address these issues, we describe a technique for applying external streamer and dart tags in situ, which uses SCUBA divers to capture and tag individual fish on the sea floor without the use of anesthetic. We demonstrate this method for Indo‐Pacific lionfish (Pterois volitans/P. miles), species which are particularly vulnerable to barotrauma when transported to and handled at the surface. To test our method, we tagged 161 individuals inhabiting 26 coral reef locations in the Bahamas over a period of 3 years. Our method resulted in no instances of barotrauma, reduced handling and recovery time, and minimal post‐tagging release displacement compared with conventional ex situ tag application. Opportunistic resighting and recapture of tagged individuals reveals that lionfish exhibit highly variable site fidelity, movement patterns, and growth rates on invaded coral reef habitats. In total, 24% of lionfish were resighted between 29 and 188 days after tagging. Of these, 90% were located at the site of capture, while the remaining individuals were resighted between 200 m and 1.1 km from initial site of capture over 29 days later. In situ growth rates ranged between 0.1 and 0.6 mm/day. While individuals tagged with streamer tags posted slower growth rates with increasing size, as expected, there was no relationship between growth rate and fish size for individuals marked with dart tags, potentially because of large effects of tag presence on the activities of small bodied lionfish (i.e., <150 mm), where the tag was up to 7.6% of the lionfish's mass. Our study offers a novel in situ tagging technique that can be used to provide critical information on fish site fidelity, movement patterns, and growth in cases where ex situ tagging is not feasible.     

Association of flower color with pollen reward may explain increased bumblebee visitation to the Scotch broom yellow morph

Given that pollinators usually visit flowers for hidden rewards, they need to rely on floral traits that indicate reward status (“honest signals”). However, the relationship between pollination, honest signals, and floral rewards is little documented in natural conditions. The Scotch broom (Cytisus scoparius) is an invasive shrub with polymorphism in the color of its flowers that can be yellow, orange, or red. In three areas dominated by the Scotch broom, we described the abundance of the floral morphs and estimated bumblebee (Bombus terrestris) visitation rate. We examined whether bumblebee visitation to the floral morphs was related to pollen reward. We collected flowers and classified their stamens according to their function: reward or pollen export. Then, we measured anther size and estimated pollen quantity. The yellow morph was more abundant and more visited by bumblebees than the orange and red morphs. The yellow flowers did indeed offer more pollen than the other morphs and this occurred only for rewarding anthers, suggesting that bumblebees could use yellow color as an honest signal to visit the most rewarding flowers. We discuss whether innate and/or learned preferences of bumblebees can explain why the yellow morph is more visited, pollinated, and abundant, while the other morphs are maintained at a lower frequency. This is one of the few field works that shows that variation in intra-specific floral traits is associated with variation in floral reward and pollinator visitation rate, helping to understand the foraging preferences of pollinators and the coexistence of floral morphs in nature.

Clinical trials registration: Not applicable.

     

Improved methods for color‐marking hummingbirds

ABSTRACT Individual color‐marking is an essential tool for studying the behavior of free‐living birds. Hummingbirds represent a particular challenge for traditional avian color‐marking techniques because of their small size and short tarsi. Although several techniques have been successfully used, the retention time of color‐markers and their safety and ease of construction could be improved. I developed two new color‐marking techniques for marking Little Hermits (Phaethornis longuemareus): (1) a plastic back tag constructed by fusing colored beads, and (2) a leg tag attached to a metal band fitted around the tarsus. Both tag designs were visible in field conditions, and neither appeared to adversely affect behavior. Both back and leg tags had high retention rates within seasons, but back tags had a poor retention rate between years. Although these marking techniques were designed for use on one species of hummingbird, they would likely also be useful with other species of hummingbirds.     

Linking frugivores to the dynamics of a fruit color polymorphism

Although fruit color polymorphisms are a widespread phenomenon, the role of frugivores in their maintenance is unknown. Selection would require that frugivores interact differentially with fruit color morphs to alter their relative fitnesses, but such a pattern has yet to be demonstrated. In a 3-yr field study, the interactions of ants and birds with Acacia ligulata, an Australian shrub with a red/yellow/ orange aril color polymorphism, were examined. Bird species fell into three feeding guilds: seed dispersers, seed predators, and aril thieves; ant species acted either as seed dispersers or aril thieves. While there was no evidence of morph bias in ants, in some years birds fed more frequently on the yellow and orange morphs. Based on patterns of seedling survival and juvenile recruitment in seed deposition sites, bird seed dispersers increased the fitness of yellow and orange morphs (relative to red) in some populations, but decreased their relative fitness in others. Bird seed predators uniformly reduced relative fitness of yellow and orange morphs, while bird aril thieves had unknown effects. Altogether, consumer biases produced spatiotemporal variability in the relative fitness of A. ligulata color morphs, a pattern qualitatively consistent with maintenance of the polymorphism.     

Growth, survival, and tag retention of steelhead trout (Oncorhynchus mykiss) and its application to survival estimates

Steelhead trout, Oncorhynchus mykiss, are known to expel acoustic tags which can negatively bias population survival estimates. Tag burden may also affect the development and behavior of smolts, thereby impacting the results of population and behavioral studies. We monitored the growth, condition, and tag expulsion rate of steelhead trout in similar-sized individuals and used these data to adjust survival rates from an acoustic telemetry study conducted in the Sacramento River. Eighty fish were surgically implanted with tags—40 with cylindrical tags of 9 mm diameter and 21 or 24 mm length (V9, Vemco Ltd) and 40 with a 7 mm diameter and 20 mm length tag (V7, Vemco Ltd)—to examine the impact of tag size on peritoneal retention and survival rate of juvenile steelhead trout. A total of 20 % (16/80) of all tags were expelled by smolts during the 143-day study. Ten V9 tags were expelled between day 18 and day 66. Six V7 tags were expelled between day 21 and day 143. A statistical difference was found for retention rate by surgeon even though the surgeons were of equal experience and received the same training. There were no significant differences in the tag retention rate in relation to the tag/body weight ratio, or in growth (weight or fork length) among the control, V7 or V9 treatment groups over the duration of the study. All individuals survived throughout the experiment. Two methods were used to adjust the survival estimates of an acoustic telemetry data set from the Sacramento River based on the tag retention study. First, a simple individual censorship approach in Program MARK was utilized and next ATLAS, a software program designed to compensate for bias in survival estimates caused by tag failures was used. The results of the adjusted survival estimates were not significantly different from the unadjusted rates suggesting that it may be more important to focus on improving surgical techniques to reduce tag expulsion rather than adjusting survival estimates dependent on the study. The surgical techniques utilized in this study did not have significant impacts on the growth rates of either of the tag treatment groups compared to the control. However, tag retention was an issue regardless of the size and weight of the implanted tag and the size of the steelhead.     

Retention of plastic-tipped dart tags in African tigerfish Hydrocynus vittatus

Estimates of tag retention and tagging-related mortality are essential for mark-recapture experiments. Mortality and tag loss were estimated from 15 tigerfish Hydrocynus vittatus marked using Hallmark model PDL plastic-tipped dart tags released into a 1 730 m² pond at Kamutjonga Inland Fisheries Institute, Namibia, and inspected bi-monthly for the presence or absence of tags. No mortality was observed during the experiment. All marked fish had lost their tags after 10 months and 50% tag loss was estimated at 3.9 months. The high tag loss rate indicates that PDL plastic-tipped dart tags are not suitable for long-term studies on this species.     

Male mate choice and the potential for complex mating dynamics in the tree lizard (Urosaurus ornatus)

A growing body of literature is recognizing that males may also play a role in the mating process by behaving non‐randomly toward potential female mates during courtship. In numerous species, discrete color polymorphisms in males are inferred to represent alternative mating tactics, which often correspond with concomitant asymmetries in ecology and behavior. In terms of their mating behavior, these ecological outcomes of a color polymorphism should affect a morph's likelihood and frequency of encountering females in a population, possibly favoring the evolution of morph‐specific mating preferences. Knowledge of how male morphs contribute to a species’ overall mating dynamics will improve our understanding of how sexual selection shapes phenotypic diversity in color polymorphic systems. We conducted a mate choice experiment to evaluate the extent and morph specificity of non‐random mating preferences by male ornate tree lizards, Urosaurus ornatus. We observed the behavior of blue and yellow males in an experimental arena in response to a choice between an orange or yellow female. We found that blue males preferred yellow females over orange females, and although yellow males visited females more often than blue males overall, their attention was not morph‐specific. Given male morph differences in choosiness, and their differences in social dominance, we conclude that female throat color may be partly under sexual selection in U. ornatus. However, a lack of concordance between male and female mating preferences (drawn from an earlier study) suggests that overall mating dynamics may serve to maintain, rather than enhance, color morph differences in this species.     

Long‐term effects of passive integrated transponder tagging (PIT tags) on the growth of the yellow European eel (Anguilla anguilla (Linnaeus, 1758))

Passive Integrated Transponder tags (PIT tags) are recommended as the most suitable method for tagging fish on the basis of their high retention and fish survival rates. The objective of this study was to determine the long‐term effects (between 1 and 11 years) of the PIT tag on the growth of the yellow European eel (Anguilla anguilla). A difference of more than 50% was observed in the growth of marked and unmarked yellow eels. If this is a general long‐term effect in all eels, it would seriously restrict the use of PIT tags for studying the dynamics of European eel populations, and so for their management.     

Intensity of male‐male competition predicts morph diversity in a color polymorphic lizard

Sexual selection is one of the main processes involved in the emergence and maintenance of heritable color polymorphisms in a variety of taxa. Here, we test whether the intensity of sexual selection, estimated from population sex ratio, predicts morph diversity in Podarcis muralis, a color polymorphic lizard with discrete white, yellow, orange, white‐orange, and yellow‐orange male and female phenotypes (i.e., morphs). In a sample of 116 Pyrenean populations and 5421 lizards, sex ratios (m/f) vary from 0.29 to 2.5, with the number of morphs for each sex ranging from 2 to 5. Male‐biased sex ratios are associated with increased morph diversity as measured with Shannon's diversity index. The main factor accounting for this relationship is male morph richness (i.e., the number of morphs). In contrast, female morph diversity is not related to sex ratio. These results suggest a relationship between the intensity of male intrasexual competition and male morph diversity. While other selective forces may interact with sexual selection in maintaining the color polymorphisms in P. muralis, this evidence suggests a complex evolutionary scenario possibly involving frequency‐dependent selection of alternative reproductive tactics and/or complex balancing selection.     

Telemetry tags increase the costs of swimming in northern fur seals,Callorhinus ursinus

Animal‐borne instruments have become a standard tool for collecting important data from marine mammals. However, few studies have examined whether placement of these data loggers affects the behavior and energetics of individual animals, potentially leading to biasing data. We measured the effect of two types of relatively small data loggers (<1% of animals’ mass and front profile) on the swimming speeds and energy expenditure of four female northern fur seals (Callorhinus ursinus) while swimming at depth. Swim speeds and rates of oxygen consumption were measured as the trained fur seals repeatedly swam an underwater circuit, with or without the tags. We found the placement of either tested tag significantly affected both the behavior and energetics of the fur seals in our study. Diving metabolic rate increased an average of 8.1%–12.3% (depending on tag type) and swim speed decreased an average of 3.0%–6.0% when wearing the tags. The combined changes in velocities and metabolic rates resulted in a 12.0%–19.0% increase in the total energy required by the fur seals to swim a set distance. The demonstrated effects of tags on behavior and energy expenditure may bias data sets from wild animals and potentially incur longer‐term impacts on the studied animals.     

Long-term retention of dummy acoustic transmitters in adult brown trout

A group of 36 1+ age class Salmo trutta were surgically implanted with dummy acoustic tags and monitored for 370 days. In total 13 tags were expelled throughout the experiment with an overall tag loss rate of c. 0^.035 tags per day. Fish length was the only explanatory variable which had a significant association with subsequent tag expulsion. The estimated probability of retaining a tag for a year for a fish of length 32 cm was 0.76, 34 cm was 0.60 and 36 cm was 0.38. The long-term tag loss patterns were examined and discussed.     

Hummingbird color preference within a natural hybrid population of Mimulus aurantiacus (Phrymaceae)

Hummingbird flowers are typically red in color but the reasons for this are not well understood. Relatively few studies have examined hummingbird flower color preferences under natural conditions in which flower color varies within a species. We recorded hummingbird visitation rates to flowers that vary in color from yellow to red in a natural hybrid population between red‐ and yellow‐flowered Mimulus aurantiacus subspecies. We also examined whether there were any correlations between color and flower size or nectar content. Finally, we reviewed the literature on hummingbird color choice tests using feeders and flowers. There were no correlations in this population between flower color and flower size, nectar volume, or sugar concentration. Nevertheless, hummingbirds undervisited the two most yellow color classes, overvisited orange flowers, and visited the two most red color classes in proportion to their frequency in the population. While Hummingbirds preferred flowers expressing red pigments to those that did not, the flowers with the most red hue were not the most attractive, as has been observed in similar studies with other species of Mimulus. While feeder studies generally fail to show hummingbird preference for red, all studies using flowers, including those that control all floral traits other than color, find consistent preference for red. Experiments are suggested that might help disentangle hypotheses for why hummingbirds exhibit this preference.     

First description of color variations in the annual killifish Millerichthys robustus,and preliminary observations about its geographical distribution

Millerichthys robustus is the only annual killifish distributed in America with phenotypic color variations, not yet described. Accordingly, we first describe the color pattern in both sexes to define its phenotypical variations. We then analyze the frequency of these phenotypes on a geographical scale, in four localities that represent opposite points of Millerichthys’s distribution in the Mexican southeast. Color analysis based on the RGB system allowed us to define five-color phenotypes in males continuously distributed in various perceptual units between two extreme colors (yellow-red): yellow, moderate orange, dark orange, strong orange and red. These color patterns found in M. robustus could be attributed to melanin, carotenoid, and pteridine pigments. The orange phenotypes was present in all localities studied. The yellow phenotype was present only in northeastern and northwestern locations, and the red phenotype was present only in northern populations. Female color variations were observed in the number of ocelli (from 1 to 15) at the base of the caudal peduncle and dorsal region. Ocelli have been associated with anti-predator functions because they resemble the eyes of vertebrates, thus shifting the target of predator attacks to less vital body parts. Females with 3 ocelli were the most frequent phenotype, and females with 13–15 ocelli occurred only in the northern populations. We concluded that male and female of M. robustus are not randomly distributed along their distribution range, which suggest that color phenotypes may react differently to biotic and abiotic factors that probably determine their distribution and frequency within the studied population.

     

Development of an automated method of detecting stereotyped feeding events in multisensor data from tagged rorqual whales

The introduction of animal‐borne, multisensor tags has opened up many opportunities for ecological research, making previously inaccessible species and behaviors observable. The advancement of tag technology and the increasingly widespread use of bio‐logging tags are leading to large volumes of sometimes extremely detailed data. With the increasing quantity and duration of tag deployments, a set of tools needs to be developed to aid in facilitating and standardizing the analysis of movement sensor data. Here, we developed an observation‐based decision tree method to detect feeding events in data from multisensor movement tags attached to fin whales (Balaenoptera physalus). Fin whales exhibit an energetically costly and kinematically complex foraging behavior called lunge feeding, an intermittent ram filtration mechanism. Using this automated system, we identified feeding lunges in 19 fin whales tagged with multisensor tags, during a total of over 100 h of continuously sampled data. Using movement sensor and hydrophone data, the automated lunge detector correctly identified an average of 92.8% of all lunges, with a false‐positive rate of 9.5%. The strong performance of our automated feeding detector demonstrates an effective, straightforward method of activity identification in animal‐borne movement tag data. Our method employs a detection algorithm that utilizes a hierarchy of simple thresholds based on knowledge of observed features of feeding behavior, a technique that is readily modifiable to fit a variety of species and behaviors. Using automated methods to detect behavioral events in tag records will significantly decrease data analysis time and aid in standardizing analysis methods, crucial objectives with the rapidly increasing quantity and variety of on‐animal tag data. Furthermore, our results have implications for next‐generation tag design, especially long‐term tags that can be outfitted with on‐board processing algorithms that automatically detect kinematic events and transmit ethograms via acoustic or satellite telemetry.     

Flower color alteration in <Emphasis Type="Italic">Lotus japonicus</Emphasis> by modification of the carotenoid biosynthetic pathway

To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding β-carotene ketolase (4,4′-β-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops.     

Degradation of C-terminal tag sequences on domain antibodies purified from E. coli supernatant

Expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. However, as the fusion peptides most often are flexible appendages at the N- or C-terminal, proteolytic cleavage may result in removal of the tag sequence. Here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. The tag sequences were inserted in fusion with the c-terminal end of a domain antibody based on the HEL4 scaffold in a phagemid vector. This particular antibody fragment was able to refold on the membrane after blotting, allowing us to detect c-terminal tag breakdown by use of protein A in combination with detection of the tags in the specific constructs. The degradation of the c-terminal tags suggested specific sites to be particularly prone to proteolytic cleavage, leaving some of the tag combinations partially or completely degraded. This specific work illustrates the importance of tag design with regard to recombinant antibody expression in E. coli, but also aids the more general understanding of protein expression.     

Effects of surgically implanted PIT tags on growth, survival and tag retention of yellow shortfin eels Anguilla australis under laboratory conditions

This study evaluated the effects of surgically implanted passive integrated transponder (PIT) tags on growth rate, survival and tag retention of yellow shortfin eels Anguilla australis with an initial mean mass of 101 g. There were no significant differences in body mass, total length, specific growth rate and survival between tagged and untagged A. australis in a 108 day laboratory trial. This tagging method was very reliable, with a tag retention of >95%.