Haruko Ando

Diet analysis by next-generation sequencing indicates the frequent consumption of introduced plants by the critically endangered red-headed wood pigeon (Columba janthina nitens) in oceanic island habitats

Oceanic island ecosystems are vulnerable to the introduction of alien species, and they provide a habitat for many endangered species. Knowing the diet of an endangered animal is important for appropriate nature restoration efforts on oceanic islands because introduced species may be a major component of the diets of some endangered species. DNA barcoding techniques together with next-generation sequencing may provide more detailed information on animal diets than other traditional methods. We performed a diet analysis using 48 fecal samples from the critically endangered red-headed wood pigeon that is endemic to the Ogasawara Islands based on chloroplast trnL P6 loop sequences. The frequency of each detected plant taxa was compared with a microhistological analysis of the same sample set. The DNA barcoding approach detected a much larger number of plants than the microhistological analysis. Plants that were difficult to identify by microhistological analysis after being digested in the pigeon stomachs were frequently identified only by DNA barcoding. The results of the barcoding analysis indicated the frequent consumption of introduced species, in addition to several native species, by the red-headed wood pigeon. The rapid eradication of specific introduced species may reduce the food resources available to this endangered bird; thus, balancing eradication efforts with the restoration of native food plants should be considered. Although some technical problems still exist, the trnL approach to next-generation sequencing may contribute to a better understanding of oceanic island ecosystems and their conservation.

Genetic and Morphological Differences among Populations of the Japanese Bush-Warbler (Aves: Sylviidae) on the Ogasawara Islands, Northern Pacific

Abstract: Cettia diphone diphone is a subspecies of the Japanese Bush-warbler that is endemic to the Bonin and Volcano Islands of the Ogasawara Islands. Although the two island groups are physically distant and have different geological histories, genetic and morphological relationships between the two populations are unknown. A few individuals of an unidentified subspecies have been observed since 2007 on Mukojima of the Bonin Islands. They were possibly wintering birds of another subspecies in light of their song pattern and appearance. We examined the genetic and morphological differences among the Bonin and Volcano populations of C. d. diphone, C. d. cantans, and C. d. sakhalinensis populations and determined the identity of the unidentified subspecies on Mukojima by comparing sequences of the CO1 region of the mitochondrial DNA (mtDNA) and conducting a canonical discriminant analysis (CDA) using five measurements per bird. Our results indicate that these populations (except C. d. sakhalinensis) are genetically and morphologically different. They did not share any haplotypes of mtDNA and indicated high discrimination rates (over 75%) based on CDA. Because the Volcano population inhabits only one island, it is a priority for conservation. The unidentified individuals on Mukojima Island are not the native subspecies. Migratory individuals might colonize Mukojima due to improved habitat conditions and an available niche. Continuous monitoring of the Mukojima population and its impact on native C. d. diphone is required.

DNA barcoding reveals seasonal shifts in diet and consumption of deep-sea fishes in wedge-tailed shearwaters

The foraging ecology of pelagic seabirds is difficult to characterize because of their large foraging areas. In the face of this difficulty, DNA metabarcoding may be a useful approach to analyze diet compositions and foraging behaviors. Using this approach, we investigated the diet composition and its seasonal variation of a common seabird species on the Ogasawara Islands, Japan: the wedge-tailed shearwater Ardenna pacifica. We collected fecal samples during the prebreeding (N = 73) and rearing (N = 96) periods. The diet composition of wedge-tailed shearwater was analyzed by Ion Torrent sequencing using two universal polymerase chain reaction primers for the 12S and 16S mitochondrial DNA regions that targeted vertebrates and mollusks, respectively. The results of a BLAST search of obtained sequences detected 31 and 1 vertebrate and mollusk taxa, respectively. The results of the diet composition analysis showed that wedge-tailed shearwaters frequently consumed deep-sea fishes throughout the sampling season, indicating the importance of these fishes as a stable food resource. However, there was a marked seasonal shift in diet, which may reflect seasonal changes in food resource availability and wedge-tailed shearwater foraging behavior. The collected data regarding the shearwater diet may be useful for in situ conservation efforts. Future research that combines DNA metabarcoding with other tools, such as data logging, may provide further insight into the foraging ecology of pelagic seabirds.

Wide-ranging Movement and Foraging Strategy of the Critically Endangered Red-headed Wood Pigeon (Columba janthina nitens): Findings from a Remote Uninhabited Island

Abstract The Red-headed Wood Pigeon, Columba janthina nitens, is an endemic and endangered subspecies of the Ogasawara Islands. This pigeon moves irregularly among island habitats. However, its range and patterns of movement, particularly between the Bonin and the Volcano Islands, which are two remote island groups approximately 150 km apart, remain unclear. In this study, we conducted a survey on the uninhabited Kita-Iwojima Island of the Volcano Islands to collect direct evidence of pigeon movement between the two island groups and to reveal their food resource availability. Pigeon food composition was also analyzed. During the study period in Kita-Iwojima, we observed two individuals banded in Chichijima in the Bonin Islands. Food composition was estimated by fecal DNA analysis and compared with a fruit census of Kita-Iwojima, which differed from fruits observed in two monitored islands of the Bonin Islands, Chichijima and Hahajima. The pigeons might move among these islands to use available food resources, reflecting limitations of food resources in a single island habitat. Fruits detected in feces of the pigeons on Kita-Iwojima were not from plants observed on the island but rather derived from plants observed on Chichijima and Hahajima, likely indicating high movement capacity of pigeons among the islands. However, the foraging habitat of the Red-headed Wood Pigeon is limited to areas of low elevation in Kita-Iwojima despite apparent food sources at higher elevations. Therefore, factors beyond food abundance, such as geographical features, might affect habitat use of pigeons on the island.

Predominance of Unbalanced Gene Flow from Western to Central North Pacific Colonies of the Black-Footed Albatross (Phoebastria nigripes)

Abstract: The Black-Footed Albatross (Phoebastria nigripes) breeds in two remote regions, approximately 4,000 km apart, in the North Pacific. The population in the central North Pacific region (Northwestern Hawaiian Islands), which contains >95% of the total population, is currently stable, although concerns exist about future declines. In contrast, the population in the western North Pacific (Izu and Ogasawara Islands in Japan) is rapidly increasing, and the breeding areas are expanding. To estimate possible gene flow caused by dispersal between populations, we performed genetic analysis on six colonies of Black-Footed Albatross using 10 microsatellite markers. The central and western North Pacific populations were genetically differentiated. However, an estimation of migrants per generation indicated directional dispersal from the western to the central North Pacific. In particular, the population on Kure Atoll, the westernmost atoll in the Hawaiian Islands in the central North Pacific, exhibited weak genetic differentiation from the western North Pacific populations, suggesting frequent immigration from the western North Pacific. The recent expansion of the western North Pacific population may be due to an increase in returning individuals, which may be caused by increased breeding success rates and/or survival rates. Range-wide and long-term monitoring of the Black-Footed Albatross population using genetic markers may help to uncover dispersal dynamics of this highly mobile but philopatric albatross species and to make appropriate conservation decisions in light of environmental changes.

Evaluation of plant contamination in metabarcoding diet analysis of a herbivore

Fecal DNA metabarcoding is currently used in various fields of ecology to determine animal diets. Contamination of non-food DNA from complex field environments is a considerable challenge to the reliability of this method but has rarely been quantified. We evaluated plant DNA contamination by sequencing the chloroplast trnL P6 loop region from food-controlled geese feces. The average percentage of contaminant sequences per sample was 1.86%. According to the results of generalized linear models, the probability of contamination was highest in samples placed in wet soil. The proportion of contaminant sequences was lowest at the earliest sampling point and was slightly higher in samples placed in open conditions. Exclusion of rare OTUs (operational taxonomic units) was effective for obtaining reliable dietary data from the obtained sequences, and a 1% cutoff reduced the percentage of contaminated samples to less than 30%. However, appropriate interpretation of the barcoding results considering inevitable contamination is an important issue to address. We suggest the following procedures for fecal sampling and sequence data treatment to increase the reliability of DNA metabarcoding diet analyses: (i) Collect samples as soon as possible after deposition, (ii) avoid samples from deposits on wet soil, and (iii) exclude rare OTUs from diet composition estimations.

Development of Microsatellite Markers for the Coastal Shrub Scaevola taccada (Goodeniaceae)

Premise of the study: Microsatellite markers were developed for the coastal shrub species Scaevola taccada to estimate the population genetic structure, which may reflect different seed dispersal patterns. Methods and Results: Thirteen microsatellite primer sets were developed for S. taccada using 454 pyrosequencing. The primer sets were tested on 64 individuals sampled from two populations in Japan. Fragments were amplified using the primers, with one to 10 alleles per locus, and the expected heterozygosity ranged from 0.00 to 0.85. Conclusions: These results indicate the utility of markers in S. taccada for broad estimations of the population genetic structure of this species.

Development of microsatellite markers for the Asian Stubtail Urosphena squameiceps by using next-generation sequencing technology

The Asian Stubtail Urosphena squameiceps winters in Southeast Asia which is facing a serious threat from deforestation and the species is categorized as a threatened species at some of the breeding populations. Monitoring population dynamics of the species in the field is difficult because they are secretive and prefer dense undergrowth. Therefore, developing genetic markers from the species is useful for detecting appropriate conservational management units and to understanding their unique breeding system. We isolated and characterized 13 microsatellite loci of the Asian Stubtail by using a next-generation sequencer. The number of alleles and the expected heterozygosity at each locus were 2–26 (mean 10.31) and 0.14–0.92 (mean 0.64), respectively. The combined first-parent non-exclusion probability was <0.005.