Seasonal Diet Partition among Top Predators of a Small Island, Iriomotejima Island in the Ryukyu Archipelago, Japan

doi:10.21203/rs.3.rs-3907562/v1

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Seasonal Diet Partition among Top Predators of a Small Island, Iriomotejima Island in the Ryukyu Archipelago, Japan

https://doi.org/10.21203/rs.3.rs-3907562/v1

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published 02 Apr, 2024

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Small islands lack predators because species at higher trophic levels often cannot survive. However, two top predators—the Iriomote cat Prionailurus bengalensis iriomotensis, and the Crested Serpent Eagle Spilornis cheela perplexus—live on small Iriomotejima Island in the Ryukyu Archipelago. To understand how these predators coexist on the island with limited resources, we focused on their seasonal diets which are considered crucial for survival in such an island ecosystem. To compare the diets of them, we used DNA metabarcoding analysis of their fecal samples. In the summer, we identified 16 prey items from Iriomote cat fecal samples, and 15 from Crested Serpent Eagle fecal samples. In the winter, we identified 37 and 14 prey items, respectively. Using a non-metric multidimensional scaling and a permutational multivariate analysis of variance, our study reveals significant differences in the diet composition at the order level between the predators during both seasons. Furthermore, although some prey items at the species-to-order level overlapped between them, the frequency of occurrence of most prey items differed in both seasons. These results suggest that this difference in diets was one of the reasons why the Iriomote cat and the Crested Serpent Eagle coexisted on such a small island.

Biological sciences/Ecology/Ecological networks

Biological sciences/Ecology/Ecosystem ecology

Biological sciences/Ecology/Molecular ecology

Island ecosystems exhibit unique ecological characteristics that are sometimes simpler or more monotonic than those of continental ecosystems. This is mainly because these islands have a limited number of species and individuals, and immigration and emigration events are less likely to occur^1,2. On such islands, predators at higher trophic levels are often absent, because predator species, such as carnivores, cannot maintain their populations without sufficient prey resources³.

However, multiple predators have been able to co-exist on Iriomotejima Island, located in the southern part of the Ryukyu Archipelago, which covers an area of approximately 289 square kilometers⁴. Two top predators are known from this island: the Iriomote cat (IRC) Prionailurus bengalensis iriomotensis, and the Crested Serpent Eagle (CSE) Spilornis cheela perplexus. The IRC is endemic to Iriomotejima Island and is a subspecies of the leopard cat P. bengalensis, which is widely distributed in East and Southeast Asia⁵. The CSE is also endemic to Iriomotejima, Ishigakijima, and Yonagunijima Islands in the Ryukyu Archipelago and is a subspecies of the Crested Serpent Eagle S. cheela, which is widely distributed in East and South Asia⁶. The mechanism of coexistence of these predators is important for understanding the ecological features of Iriomotejima Island, which has limited resources. To address this question, we focused on the seasonal diets of the IRC and CSE, as these are thought to be one of the most important factors for their survival in a small island ecosystem.

When multiple top predators are sympatric on a small island with restricted food resources, their feeding habits are expected to differ from each other⁷. Although a few studies have compared the diet of sympatric animals that exhibit similar ecological niches^8–10, few studies have focused on sympatric predators endemic to small islands such as Iriomotejima Island¹¹.

Previous studies on the diets of IRC and CSE on Iriomotejima Island have shown that both are carnivorous and feed on a variety of prey animals, such as mammals, birds, reptiles, amphibians, fishes, insects, and crustaceans^12,13. Most of these prey animals overlapped between the two species^12–17. The diet of the IRC was analyzed based on the morphological observation of fecal and stomach contents of dead individuals^13–17, although it is often difficult to identify prey animals at the species level from such digested items. Analysis of the CSE fecal content is more challenging, as carnivorous birds often spit out undigested items such as pellets, and only soluble materials are excreted as feces. Therefore, previous studies on the CSE diet have largely been limited to stomach content analysis of dead individuals and direct observation of their feeding behaviors^12,16. However, in these studies, the sample size was insufficient because of the difficulty in obtaining dead individuals or the limited frequency of direct observations.

In this study, we performed DNA barcoding analysis of feces to examine the prey of the IRC and CSE. DNA barcoding generally provides a higher taxonomic resolution than the morphological observation of fecal and stomach samples and has recently become a major method for animal diet analysis^18,19. This method enabled us to analyze CSE diets using fecal samples with larger sample sizes than stomach content analysis or direct observations. Furthermore, it allows a direct comparison of the prey animals of birds and mammals based on the same method. Some studies in Pakistan and China conducted DNA barcoding to investigate the diets of other subspecies of the leopard cat^20–23, while no studies have addressed any CSE subspecies.

In the present study, we aimed to elucidate the coexistence mechanism of the two top predators, IRC and CSE, living on Iriomotejima Island. Our goal was to examine their competition for limited food resources on a small island. To achieve this, we utilized DNA barcoding to analyze their diets, providing higher resolution (Fig. 1).

Fecal sampling and DNA sequencing results

We obtained 38 and 76 fecal samples from the IRC in the summer and winter, respectively. Similarly, we collected 24 and 12 CSE fecal samples in the summer and winter, respectively. The numbers of IRC samples that contained DNA sequences from more than one prey item were 31 (81.6%) and 64 (90.1%) in the summer and winter, respectively. The numbers were 21 (87.5%) and nine (75.0%) among CSE samples in the summer and winter, respectively. These samples were used for subsequent analyses.

Using the DNA metabarcoding sequencing results, we obtained 333 sequencing reads using the COI#1; 10,335 sequencing reads using the COI#2; 208,673 sequencing reads using the Ecoprimer; and 74,483 sequencing reads using the MtAnr. These sequences were derived from 55 different prey items, including five species of Mammalia; 17 species, one genus, one order, and one family of Aves; 10 species and one genus of Reptilia; seven species and one family of Amphibia; one species of Osteichthyes; three species of Malacostraca; four species and one order of Insecta; and two families of Chilopoda (Supplementary Table 2). We could identify 83.5% of the MOTUs at the species level.

Characteristics of detected prey species by each primer set

The number of sequencing reads for the detected prey taxa varied remarkably among primer sets. Invertebrates were detected using COI#1 and COI#2, whereas vertebrates were detected using all primer sets (Fig. 3). The COI#1 detected the Brown-eared Bulbul Hypsipetes amaurotis stejnegeri which was not detected by any other primer sets. COI#2 detected a relatively large number of invertebrates, such as the mudflat crab Chiromantes dehaani, the blue land crab Discoplax hirtipes, the vine hawkmoth Hippotion celerio, the bush cricket Mecopoda elongate, the privet hawkmoth Psilogramma menephron, and centipedes Scolopendromorpha sp., that were not detected by any other primer sets. The Ecoprimer detected each class of land vertebrates almost evenly. In particular, Osteichthyes (the barred mudskipper Periophthalmus argentilineatus) was not detected by any other primer sets, and was only detected in one fecal sample (Fig. 3). The sequencing reads derived from Amphibia accounted for 97.2% of the total number of sequencing reads obtained by the MtAnr. The Yaeyama kajika frog Buergeria choui and Utsunomiya's tip-nosed frog Odorrana utsunomiyaorum were detected only using this primer set.

The estimated diet composition of IRC and CSE

The number of prey items detected from the fecal samples of each predator during each season was as follows—summer samples of IRC: 14 species, one genus, and one order belonging to five classes, nine orders, 12 families, and 15 genera; summer samples of CSE: 12 species, one genus, and two families belonging to five classes, five orders, 11 families, and 12 genera; winter samples of IRC: 30 species, two genera, three families, and two orders belonging to eight classes, 16 orders, 23 families, and 30 genera; winter samples of CSE: 12 species and two families belonging to six classes, seven orders, 12 families, and 12 genera (Fig. 7, Supplementary Table 2). The prey taxonomic richness for each predator showed no significant difference in both seasons (Supplementary Fig. 1).

Based on a comparison of the FOO values of IRC prey items obtained in this study with those obtained in a previous study¹³, except for Aves and Insecta, there were no significant differences in the FOO values of Mammalia, Reptilia, Amphibia, and Osteichthyes (Fig. 4). It should be noted that Aves and Insecta were significantly less frequent in this study than in the previous study (Fig. 4; p < 0.05 and p < 0.01, respectively).

Regarding the detected prey classes, Mammalia was not detected in any CSE samples, while they were frequently detected in IRC samples (Fig. 5). The FOO values of the Mammalia were significantly different between the two predators in winter (p < 0.05). In contrast, Malacostraca and Chilopoda were rarely detected in IRC samples while they were frequently detected in CSE samples (Fig. 5). These difference between CSE and IRC was significant in both seasons (p < 0.01 in Malacostraca in summer and Chilopoda in winter, p < 0.05 in Chilopoda in summer and Malacostraca in winter). Reptilia were significantly more frequent in IRC samples than in CSE samples in summer (p < 0.05), whereas the difference was not statistically significant in winter. On the other hand, Amphibia were significantly more frequent in CSE samples than in IRC samples in summer (p < 0.05), whereas this trend was not statistically significant in winter. Within each predator, the FOO of Mammalia and Reptilia detected in IRC feces showed significant differences between seasons (p < 0.05, p < 0.01 in Mammalia and Reptilia, respectively). In addition, the FOO of Amphibia detected in CSE showed a significant difference between seasons (p < 0.05).

The NMDS and PERMANOVA analyses for comparison of diet composition between IRC and CSE showed that the prey composition at the order level was significantly different between them in both seasons (Fig. 6; p < 0.001) and the differences were not caused by different degrees of variation among the samples of IRC and CSE (p = 0.4899 in summer; p = 0.7077 in winter).

When prey species at the species-to-order level were sorted by FOO and wPOO values, there was little difference in the ranking of prey species, with the exception of winter CSE, which had a small sample size (Fig. 7).

In Mammalia, the black rat Rattus rattus, had the highest FOO and wPOO values in the IRC samples in winter (Fig. 7), although FOO was not significantly higher in the IRC samples than in the CSE samples (Supplementary Table 2). In both seasons, black rats were the most frequently detected mammals in the IRC samples (Fig. 7), and their FOO was significantly higher in winter than in summer (Table S2; P < 0.05).

In Reptilia, the FOO and wPOO values of Japanese skinks Plestiodon sp., were the highest in the detected IRC prey items in summer (Fig. 7), and its FOO was significantly higher in IRC samples than in CSE samples (Supplementary Table 2; p < 0.05). Within the IRC samples, the FOO value of Japanese skinks was significantly higher in summer than in winter (Supplementary Table 2; p < 0.01). Similarly, the Sakishima beauty snake Elaphe taeniura schmackeri, showed a significantly higher FOO in summer than in winter in the IRC samples (Supplementary Table 2; p < 0.05). The detection of Sakishima smooth skink Scincella boettgeri was significantly more frequent in the CSE samples than in the IRC samples in winter (Supplementary Table 2; p < 0.01), and was significantly more frequent in winter than in summer within the CSE samples (Supplementary Table 2; p < 0.05).

In Amphibia, the Sakishima rice frog Fejervarya sakishimensis, had the highest FOO and wPOO values in the detected CSE prey items in summer (Fig. 7). The FOO value of this frog was significantly higher in CSE samples than in IRC samples (Supplementary Table 2; P < 0.01), although this frog also had the second highest FOO and wPOO values in the detected IRC prey items during this season (Fig. 7, Supplementary Table 2). On the other hand, in winter, this frog was not detected in CSE samples, and was detected in IRC samples at a lower frequency (Fig. 7), exhibiting no significant difference between IRC and CSE in the Sakishima rice frog in winter (Supplementary Table 2). Within the CSE, the FOO of this frog was significantly higher in summer than in winter (Supplementary Table 2; P < 0.01). In the IRC samples in winter, the greater tip-nosed frog Odorrana supranarina, Sakishima rice frog F. sakishimensis, Owston’s green tree frog Rhacophorus owstoni, and Yaeyama Narrow-mouthed toad Microhyla kuramotoi were the second- to fifth-ranking prey in terms of the FOO value (Fig. 7). Within these Anura species, Owston’s green tree frog and Yaeyama Narrow-mouthed toad were detected also in CSE samples in winter (Fig. 7, Supplementary Table 2).

Among invertebrates, only crabs Decapoda sp., were detected as Malacostraca and only centipedes Scolopendromorpha sp., were detected as Chilopoda in the CSE samples (Supplementary Table 2). The FOO value of the black cicada Cryptotympana facialis was significantly higher in CSE samples than in IRC samples in summer (Supplementary Table 2; p < 0.05).

Because both IRC and CSE are known to feed on a wide range of prey animals, including invertebrates and vertebrates^12–17, this study aimed to identify prey items from various taxonomic groups using multiple primer sets. By integrating the results of each primer set, we successfully identified various prey species from a wide range of taxa. A previous study that utilized visual analysis of stomach contents, which is thought to provide higher resolution of prey identification than that of fecal contents²⁴, identified approximately 61.6% of prey items at the species level¹⁴. The present fecal DNA metabarcoding analysis identified 85.5% of prey items at the species level (Supplementary Table 2). Consequently, DNA-based methods appear to be well-suited for conducting detailed dietary analyses.

A comparison of the FOO of IRC prey items obtained in this study with that obtained in a previous study¹³suggests that for Mammalia, Reptilia, Amphibia, and Osteichthyes, DNA metabarcoding demonstrates the same level of detectability as visual analysis of fecal contents at the class level when identifying IRC prey items (Fig. 4). However, Aves and Insecta were detected less frequently in this study for several reasons. According to Watanabe (2012)¹³, winter birds, which visit Iriomotejima Island from autumn to winter, account for 40% of the bird species detected in fecal samples of IRC. In contrast, in this study, only 17.6% of the bird species were identified as winter birds. The number of migratory birds arriving in a particular area can vary significantly from year to year owing to various environmental changes^25,26. Therefore, the annual variation in the number of winter bird visits between the samples analyzed in this study and those used by Watanabe (2012)¹³ may have influenced the results.

Insecta may not have been detected adequately using our method. One possible explanation for this is the insufficient accumulation of reference sequencing data in NCBI²⁷. In particular, the sequence data of insects on Iriomotejima Island are still poor. Sufficient reference database is important to use the COI region as a barcode region due to the high genetic variation in genes that encode proteins in the COI region^28,29. Indeed, many cockroaches Rhabdoblatta sp. were identified in a study conducted by Watanabe (2012)¹³, whereas they were not detected in the present study. The absence of sequence information for Rhabdoblatta sp. from Iriomotejima Island leads to the possibility that ambiguity in taxonomic assignment, or the failure to amplify its sequence due to mutations in primer biding region, contributed to the non-detection of Rhabdoblatta sp. Previous study that used a primer set amplifying the same region as the current study also noted the presence of uncertain taxonomic assignment resulting from an insufficient insect database³⁰. Collecting sequence information on insects from Iriomotejima Island is a necessary step for the future.

We will not discuss details regarding Aves and Insecta below because the reasons for the limited detection of these taxa remain unclear; however, it is noteworthy that a significant comparison of diet between IRC and CSE was achieved in this study using the same methods for both species.

Additionally, the ranking of CSE prey frequency in winter was not accurately determined in this study due to the difference in prey species ranking by FOO and wPOO values. The inconsistency in rankings may be the result of the insufficient sample size of CSE feces in winter. However, the comparison of the frequency of each prey species between predators and seasons is meaningful.

Based on the results of the NMDS and PERMANOVA analyses at the order level (Fig. 6), significant differences were found between IRC and CSE in the composition of prey items in both summer and winter. To consider how prey animals differed between the two predators, we focus on each prey taxon.

Mammalian prey was detected in IRC feces in both seasons. Malacostraca (crabs) and Chilopoda (centipedes) were detected with higher FOO in CSE compared to IRC feces (Fig. 5). Previous studies have shown that Mammalia and crabs are the main prey animals of IRC and CSE, respectively, with FOO values of 30.9% in the visual analysis of IRC fecal contents and 57.1% in the analysis of CSE stomach contents^13,16. While predation on centipedes by CSE has been observed visually¹², it was not detected in their stomach contents presumably due to digestion¹⁶. Furthermore, although IRC have been shown to prey on Malacostraca and Chilopoda¹³, FOO values were low in the present study with 4.22% and 0.11%, respectively, in the visual analysis of their fecal contents. Previous studies also reported that the black rat could be a target of CSE predation¹². However, the predation frequency of the black rat by CSE remains undetermined, and it was not detected in feces in the present study. In summary, it appears as though IRC feeds more frequently on Mammalia and that CSE feeds more frequently on crabs and centipedes. As the FOO values of Mammalia were not significantly different between the two predators in the summer season, additional research is required to examine the predation of Mammalia by CSE, especially the black rat.

Differences in the prey animals of IRC and CSE may be attributed to their distinct feeding behaviors. Iriomote cat actively hunts animals walking around the ground^15,17, whereas CSE exhibits a sit-and-wait, or passive, foraging strategy based on perching and searching for prey animals on the ground^12,31. The latter passive strategy of CSE may make it difficult to capture swiftly moving, larger-sized animals such as mammals which rarely appear in open areas. The frequent detection of crabs and centipedes in CSE feces may be explained by the fact that crabs and centipedes appear more frequently in open areas where CSE tend to hunt and exhibit slower movements.

The detection of Reptilia was significantly more frequent in IRC samples than in CSE samples during the summer. In particular, Japanese skink Pleistodon sp. exhibited the highest FOO and wPOO values among the detected IRC prey items, and the FOO was significantly higher than that in the CSE samples (Fig. 7 and Supplementary Table 2). This finding is concordant with that of a previous study that identified skink as a major prey for the IRC in summer¹³. However, in winter, the FOO of Reptilia was not significantly different between the two predators, probably due to the decreased FOO of Sakishima beauty snakes as well as skinks in the IRC samples during this season (Fig. 7 and Supplementary Table 2). In winter, the poikilothermic reptiles exhibit decreased activity owing to lower temperatures and appear only during warm daytime periods. Thus, the nocturnal predator IRC would have fewer opportunities to feed on reptiles¹³. On the other hand, CSE did not significantly affect the FOO of Reptilia between the two seasons. It is plausible that diurnal CSE has a sufficient chance of finding skinks in both seasons. Additionally, the detection of Sakishima smooth skink was significantly more frequent in the CSE in winter than in summer, and was not observed in the IRC (Fig. 7 and Supplementary Table 2). The potential for frequent feeding on Sakishima smooth skinks in CSE is the first finding of this study and requires further investigation.

IRC and CSE samples exhibited relatively high FOO and wPOO values for amphibians in both seasons (Fig. 5). Sakishima rice frogs were detected more frequently in the summer for both predators (Fig. 7 and Supplementary Table 2). This is possible because the biomass of the Sakishima rice frog is the highest on Iriomotejima Island, and its activity increases during the summer^13,32. In CSE samples, the FOO and wPOO of this frog were the highest among all prey species in summer (Fig. 7), although frogs are generally nocturnal animals, and the FOO was significantly higher than that of the IRC samples. This may be due to the relative increase in daytime activity of the sakishima rice frog during summer. In winter, the FOO of Sakishima rice frogs decreased significantly in the CSE samples, and the difference between the two predators was not significant. This may be related to the decreased activity of Sakishima rice frogs during winter¹³. Other amphibian prey species, the Yaeyama narrow-mouthed toad and the Owston’s green tree frog, were detected for both predators in winter (Fig. 7 and Supplementary Table 2). The supposed competition for the frogs between IRC and CSE in winter requires further discussion, particularly with a larger CSE sample size.

In summary, despite previous studies indicating an overlap in prey items between the IRC and CSE^12–17, we found differences in diets between the IRC and CSE in winter and summer, respectively. We performed fecal DNA barcoding analysis of each predator and calculated the frequency of detection of each prey item as an indicator. This enables a comparison between IRC and CSE diets for the first time. The proposed differential feeding habits of the two predators could be attributed to fundamental ecological differences such as activity patterns, foraging strategies, and seasonal variations in the activity of prey animals. It is noteworthy that prey animals with high biomass were shared by both IRC and CSE; however, we found at least partially significant differences in the FOO of each prey animal between predators. These quantitatively different diets of the IRC and CSE may contribute to distinguishing feeding habits between them. This could potentially allow both predators to survive on a small island with limited resources and challenging environmental conditions.

IRC and CSE subspecies in the Eurasian continent, that is, the leopard cat and the Crested Serpent Eagle, predominantly feed on rodents and snakes^{10,11,33–37}. Dietary analysis of the subspecies of IRC in the Korean Peninsula, Prionailurus bengalensis euptilurus, revealed that rodents accounted for over 90% of their diet based on morphological observations of fecal samples³⁵. Similarly, regarding the subspecies of CSE in India, Spilornis cheela melanotis, snakes constituted over 70% of their diet, based on direct visual observation of their feeding behavior³³. In contrast, both IRC and CSE on Iriomotejima Island exhibited relatively lower FOO values for rodents (25.3%) and snakes (7.3%) (Supplementary Table 2). These island subspecies tended to feed on more diverse prey species than their continental counterparts (Fig. 8). This finding suggests that the unique small island ecosystem of Iriomotejima Island has influenced the ecology of IRC and CSE to adapt to their available prey, along with other external factors, resulting in dietary habits distinct from those of their continental counterparts.

Study Area

Iriomotejima Island has an area of approximately 284 square kilometers (24° 15–25’ N, 123° 40–55’ E), and is located in the southernmost part of the Ryukyu Archipelago, Japan (Fig. 2). The climate is subtropical, and dominated by Castanopsis sieboldii and Quercus miyagii occupy most of the mountainous areas, with the highest elevation at 469 m. Human habitats are limited to lowland areas along the coast, and there are some cultivated areas around villages as well as swampy and mangrove forests along several rivers. The average temperature is 19.2°C in winter (December to February) and 28.4°C in summer (June to August), and the average amounts of rainfall are 483.0 mm and 597.2 mm in winter and summer, respectively (1991 to 2020, Japan Meteorological Agency).

Fecal sampling

We collected feces from the IRC and CSE once a day in the transects shown in Fig. 2 from November 2018 to February 2019 (winter) and June to September 2019 (summer). The IRC and CSE feces were determined from their shapes. Fresh feces of CSE were collected immediately after excretion was observed from an adequate distance by the author (A.T.). Prey species found in the feces were confirmed through DNA barcoding, as described in a later section. Adherent uric acid mucus was manually removed to prevent PCR inhibition^38–40. Subsequently, samples were immediately preserved in a 100 mL wide-mouth bottle or 2 mL tube filled with 99% ethanol. We also analyzed IRC feces collected and preserved by the Monitoring Program of Okinawa District Forest Office of Forest Agency and Ministry of the Environment of Japan, and CSE feces collected from individuals injured in traffic accidents and rescued by the Iriomote Wildlife Conservation Center of the Ministry of the Environment of Japan.

DNA metabarcoding analysis

A total of 100–200 mg of small blocks of feces were evenly sampled from each feces. The samples were homogenized using a Micro Homogenizing System Micro Smash MS-100R (TOMY SEIKO, Tokyo, Japan) with sterilized 3.175 mm stemless beads. Each homogenate was then incubated at 50°C for 120 min with the addition of 5.0 µL of proteinase K solution. Total DNA was extracted from each processed homogenate using a DNeasy PowerSoil Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol. Finally, 50 µL of total DNA solution was obtained from each sample.

First-round PCR was performed for each extracted DNA sample using universal primer sets to amplify partial mitochondrial genes derived from IRC and CSE prey species. Since the prey animals of these species are supposed to be diverse including vertebrates and invertebrates, we used four primer sets targeting different gene regions: Ecoprimer⁴¹ to amplify the vertebrate partial mitochondrial 12S ribosomal RNA (rRNA) gene, COI#1 (mlCOIintF and dgHCO2198)^42,43for vertebrate and invertebrate partial mitochondrial cytochrome oxidase C subunit 1 (COI) genes, COI#2 (dgLCO1490 and COI-CFMRa)^44,45for the insect partial COI gene, and MtAnr for the anuran partial mitochondrial 16S rRNA gene (Supplementary Table 1). All PCR reactions were conducted under the same conditions:10 µL mixtures were prepared for each PCR amplification, containing 1.0 µL of 10×Ex Taq Buffer (TaKaRa, Shiga, Japan), 0.80 µL of dNTP Mixture (TaKaRa), 0.10 µL of Ex Taq HS (TaKaRa), 0.60 µL (10 µM) of each primer, 1.0 µL of the extracted DNA solution, and 5.9 µL of nuclease-free water (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions were as follows as Hebert et al. (2003)⁴⁶: an initial denaturation at 94°C for 1 min, followed by five cycles of 94°C for 1 min, 45°C for 1 min 30 sec, and 72°C for 1 min 30 sec, followed by 35 cycles of 94°C for 1 min, 50°C for 1 min 30 sec, and 72°C for 1min, and a final incubation at 72°C for 5 min. All PCR reactions included negative controls.

Second-round PCR was conducted to add MiSeq (Illumina, San Diego, CA, USA) adaptor sequences and 8 bp index sequences to both ends of the first-round PCR products, as described by Miya et al. (2015)⁴⁷. The PCR mixtures were prepared in a total volume of 10 µL, containing 1.0 µL of 10×Ex Taq Buffer (TaKaRa), 0.80 µL of dNTP Mixture (TaKaRa), 0.050 µL of Ex Taq HS (TaKaRa), 1.5 µL (10 µM) of each index primer, 1.0 µL of diluted 1st-round PCR products (30x) with nuclease-free water (Thermo Fisher Scientific), and 4.15 µL of nuclease-free water (Thermo Fisher Scientific). The PCR conditions were as follows: an initial denaturation at 98°C for 1 min, followed by 12 cycles of 98°C for 30 sec, 65°C for 30 sec, 72°C for 30 sec, and final incubation at 72°C for 5 min. We then pooled 3.0 µL of each 2nd-round PCR product for each respective primer set. The pooled samples were separated using 1.5% agarose gel electrophoresis using L03 Agarose (TaKaRa), and the DNA bands of the expected PCR target size for each primer set were dissected manually from the gel using a sterilized disposable scalpel. DNA was purified using a MinElute Gel Extraction Kit (Qiagen) following the manufacturer’s protocol. After purification using Agencourt AMPure XP (Beckman Coulter, Brea, California,USA) according to the standard protocol, the sample DNA concentration was quantified using the Qubit dsDNA HS assay kit and Qubit 4 Fluorometer (Thermo Fisher Scientific). DNA concentration was adjusted to 6.0 nM based on the expected amplicon size using nuclease-free water (Thermo Fisher Scientific). Finally, the prepared DNA library was subjected to 250 bp pair-end sequencing using MiSeq (Illumina), MiSeq Reagent Kits v2 (Illumina), and Micro flow cell (Illumina).

Sequencing data analysis and species identification

From the obtained DNA sequence data, low quality 3' ends of the nucleotide sequences (Phred score < 10) were removed using the software DynamicTirm⁴⁸. Paired-end reads were connected using the program FLASH⁴⁹, and merged sequences less than 90 bp were discarded. Singleton sequences were removed using the program Uclust⁵⁰, and the remaining sequences with more than two sequence counts were referred to as molecular operational taxonomic units (MOTUs). MOTUs were further filtered by their sequencing read numbers with a cut-off threshold of 0.01% of the total number of sequences in each sample. Sequence similarity-based Blast analysis⁵¹ was performed using the reference nucleotide database of NCBI (National Center for Biotechnology Information⁵²). The Blast hit record was adapted as a taxon of each MOTU with the following assignment methods: If the sequence similarity was (1) 99% or higher, the top-hit species was adopted, (2) 98% or higher but less than 99%, the top-hit genus was adopted, and (3) 98% or higher but less than 90%, the top-hit family was adopted⁵³. If the multiple species were identified as the top-hit, the highest taxonomic rank below (1) genus, (2) order, and (3) family shared by all top-hit species were adopted⁵³. Taxa and MOTUs were excluded if they did not inhabit Iriomotejima Island or if they were difficult to consider as prey items for IRC and CSE. These include IRC and CSE (the source of feces), fungi, bacteria, humans, and insects less than 2 cm. The source species of each fecal sample was estimated based on the taxonomic assignment results of the Ecoprimer PCR products. MOTUs assigned to dogs and cattle were also excluded because they were considered environmental contaminants. We also excluded samples that did not contain any prey items from subsequent analyses as described in the results section.

Statistical analysis

All statistical analyses were performed using R version 4.3.0 (R Core Team 2023). The quantitative data derived from the sequencing read number of each prey item were used to confirm the detectability of each primer set. The presence/absence data for each prey item were used for other analyses. The prey taxonomic richness was estimated with accumulation and extrapolation curves using ‘iNEXT’^54–56. We utilized two metrics, frequency of occurrence (FOO) and weighted percentage of occurrence (wPOO)⁵⁷ to conduct multivariate and statistical analyses. The results obtained from this study were examined by comparing the FOO values of each prey item with those obtained from a previous study that utilized morphological observations of fecal content¹³. Fisher’s exact test was used to examine the non-random relationships between the presence/absence ratio of each prey item for each predator to determine whether the differences between them were statistically significant. The weighted POO was not used in the comparative analysis because it was not suitable for interspecific comparisons⁵⁸. To compare the diet composition of the two predators based on the Jaccard Index of the dissimilarity of the presence/absence data for prey items at the order level, we performed a non-metric multidimensional scaling (NMDS) analysis using the ‘vegan’ package⁵⁹. The differentiation of the diet composition between these two species was confirmed by performing a permutational multivariate analysis of variance (PERMANOVA⁶⁰) using the ‘adonis’ function. The ‘betadisper’ and ‘anova’ functions were also used to examine whether the differences were caused by different degrees of variation among the samples of IRC and CSE or by the different diet composition between these two species.

Acknowledgments

We extend our sincere gratitude to Ryosuke Terada, Shun Kobayashi, and the entire staff of the Faculty of Science at the University of the Ryukyus for their support in conducting field surveys and experimental work. We also appreciate the support in the Iriomotejima Island by Hiroyoshi Kohno and Akira Mizutani from the Okinawa Regional Research Center at Tokai University, Kazumasa Ohama and the dedicated staff at Ohama Farm, as well as Miki Sugiyama and Chieko Matsumoto. Additionally, we would like to thank to Mamoru Toda at the Tropical Biosphere Research Center, University of the Ryukyus for supplying us with the experimental information. Miho Murayama-Inoue and Kristin Havercamp from the Wildlife Research Center, Kyoto University reviewed and advised on the manuscript. We thank the Okinawa District Forest Office of the Forest Agency, Ministry of the Environment of Japan, and the Iriomote Wildlife Conservation Center, Ministry of the Environment of Japan, for generously providing us with valuable samples. Each affiliation is current at the time of the study.

Author contributions

A.T. designed the study, and conceptual the main ideas. A.T. and N.N. conducted the field work on Iriomotejima Island and collected the samples. Y.S. and N.W. designed the experiments. A.T., N.W. and Y.S. performed the laboratory experiments and data analyses. A.T. visualized the outputs from data analyses. M.I., N.N., Y.S. and N.W. aided in interpreting the results. A.T., Y.S. and M.I. worked on the manuscript. M.I. supervised the project. All authors contributed input to the draft and final versions of the manuscript.

Funding

This work was supported by Pro Natura Foundation Japan’s 29th Pro Natura Fund. The authors have declared that no competing interests exist.

Data availability

The MiSeq-generated raw sequence reads from this study can be found in the DDBJ Sequence Read Archive (DRA) under the accession numbers DRA017514. The other data and computer code are available on Data Dryad review sharing link (https://datadryad.org/stash/share/ef8jckjnMMXqvQ9EnHaTJfoFJlKbeE28MNv-ioAnNeg). The private DOI is only available for review as the manuscript is under review.

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No competing interests reported.

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Seasonal Diet Partition among Top Predators of a Small Island, Iriomotejima Island in the Ryukyu Archipelago, Japan

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Abstract

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Introduction

Results

Fecal sampling and DNA sequencing results

Characteristics of detected prey species by each primer set

The estimated diet composition of IRC and CSE

Discussion

Methods

Study Area

Fecal sampling

DNA metabarcoding analysis

Sequencing data analysis and species identification

Statistical analysis

Declarations

Data availability

References

Additional Declarations

Supplementary Files

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