Identification of Lagopus muta japonica plant food resources in the Northern Japan Alps using DNA metabarcoding

DNA metabarcoding was employed to identify plant-derived food resources of the Japanese rock ptarmigan (Lagopus muta japonica), registered as a natural living monument in Japan, in the Northern Japanese Alps in Toyama Prefecture, Japan, in July to October, 2015-2018. By combined use of rbcL and ITS2 local databases of 74 alpine plant species found in the study area, a total of 43 plant taxa were identified and could be assigned to 40 species (93.0%), two genera (4.7%), and one family (2.3%). Rarefaction analysis of each sample collection period showed that this study covered more than 90% of the plant food resources found in the study area. Of the 21 plant families identified using the combined rbcL and ITS2 local databases, the most dominant families were Ericaceae (98.1% of 105 fecal samples), followed by Rosaceae (42.9%), Apiaceae (35.2%), and Poaceae (19.0%). In all fecal samples examined, the most frequently encountered plant species were Vaccinium ovalifolium var. ovalifolium (69.5%), followed by Empetrum nigrum var. japonicum (68.6%), Vaccinium sp. (54.3%), Kalmia procumbens (42.9%), and Tilingia ajanensis (34.3%). Rarefaction analysis of each collection period in the study revealed that this study covered more than 90% (from 91.0% in July to 97.5% in September) of the plant food resources found in the study area, and 98.1% of the plant food taxa were covered throughout the entire study period. Thus, DNA metabarcoding using the rbcL and ITS2 local databases of alpine plants in combination and rarefaction analysis are considered to be well suited for estimating the dominant food plants in the diet of Japanese rock ptarmigans. Further, the local database constructed in this study can be used to survey other areas with similar flora.


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The rock ptarmigan (Lagopus muta) is a medium-sized grouse that is widely found 49 in subarctic regions of Eurasia and North America [1]. The Japanese rock ptarmigan (L. 50 m. japonica), a subspecies of the rock ptarmigan, is endemic to Japan and resides in the 51 southernmost region of the rock ptarmigan's habitat [1,2]. The Japanese rock ptarmigan 52 is a relict species that remained on the Japanese archipelago after the last glacial stage. 53 The Japanese rock ptarmigan inhabits the alpine meadow zone, which is beyond the forest 54 limit and scattered with Pinus pumila, at altitudes ranging from 2,000 to 3,000 m above 55 sea level (a.s.l.) on the Japanese mainland [1,3] and feeds mainly on alpine plants found 56 in the region. During the breeding season (April to May), the male occupies its territory 57 of 0.015 to 0.072 km 2 [3]. The female builds her nest at the base of P. pumila from June 58 to July, and lay eggs in July. The female alone incubates the eggs and raises the chicks 59 until October, at which time the chicks become independent [4]. When their habitat 60 (alpine meadow zone) is covered with snow (November to April), the Japanese rock 61 ptarmigan migrates to the forest zone, returning to the alpine meadow zone when the 62 snow begins to melt [5]. 63 Although the Japanese have traditionally protected the Japanese rock ptarmigan as 64 a worship object since ancient times by not allowing their capture or hunting, since the 65 1930s, populations of this species on several mountains have either declined or become 66 locally extinct [1]. In 1955, the Japanese rock ptarmigan was registered as a natural living 67 monument in Japan because of its worldwide scarcity, and it is the focus of conservation population of Japanese rock ptarmigans was estimated to be 1,700 [1]. The reasons for 71 this decline are poorly understood; however, loss of alpine meadow vegetation and 72 damage by Sika deer (Cervus nippon) are thought to be primarily responsible [7]. In 73 addition, the alpine meadow vegetation has been damaged extensively by mountain 74 climbers in the alpine belt [8]. Recently, concerns have been raised about habitat 75 destruction due to climate change. It is predicted that the habitat will be drastically 76 reduced from 2081 to 2100 and that the future risk of extinction is very high [3]. To 77 address these issues, restoration and management of the alpine meadow flora is being 78 carried out in various alpine areas in Japan [9, 10]. However, none of these efforts has 79 taken into consideration the impact of plant foods utilized by the Japanese rock ptarmigan, 80 primarily because few surveys of the plant foods of this species have been conducted to 81 date. In addition, breeding programs have been implemented in an attempt to increase the 82 population; however, chicks raised in captivity are suggested to suffer high mortality as 83 a result of inappropriate dietary composition [11,12]. For these reasons, detailed 84 understanding of the alpine meadow vegetation as a food source for Japanese rock 85 ptarmigans is important for conservation of this species [13]. 86 Given this background, several methods have been employed to qualitatively or 87 quantitatively elucidate the dietary composition of the Japanese rock ptarmigan.  Therefore, it is necessary to establish a stress-free survey method to determine the plant 103 food resources of the Japanese rock ptarmigan at the species level more rapidly than the 104 previously reported survey methods [13][14][15][16][17].

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The DNA barcoding method has been successfully applied to food residues in feces 106 and gastric contents to identify dietary composition in wildlife [18][19][20][21][22][23][24][25][26][27][28][29][30]. We also reported 107 the first application of DNA barcoding by the cloning method and using the partial 108 sequence of the chloroplast rbcL gene (rbcL) to plant food residues in the feces of the In the short rbcL region used in our previous studies, closely related plant species, e.g., 119 Asteraceae, Vaccinium sp., and Rhododendron sp., with no polymorphisms in this region 120 have the same homology. Therefore, these taxa could not be identified at the species level.

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In addition, the rarefaction and extrapolation sampling curves revealed that this survey database. In this study, we used these methods to analyze the major plant food resources 139 of the Japanese rock ptarmigan during the breeding season (July to October) in Japan's 140 Northern Alps, which is at the center of their distribution area, and the effectiveness of 141 these methods was evaluated. In addition, in order to evaluate the completeness of our            were collected from the study area (Table 2) and used to construct the local rbcL and ITS2 300 databases. Although rbcL had already been successfully amplified from all 74 plant 301 specimens in our previous study (  Table).   Tilingia ajanensis (34.3%) ( indicating that these three plant taxa were the most important food resources in the study 382 area (Fig 3). The subdominant plants (more than 10% to less than 30% of 105 fecal subsp. japonicum (11.4%) and P. juniperinum (10.5%) ( August (Fig 3 and    rbcL (34 species) was higher than that by ITS2 (26 species) (S5 Table and S6 Table); 427 however, ITS2 was used to supplement the plants that could not be identified by rbcL 428 (Table 5). In this study, the number of identified plant food resources by using the 429 combination of rbcL and ITS2 local databases (40 species) was higher than that obtained 430 using the rbcL (34 species) or ITS2 (26 species) local database alone (Table 5).  were not detected in any of the fecal samples collected in this study ( three Vaccinium species at the species level in this study, as these three species have the 493 same rbcL and ITS2 sequences. Therefore, it is necessary to select a region other than the 494 rbcL and ITS2 regions for identification of such plant foods.

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Of the subdominant plant foods (more than 10% to less than 30% of the 105 496 fecal samples), S. commixta (15.2%) and S. kurilensis (14.3%) were abundantly found in 497 August (Fig 3 and Table 5). Since S. commixta is in full bloom and S. kurilensis is

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In general, P. pumila can be found in all Japanese rock ptarmigan habitats in 504 Japan; however, individual habitats are characterized by their own unique alpine 505 vegetation, e.g., E. nigrum var. japonicum is a dominant species in the study area of the 506 Northern Japanese Alps, but rarely grows in the Japanese rock ptarmigan habitat of the 507 Southern Japanese Alps [1]. The major plant food species for the Japanese rock ptarmigan 508 may differ according to the mountain region [1]. Therefore, conservation of the Japanese