Introduction

Amphibians are the most abundant vertebrates in wetland ecosystems, although they are relatively inconspicuous animals because of their small sizes, nocturnal activities, and preferred habitats in the ground or under rocks (deMaynadier Hunter, 1995). During ontogenetic development, tadpoles and adults occupy two distinct habitats after metamorphosis. Therefore, habitat loss and fragmentation are fatal to amphibians moving between aquatic and terrestrial ecosystems. These are the most critical reasons why amphibian populations are declining worldwide (Becker et al., 2007).

A restoration program consists of three stages: stage 1, preparation; stage 2, implementation, and stage 3, post-restoration. In the preparation stage, the appropriateness of the restoration project is evaluated, the project is decided, and items for the project are prepared. The implementation stage is the actual implementation of the project. Finally, the post-restoration stage is conducted with post-restoration monitoring, evaluation, and public relations promotion (Gascon et al., 2007; International Union for Conservation of Nature (IUCN), Species Survival Commission (SSC), 2013; Ra, 2010). A monitoring period longer than 5 years is required to adequately assess site restoration regarding demographic responses due to amphibian characteristics such as significant population lags and sensitivity to site disturbances (Petranka et al., 2003).

Global amphibian populations have sharply decreased (IUCN, 2020). Major threat factors are habitat degradation, diseases, exotic species, pollution, and over-exploitation (Sung et al., 2009). Pelophylax chosenicus is designated as an endangered species by the Ministry of Environ ment, Korea. It is categorized as vulnerable by the IUCN. National Institute of Biological Resources (NIBR) (2018) has revealed that this species is also gradually decreasing for similar reasons such as habitat loss and destruction due to civilization, road construction, water quality degradation, pesticides, and exotic bullfrogs. Studies on P. chosenicus have been actively conducted in various fields, such as food sources (Yoon et al., 1998), habitats and behaviors (Park et al., 2018; Ra, 2010; Sung et al., 2009), genetic diversity (Park et al., 2009), and water quality (Borzée et al, 2018). However, no study related to restoration has been reported to date.

Reintroduction has been increasingly selected as one restoration method (Fischer Lindenmayer, 2000; Griffith et al., 1989; Wolf et al., 1996). In several species with a limited ability to move like amphibians, reintroduction is required to repopulate sites where extirpation has already occurred (Blaustein et al., 1994). Post-release monitoring is an important stage in the restoration of a target species. Using monitoring data, we can estimate, modify, and complement a restoration program. Monitoring the current status of a reintroduced population is needed to demonstrate restoration success.

In this study, we conducted a reintroduction program for captive-reared P. chosenicus. After reintroduction, we performed six post-release monitoring sessions to investigate its successful settlement and estimate applicability of the reintroduction method to restore frogs in Korea.

Materials and Methods

Captive-breeding of P. chosenicus

P. chosenicus is designated as endangered species class II by the Ministry of Environment, Korea. We obtained permission from a regional environment office by law (Geum River Basin Environment Office, 2018-33). A total of 20 (male to female ratio = 1:1, classified by eye size vs. eardrum size) adults were collected from Asan City in September 2018 and transferred to the Endangered Species Restoration Center of the National Institute of Ecology (NIE). Collected frogs were reared in two PVC aquaria (99.0 cm in length× 45.0 cm in width × 40.0 cm in height). In these aquaria, a proper artificial habitat was set to rear P. chosenicus. A 10 cm depth peat moss mound with a sponge was provided to make a small land. The water depth was maintained at 5 cm. The water temperature was maintained at 27.0 ± 1.0°C using a titanium heater (OKE-HE185, Sewon, Korea) and a temperature regulator (OKE-6428HC, Sewon, Korea). The air temperature was regulated at 23.0 ± 1.0°C using an air temperature controller. During the daytime, additional light was supplied using a spot lamp to prevent light shortage. Adult crickets were provided as food once daily (two crickets per frog). During January 2019, captive-reared frogs spawned naturally in these aquaria [Certification of artificial breeding (Daegu Regional Environment Office, 2019-04)]

Selection of release site and reintroduction

The release site was selected by site observations and expert consultation. Among five candidate sites (e.g., paddy fields in Asan, Asan ecological environment insect museum, Duung wetland in Taean, wetland near the subway station in Ansan, and NIE), two sites (Asan ecological environment insect museum and the NIE) were selected as candidates for reintroduction. Finally, the NIE was selected as the best release site considering habitat conditions, threat factors, management, and SWOT analysis (Fig. 1). The release site, an aquatic garden, was in the NIE. This site had many advantages for managing our frog restoration program. Additionally, P. chosenicus was once inhabited the release site. Therefore, this site was ideal for reintroduction.

Post-release monitoring

Releases were conducted three times (August 19, August 29, and September 18) on the aquatic garden in the NIE. A total of 600 individuals were released (200 individuals per release). After reintroduction, post-release monitoring was conducted six times from August 2019 to May 2020 (1st: August 29; 2nd: September 4; 3rd: September 18; 4th: October 7; 5th: November 5, 2019; 6th: May 7, 2020). Monitoring was performed by counting calls, observation, and capture during sunset to midnight near the release site with elimination of potential threat factors. After measuring snout-vent length (SVL) and body weight (BW) of each recaptured individual, these individuals were then immediately released. Condition factor (CF) was calculated to estimate the nutritional status of P. chosenicus (Froese, 2006; Zhelev et al., 2017) using the following formula:

Condition Factor = (BW/SVL3) × 102

Statistical analyses

Linear regression analysis was conducted to identify growth patterns of recaptured individuals. Monitoring date was converted to Julian date before the calculation. To investigate size differences of captured frogs, Kruskal-Wallis tests were conducted. All statistical analyses were performed using Excel 2013 (Microsoft Corperation, USA) and R 4.0.2 (R Core Team, 2020).

Results and Discussion

Recapture and dispersion

We recaptured 27 individuals (recapture rate = 13.5%) among 200 individuals released during the 1st monitoring. During the 2nd and the 3rd monitoring, 39 (9.75%) and 24 (6.0%) individuals were recaptured of 400 released individuals, respectively. During the 4th, 5th, and 6th monitoring, 46 (7.7%), 7 (1.2%), and 25 (4.2%) individuals were recaptured among 600 released individuals, respe- The aquatic garden of the NIE is a well-managed place for reintroduction. However, several American bullfrogs (Lithobates catesbeianus), a predator of P. chosenicus, were present. Therefore, bullfrogs were continuously removed to stabilize the released P. chosenicus population during the entire monitoring period. A total of 592 bullfrog tadpoles and adults were removed from the 1st monitoring to the 6th monitoring. Additionally, one large individual of a largemouth bass (Micropterus salmoides) was ctively (Table 1). The average recapture rate was 4.66%, which was higher than that reported previously (Cheongwon: 0.5%, Taean: 0.3%; Ministry of Environment, Kangwon National University-Industry Cooperation Foundation, 2009).

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Table 1.
Comparison of recapture rate of Pelophylax chosenicus in this study and references
This study Reference
1st 2nd 3rd 4th 5th 6th Total(average) Cheongwon-gun (abandoned Paddy Field) Taean-gun (duung-wetland)
Number of release/ recapture 200/27 400/39 400/24 600/46 600/7 600/25 600/28 700/4 300/1
Recapture rate (%) 13.5 9.75 6.0 7.7 1.2 4.2 4.66 0.57 0.33

The aquatic garden of the NIE is a well-managed place for reintroduction. However, several American bullfrogs (Lithobates catesbeianus), a predator of P. chosenicus, were present. Therefore, bullfrogs were continuously removed to stabilize the released P. chosenicus population during the entire monitoring period. A total of 592 bullfrog tadpoles and adults were removed from the 1st monitoring to the 6th monitoring. Additionally, one large individual of a largemouth bass (Micropterus salmoides) was captured and removed during the monitoring period.

Reintroduced P. chosenicus individuals were widely dispersed in aquatic gardens. The first reintroduced individuals released in the amphibian wetland moved to the aquatic garden. No individual was observed in the amphibian wetland. The aquatic garden is wide and filled with many aquatic plants, which are considerably useful as covers of frogs (Fig. 1).

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Fig. 1.
Maps of aquatic garden in National Institute of Ecology
PNIE6-f1.png
Body size changes of recaptured individuals

Both SVL and BW of recaptured individuals increased slightly during the monitoring period (Fig. 2). However, there was no significant difference in SVL (Kruskal-Wallis test, X2 = 125.66, P = 0.32), BW (X2 = 120.33, P = 0.28), or CF (X2 = 126.04, P = 0.38) according to the order of monitoring (Table 2). Hibernation has evolved in many amphibians as a protective mechanism in response to cold temperatures, playing a prominent role in amphibian life-history strategies to survive under adverse environmental conditions (Pinde et al., 1992; Wells, 2007). Temperature is a major environmental factor that determines the hibernation period. Sex and age are key factors in phonological responses of P. nigromaculatus. Particularly, males emerge from hibernation earlier than females and old ones enter and emerge from hibernation earlier than young ones (Gao et al., 2015). Willis et al. (1956) have also demonstrated that larger individuals of L. catesbe begin hibernating earlier than smaller individuals. In contrast to the above studies, we found that P. chosenicus had greater SVL and BW at the 5th monitoring session (November 5, hibernating phase) than those at the 4th session (October 5) (Fig. 3). This meant that larger individuals of P. chosenicus began to hibernate later than smaller individuals. This result might indicate that older P. chosenicus were more adaptable than smaller individuals at the NIE in lower environmental conditions. They may feed to actively accumulate energy before entering hibernation. Future further release and monitoring require individual identification using VIE tags, PIT tags, and others. After the hibernating phase, sub-adults of P. chosenicus reintroduced in 2019 were detected on the 6th morning (May 2020). This finding indicated that reintroduced P. chosenicus individuals had successfully hibernate

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Fig. 2.
Relationships between date of monitoring (Julian date) and (A) Snout-vent length (SVL), (B) Bodyweight (BW) of Pelophylax chosenicus on aquatic garden in National Institute of Ecology.
PNIE6-f2.png
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Table 2.
Mean (± SD) snout-vent length (SVL), body weight (BW), and condition factor (CF) of Pelophylax chosenicus on aquatic garden in National Institute of Ecology according to monitoring order.
Monitoring X2 P
1st 2nd 3rd 4th 5th 6th
SVL 35.79±6.77 35.90±5.74 37.61±5.84 36.21±8.12 49.50±6.72 36.34±2.28 125.66 0.32
BW 5.92±3.03 5.03±2.18 5.67±2.79 6.36±3.92 13.28±4.96 5.79±1.23 120.33 0.28
CF 12.93±2.72 10.87±1.33 10.66±1.17 13.39±0.91 10.95±0.86 12.05±1.01 126.04 0.38
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Fig. 3.
Size distributions of Pelophylax chosenicus on aquatic garden in National Institute of Ecology ((A) Snout-vent length (SVL), (B) Body weight (BW)) by date of monitoring.
PNIE6-f3.png

There are two levels of indicators of restoration success. One is an early indicator related to the survival of eggs and larvae through metamorphosis, recruitment of juveniles into adults, and breeding activity. The breeding activity was demonstrated by egg-carrying females, calling males, amplexus, and spawning. The other is a longterm indicator related to the establishment of adult population size (excess of 50 individuals within 5-10 years), mixed population structure (with juveniles and adults regularly recorded, and regular breeding success), and progressive colonization into multiple ponds (establishment of a robust meta-population structure at least five over 5-10 years) (Baker Foster, 2015).

P. chosenicus takes at least 2-3 years to mature sexually (NIE, 2018). In a future study, sexual maturity of P. chosenicus reintroduced in 2019 should be observed by monitoring their eggs and tadpoles through their breeding activities. A normal growth of their offspring in the NIE would indicate the success of restoration by satisfying the early indicator. Annual monitoring results should be assessed against the success criteria described above as restoration projects develop. Post-reintroduction monitoring periods commonly last 3 and 5 years following mitigation of the restoration of a target species (National Research Council, 2001).

To achieve restoration success of P. chosenicus, continuous monitoring is required for at least 3 years. Therefore, more post-release monitoring of P. chosenicus should be conducted in the aquatic garden of the NIE. Findings of the current study provide fundamental information for restoration and conservation strategies throughout post-release monitoring after reintroduction of P. chosenicus.

Confict of Interest

The authors declare that they have no competing interests.

Acknowledgments

This work was supported by a grant (NIE-B-2021-49) from the National Institute of Ecology (NIE) funded by the Ministry of Environment (MOE), Republic of Korea. interests.

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