EcoHealth

, Volume 11, Issue 2, pp 241–250

Retrospective Survey of Museum Specimens Reveals Historically Widespread Presence of Batrachochytrium dendrobatidis in China

Authors

  • Wei Zhu
    • Key Laboratory of Animal Ecology and Conservation Biology, Institute of ZoologyChinese Academy of Sciences
    • University of Chinese Academy of Sciences
  • Changming Bai
    • Key Laboratory of Animal Ecology and Conservation Biology, Institute of ZoologyChinese Academy of Sciences
    • University of Chinese Academy of Sciences
    • Yellow Sea Fisheries Research InstituteChinese Academy of Fishery Sciences
  • Supen Wang
    • Key Laboratory of Animal Ecology and Conservation Biology, Institute of ZoologyChinese Academy of Sciences
    • University of Chinese Academy of Sciences
  • Claudio Soto-Azat
    • Facultad de Ecología y Recursos NaturalesUniversidad Andres Bello
  • Xianping Li
    • Key Laboratory of Animal Ecology and Conservation Biology, Institute of ZoologyChinese Academy of Sciences
    • University of Chinese Academy of Sciences
  • Xuan Liu
    • Key Laboratory of Animal Ecology and Conservation Biology, Institute of ZoologyChinese Academy of Sciences
    • Key Laboratory of Animal Ecology and Conservation Biology, Institute of ZoologyChinese Academy of Sciences
Original Contribution

DOI: 10.1007/s10393-013-0894-7

Cite this article as:
Zhu, W., Bai, C., Wang, S. et al. EcoHealth (2014) 11: 241. doi:10.1007/s10393-013-0894-7

Abstract

Chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has been implicated in amphibian population declines worldwide. However, no amphibian declines or extinctions associated with Bd have been reported in Asia. To investigate the history of this pathogen in China, we examined 1,007 museum-preserved amphibian specimens of 80 species collected between 1933 and 2009. Bd was detected in 60 individuals (6.0%), with the earliest case of Bd infection occurring in one specimen of Bufo gargarizans and two Fejervarya limnocharis, all collected in 1933 from Chongqing, southwest China. Although mainly detected in non-threatened native amphibians, Bd was also found in four endangered species. We report the first evidence of Bd for Taiwan and the first detection of Bd in the critically endangered Chinese giant salamander (Andrias davidianus). Bd appears to have been present at a low rate of infection since at least the 1930s in China, and no significant differences in prevalence were detected between decades or provinces, suggesting that a historical steady endemic relationship between Bd and Chinese amphibians has occurred. Our results add new insights on the global emergence of Bd and suggest that this pathogen has been more widely distributed in the last century than previously believed.

Keywords

Andrias davidianusBatrachochytrium dendrobatidisChinaChytridiomycosisMuseum specimens

Introduction

Amphibian populations have experienced recent population declines in many regions of the world (Stuart et al. 2004). Chytridomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (hereafter Bd), is considered to be an important driver of dramatic amphibian population declines and extinctions in Australia, Central and North America, and Europe (Berger et al. 1998; Longcore et al. 1999; Walker et al. 2008; Vredenburg et al. 2010). The decline of amphibians due to chytridiomycosis is considered “the most spectacular loss of vertebrate biodiversity due to disease in recorded history” (Skerratt et al. 2007). Bd infection has been demonstrated in over 500 species of amphibians from all continents where amphibians exist (Olson et al. 2013). In postmetamorphic susceptible amphibians, Bd infects the epidermis, causing hyperkeratosis, electrolyte and osmotic imbalances that can lead to death (Berger et al. 1998; Longcore et al. 1999; Voyles et al. 2009).

Despite considerable research efforts, the geographic origins of this pathogen and its patterns of global spread have still not been determined (Farrer et al. 2011). Two hypotheses have been proposed to explain the origin of Bd: the novel and the endemic pathogen hypotheses (Rachowicz et al. 2005). Early genetic studies supported the first, based on extremely low genetic diversity detected in strains of Bd collected from disparate regions of the world (Morehouse et al. 2003; James et al. 2009), and the rapid decline of amphibians coincident with the apparent introduction of Bd (Lips et al. 2006; Ryan et al. 2008; Cheng et al. 2011). For instance, a retrospective survey using museum specimens revealed that the arrival of a “Bd epidemic wave” that began in Mexico in the 1980s and subsequently spread to Central America was responsible for documented declines in amphibians from southern Mexico, Guatemala, and Costa Rica (Cheng et al. 2011). However, recent genomic data have demonstrated a higher genetic differentiation than previously recognized, including the existence of a globally widespread and several regional endemic strains of Bd (Goka et al. 2009; Farrer et al. 2011; Bai et al. 2012; Schloegel et al. 2012; Bataille et al. 2013, Rosenblum et al. 2013). Farrer et al. (2011) characterized three deeply diverged lineages of Bd: one of which may be endemic to Europe (BdCH); one endemic to South Africa and then introduced to Mallorca (BdCAPE); and a global pandemic lineage (BdGPL). Additional studies have also found endemic strains associated with Asian native amphibians (Goka et al. 2009; Bai et al. 2012; Bataille et al. 2013) and Schloegel et al. (2012) characterized a new lineage of Bd (BdBrazil), which has been found widespread in Brazil, USA, and Japan. More recently, Martel et al. (2013) isolated and characterized a well-supported new species of chytr id fungus, Batrachochytrium salamandrivorans, which has been associated with lethal chytridiomycosis and severe population decline of fire salamanders (Salamandra salamandra) in the Netherlands.

Weldon et al. (2004) first proposed that Bd originated in Africa and that the international trade in the African clawed frog (Xenopus laevis) might have contributed to the global spread of Bd, giving rise to the Bd “out of Africa” hypothesis. Currently, the earliest known record of Bd is from a specimen of the Fraser’s African clawed frog (Xenopus fraseri) collected from Cameroon in 1933 (Soto-Azat et al. 2010). Both of these studies have also shown a consistently low historical prevalence of Bd in Xenopus spp. throughout the 20th century, suggesting a long history of coevolution between Xenopus spp. and Bd in Africa (Weldon et al. 2004; Soto-Azat et al. 2010).

In Asia, a potential historical Bd infection from museum specimens has been suggested to date back to as early as 1902, based on a Bd positive case in a museum specimen of the giant salamander (Andrias japonicus) from Japan (Goka et al. 2009), although this case has not been confirmed by PCR. Additionally, by sequencing the ribosomal DNA of Bd, three population genetic studies have found endemic lineages of Bd in Japan (Goka et al. 2009), China (Bai et al. 2012) and South Korea (Bataille et al. 2013). These results imply that Bd has had a long historical association with Asia; an argument from which a Bd “out of Asia” hypothesis has also been suggested (Fisher 2009; Goka et al. 2009; Bai et al. 2012). Three lines of evidence further sustain this hypothesis: (1) a low prevalence of Bd in wild amphibian populations, (2) all amphibians infected with Bd have shown no clinical signs of disease, and (3) no substantial chytridiomycosis-caused population declines have been reported in Asia (Kusrini et al. 2008; Goka et al. 2009; Yang et al. 2009; Rowley et al. 2010; Bai et al. 2010, 2012; Savage et al. 2011; Swei et al. 2011; Bataille et al. 2013).

However, the epidemic history of Bd in Asia is little known. Three PCR-based studies have assayed for Bd in archived amphibian specimens collected in Asia (Ouellet et al. 2005; McLeod et al. 2008; Zeng et al. 2011). Only one of these found Bd, detecting it in Asiatic toad (Bufo gargarizans) specimens, originally collected in 1980 in China (Zeng et al. 2011). To investigate the epidemic history of Bd, we undertook a widespread retrospective survey for Bd infection on more than 1,000 museum amphibian specimens collected in China in the last 80 years. We investigated the prevalence of Bd in museum specimens using a nested PCR assay and compared the differences in Bd infection between different time periods and provinces.

Materials and Methods

A total of 1,007 postmetamorphic wild-caught amphibians of 80 species collected between 1933 and 2009 were examined for evidence of Bd infection. These included 79 native and one alien species (Lithobates catesbeianus), which had been collected by many people for different purposes other than disease surveillance (Table 1). Fixation history of specimens is as follows: 967 specimens were always preserved in 10% buffered formalin, 39 specimens were originally fixed in 10% buffered formalin and then transferred into 95% ethanol (EtOH) in 2006, and only one specimen was preserved in ethanol since collection in 2009. Amphibians were collected from 14 provinces in China, mainly from the southwest regions of the country, where the highest amphibian diversity is found, but regions in the north, west, central, and east, and including the islands of Hainan and Taiwan were also represented.
Table 1

Summary data on 1,007 archived amphibians collected from 14 provinces in China, examined for Batrachochytrium dendrobatidis (Bd) infection.

Taxon

Province

Sampling period

No. positives/ No. examined

Conservation status#

Amolops chunganensis

Sichuan

1974

0/4

LC

Amolops mantzorum

Sichuan, Yunnan

1962–1992

0/19

LC

Amolops ricketti

Fujian, Guangxi, Sichuan.

1964–1991

0/12

LC

Amolops torrentis

Hainan

1964

0/1

VU

Amolops viridimaculatus

Yunnan

1987

0/3

NT

Andrias davidianus

Chongqing, Guangdong, Sichuan.

1954–1986s, ND

1/17

CR

Babina adenopleura

Taiwan

no data

1/3

LC

Babina daunchina

Sichuan

1933–1991

1/28

LC

Babina pleuraden

Yunnan

1965–1989

1/2

LC

Batrachuperus pinchonii

Hubei, Sichuan

1979–1992

0/12

VU

Batrachuperus tibetanus

Sichuan

1941–1992

0/22

VU

Bombina maxima

Sichuan

1942–1992

0/13

NE

Buergeria japonica

Taiwan

no data

0/3

LC

Bufo bankorensis

Taiwan

no data

0/2

LC

Bufo gargarizans

Hubei, Sichuan

1933–1985

8/46

LC

Bufo tibetanus

Sichuan

1973

0/1

LC

Calluella yunnanensis

Guizhou

1959

0/1

LC

Chiromantis doriae

Hainan

1964

0/1

LC

Cynops cyanurus

Yunnan

1987

0/5

LC

Cynops orientalis

Anhui

1975

0/2

LC

Fejervarya limnocharis

Guangxi, Sichuan

1933–1996

5/36

LC

Hoplobatrachus tigerinus

Sichuan

1979

0/2

LC

Hyla annectans

Chongqing, Hubei, Sichuan, Yunnan

1935–2000

6/39

LC

Hyla immaculata

Guizhou

1963

1/5

LC

Hyla tsinlingensis

Chongqing, Hubei, Sichuan

1964–1998

0/9

LC

Hylarana guentheri

Guizhou, Sichuan

1933–1996

0/35

LC

Ichthyophis bannanicus

Guangxi

2006–2009

0/3

LC

Kaloula rugifera

Sichuan

1961

0/2

LC

Kaloula verrucosa

Sichuan, Yunnan

1942–1982

0/4

LC

Leptobrachium boringii

Sichuan

1957–1973

0/20

EN

Leptolalax alpinus

Yunnan

1987

0/5

EN

Leptolalax oshanensis

Sichuan

1966–1990

0/4

LC

Lithobates catesbeianus

Sichuan

1988

0/2

LC

Liua shihi

Chongqing, Hubei, Sichuan

1934–1998

2/26

NT

Microhyla butleri

Sichuan, Yunnan

1973–1991

0/17

LC

Microhyla heymonsi

Fujian, Taiwan

1964–1995

0/3

LC

Microhyla mixtura

Chongqing, Hubei, Sichuan

1979–1998

1/14

LC

Microhyla ornata

Guizhou, Sichuan, Yunnan

1943–1991

0/55

LC

Microhyla pulchra

Hainan

1964

0/1

LC

Nanorana maculosa

Yunnan

1982

0/1

EN

Nanorana parkeri

Tibet

1973

0/1

LC

Nanorana pleskei

Sichuan

1973–1987

0/6

NT

Nanorana quadranus

Hubei, Sichuan

1965–1998

1/21

NT

Nanorana unculuanus

Yunnan

1982

2/4

EN

Nanorana yunnanensis

Sichuan, Yunnan

1965–1992

1/12

EN

Occidozyga martensii

Hainan

1964

0/1

LC

Odorrana grahami

Sichuan

1934–1992

2/20

NT

Odorrana cf. livida*

Sichuan

1991

0/10

DD

Odorrana margaretae

Sichuan

1943–1991

2/36

LC

Odorrana schmackeri

Chongqing, Guangxi, Sichuan

1973–1986

4/27

LC

Oreolalax xiangchengensis

Sichuan

1992

0/9

LC

Paramesotriton caudopunctatus

Guizhou

1981

0/1

NT

Paramesotriton chinensis

Guizhou

1981

0/1

LC

Pelophylax hubeiensis

Hubei

1979

0/5

LC

Pelophylax nigromaculatus

Chongqing, Hubei, Sichuan, Yunnan

1934–1996

6/113

NT

Polypedates megacephalus

Sichuan, Yunnan

1963–2000

0/49

LC

Pseudepidalea viridis

Xinjiang

1962

0/1

LC

Pseudohynobius flavomaculatus

Hubei

1979

0/2

VU

Quasipaa boulengeri

Chongqing, Sichuan

1974–2000

2/34

EN

Quasipaa exilispinosa

Sichuan

1991

0/5

VU

Quasipaa spinosa

Sichuan

1942

0/3

VU

Rana chensinensis

Chongqing, Hubei, Shanxi, Sichuan

1933–1998

5/32

LC

Rana omeimontis

Guizhou, Sichuan

1941–1999

7/42

LC

Rana sauteri

Taiwan

1997

0/3

EN

Rana shuchinae

Yunnan

1982

0/2

LC

Rhacophorus chenfui

Sichuan

1973–1990

0/7

LC

Rhacophorus dennysi

Guangxi, Guizhou

1963–1986

1/2

LC

Rhacophorus dugritei

Chongqing, Hubei, Sichuan, Yunnan

1973–1998

0/13

LC

Rhacophorus omeimontis

Sichuan

1954–1990

0/10

LC

Scutiger boulengeri

Sichuan

1975

0/1

LC

Scutiger glandulatus

Sichuan

1987

0/1

LC

Scutiger mammatus

Sichuan

1975

0/1

LC

Scutiger muliensis

Sichuan

1992

0/9

EN

Tylototriton kweichowensis

Guizhou

No data

0/2

VU

Tylototriton verrucosus

Yunnan

1958–1982

0/5

LC

Xenophrys minor

Sichuan

1989–1996

0/10

LC

Xenophrys nankiangensis

Sichuan

1986

0/1

VU

Xenophrys omeimontis

Sichuan

1989

0/10

NT

Xenophrys shapingensis

Sichuan

1965

0/1

LC

Xenophrys spinata

Sichuan

1990–1991

0/14

LC

Total

  

60/1,007

 

# Based on the IUCN Red List of Threatened Species, Version 2012.2.

* According to Amphibian species of the World (ASW ver. 5.6, 2013), Odorrana livida is known only from the type locality (Myanmar) near the Thai border. So we use Odorrana cf. livida here instead.

NE not evaluated, DD data deficient, LC least concern, NT near threatened, VU vulnerable, EN endangered, CR critically endangered.

Each specimen was handled using a new pair of disposable latex gloves to prevent possible cross-contamination. To decrease the chances of cross-contamination between specimens preserved in the same jars, each individual was rinsed with 70% EtOH before sampling. Each specimen was swabbed 30 times: five times on the ventral surface of the body, pelvic area, each ventral hind limb, and the plantar surface of each hind foot. After sampling, swabs were stored in 1.5 ml microcentrifuge tubes at −20°C.

DNA was extracted following the procedure described by Goka et al. (2009). Each swab was put into a microtube containing 150 μl of lysis buffer, which was prepared with the following proportions: 1 mg/ml proteinase K, 0.01 M NaCl, 0.1 M EDTA, 0.01 M Tris–HCl (pH 8.0), and 0.5% Nonidet P-40. Each tube was shaken for 1 min using a vortex mixer and then centrifuged for 5 s at 2,000 rpm. After removing the swabs, the tubes were centrifuged again for 5 s and subsequently incubated first at 50°C for 2 h and later at 95°C for 20 min. After incubation, 10 μl of the supernatant was deposited in a 0.5 ml microcentrifuge tube containing 90 μl of TE buffer and then used as a DNA template for the PCR assay.

The DNA template was amplified using a nested PCR assay (Gaertner et al. 2009; Goka et al. 2009; Bai et al. 2012). The primers for the first amplification were ITS1f and ITS4, which amplify the 5.8S rRNA gene along with the flanking internal transcribed spacer (ITS) of all fungi (White et al. 1990; Gaertner et al. 2009). In the second amplification step, we used Bd1a and Bd2a to amplify the first-round PCR products (Annis et al. 2004).

The nested PCR assay was performed following the procedure described by Bai et al. (2012). This PCR procedure was optimized to achieve a sensitivity able to detect as little as 0.1 Bd zoospore equivalents per μl of extracted DNA (amount above which infection is indicated). Total reaction volumes were 25 μl, consisting of 2 μl of DNA template, 10× PCR Buffer (200 mM Tris–HCl [pH 8.4], 200 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, and PCR enhancer), 0.4 mM of each primer, 0.2 mM of each dNTP, and 1.25 units of TransStart Taq DNA polymerase (Beijing TransGen Biotech, Beijing, China).

For the first amplification, the conditions were an initial denaturation for 5 min at 94°C; 30 cycles of 30 s at 94°C, 30 s at 59°C, and 1 min at 72°C and a final extension for 10 min at 72°C. For the second amplification, the conditions were an initial denaturation for 5 min at 94°C; 30 cycles of 30 s at 94°C, 30 s at 65°C and 30 s at 72°C, and a final extension for 5 min at 72°C.

For each amplification, we included a positive control using DNA template solution containing 0.1 zoospore equivalents per μl and a negative control using TE buffer without any DNA. We then separated the PCR products (about 300 bp) on agarose gel electrophoresis (1.5% agarose gels). Each sample was tested in duplicate. Samples were regarded as being Bd positive if one out of two replicates returned a positive result (Cheng et al. 2011). Negative controls were run to avoid false positive.

Pearson χ2 analyses were used to test differences in Bd prevalence between time periods and between Chinese provinces from which amphibians were collected. Confidence intervals (CI) were calculated by exact binomial probabilities. Statistical analyses were carried out using SPSS (v. 19.0). A map showing sample locations and our data on historical Bd distributions was produced using ArcGIS (v 9.3).

Results

Bd infection was detected in 60 of 1,007 (6.0%) amphibians examined, representing 21 of 80 (26.3%) species investigated. We report the first detection of Bd in 16 Chinese amphibian species, including the first detection of Bd in an amphibian collected from Taiwan (Table 1). Bd was previously known from Babina pleuraden, Bufo gargarizans, Fejervarya limnocharis, Hyla annectans, Pelophylax nigromaculatus (Bai et al. 2010, 2012). The earliest cases of Bd were found in one specimen of B. gargarizans and two specimens of the Asian grass frog (Fejervarya limnocharis), both collected from Chongqing in 1933.

All studied decades presented Bd-positive individuals, and significant differences between Bd prevalence and time periods were not found (x2 = 9.41, P = 0.23; Table 2). Infected amphibians were found in 7 of the 14 studied provinces in China. Bd was primarily detected in provinces located in the southwest of the country: Sichuan (42 of 775 individuals tested), Chongqing (4 of 61), Yunnan (4 of 57), and Guizhou (1 of 19), but it was also found in the central region of Hubei (5 of 41), the south region of Guangxi (2 of 18), and the island of Taiwan on the east coast of the country (1 of 13; Fig. 1). Differences in the prevalence of Bd infection among the 14 provinces surveyed were not significant (x2 = 5.33, P = 0.97; Table 3). Additionally, according to the IUCN Red List of Threatened Species (2012), four of the 21 infected species are classified as threatened. These include the Critically Endangered (CR) Chinese giant salamander (Andrias davidianus) and another three species of Endangered (EN) amphibians (Table 1).
Table 2

Batrachochytrium dendrobatidis positive Chinese archived amphibian specimens classified by time period.

Time period

No. examined

No. positives

% positives (95% CI)

1930–1939

67

6

9.0 (3.4–18.5)

1940–1949

63

3

4.8 (1–13.3)

1950–1959

18

1

5.6 (0.1–27.3)

1960–1969

28

1

3.6 (0.1–18.3)

1970–1979

174

6

3.4 (1.3–7.4)

1980–1989

293

26

8.9 (5.9–12.7)

1990–1999

279

12

4.3 (2.2–7.4)

2000–2009

22

1

4.5 (0.1–22.8)

Total

944a

56

5.9 (4.5–7.6)

aThe sampling dates of 63 specimens are unknown.

CI confidence interval.

https://static-content.springer.com/image/art%3A10.1007%2Fs10393-013-0894-7/MediaObjects/10393_2013_894_Fig1_HTML.gif
Figure 1

Map of China showing historical distribution of Batrachochytrium dendrobatidis (Bd) infection detected from amphibian museum specimens. Filled circles indicate sites where Bd was detected, and open circles indicate sites where Bd was not detected. AH Anhui, FJ Fujian, GD Guangdong.

Table 3

Batrachochytrium dendrobatidis positive archived Chinese amphibian specimens classified by province.

Province

No. examined

No. positives

% positives (95% CI)

Sichuan

775

42

5.4 (3.9–7.3)

Chongqing

61

4

6.6 (1.8–15.9)

Yunnan

57

4

7.0 (1.9–17.0)

Hubei

41

5

12.2 (4.1–26.2)

Guizhou

19

1

5.3 (0.1–26.0)

Guangxi

18

2

11.1 (1.4–34.7)

Taiwan

13

1

7.7 (0.2–36.0)

Hainan

4

0

0.0 (0.0–60.2)

Guangdong

3

0

0.0 (0.0–70.8)

Anhui

2

0

0.0 (0.0–84.2)

Fujian

2

0

0.0 (0.0–84.2)

Shanxi

2

0

0.0 (0.0–84.2)

Tibet

1

0

0.0 (0.0–97.5)

Xinjiang

1

0

0.0 (0.0–97.5)

Total

999a

59

5.9 (4.5–7.6)

aThe locations of eight specimens are unknown.

CI confidence interval.

Discussion

Our study expands the historical distribution of Bd in China based on a retrospective survey of amphibians over a long period (1933–2009). Although a low proportion of Bd infection was found (6.0%), this is remarkably similar to the overall prevalence (7.6%) detected from wild Chinese living amphibians in a previous study (Bai et al. 2012). Despite most of the examined specimens were entirely or at least initially fixed in formalin, the DNA extraction protocol and nested PCR assay used in this study, were successful in recovering and amplifying Bd genetic material from archived amphibians. The oldest Bd-positive specimens found in this study (three individuals collected in 1933) were stored in formalin for 79 years prior to testing. Similar results have been described by other recent studies (Cheng et al. 2011; Richards-Hrdlicka 2012), however, time of formalin fixation before testing has been considerably smaller (39 and 44 years, respectively). Formalin is known to be capable of degrading DNA, possibly reducing the likelihood of Bd detection (Soto-Azat et al. 2009); our study might, therefore, underestimate the true historical prevalence of Bd in native amphibians in China, but this only strengthens our findings on the historical widespread and long presence of Bd in the country.

We found the earliest known case of Bd infection in mainland Asia, in three specimens of two common and widespread Asian amphibians (B. gargarizans and F. limnocharis) collected in 1933 from southwest China. These three specimens were collected from the same area, but only the two F. limnocharis were collected during the same collection session and, therefore, kept in the same jar. It is possible that cross-contamination occurred between these individuals, either during field collection or while fixed and subsequently preserved, however, as the amount of amplified Bd DNA from museum amphibians is generally very low particularly if fixed in formalin (Soto-Azat et al. 2009, 2010; Vredenburg et al. 2013) and due to the measures put in practice to prevent it, probability of cross-contamination appears to be minimal.

Our study is also the first report of the presence of Bd in Taiwan, in a specimen of the olive frog (Babina adenopleura) collected from Yilan County in the north of the island. Only one previous study has assayed for Bd on amphibians in Taiwan, which investigated 20 frogs of 12 species in 2006; no Bd-positive result was obtained (Lehtinen et al. 2010). Nonetheless, the date of collection of the studied Taiwanese B. adenopleura specimens is unknown; thus, further retrospective and prospective epidemiological studies are required to determine the current and historical Bd status in Taiwan.

We also found a consistently low prevalence of Bd infection in Chinese amphibians over time, suggesting that a historical steady endemic relationship between Bd and native amphibians has occurred. The infection was not detected in seven provinces from which collected amphibians were available (Hainan, Guangdong, Anhui, Fujian, Shanxi, Tibet, Xinjiang),, however, each of these was represented by small sample sizes (<5 examined specimens). As a consequence, the absence of Bd from these and other non Bd-surveyed provinces of China cannot be ruled out. No significant differences were observed in the proportion of amphibians infected between provinces, indicating that Bd has been evenly distributed across much of China post 1933.

Bd infection was also detected for the first time in four rapidly declining Asian amphibian species: A. davidianus (CR), Nanorana unculuanus (EN), N. yunnanensis (EN), and Quasipaa boulengeri (EN). Whether chytridiomycosis is implicated as a contributing factor in the population declines described in these species is not known. Goka et al. (2009) found a high proportion of Bd infection in wild A. japonicus in Japan, with an apparently non-pathogenic species-specific endemic genotype of Bd. A drastic population decline, calculated to be >80% over the last three generations, has been described in A. davidianus (Gang et al. 2004). Whether wild A. davidianus in China are infected with endemic or a more virulent introduced pathogenic strain is unknown, therefore, an assessment of Bd as a threat to the world’s largest amphibian species should be matter of future research.

Historical Bd infections found on archived specimens collected in Africa and Asia (Weldon et al. 2004; Goka et al. 2009; Soto-Azat et al. 2010) about 40 years before the beginning of the global amphibian population decline phenomenon (Stuart et al. 2004), should be considered as indicators of regions that might harbor novel endemic strains of Bd (Schloegel et al. 2012). Bd present in China since at least 1933, is unlikely to have been introduced from abroad. The international trades in X. laevis and L. catesbeianus are considered two important global vectors of Bd (Weldon et al. 2004; Garner et al. 2006; Liu et al. 2013). Goka et al. (2009) found that many alien strains of Bd including BdGPL have been introduced into Japan via imported amphibians such as L. catesbeianus. It is also possible that L. catesbeianus vectored exotic Bd genotypes such as BdGPL or BdBrazil into South Korea and China when they were introduced (Bai et al. 2012; Schloegel et al. 2012; Bataille et al. 2013). As a result of bullfrog invasions, native frog abundance and species richness have declined on the Zhoushan Archipelago, China, though the role of Bd in these declines is not yet clear (Yiming et al. 2011).

The first reports of L. catesbeianus trade into China date back to the late 1950s and first establishments of wild populations did not occur until the early 1960s (Liu et al. 2010). In total, we detected 10 cases of Bd infection from museum specimens collected between 1933 and 1959. These early positive cases are consistent with infections with endemic strains of Bd (Bai et al. 2012), however, to clarify the origin of these, Bd DNA sequencing from museum-preserved material is required. Additionally, future retrospective studies on archived wild-caught L. catesbeianus might be useful to date the epidemic history of exotic Bd strain introductions to China.

Conclusion

Retrospective studies on archived amphibians can provide an important basis for our understanding of the global emergence and epidemiology of Bd. This study found the earliest known case of Bd infection on mainland Asia and confirms a historical widespread presence of Bd in China. We also found a consistently low prevalence of Bd infection in Chinese amphibians over time, suggesting that a historically steady endemic relationship between Bd and native amphibians has occurred. Independent of the strain of Bd involved, this pathogen appears to be more widely distributed in the last century than previously believed. Future studies on the historical and current presence of endemic and exotic strains of Bd, including isolation, partial DNA or whole genome sequencing, and virulence testing are required to understand the impacts of chytridiomycosis to native amphibians in China.

Acknowledgements

We thank Enqi Ye of the National Zoological Museum (Beijing) and Zhiqing Xu of the Chongqing Museum of Natural History (Chongqing) for assistance with sampling. We are also grateful to Kris Murray and two anonymous reviewers for helpful comments on the draft of the manuscript. This research was supported by grants from the National Natural Science Foundation of China (code: 31172111, 31200416) and the Beijing Natural Science Foundation (code: 5132026).

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© International Association for Ecology and Health 2013