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\begin{document}

\title{Climate as a gatekeeper: Unexpected absence of \emph{Batrachochytrium
salamandrivorans} in an Asian Chytrid hotspot suggests a narrow climatic
niche for the pathogen}



\author[1]{Dan Sun}%
\author[1]{Gajaba Ellepola}%
\author[1]{Jayampathi Herath}%
\author[1]{Madhava Meegaskumbura}%
\affil[1]{Guangxi University}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
The chytrid fungal pathogens \emph{Batrachochytrium salamandrivorans}
(\emph{Bsal}) and \emph{B. dendrobatidis} (\emph{Bd}) are driving
amphibian extinctions and population declines worldwide. They are
thought to have originated in East/Southeast Asia, and hence Asia
remains crucial for understanding the ecology of these pathogens.
However, \emph{Bsal} screening is relatively limited in this region,
particularly in hotspots where \emph{Bd} lineage diversity is high. To
address this gap, we conducted an extensive survey across the Guangxi
region of Southern China, now considered a \emph{Bd} hotspot. We
examined 1230 individuals from 38 amphibian species and 36 environmental
water bodies sampled during mostly the middle spring and summer months
of 2019-21. However, PCR testing revealed absence of \emph{Bsal} in our
samples. A subsequent niche modeling analysis for \emph{Bsal} suggested
that the bioclimatic conditions of much of the region may not be
conducive to \emph{Bsal}. Although our findings show absence of
\emph{Bsal} in the surveyed habitats, periodic monitoring in amphibians
is still needed, particularly across seasons. This is because
\emph{Bsal} pathogenicity could increase at relatively lower
temperatures, and \emph{Bsal} may be partitioning its niche with
\emph{Bd} across seasons. Our results suggest that the climatic
conditions of the karstic Guangxi plains and perhaps competition from
other \emph{Bd} lineages may suppress \emph{Bsal} establishment in the
region. Our study provide interesting insights into \emph{Bsal} niche
dynamics and the knowledge generated will facilitate the conservation
efforts in amphibian populations devastated by chytrid pathogens across
other regions of the world.%
\end{abstract}%



\sloppy


\textbf{Climate as a gatekeeper: Unexpected absence
of\emph{Batrachochytrium salamandrivorans} in an Asian Chytrid hotspot
suggests a narrow climatic niche for \emph{Batrachochytrium
salamandrivorans}}

Dan Sun\textsuperscript{1}, Gajaba Ellepola\textsuperscript{1, 2},
Jayampathi Herath\textsuperscript{1, 3}, Madhava
Meegaskumbura\textsuperscript{1,*}

1 Guangxi Key Laboratory for Forest Ecology and Conservation, College of
Forestry, Guangxi University; Nanning, Guangxi 530000, People's Republic
of China

2 Department of Zoology, Faculty of Science, University of Peradeniya,
Peradeniya, KY20400, Sri Lanka

3 School of Biomedical Sciences, International Institute of Health
Sciences (IIHS), No 704 Negombo Rd, Welisara 71722, Sri Lanka

* Corresponding Author E--mail: madhava\_m@mac.com

\textbf{Abstract}

The chytrid fungal pathogens \emph{Batrachochytrium
salamandrivorans}(\emph{Bsal} ) and \emph{B. dendrobatidis} (\emph{Bd} )
are driving amphibian extinctions and population declines worldwide.
They are thought to have originated in East/Southeast Asia, and hence
Asia remains crucial for understanding the ecology of these pathogens.
However, \emph{Bsal} screening is relatively limited in this region,
particularly in hotspots where \emph{Bd} lineage diversity is high. To
address this gap, we conducted an extensive survey across the Guangxi
region of Southern China, now considered a \emph{Bd} hotspot. We
examined 1230 individuals from 38 amphibian species and 36 environmental
water bodies sampled during mostly the middle spring and summer months
of 2019-21. However, PCR testing revealed absence of \emph{Bsal} in our
samples. A subsequent niche modeling analysis for \emph{Bsal} suggested
that the bioclimatic conditions of much of the region may not be
conducive to \emph{Bsal} . Although our findings show absence
of\emph{Bsal} in the surveyed habitats, periodic monitoring in
amphibians is still needed, particularly across seasons. This is
because\emph{Bsal} pathogenicity could increase at relatively lower
temperatures, and \emph{Bsal} may be partitioning its niche
with\emph{Bd} across seasons. Our results suggest that the climatic
conditions of the karstic Guangxi plains and perhaps competition from
other \emph{Bd} lineages may suppress \emph{Bsal} establishment in the
region. Our study provide interesting insights into \emph{Bsal} niche
dynamics and the knowledge generated will facilitate the conservation
efforts in amphibian populations devastated by chytrid pathogens across
other regions of the world.

\textbf{Keywords}

\emph{Batrachochytrium salamandrivorans} ; Amphibians; Asia; Niche;
Temperature; Geographic Distribution; Seasonality



\textbf{Introduction}

Amphibians worldwide face considerable threats, including habitat loss,
climate change, and disease, which contribute to severe population
declines and biodiversity loss (Amphibiaweb, 2023). Chytridiomycosis, a
disease caused by the chytrid fungi \emph{Batrachochytrium
dendrobatidis} (\emph{Bd} ) and \emph{B. salamandrivorans} (\emph{Bsal}
), is a major driver of these declines (Longcore et al., 1999; Martel et
al., 2013; Scheele et al., 2019). Both \emph{Bd} and \emph{Bsal} are
thought to have originated in Asia, with \emph{Bd} portraying multiple
lineages in the region (Martel et al., 2014; O'hanlon et al.,
2018).\emph{Bd} affects all three amphibian orders and has caused
widespread declines, particularly in anuran species across all
amphibian-inhabited continents (Scheele et al., 2019). In contrast,
although present in Asia, \emph{Bsal} has led to severe declines of
native salamanders only in Europe so far (Fisher \& Garner, 2020),
however, the impending threat for the highly diverse Northern American
and Neotropical urodelans is significant (Carter et al., 2020; Crawshaw
et al., 2022; Garc\selectlanguage{ngerman}ía-Rodríguez et al., 2022; Moubarak et al., 2022;
North American\emph{Bsal} Task Force, 2022; Yap et al., 2015).

Although \emph{Bsal} primarily infects urodelans, recent studies show
that anurans can act as reservoir hosts or as transmitters (Lötters et
al., 2020; Nguyen et al., 2017; Schulz et al., 2020; Stegen et al.,
2017; Towe et al., 2021). This role increases disease risk to sensitive
salamander species and populations. Therefore, identifying reservoir
hosts is crucial for understanding infection dynamics and potential
occurrences as \emph{Bsal} continues to expand its range and threaten
biodiversity (Martel et al., 2020; Spitzen-Van Der Sluijs et al., 2016).

\emph{Bsal} has restricted distributional areas, co-occurring
with\emph{Bd} in several microhabitats (Laking et al., 2017; Lötters et
al., 2020). \emph{Bd} -\emph{Bsal} occurrences can potentially generate
new genotypes with heightened pathogenicity (Farrer et al., 2011; Fisher
\& Garner, 2020; Wacker et al., 2023). \emph{Bsal} has stronger
pathogenicity compared to \emph{Bd} within a shared host species,
leading to more severe infection outcomes (Farrer et al., 2017). Out of
documented 67 susceptible host species for \emph{Bsal} , nearly 32
species also can be infected by \emph{Bd} (Castro Monzon et al., 2023;
Olson et al., 2021), these shared species could serve as the
hyper-reservoirs or gain greater impacts from both pathogens.
Importantly, \emph{Bd} -\emph{Bsal} coinfections have been reported in
three urodelean species in natural habitats (Lötters et al., 2020;
Lötters et al., 2018), and such coinfections are known to increase
infection severity and disease risk under the laboratory experimental
conditions (Longo et al., 2019; Ribas et al., 2022). These emphasize the
importance of \emph{Bsal} screening in natural habitats where
\emph{Bd}occurs.

In Southeast Asia (Vietnam), \emph{Bsal} co-occurs together with the
globalized genotype \emph{Bd} GPL (Laking et al., 2017). However,
knowledge on \emph{Bsal} infection is scarce in other Asian amphibian
hotspots where different \emph{Bd} lineages are found. South China, for
example, harbors the unique Asian genotype \emph{Bd} Asia3
alongside\emph{Bd} GPL (Byrne et al., 2019).

Here, we hypothesize that \emph{Bsal} co-occurs together with
\emph{Bd}populations in South China. To investigate this, we screened
for\emph{Bsal} in salamanders and potential anuran reservoir hosts
within regional amphibian hotspot in South China, which is recognized
for its diverse \emph{Bd} genotypes spanning various natural habitats.
We also assessed the influence of climatic factors on \emph{Bsal}
distribution by estimating its potential range under the current
climatic conditions. Analyzing the presence of \emph{Bsal} in areas with
high \emph{Bd}diversity will enhance our comprehension of \emph{Bsal}
niche dynamics and pathogenicity. This has significant implications for
amphibian conservation in the context of pathogen virulence evolution.

\textbf{Materials and Methods}

\textbf{Sample collection}

From spring 2019 to early winter 2021, a total of 31 sites (27 field
sites and 4 captive sites) were surveyed throughout the Guangxi region
in China (Fig. 1A), which shares its southwestern border with Vietnam, a
country known to harbor \emph{Bsal} (Laking et al., 2017). The pathogen
was detected in an individual of a salamander species, \emph{Pachytriton
wuguanfui} in Guangxi region (Yuan et al., 2018). It is now known that
basal Asian and global lineages of \emph{Bd} exist in this region (Fig.
1B, Sun et al., 2023). Occasionally, \emph{Bsal} can spill over from
captive populations into wildlife communities (Cunningham et al., 2015;
Fitzpatrick et al., 2018; Sabino-Pinto et al., 2015). To account for
this, random samples were collected from pet markets and frog farms
within the region. Skin swabs were collected following the sampling
protocol for chytrid pathogens in amphibians, as described in Hyatt et
al. (2007). Water samples were collected from bodies of water
for\emph{Bsal} detection in relation to local populations, to be used
for environmental DNA (eDNA) analysis; eDNA can be used for pathogen
detection in the absence of hosts (Kirshtein et al., 2007; Schulz et
al., 2020). For this, approximately 1 L of water was sampled for each
water body, then filtered through the 0.40 \selectlanguage{greek}μ\selectlanguage{english}m polycarbonate filter
membrane using a sterile 100 ml syringe.

Ethical clearance for this study was obtained from the Institutional
Animal Care and Use Committee of Guangxi University (GXU2018-048, with
an extension of GXU2020-501).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 1. Study region, sampling sites and climatically suitable
regions for \emph{Bsal}. (A)} Site distribution and climatically
suitable habitats for \emph{Bsal} in Guangxi region.
\textbf{(B)}Documented sites for \emph{Bd} presence in the region (Sun
et al., 2023) over a background of amphibian species richness (IUCN
2015) .

\textbf{Detection of \emph{Bsal} infection}

The genomic DNA for skin swabs was extracted using the PreMan Ultra
(Applied Biosystems) (Boyle et al., 2004) and Qiagen DNeasy Blood and
Tissue Kit, following animal tissue part in the modified manufacturers'
protocols (Kosch \& Summers, 2012). The environmental DNA for filters
was extracted using Qiagen DNeasy PowerSoil Kit.

We used nested PCR to detect \emph{Bsal} on DNA extracts from skin swabs
(Gaertner et al., 2009; Zhu et al., 2014). For the first amplification,
the primers ITS1f and ITS4 were used which specifically combined the 18S
and 28S rRNA genes. The PCR amplification conditions, 4 min at 94\selectlanguage{ngerman}°C,
followed by 30 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C
and a final 10 min at 72°C were implemented. For the second
amplification, the specific primers (STerF and STerR) for \emph{Bsal}
were used to amplify a fragment gene of the ITS-5.8S rRNA region (Martel
et al., 2013). The conditions of PCR amplification included 4 min at
94°C, followed by 30 cycles of 30 s at 94°C, 60°C for 30s, and 1 min at
72°C and a final 10 min at 72°C. PCR amplification products were
visualized by 1.5\% agarose gel electrophoresis.

Environmental samples were tested for \emph{Bsal} using the RT-PCR
method described by Blooi \emph{et al} . (2013, 2016). We used a cycle
of an initial step at 95°C for 10 min, followed by 50 cycles of 95°C for
10 s and 60°C for 1 min on Light Cycler® 480 System (Roche, Mannheim,
Germany). Each water sample was run in duplicate.

We used the synthetic DNA sequences of \emph{Bsal} as positive controls
(Pisces Molecular, Boulder, Colorado, USA) and two negative controls in
each plate. If nested PCR and RT-PCR products yielded positive results,
Sanger sequencing was conducted to verify if the amplified DNA fragments
were indeed associated with \emph{Bsal} .

\textbf{Analyses of \emph{Bsal} prevalence}

\emph{Bsal} prevalence, defined as the ratio of infected amphibians to
the total population, was calculated for each species, including 95\%
binomial confidence intervals (CI). The confidence intervals were
derived using the ''binom'' package (Dorai-Raj, 2022) in R 4.2.2 (R Core
Team, 2022).

\textbf{Climatic suitability for \emph{Bsal}}

To understand how environmental conditions affect the distribution
of\emph{Bsal} , we used Generalized Linear Modeling (GLM) and Maximum
Entropy Modeling (MaxEnt) to estimate its habitat suitability (Breiner
et al., 2015; Phillips et al., 2006). If new positive locations
of\emph{Bsal} were detected in our study\emph{,} they were to be
combined with the previous verified presence records in native Asia to
build the niche model. Previous present records of \emph{Bsal} for
Thailand, Vietnam, China and Japan in Asia (n=34), were attained from
Basanta et al. (2019), Beukema et al. (2018), Laking et al. (2017),
Lötters et al. (2020), and Yuan et al. (2018). We used the ecoregions
(Dinerstein et al., 2017) of these \emph{Bsal} occurrences as background
to improve model calibration. Nineteen bioclimatic variables
representing climatic conditions were download from WorldClim version
2.1 (Fick \& Hijmans, 2017) as environmental predictors, at the spatial
resolution of 30 seconds (\textasciitilde{}1 km2). To exclude the
effects of high collinearity between predictors, we used the bioclimatic
variables with Pearson's r \textless{} 0.7, where the correlation was
calculated in the ENMTools (Warren et al., 2010). The final predictor
set included 6 variables: Mean Diurnal Range (BIO2), Maximum Temperature
of Warmest Month (BIO5), Temperature Annual Range (BIO7), Annual
Precipitation (BIO12), Precipitation of Warmest Quarter (BIO18), and
Precipitation of Coldest Quarter (BIO19). To trim duplicate observation
records, a single coordinate for each grid cell was retained in our
model predictions. The model performance for \emph{Bsal} was evaluated
using the AUC (Lobo et al., 2008), which was calculated by splitting the
training (70\%) and testing (30\%) observations. The final ensembled
model contained ten replications for each modeling, and were run in
``sdm'' package (Naimi \& Araújo, 2016).

\textbf{Results}

A total of 1230 individuals including 1195 wild individuals and 95
captivities (0\% prevalence, 95\% CI of 0.00\%-0.31\%; Table 1),
representing 38 amphibian species, were sampled during the study. The
thirty-six water bodies sampled were from artificial lakes, natural
pools, and slow flow streams (Fig. 1A). However, \emph{Bsal} was not
detected in any of the sampled individuals or water bodies.

\textbf{Table 1} Individual numbers and amphibian species detected in
this study in Guangxi Region. Bold fonts represent species which have
been recorded positive to \emph{Bd} infection in previous studies from
other regions (Bai et al., 2012; Gaertner et al., 2011; Sun et al.,
2023; Wang et al., 2018; Zhu et al., 2014)
;\selectlanguage{english}\emph{\textsuperscript{\textbf{*}}} denote samples collected in
captivity including a pet market and three frog farms.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
Family Fly Genus Species N 95\%CI Ranidae Amolops Amolops chunganensis
23 0.00\%-14.31\% Ranidae Amolops Amolops ricketti 272 0.00\%-1.39\%
Megophryidae Boulenophrys Boulenophrys brachykolos 6 0.00\%-39.03\%
Megophryidae Boulenophrys Boulenophrys mirabilis 1 0.00\%-79.35\%
Megophryidae Brachytarsophrys Brachytarsophrys carinense 2
0.00\%-65.76\% Dicroglossidae Fejervarya Fejervarya multistriata 26
0.00\%-12.87\% Dicroglossidae Hoplobatrachus Hoplobatrachus chinensis 19
0.00\%-16.82\% Ranidae Hylarana Hylarana latouchii 4 0.00\%-48.99\%
Ranidae Hylarana Hylarana maosonensis 17 0.00\%-18.43\% Ranidae Hylarana
Hylarana guentheri 43 0.00\%-8.20\% Microhylidae Kalophrynus Kalophrynus
interlineatus 1 0.00\%-79.35\% Microhylidae Kaloula Kaloula pulchra 1
0.00\%-79.35\% Rhacophoridae Kurixalus Kurixalus odontotarsus 69
0.00\%-5.27\% Megophryidae Leptobrachella Leptobrachella liui 19
0.00\%-16.82\% Megophryidae Leptobrachella Leptobrachella alpina 4
0.00\%-48.99\% Megophryidae Leptobrachella Leptobrachella
shiwandashanensis 7 0.00\%-35.43\% Megophryidae Leptobrachium
Leptobrachium guangxiense 2 0.00\%-65.76\% Dicroglossidae Limnonextes
Limnonectes bannaensis 9 0.00\%-29.91\% Rhacophoridae Liuixalus
Liuixalus shiwandashan 3 0.00\%56.15\% Microhylidae Microhyla Microhyla
heymonsi 28 0.00\%-12.06\% Microhylidae Microhyla Microhyla pulchra 15
0.00\%20.39\% Ranidae Odorrana Odorrana exiliversabilis 12
0.00\%-24.25\% Ranidae Odorrana Odorrana graminea 54 0.00\%-6.64\%
Ranidae Odorrana Odorrana lungshengensis 22 0.00\%-14.87\% Ranidae
Odorrana Odorrana nasuta 46 0.00\%-7.71\% Megophryidae Ophryophryne
Ophryophryne microstoma 12 0.00\%-24.25\% Salamandridae Pachytriton
Pachytriton inexpectatus 15 0.00\%-20.39\% Salamandridae Pachytriton
Pachytriton moi 16 0.00\%-19.36 Rhacophoridae Polypedates Polypedates
megacephalus 138 0.00\%-2.71\% Dicroglossidae Quasipaa Quasipaa
boulengeri 34 0.00\%-10.15\% Dicroglossidae Quasipaa Quasipaa spinosa 70
0.00\%-5.20\% Rhacophoridae Rhacophorus Rhacophorus laoshan 16
0.00\%-19.36\% Rhacophoridae Theloderma Theloderma rhododiscus 38
0.00\%-9.18\% Rhacophoridae Liuixalus Theloderma albopunctatum 2
0.00\%-65.76\% Salamandridae Tylototriton Tylototriton asperrimus 13
0.00\%-22.81\% Salamandridae Tylototriton Tylototriton spp. 3
0.00\%-56.15\% Megophroridae Xenophrys Xenophrys major 3 0.00\%-56.15\%
Rhacophoridae Zhangixalus Zhangixalus dennysi 32 0.00\%-10.72\%
Rhacophoridae Zhangixalus Zhangixalus leucofasciatus 37 0.00\%-9.41\%
Rhacophoridae Zhangixalus Zhangixalus pinglongensis 1
0.00\%-79.35\%Family\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
Species\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
N\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
95\% CI\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ranidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Amolops chunganensis\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
23\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-14.31\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ranidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Amolops ricketti\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
272\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-1.39\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Megophryidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Boulenophrys brachykolos}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-39.03\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Megophryidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Boulenophrys mirabilis}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-79.35\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Megophryidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Brachytarsophrys carinense}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-65.76\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Dicroglossidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Fejervarya multistriata}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
26\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-12.87\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Dicroglossidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Hoplobatrachus chinensis}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
19\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-16.82\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ranidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Hylarana latouchii}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-48.99\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ranidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Hylarana maosonensis}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
17\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-18.43\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ranidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Hylarana guentheri\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
43\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-8.20\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Microhylidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Kalophrynus interlineatus}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-79.35\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Microhylidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Kaloula pulchra\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-79.35\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Rhacophoridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Kurixalus odontotarsus}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
69\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-5.27\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Megophryidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Leptobrachella liui\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
19\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-16.82\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Megophryidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Leptobrachella alpina}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-48.99\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Megophryidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Leptobrachella shiwandashanensi}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-35.43\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Megophryidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Leptobrachium guangxiense}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-65.76\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Dicroglossidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Limnonextes bannaensis}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
9\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-29.91\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Rhacophoridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Liuixalus shiwandashan}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%56.15\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Microhylidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Microhyla heymonsi}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
28\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-12.06\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Microhylidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Microhyla pulchra\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
15\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%20.39\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ranidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Odorrana exiliversabilis\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
12\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-24.25\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ranidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Odorrana graminea\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
54\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-6.64\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ranidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Odorrana \textbf{lungshengensis}}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
22\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-14.87\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ranidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Odorrana nasuta}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
46\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-7.71\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Megophryidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Ophryophryne microstoma}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
12\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-24.25\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Salamandridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Pachytriton inexpectatus\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
15\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-20.39\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Salamandridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Pachytriton moi}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
16\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-19.36\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Rhacophoridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Polypedates megacephalus\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
138\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-2.71\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Dicroglossidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Quasipaa boulengeri\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
34\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-10.15\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Dicroglossidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Quasipaa spinosa}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
70\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-5.20\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Rhacophoridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Rhacophorus laoshan}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
16\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-19.36\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Rhacophoridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Theloderma rhododiscus\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
38\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-9.18\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Rhacophoridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Liuixalus albopunctatum}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-65.76\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Salamandridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Tylototriton asperrimus}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
13\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-22.81\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Salamandridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Tylototriton spp.}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-56.15\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Megophroridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Xenophrys major}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-56.15\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Rhacophoridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Zhangixalus dennysi\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
32\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-10.72\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Rhacophoridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Zhangixalus leucofasciatus}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
37\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-9.41\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Rhacophoridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Zhangixalus pinglongensis}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-79.35\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Salamandridae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Andrias davidianus (Larva)\textsuperscript{*}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-43.45\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ranidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Lithobates catesbeiana\textsuperscript{*}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
75\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-4.87\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Dicroglossidae\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Hoplobatrachus chinensis\textsuperscript{\textbf{*}}}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
15\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.00\%-20.39\%\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

The average AUC value for the \emph{Bsal} distribution model was 0.917
(SD \selectlanguage{ngerman}± 0.047), signifying strong model performance. The top three
variables for model predictions included mean diurnal range, maximum
temperature of the warmest month, and precipitation during the coldest
quarter (Fig. 1A).

The modeling predictions estimated that only a small fraction of
suitable habitats for \emph{Bsal} occurrences exists under current
climate conditions, with patchy distribution in the subtropical
evergreen forests of the Guangxi region. (Fig. 1A). The anticipated
habitats for \emph{Bsal} occurrence were situated in southwestern areas
near the coastlines. Among the 27 wild sampling sites, 14 were deemed
suitable for \emph{Bsal} , with suitability levels ranging from low to
high.

\textbf{Discussion}

We analyzed a wide range of species and environmental reservoirs across
different seasons to evaluate the infection patterns of \emph{Bsal} in
relation to geographic and environmental factors in Guangxi region. We
did not detect \emph{Bsal} in any of the samples from wild and captive
animals or sampled water bodies. Our species distribution model
estimated a small fraction of suitable habitats in this region under the
current climatic conditions, but with patchy distributions. These
results suggest that climate is a potential barrier, limiting\emph{Bsal}
spread into the surveyed sites. However, there is a possibility that it
could be present at undetectable levels, or occur in specific
microhabitats that were not sampled in our study.

Our niche model predicted that climatically suitable areas
for\emph{Bsal} in the Guangxi region are less and fragmented, generally
low suitability. The model identified mean diurnal range as the most
crucial variable in our models. This corroborates the findings from
previous\emph{Bsal} distribution models in its native Asian range
(Moubarak et al., 2022). The relatively high variations in mean diurnal
range within the study area seem to limit the potential distribution of
\emph{Bsal} , emphasizing that environmental temperature is a primary
factor influencing \emph{Bsal} occurrences (Martel et al., 2013).

\emph{Bsal} prefer lower thermal conditions for their existence, the
temperatures above 22°C adversely affects its growth and diminish its
pathogenicity to hosts (Carter et al., 2021; Martel et al., 2013).
Previous studies have proposed that temperature is a key factor behind
the low prevalence of \emph{Bsal} in Asian amphibians, as the
environmental as well as the water temperatures in these regions are
generally considered suboptimal (\selectlanguage{english}[?] 15degC) for\emph{Bsal} 's survival and
growth (Laking et al., 2017; Martel et al., 2013; Yuan et al., 2018). It
has also been documented that warmer temperatures can potentially reduce
intraspecific transmission rates, especially when temperatures surpass
25degC (Blooi et al., 2015) .

Furthermore, the precipitation of the coldest quarter plays a
contributory role in modeling predictions for \emph{Bsal} occurrences in
Asia which shows the same pattern as of European \emph{Bsal} presence
which requires moist, relatively cool and stable conditions (Beukema et
al., 2021), the high fluctuations of precipitation in cool periods
weaken its occurrences.

The absence of \emph{Bsal} in northern sites in our study might partly
result from competition with other pathogens that share a similar
climatic niche, such as \emph{Bd} . It was identified at ten of the
surveyed sites (Fig. 1B), displaying a high prevalence in the northern
locales of the Guangxi region (Sun et al., 2023). One of 38 species we
examined, \emph{A. davidianus} (positive to \emph{Bd} in our subsequent
detections), have been reported as \emph{Bsal} -positive species (Fig.1;
Laking et al., 2017; Yuan et al., 2018), but it was negative
to\emph{Bsal} infection in our present study. These patterns suggest
that Asian \emph{Bd} lineages might outcompete \emph{Bsal} when
site-specific conditions and species susceptibility enhance the
pathogenic performance of \emph{Bd} lineages as pathogenicity of both
pathogens varies with species and environmental conditions (Fisher \&
Garner, 2020; Martel et al., 2014). This competition could occur because
\emph{Bd} infection can negatively impact \emph{Bsal} infection under
certain circumstances (Direnzo et al., 2021).

Considering the possibility that \emph{Bsal} has not yet been
transmitted to the current sampling sites, particularly in highly
suitable habitats (e.g., southwestern sites) where \emph{Bd} is present
(Fig. 1), the coexistence of \emph{Bd} and \emph{Bsal} in the same
microhabitats could result in severe coinfections or reinfections and
potentially generate new, highly virulent genotypes within the same host
once the pathogen is introduced to these sites (Greenspan et al., 2018).
Among the 38 amphibian species tested for \emph{Bsal} , none were found
to be infected. However, 16 of these species tested positive
for\emph{Bd} infection (Table 1), highlighting the need for further
investigation into the potential co-occurrence of these fungal
pathogens. Specifically, we recommend investigating predicted suitable
areas for \emph{Bsal} where \emph{Bd} is present, in order to better
understand the ecological interactions between these pathogens and their
impact on amphibian species.

Geographic barriers such as drier plains which prevents the migration of
susceptible species could be another potential reasons for absence
of\emph{Bsal} (restricted slowly dispersal) in the region. This may
inhibit the host-dependent intraspecific and interspecific as well as
human-mediated transmissions of this pathogen within landscapes (Malagon
et al., 2020; Spitzen-Van Der Sluijs et al., 2018; Tompros et al.,
2021). In addition, the negative result of \emph{Bsal} is consistent
with previous studies of this pathogen in Asia that primarily detected
the fungus in anuran species (Wang et al. 2017; Zhu et al. 2014), which
was thought to lead to transient persistence for \emph{Bsal} and
decreased transmission rates due to low galactose proportions in host
skin (Stegen et al., 2017; Wang et al., 2021). However, it is possible
that \emph{Bsal} could be present sub clinically at low prevalence in
these studied amphibians, with fungal loads below the thresholds of
detection. As growth of \emph{Bsal} could also depend on relevant plant
materials in water (Kelly et al., 2021), extending the range of sampling
efforts in environmental reservoirs can improve our understanding the
existence of \emph{Bsal} in the region.

Seasonality mainly explained by variations in temperature and
precipitation, may influence \emph{Bsal} occurrences and host
susceptibility (Bozzuto \& Canessa, 2019; Carter et al., 2021). Our
study primarily surveyed amphibians that are reproductively active
during spring and summer, with a limited number of individuals sampled
in autumn. We were unable to conduct any sampling of wild host species
during winter, which limits our understanding of \emph{Bsal} infection
across seasons. Notably, autumn and winter conditions in this
subtropical region might be more conducive for \emph{Bsal} prevalence.

It is important to understand the resource partitioning and natural
behaviors of amphibians across seasons. One such factor would be
overwintering and potential attenuation of defensive mechanisms such as
reduced skin bacterial diversity during hibernation (Tong et al., 2019).
This would lead the pathogen to cause sub-lethal infections in
susceptible amphibians during autumn and winter. Hence, we recommend
integrating experimental and field studies in both summer and winter, to
enhance our understanding of the effects of \emph{Bsal} infection on
amphibians in specific ecological contexts.

In conclusion, the absence of \emph{Bsal} in our study should not
undermine the importance of proactive management and conservation
strategies aimed at minimizing the risk of \emph{Bsal} transmission in
the region. This is due to the limited presence of climatically suitable
areas for \emph{Bsal} in this region and the isolated suitable sites
within mountains could provide an important refuge for
\emph{Bsal}(Beukema et al., 2021; Lotters et al., 2020). Implementing
stringent biosecurity measures, such as regulating the trade of
amphibians can help reduce the chances of pathogen introduction. Regular
monitoring and surveillance programs are crucial for early detection of
\emph{Bsal} , which could allow for timely interventions to prevent
severe declines in amphibian populations (Thomas et al., 2019). In
addition, further research into the ecology, transmission dynamics, and
host-pathogen interactions of \emph{Bsal} is essential to generate
knowledge that will inform targeted conservation.

\textbf{Author contributions}

\textbf{Dan Sun:} Conceptualization (lead); data curation (lead); formal
analysis (lead); investigation (lead); methodology (lead); resources
(equal); software (lead); visualization (lead); writing-original draft
(lead); writing-review and editing (lead).\textbf{Gajaba Ellepola:} Data
curation (supporting); methodology (supporting); visualization
(supporting); validation (equal); writing-original draft (supporting);
writing-review and editing (equal).\textbf{Jayampathi Herath:} Data
curation (supporting); methodology (supporting); validation (equal);
writing-original draft (supporting); writing-review and editing (equal).
\textbf{Madhava Meegaskumbura:}Conceptualization (equal); funding
acquisition (lead); resources (lead); software (supporting); project
administration (lead); supervision (lead), validation (equal);
writing-original draft (equal); writing-review and editing (equal).

\textbf{Acknowledgements}

We thank Guangxi University Laboratory Startup Funding (MM). We thank YW
Liu and EM Hu for their assistance in field work. We thank John Wood for
his advices in RT-PCR method for the detection of amphibian chytrid
fungi.

\textbf{Conflict of interest statement}

The authors declare no conflict of interest.

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