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Detection of Batrachochytrium dendrobatidis in Amphibians from Northeastern

Colombia

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220 AMPHIBIAN AND REPTILE DISEASES
for establishing the current distribution of pathogens in a par-
ticular region, but to understand the complete story of Bd, it is
necessary to couple modern efforts with the information from
the past that historical specimens have to offer.
Acknowledgments.—We thank S. Fox and E. Santoyo-Brito
(Oklahoma State University) and J. Jacobs (Smithsonian National
Museum of Natural History) for loaning specimens to RLF and JLW.
Funding support for these analyses was provided, in part, by the
Oklahoma Department of Wildlife Conservation (F14F01225 [T-80-
1]) and the Oklahoma City Zoo and Botanical Gardens Conserva-
tion Action Now (CAN) grant to CDS and JLW. RLF would like to
thank the SSAR Student Travel Award to allow her to attend SSAR
2015 in Lawrence, Kansas to present preliminary findings of this
project; MJL and MLP would like to thank the University of Okla-
homa First Year Research Experience (FYRE) program for support-
ing their involvement in undergraduate research in the Siler Lab. All
swabbed specimens were collected under relevant permits and IA-
CUC approvals necessary at the time of collection. We thank mem-
bers of the Siler Lab for reading early versions of this manuscript
and anonymous reviewers for helpful comments.
Literature Cited
Bd-maPs. 2015. Available at http://www.bd-maps.net (Accessed 14
October 2015).
CHeng, t. L., s. m. rovito, d. B. wake, and v. t. vredenBurg. 2011. Co-
incident mass extirpation of neotropical amphibians with the
emergence of the infectious fungal pathogen Batrachochytrium
dendrobatidis. Proc. Nat. Acad. Sci. 108:9502–9507.
Herpetological Review, 2016, 47(2), 220–226.
© 2016 by Society for the Study of Amphibians and Reptiles
Detection of Batrachochytrium dendrobatidis in
Amphibians from Northeastern Colombia
The effects of anthropogenic activities clearly threaten am-
phibians, yet emergent diseases like chytridiomycosis, caused
by the fungal pathogens Batrachochytrium dendrobatidis (Bd)
(Longcore et al. 1999) and B. salamandrivorans (Bsal) (Martel
ALDEMAR A. ACEVEDO*
MÓNICA P. MARTÍNEZ
Grupo de Ecología y Biogeografía, Universidad de Pamplona,
Pamplona, AA 543050, Colombia
LUIS ORLANDO ARMESTO
Grupo de Ecología y Biogeografía, Universidad de Pamplona,
Pamplona, AA 543050, Colombia; Departamento de Investigación,
Universidad Simón Bolívar–Cúcuta, Colombia
LILIANA SOLANO FLOREZ
Grupo de Biología Evolutiva, Universidad de Sucre, Sincelejo,
Colombia
KAREN SILVA PÉREZ
Grupo de Ecología y Biogeografía, Universidad de Pamplona,
Pamplona, AA 543050, Colombia
DIEGO J. LIZCANO
Departamento Central de Investigación, Universidad Laica Eloy
Alfaro de Manabí, Manta, Ecuador
*Corresponding author; e-mail: bioaldemar@gmail.com
Herpetological Review 47(2), 2016
Fong, J. J., t. L. CHeng, a. BataiLLe, a. P. Pessier, B. waLdman, and v. t.
vredenBurg. 2015. Early 1900s detection of Batrachochytrium den-
drobatidis in Korean amphibians. PLOS ONE 10:e0115656.
kerBy, J. L., a. sCHieFFer, J. r. Brown, and s. wHitFieLd. 2013. Utilization
of fast qPCR techniques to detect the amphibian chytrid fungus:
a cheaper and more efficient alternative method. Methods Ecol.
Evol. 4:162–166.
Lannoo, m. J., C. Petersen, r. e. LoviCH, P. nanJaPPa, C. PHiLLiPs, J. C.
mitCHeLL, and i. maCaLLister. 2011. Do frogs get their kicks on Route
66? Continental U.S. transect reveals spatial and temporal patterns
of Batrachochytrium dendrobatidis infection. PLOS ONE 6:e22211.
okLaHoma dePartment oF wiLdLiFe Conservation. 2015. Oklahoma Com-
prehensive Wildlife Conservation Strategy. Available at http://
www.wildlifedepartment.com/CWCS.htm (Accessed 15 January
2016).
oueLLet, m., i. mikaeLian, B. d. PauLi, J. rodrigue, and d. m. green.
2005. Historical evidence of widespread chytrid infection in North
American amphibian populations. Conserv. Biol. 19:1431–1440.
sHaw, s. d., L. F. skerratt, a. HaigH, B. d. BeLL, L. dagLisH, P. J. BisHoP, r.
summers, v. moreno, s. meLZer, m. oHmer, s. HerBert, d. gLeeson, L.
rowe, and r. sPeare. 2013. The distribution and host range of Batra-
chochytrium dendrobatidis in New Zealand, 1930–2010. Ecology
94:2108–2111.
steiner, s. L., and r. m. LeHtinen. 2008. Occurrence of the amphibian
pathogen Batrachochytrium dendrobatidis in Blanchard’s Cricket
Frog (Acris crepitans blanchardi) in the U.S. Midwest. Herpetol.
Rev. 39:193–196.
taLLey, B. L., C. r. muLetZ, v. t. vredenBurg, r. C. FLeisCHer, and k. r.
LiPs. 2015. A century of Batrachochytrium dendrobatidis in Illinois
amphibians (1888–1989). Biol. Conserv. 182:254–261.
et al. 2013), are probably the most decisive factor in amphib-
ian population declines (e.g., Berger et al. 1998; Bosch 2003;
Bonaccorso and Guayasamin 2003; Lips et al. 2006). Moreover,
Bd shows a rapid transmission, reported in 71 of 105 countries
sampled (Olson and Ronnenberg 2014), which has generated an
alert in terms of conservation worldwide (Kriger and Hero 2009;
Berger et al. 2016). In Central and South America, the decline of
at least 56 species of amphibians have been associated with the
presence of Bd (Berger et al. 1998; Lips 1999; Bonaccorso and
Guayasamin 2003; Burrowes et al. 2004; La Marca et al. 2005;
Lampo et al. 2006; Lips et al. 2006).
The presence of Bd has not been exhaustively assessed in
many areas of Colombia, despite its high biodiversity with over
791 species of amphibians (Acosta-Galvis and Cuentas 2016).
Thirty species of amphibians have been reported to be infected
with Bd from the Central and Western Cordilleras (Velásquez et
al. 2008; Urbina and Galeano 2011, Flechas et al. 2012, 2015); for
the Eastern Cordillera 18 species have been reported to be in-
fected (Ruíz and Rueda-Almonacid 2008, Vásquez-Ochoa et al.
2012, Acevedo et al. 2011, 2013, 2014, 2016). The lack of informa-
tion on Bd contributes to the large number of species classified
as data deficient (DD: 129) in the country (IUCN 2015). Our aim
was to assess Colombian amphibians for Bd infection in previ-
ously unexplored areas, sampling in the northeastern corner of
 AMPHIBIAN AND REPTILE DISEASES 221

the Eastern Cordillera, along a wide altitudinal range and diffe-

rent vegetation types, as part of an ongoing plan for monitoring
and conserving regional amphibians.
Between May 2010 and June 2015, we sampled amphibians
from 20 localities ranging from 200 to 3,600 m elevation in the
department of Norte de Santander, northeastern Colombia, near
the border with Venezuela (Fig. 1); We visited every locality twice,
except for Villa Marina, Los Estoraques and El Zulia, where only
one sampling session was possible. We swabbed every individual
over the ventral surface, in the groin, palms, fingers and legs 30
times using a sterile swab (MWE100). Samples were stored at 4°C
in Eppendorf tubes with 70% ethanol (Hyatt et al. 2007). We fol-
lowed internationally accepted biosecurity protocols for animal
handling and sampling procedures to avoid accidental spread of
pathogens between amphibians and places (Phillott et al. 2010).
We kept individuals in separate plastic bags and used fresh gloves
to handle each individual. All materials and clothing were disin-
fected with a solution of bleach before leaving each study site.
Samples were processed in the Laboratory of Ecophysiology,
Behavior and Herpetology - GECOH at the Universidad de los An-
des and at the Laboratory of Ecology and Biogeography - GIEB at
the Universidad de Pamplona in Colombia. Conventional poly-
merase chain reaction (PCR) was performed to diagnose Bd, us-
ing the primers developed by Annis et al. (2004) to amplify the
ITS1-ITS2 region specifically in B. dendrobatidis: Bd1a (5’- CAGT-
Fig. 1. Prevalence of Batrachochytrium dendrobatidis in northeast-
GTGCCATATGTCACG-3’) and Bd2a (5’-CATGGTTCATATCTGTC- ern Colombia. Numbers on the map indicate the localities, following
CAG-3’). To calculate the prevalence of Bd per species we divided Table 1. Size of circles depict the abundance of samples diagnosed
the number of Bd-positive individuals by the total number of in- for Bd.
dividuals sampled. We assessed relationships between Bd preva-
lence and the vegetation of the site (Spearman rank correlation
analysis); we classified the sampled areas as one of four types
of natural or anthropogenically altered environments (Figure 2)
using vegetation data from every locality and spatial layers of land
cover (scale 1:100,000) (IDEAM 2010). We also assessed the rela-
tionship between Bd prevalence and altitudinal range category
(four categories, Figure 2) (Spearman rank correlation analysis).
We detected Bd infections in 161 of 803 (20%) individuals,
corresponding to 34 of 43 (79%) species sampled (Table 1). At
most sites, few individuals per species were sampled, precluding
an adequate assessment of occurrence if Bd occurred at a low Fig. 2. Associations of Bd prevalence with: A) vegetation types (AG:
rate of prevalence (Skerratt et al. 2008). Fourteen of 20 localities Agricultural areas, PF: Primary forest, FF: Fragmented forest, PS:
were Bd-positive, with at least one Bd-positive record. Relative to Paramo and subparamo); and B) altitudinal range. Spearman rank
altitude, sites within the range between 200 and 1,000 m, domi- correlation results shown.
nated by the biomes tropical dry forest and xerophytic forest,
showed the highest number of infected individuals and species, However, our result for intermediate altitudes does not support
with 48 of 119 (40%) individuals from 16 of 26 (61%) species be- the contention by Pounds et al. (2006), who proposed that am-
ing Bd-positive. Between 2,000 and 3,000 m elevation, we found phibians from intermediate altitudes are more likely to become
73 of 399 (18%) individuals infected, from 9 of 12 (75%) species infected because the high day-time temperatures in lowlands
sampled; this elevation range was mainly covered by Andean and and very cold temperatures at night in higher altitudes suppress
high Andean forests. At intermediate altitudes (1,000 – 2,000 m) the development of the chytrid fungus. In Colombia, amphib-
there were only 35 of 163 (21%) individuals infected from 12 of ians in the region we sampled may be potentially susceptible to
16 (75%) species sampled. Finally, in the paramo areas, Bd oc- multiple threat factors and their interactions, possibly clouding
currences were more limited, only 5 of 122 (4%) individuals were such a pattern. Every locality we visited exhibited accelerated
infected from 2 of 4 (50%) species sampled. In the Almorzadero processes of habitat fragmentation; for example, localities with
complex (Siagá, Roble, Presidente, Baraya), that mainly corre- forests were immersed in a matrix of agricultural zones. Most
sponds to paramo, there were no Bd-positive records (64 indi- of the Bd-positive records were associated with those areas. We
viduals sampled). These results could be a consequence of the observed a significant association between vegetation and Bd
very low temperatures (4–8°C) that prevent the transmission or prevalence, with higher prevalence in agricultural areas and
survival of the fungus in the high mountain areas (Muths et al. fragmented forests, and lower prevalence in pristine forests and
2008; Pounds et al. 2006) (Table 1). areas of paramo and sub-paramo (F = 11.31, df = 3, p = 0.001,
Our data supports the notion that altitude plays an important Fig. 2A). Also, we observed a significant correlation between Bd
role in shaping Bd infection patterns (Berger et al. 1998, 2016). prevalence and altitude, where prevalence was higher at lowland

Herpetological Review 47(2), 2016

 222 AMPHIBIAN AND REPTILE DISEASES

taBLe 1. Results of sampling for Batrachochytrium dendrobatidis (Bd) in amphibian species from northeastern Colombia. CI = 95% binomial
confidence interval for Bd prevalence (%). Vegetation cover: AG: Agricultural areas, PF: Primary forest, FF: Fragmented forest, PS: Paramo and
subparamo. Numbers after localities correspond to Fig. 1.

Locality Species / Coordinates Vegetation Year Elevation Bd positives/ CI

cover (m) No. sampled

Estoraques (1) Rhinella humboldti

8.38076°N, 73.066139°W AG 2012 639 1/1 3–100

El Zulia (2) Leptodactylus colombiensis

7.9361556°N, 72.624435°W AG 2015 366 1/1 3–100

Rhinella humboldti
7.9205137°N, 72.634735°W AG 2015 264 1/1 3–100

Villa del Rosario (3) Rhinella marina

7.8433157°N, 72.4792098°W AG 2013 481 1/1 3–100

Cúcuta (4) Hypsiboas crepitans

7.86474218°N, 72.50598907°W AG 2013 347 1/1 3–100

La Garita (5) Trachycephalus typhonius

7.745012°N, 72.560577°W AG 2015 540 0/1 0–98

Scinax rostratus
7.727333°N, 72.578444°W PF 2015 619 1/2 1.3–99

Rhinella marina
7.726639°N, 72.575028°W PF 2015 583 0/3 0–71

Rheobates sp. 1
7.761694°N, 72.568917°W PF 2015 837 1/4 1–81

Pristimantis sp. 5
7.727194°N, 72.577528°W PF 2015 604 0/1 0–98

Pristimantis sp. 2
7.761694°N, 72.568917°W PF 2015 837 0/1 0–98

Lithodytes lineatus
7.761917°N, 72.570083°W PF 2015 851 0/1 0–98

Lithobates palmipes
7.727333°N, 72.578444°W PF 2015 619 0/1 0–98

Leptodactylus poecilochilus
7.726639°N, 72.575028°W PF 2015 583 1/3 1–91

Leptodactylus colombiensis
7.727194°N, 72.577528°W PF 2015 604 0/1 0–98

Hypsiboas crepitans
7.727472°N, 72.576611°W PF 2015 596 0/4 0–60

Engystomops pustulosus
7.743639°N, 72.561611°W AG 2015 599 0/2 0–84

Dendropsophus microcephalus
7.743639°N, 72.561611°W AG 2015 599 3/4 19–99

Phyllomedusa venusta
7.743639°N, 72.561611°W AG 2015 599 1/1 3–100

Naranjal (6) Pristimantis sp. 5

7.6845°N, 72.6194°W AG 2015 1098 0/10 0–31

Pristimantis sp. 2
7.6852°N, 72.6188°W AG 2015 1076 0/2 0–84

Lithodytes lineatus
7.6832°N, 72.6173°W AG 2015 1079 2/4 7–93

Herpetological Review 47(2), 2016

 AMPHIBIAN AND REPTILE DISEASES 223

taBLe 1. Continued.

Locality Species / Coordinates Vegetation Year Elevation Bd positives/ CI

cover (m) No. sampled

Leptodactylus poecilochilus
7.6852°N, 72.6188°W AG 2015 1076 0/8 0–37

Engystomops pustulosus
7.6832°N, 72.6175°W AG 2015 1072 1/4 1–81

Aromobates aff. saltuensis

7.6849°N, 72.6175°W AG 2015 1074 1/6 0.4–64

Villa Marina (7) Hypsiboas crepitans

7.54225°N, 72.63305°W AG 2010 1211 2/3 9–99

La Cocalina (8) Tachiramantis lentiginosus

7.460417°N, 72.6095°W PF 2015 1999 2/17 2–36

Scinax manriquei
7.461417°N, 72.614361°W PF 2015 1867 0/6 0–46

Leptodactylus colombiensis
7.461417°N, 72.614361°W PF 2015 1867 1/4 1–81

Hyloscirtus platydactylus
7.461417°N, 72.614361°W PF 2015 1867 1/2 1–99

Centrolene daidaleum
7.460694°N, 72.61075°W PF 2015 1966 0/3 0–71

Orocué (9)* Tachiramantis douglasi

7.4295833°N, 72.44533°W FF 2011 2418 25/160 10–22

Pristimantis sp. 3
7.4295833°N, 72.44533°W FF 2011 2418 4/12 1–65

Pristimantis melanoproctus
7.4223611°N, 72.443138°W PF 2011 2455 1/11 0.2–41

Dendropsophus pelidna
7.4223611°N, 72.443138°W PF 2011 2455 3/18 4–41

Pamplona (10) Dendropsophus labialis

7.3729722°N, 72.66325°W AG 2014 2569 2/2 16–100

Chichira (11) Pristimantis anolirex

7.334508°N, 72.573453°W PS 2015 3487 0/4 0–60

Páramo Tamá (12)* Pristimantis sp. 3

7.3900861°N, 72.3945°W PS 2010 3161 0/7 0–41

Pristimantis nicefori
7.3900861°N, 72.3945°W PS 2010 3161 0/12 0–26

Pristimantis anolirex
7.3900861°N, 72.3945°W PS 2010 3161 3/14 5–51

Gastrotheca helenae
7.3900833°N, 72.39397°W PS 2012 3167 2/18 1.4–35

Santa Isabel (13)* Gastrotheca helenae

7.358381°N, 72.34977°W PS 2015 3135 0/3 0–71

Remansos (14)* Pristimantis mondolfii

7.3358611°N, 72.469305°W FF 2011 2193 5/18 10–53

Pristimantis melanoproctus
7.3360833°N, 72.485972°W FF 2011 1739 3/29 2–27

Pristimantis gryllus
7.3358611°N, 72.469305°W FF 2011 2193 7/19 16–62

Herpetological Review 47(2), 2016

 224 AMPHIBIAN AND REPTILE DISEASES

taBLe 1. Continued.

Locality Species / Coordinates Vegetation Year Elevation Bd positives/ CI

cover (m) No. sampled

Pristimantis frater
7.3358611°N, 72.469305°W FF 2011 2193 0/8 0–37

Dendropsophus labialis
7.3360833°N, 72.485972°W AG 2011 1739 5/24 7–42

Centrolene daidaleum
7.3360833°N, 72.485972°W FF 2011 1739 5/9 21–86

Bolitoglossa tamaense
7.3360833°N, 72.485972°W FF 2011 1739 11/31 19–55

Asiria de Belén (15)* Pristimantis sp. 1

7.3223888°N, 72.364972°W PF 2011 2753 0/11 0–28

Pristimantis nicefori
7.3223888°N, 72.364972°W PF 2011 2753 0/11 0–28

Pristimantis mondolfii
7.3223888°N, 72.364972°W PF 2011 2753 10/31 17–51

Pristimantis melanoproctus
7.3068888°N, 72.382138°W FF 2011 2728 7/37 8–35

Pristimantis gryllus
7.3223888°N, 72.364972°W PF 2011 2753 2/13 2–45

Pristimantis frater
7.3068888°N, 72.382138°W FF 2011 2728 2/12 2–48

Gastrotheca helenae
7.3223888°N, 72.364972°W PS 2011 2753 0/12 0–26

Bolitoglossa tamaense
7.3068888°N, 72.382138°W FF 2011 2728 5/24 7–42

Siagá (16) Pristimantis anolirex

7.150928°N, 72.633506°W PS 2015 3112 0/6 0–46

Roble (17) Pristimantis anolirex

7.104278°N, 72.721139°W PS 2015 3103 0/31 0–11

Pristimantis nicefori
7.077972°N, 72.712806°W PS 2015 3636 0/2 0–84

San Antonio (18) Allobates sp. 1

7.15425°N, 72.22830°W PF 2010 725 2/4 7–94

Scinax manriquei
7.1570777°N, 72.222°W AG 2010 829 3/3 29–100

Rhaebo glaberrimus
7.1608055°N, 72.22811°W AG 2010 658 0/4 0–60

Pristimantis sp. 4
7.1540388°N, 72.221277°W FF 2010 874 0/1 0–98

Leptodactylus poecilochilus
7.1529722°N, 72.221583°W AG 2010 846 0/2 0–85

Leptodactylus colombiensis
7.1546666°N, 72.219694°W AG 2010 806 20/39 35–68

Hypsiboas lanciformis
7.1514166°N, 72.22027°W AG 2010 801 3/5 15–95

Hypsiboas crepitans
7.1540388°N, 72.22127°W AG 2010 874 2/7 4–71

Herpetological Review 47(2), 2016

 AMPHIBIAN AND REPTILE DISEASES 225

taBLe 1. Continued.

Locality Species / Coordinates Vegetation Year Elevation Bd positives/ CI

cover (m) No. sampled

Flectonotus pygmaeus
7.15025°N, 72.22177°W FF 2010 780 1/2 1.3–99

Espadarana andina
7.1602611°N, 72.229583°W AG 2010 634 2/4 7–94

Dendropsophus sp. 1
7.1624722°N, 72.220944°W AG 2010 758 1/2 1.3–99

Caecilia subnigricans
7.1569166°N, 72.212472°W PF 2010 714 0/1 0–98

Bolitoglossa leandrae
7.1531388°N, 72.227305°W PF 2010 722 1/3 1–91

Aromobates aff. saltuensis

7.1603611°N, 72.22855°W PF 2010 646 2/9 3–60

Presidente (19) Pristimantis anolirex

7.010244°N, 72.681878°W PS 2015 3414 0/5 0–52

Pristimantis nicefori
7.010214°N, 72.682033°W PS 2015 3417 0/10 0–31

Baraya (20) Pristimantis anolirex

6.971458°N, 72.83875°W PF 2015 3194 0/10 0–31

* Localities reported in Acevedo et al. 2016.

and intermediate elevation sites than at higher elevations (F =
3.83, df = 3, p = 0.013, Fig. 2B).
Our results showed that along the altitudinal gradient we
sampled, zones that were highly affected by anthropogenic ac-
tivities (i.e., agricultural zones) registered the highest Bd-preva-
lence. In particular, this pattern was evident in dry and sub-an-
dean forests. Although the relationship between anthropogenic
hábitat fragmentation and Bd occurrence warrants further study,
this area may represent a conservation priority for the region
because there are no protected areas that might help mitigate
risks. Species potentially susceptible to losses occur in this araa,
such as Bolitoglossa leandrae, whose abundances were very low
(Acevedo et al. 2013; 2016). We would like to clarify, however, that
most of the species reported as infected with Bd in zones with
high anthropogenic impact are species common in open areas,
nevertheless their abundances did not appear to be as high as ex-
pected. On the other hand, many species from higher elevations
could be affected by local Bd-infection episodes, despite the pos-
sible climatic restrictions such areas may represent to the gene-
ral prevalence of the fungus. The majority of these high altitude
species are endemic or have reduced distributions (including
Bolitoglossa tamaense, Gastrotheca helenae and the high Andean
species of Pristimantis), possibly increasing their vulnerability to
such local risks.
Nevertheless, we have a poor understanding of Bd’s occur-
rence, effects, and potential synergisms with anthropogenic
habitat fragmentation, pollution, and invasive species, especially
in the Andean region. This highlights the need for surveillance
and monitoring of infections in natural populations in vulner-
able areas (Daszak et al. 2007; Sánchez et al. 2008; James et al.
2015) such as northeastern Colombia, with implementation of
biosecurity programs (Berger et al. 2016). It is relevant to com-
pare Bd occurrence here with that in the Andes of Venezuela
(Lampo et al. 2006), and the central and western cordilleras of
Colombia, in order to advance our understanding on the patho-
gen distribution and its potential threat to the regional biodiver-
sity; the latter will allow us, for example, to identify hotspots and
coldspots, and therefore to find areas of high prevalence of Bd or
those with environmental restrictions to its development (James
et al. 2015).
Acknowledgments.—This study was funded by the Conservation
Leadership Programme, Save Our Species, Idea Wild, Colciencias
(Convocatoria-Bio 659-2014) and the Universidad de Pamplona.
We thank Diego Carrero for statistical support, Rosmery Franco for
collaboration in the “Proyecto de Anfibios del Tamá,” James Cabrera
for collaboration in the “Proyecto de diversidad filogenética de anfi-
bios,” Sandra V. Flechas for collaboration in the diagnosis of Bd, and
John D. Lynch for verifying the taxonomic identification of the col-
lected specimens.
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