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Population Connectivity Predicts Vulnerability to White-Nose Syndrome in the Chilean Myotis ( Myotis chiloensis ) - A Genomics Approach

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  • ConserBat EIRL

Abstract and Figures

Despite its peculiar distribution, the biology of the southernmost bat species in the world, the Chilean myotis ( Myotis chiloensis ), has garnered little attention so far. The species has a north-south distribution of c. 2800 km, mostly on the eastern side of the Andes mountain range. Use of extended torpor occurs in the southernmost portion of the range, putting the species at risk of bat white-nose syndrome (WNS), a fungal disease responsible for massive population declines in North American bats. Here, we examined how geographic distance and topology would be reflected in the population structure of M. chiloensis along the majority of its range using a double digestion RAD-tag method. We sampled 66 individuals across the species range and discovered pronounced isolation-by-distance. Furthermore, and surprisingly, we found higher degrees of heterozygosity in the southernmost populations compared to the north. A coalescence analysis revealed that our populations may still not have reached secondary contact after the Last Glacial Maximum. As for the potential spread of pathogens, such as the fungus causing WNS, connectivity among populations was noticeably low, especially between the southern hibernatory populations in the Magallanes and Tierra del Fuego, and more northerly populations. This suggests the probability of geographic spread of the disease from the north through bat-to-bat contact to susceptible populations is low. The study presents a rare case of defined population structure in a bat species and warrants further research on the underlying factors contributing to this.
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INVESTIGATION
Population Connectivity Predicts Vulnerability to
White-Nose Syndrome in the Chilean Myotis (Myotis
chiloensis) - A Genomics Approach
Thomas M. Lilley,*
,
1
,
2
Tiina Sävilammi,
Gonzalo Ossa,
,
§
Anna S. Blomberg,
Anti Vasemägi,**
Veronica Yung,
††
David L. J. Vendrami,
‡‡
and Joseph S. Johnson
§§
*Finnish Museum of Natural History, University of Helsinki, Helsinki, Finland, Department of Biology, University of Turku,
Finland, ConserBat EIRL, San Fabian, Chile, §Programa para la Conservación de los Murciélagos de Chile, Santiago, Chile,
**Department of Aquatic Resources, Swedish University of Agricultural Sciences, Uppsala, Sweden, ††Sección Rabia,
Subdepartamento de Enfermedades Virales, Instituto de Salud Pública, Santiago, Chile, ‡‡Department of Animal Behavior,
University of Bielefeld, Germany, and §§Department of Biological Sciences, Ohio University, Athens, Ohio
ORCID IDs: 0000-0001-5864-4958 (T.M.L.); 0000-0001-9836-3843 (T.S.); 0000-0002-6754-4948 (A.S.B.); 0000-0001-9409-4084 (D.L.J.V.);
0000-0003-2555-8142 (J.S.J.)
ABSTRACT Despite its peculiar distribution, the biology of the southernmost bat species in the world, the Chilean
myotis (Myotis chiloensis), has garnered little attention so far. The species has a north-south distribution of
c. 2800 km, mostly on the eastern side of the Andes mountain range. Use of extended torpor occurs in the
southernmost portion of the range, putting the species at risk of bat white-nose syndrome, a fungal disease
responsible for massive population declines in North American bats. Here, we examined how geographic distance
and topology would be reected in the population structure of M. chiloensis along the majority of its range using a
double digestion RAD-seq method. We sampled 66 individuals across the species range and discovered pro-
nounced isolation-by-distance. Furthermore, and surprisingly, we found higher degrees of heterozygosity in the
southernmost populations compared to the north. A coalescence analysis revealed that our populations may still not
have reached secondary contact after the Last Glacial Maximum. As for the potential spread of pathogens, such as
the fungus causing WNS, connectivity among populations was noticeably low, especially between the southern
hibernatory populations in the Magallanes and Tierra del Fuego, and more northerly populations. This suggests the
probability of geographic spread of the disease from the north through bat-to-bat contact to susceptible populations
is low. The study presents a rare case of dened population structure in a bat species and warrants further research on
the underlying factors contributing to this. See the graphical abstract here. https://doi.org/10.25387/g3.12173385
KEYWORDS
Population
genetics
population
connectivity
population
structure
chiroptera
disease spread
Transmission of infectious diseases has garnered attention as one
of the greatest risks to human, agriculture and wildlife health over
the last decade (Cangelosi et al. 2004; Semenza and Menne 2009).
Previous research demonstrates that the emergence of previously
unknown diseases often results from a change in the ecology of
the host, pathogen, and/or their environment (Scholthof 2007).
An example of this is white-nose syndrome (hereafter WNS), an
epizootic disease that emerged in North America in 2006 (Blehert
et al. 2009). The disease is caused by the fungus, Pseudogymnoascus
destructans, which infects insectivorous bats during the hiberna-
tion period at latitudes where prey are not widely available
during winter (Lorch et al. 2011). Populations of highly suscep-
tible species, especially from the genus Myotis, have declined
by .90% in areas affected by WNS (Frick et al. 2015). The
opportunistic pathogen can utilize alternative carbon sources
(Raudabaugh and Miller 2013) and can persist in the cold, humid
environment within hibernacula in the absence of bat hosts
(Lorch et al. 2013; Hoyt et al. 2014).
Copyright © 2020 Lilley et al.
doi: https://doi.org/10.1534/g3.119.401009
Manuscript received December 21, 2019; accepted for publication April 21, 2020;
published Early Online April 22, 2020.
This is an open-access article distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://creativecommons.org/
licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.
Supplemental material available at gshare: https://doi.org/10.25387/g3.12173385.
1
Present address: Finnish Museum of Natural History, P. Rautatiekatu 13, University
of Helsinki, PL 17, 00014 Helsinki, Finland.
2
Corresponding author: Luonnontieteellinen keskusmuseo, Helsingin yliopisto,
P. Rautatiekatu 13, PL17 00100 Helsinki, Finland. E-mail: thomas.lilley@helsinki.
Volume 10 | June 2020 | 2117
P. destructans is native to Eurasia, where it has a large geographic
range, with transmission to North America likely facilitated by
humans (Warnecke et al. 2012; Leopardi et al. 2015). In North
America, bats suffering from WNS were rst detected in the state
of New York during the winter of 20062007 (Blehert et al. 2009).
The fungus has since spread across North America, with records
of prevalence in 33 U.S. states and 7 Canadian provinces. So far,
P. destructans has been detected on 17 species of bats, with more
species likely to follow. While human-assisted transmission of
P. destructans likely has contributed to this spread, the ecology and
behavior of cave-hibernating bats in North America also makes them
efcient vectors over large geographic areas (Wilder et al. 2015).
Because WNS affects bats during extended bouts of torpor, at low
temperatures where the fungus is able to grow and infect the host,
there has been speculation over how far into the southern North
America the disease will spread (Verant et al. 2012; Meierhofer et al.
2019). Although bats inhabiting lower latitudes may suffer less from
WNS, P. destructans conidia may be able to survive on the body
of bats for extended periods of time, even at temperatures up to
37°(Campbell et al. 2020). This could facilitate the movement of
WNS across Mesoamerica and the tropics, to arrive to high southern
latitudes where bats may be susceptible (Holz et al. 2019; Turbill and
Welbergen 2019).
Of species known to harbor the WNS fungus, Tadarida brasi-
liensis is of particular interest. As a long-range migratory species, with
movements spanning thousands of kilometres (Cockrum 1969; Glass
1982), T. brasiliensis may be an important vector for spreading
P. destructans into the southern hemisphere (Ommundsen et al.
2017; McCracken et al. 2018). Ecological niche models predict suit-
able habitat for the proliferation of P. destructans in South America,
highlighting the need to understand vectors such as T.brasiliensis
as well as human transmission (Escobar et al. 2014). However, once
P. destructans arrives in South America, its spread will not necessarily
resemble that seen in North America, as it is likely to be inuenced by
differing geology and species ecology.
The Chilean myotis (Myotis chiloensis) is the most Southerly
distributed species of bat in the world, together with the southern
big-eared brown bat (Histiotus magellanicus, Koopman 1967; Gardner
2007). Myotis chiloensis has a vast north-south distribution that
includes forested areas on both sides of the Andes from the northern
shore of Navarino Island to the southern border of the Atacama
desert in Chile (Ossa and Rodriguez-San Pedro 2015). Most of
the distribution range of M. chiloensis overlaps with the distribution
of T. brasiliensis, from the north, where M. chiloensis is not believed to
hibernate, to 45°S of latitude, where M. chiloensis possibly hibernate
and may therefore be susceptible to WNS (Bozinovic et al. 1985).
However, there is no information available on the population structure
of M. chiloensis, precluding an understanding of how P. destructans
could be transported from the northern edge of its range to more
southern, and vulnerable, populations. The connectedness of individ-
uals across the range of the species will determine the speed and
intensity of potential spread. Population structuring in bats is often
relatively low because of their efcient mode of dispersal, ight (Laine
et al. 2013). An ability to disperse more efciently results in decreased
population differentiation (Bohonak 1999) to the extent that some
bat species are panmictic across their range (Burland and Wilmer
2001; Laine et al. 2013). Such high dispersal would likely result in
rapid spread of P. destructans. However, bats in the genus Myotis
show instances of pronounced population structure which may
hinder the spread of the fungus. For instance, the Gibraltar Strait,
which separates the Iberian Peninsula from the Maghreb in Morocco
by a minimum gap of 14 km of the open sea, represents a barrier for
gene ow for M. myotis (Castella et al. 2000). Chile is littered with
such potential barriers to gene ow, such as the Atacama Desert,
glaciers, ice elds, the Andes Mountains, and the Magellan Strait,
which in turn can hinder the potential spread of P. destructans.
Furthermore, populations may still be affected by the Last Glacial
Maximum, which covered a large part of Patagonia under ice until c.
10000 years bp (Sérsic et al. 2011; Mansilla et al. 2018).
With tourism in southern Chile expected to increase (e.g.,.http://
www.conaf.cl/parques-nacionales/visitanos/estadisticas-de-visitacion/),
and migratory species such as T. brasiliensis capable of carrying
spores across large distances, there is a serious need to better
understand the population structure of Patagonian hibernatory
bat species before WNS spreads to the region. The lack of knowledge
on the extent of migration and mixing, and life history traits in
general, means that research in this area is now urgent and essential
(Ossa and Rodriguez-San Pedro 2015; Ossa 2016; Ossa et al. 2019).
Studying population ecology through molecular genetic methods
allows for the identication of more accurate population bound-
aries, which is important when assessing conservation in response
to threats of disease and dramatic declines in population size
(Moritz 1994). This study will therefore aim to describe popu-
lation structure and isolation-by-distance in M. chiloensis across
the range of the species. By studying M. chiloensis along 2400 km
of latitudinal gradient using genome-wide SNP markers, we aimed to
test if geography and the Last Glacial Maximum inuence genetic
isolation patterns.
MATERIALS AND METHODS
Sample collection and DNA extraction
To describe population genetic structure in M. chiloensis, we obtained
wing tissue samples of 66 bats from two sources. A portion were
obtained from live bats captured in the eld during November and
December 2017 (i.e., austral spring) from two localities: Chicauma,
Metropolitana region (33 °S70°W) and Karukinka Reserve, Tierra
del Fuego (64 °S78°W) respectively (Capture permit: 4924_2017,
Figure 1 A, Table S1). We used disposable biopsy punches (5 mm,
MLT3335, Miltex Instrument Co, Plainsboro, New Jersey) to collect
tissue samples from the plagiopatagium of captured, live bats. The
sampled bats were released at the capture site. Additional samples
were obtained from dead bats submitted to the Public Health Institute
of Chile for rabies testing. Submitted bats included latitude and
longitude locations of origin (Figure 1 A, Table S1). Tissue samples
from the bats submitted for rabies testing were obtained from the
plagiopatium using sterile scalpels. To determine if P. destructans had
already spread to Chile, we swabbed the nose and wings of all bats in
the eld and at the Public Health Institute of Chile with a sterile
polyester swab (Puritan 25-806 1PD, Guildford ME, USA) which was
stored at -20°until analysis.
We divided samples into four populations according to their
geographic origin, with sub-regions within each group to assist in
further analyses. Sampling locations are presented in Figure 1 A
and details of populations and samples are provided in Table 1 and
Table S1. Tissue samples were stored in 1.5 ml tubes with 95% EtOH
and stored at -20°until further analysis. Fungal spore samples were
stored in 1.5 ml tubes and stored at -20°until further analysis. We
extracted DNA from tissue samples using QIAmp DNA Mini Kits
(Qiagen, Hilden Germany) and stored DNA at -80°. DNA from
fungal swabs was extracted using QIamp DNA Micro Kits (Qiagen,
Hilden Germany).
2118 | T. M. Lilley et al.
The amount of DNA in the nal solution of each sample was
tested and quantied using the Thermo Scientic Nanodrop spec-
trophotometer, giving a result for the amount of DNA in ng/mL.
Samples were frozen between DNA extraction and analysis.
Identication and quantication of P. destructans
Quantication of P.destructans load by qPCR was completed as
described previously in Johnson et al. (2015) with the exception of
using 1 ml sample in the reaction, Roche Fast Start Essential DNA
Probe Master, and a Roche Lightcycler 480 instead of a BioRad
iCycler.
RAD sequencing
We sequenced 66 individuals in total. Duplicate samples from
30 individuals were additionally sequenced to estimate repeat-
ability and error rate of the called genotypes. DNA was prepared
for genotyping-by-sequencing using a double digestion RAD-seq
method as described in Elshire et al. (2011). PstI-BamHI-digested
libraries were prepared by the Center of Evolutionary Applications
(University of Turku; see Lemopoulos et al. 2017 and references
therein for further details) and sequenced in an Illumina HiSeq2500
run (100 bp single-end reads and pooling 96 barcoded samples on a
lane) at Finnish Functional Genomics Centre (Turku Bioscience).
Yield comparison
As the amount of DNA available may often be very limited in studies
where preserved samples from the rabies laboratory are utilized, we
wanted to estimate the effect of initial DNA concentration to the
resulting read coverage. We compared the total read coverages of the
replicate samples with Pearsons correlation.
The resulting fastq reads, separated by barcode for each sample,
were quality-controlled with FastQC v. 0.11.8 and low-quality bases
were trimmed with ConDeTri v. 2.3 with parameter minlen = 30 (min.
length of a trimmed read) followed by adapter-trim with Cutadapt
v. 1.10 for Illumina universal adapters from the end of the reads
(identied with FastQC quality control in some of the samples).
Then, reads were mapped against M. lucifugus genome BioProject ID:
PRJNA16951, using BWA mem 0.7.17 with parameters -B 3 -O 5 -k
15. After mapping the reads against the reference, we used SAMtools
v. 1.4 and the associated bcftools for calling genotypes for SNPs
and for ltering SNPs based on minimum of 40% of the samples
genotyped, at least eight alternative alleles detected and SNP
quality $20 (bcftools lter). We further ltered the SNPs based
on exactly two alleles detected and excluded SNPs with particularly
low (#5) or unusually high ($125) mean coverage based on visual
inspection of the mean coverage distribution.
Population genetic analysis
For the nal SNP dataset, which only included unduplicated samples,
we performed principal component analysis implemented in prcomp
function of R stats package v. 3.5.2, and calculated Euclidean genetic
coordinates for each individual from PC1 and PC2. The four pre-
determined ancestral populations based on the sampling regions
of each individual were conrmed by hierarchical clustering of
the Euclidean distance, calculated from PC1 and PC2. Individuals
clustering to a neighboring population were assumed to have dis-
persed from their natal populations and were reassigned for the later
analysis. We further investigated the relative contributions of the
ancestral populations inhabiting regions 1-4 in the present-day
nucleotide variation using ADMIXTURE (Alexander et al. 2009)
analysis for the A-samples, run with four expected populations
(parameter K = 4) based on prior knowledge of the population
structure, and with quasi-Newton convergence acceleration method.
We were particularly interested in identifying possible hybrid
individuals.
After assigning each individual to the nal regions, we calculated
Neis pairwise F
ST
using hierfstat v. 0.04.22 function pairwise.fst
for A-samples for SNPs with no missing values. To assess the
signicance of genetic differentiation, we compared the actual
obtained F
ST
estimates to the null distributions of F
ST
values under
panmixia, obtained from a hundred random permutations of alleles.
Latitude and longitude coordinates of the sampling locations were
used to calculate pairwise geographic distances between individuals
in kilometres using Haversine method assuming a spherical earth,
implemented in function distm in the R package geodist v. 1.5.10.
We estimated Isolation-by-distance with two methods: a Mantel
test with complete permutations and a linear model geographic
distance genetic distance. We used pairwise F
ST
as a measure for
the genetic distances and mean of between-individual distances as
geographic coordinates for populations inhabiting each of the four
regions. To study the population structure in more detail, we repeated
the isolation-by-distance analysis for the genetic vs. geographic
distances from the most extreme individual (sample ID 700) using
a Mantel test with all possible permutations, and a linear model to
identify individuals with unusually high or low genetic differentia-
tion. We then studied the systematic differentiation of the individ-
uals sampled in different regions by assessing the differences of the
residual distributions of each of the four populations from zero using
t-tests and Bonferroni-correction of the P-values.
We also calculated mean observed heterozygosity within the
variable loci in Hardy-Weinberg equilibrium (FDR$0.05) in each
of the four study regions and compared the observed values to the
mean expected heterozygosities using inbreeding coefcient F,
calculated as the difference between expected and observed hetero-
zygosity divided by expected heterozygosity (Serre 2006). The 95%
condence intervals for the heterozygosity estimates were found by
randomly sampling the variable loci for 1000 times and extracting
the distributions of bootstrap means. The deviations of Fstatistics
from zero were detected using single-sample Wilcoxon tests. The
signicances of the regional differences in the observed and expected
heterozygosity distributions, and in the Fstatistics, were tested both by
inspecting the overlaps in the bootstrapped condence intervals and
using analysis of variance and Tukeyspost hoc tests. The signicances of
within-region differences between the expected and observed heterozy-
gosities were tested both by comparing the bootstrapped condence
intervals and by pairwise t-tests and Bonferroni-corrected P-values.
Finally, the fraction of SNPs unique to any one region, and the number
of SNPs shared between all regions, were calculated from the obser-
vations of variable and non-variable loci within regions.
Demographic modeling
To formally test whether any of the studied populations experienced
secondary contact following glaciation, we implemented a demo-
graphic analysis using the software fastsimcoal2 (Excofer et al. 2013)
in combination with the folded site frequency spectra (SFS) calculated
from our data using easySFS.py utility (available from https://github.com/
isaacovercast/easySFS). Specically, we evaluated support for two
alternative models (Supplemental Material, Figure S6). The rst
model, representing our null hypothesis of no secondary contact
among populations, specied four distinct lineages (region 1, region 2,
region 3 and region 4) corresponding to the populations inhabiting
Volume 10 June 2020 | Population Structure in M. chiloensis |2119
the four geographic regions sampled in the present study. These diverged
from each other at the time points T1, T2 and T3 as presented in Figure
S6A, and exchanged no migrants. Region 1 was used as the lineage from
which the other three populations emerged, as this was determined to be
the population which is closest to the ancestral M. chiloensis population.
The second model, representing our alternative hypothesis of secondary
contact among populations, was identical to the rst model with the
exception that symmetric migration was present between population pairs
region 1-2 and region 3-4, and asymmetric migration was implemented
from region 2 to region 3 (Figure S6B). In addition to identifying the
model that was best supported by our data, we also estimated divergence
times (T1, T2 and T3) and effective population sizes (region 1, region 2,
region 3 and region 4) for the four modeled lineages, as well as migration
rates (Mig12, Mig21, Mig34, Mig43 and Mig32).
We performed 50 independent fastsimcoal2 runs for each model,
with 100,000 simulations and 40 cycles of the likelihood maximization
algorithm. We then calculated AkaikesInformationCriteria(AIC)from
the fastsimcoal2 runs which yielded the highest maximum likelihood for
each model and used these values for model comparison. Finally, we
extracted parameter estimates from the best run of the most supported
model and calculated 95% condence intervals based on 100 parametric
bootstrap replicates, as described in Excofer et al. (2013).
Data availability
The RAD sequencing reads were deposited at NCBI SRA under
BioProject ID PRJNA596389. R scripts are available at https://
github.com/tiinasa/mchilorad. Supplemental material and graphical
abstract available at gshare: https://doi.org/10.25387/g3.12173385.
RESULTS
Filtering
Ninety-one of the 96 samples (66 individuals and 30 duplicates) had
reads matched with a barcode. Of the obtained genotypes, 54846 were
biallelic and used in the later analysis, while we excluded 88882 non-
variable (homozygous to alternative) variants and 1708 variants with
more than 2 alleles. After ltering, the mean SNP coverage ranged from
0.5 to 257.0 (Figure S1). We excluded tags with ,5or.125 mean
coverage, leaving 47079 tags. The average rate of missing SNPs among
the nal unique samples was 6.2%, ranging from 0 to 24%.
Replicate samples
The correlation between input DNA concentration and the resulting
mean per-sample read coverage was only moderate (cor = 0.3394,
t
61
= 2.8178, P= 0.0065, Figure S2 A, Figure S3 A-B). In contrast, we
found strong and negative association between mean read coverage
after sequence assembly and the number of missing genotype calls
(cor = -0.9763, t
61
= -35.223, P,2.2e-16, Figure S2 B). Furthermore,
read coverages were very similar between the technical replicate
samples (cor = 0.8736, t
26
= 9.1542, P= 1.292e-09, Figure S2 C),
allowing us to omit Bsamples (replicated). The removal of the
biological replicates was conducted to minimize the possible SNP
calling bias induced by some samples having approximately twice the
amount of sequence data compared to the others, if replicate samples
had been combined. Identical genotype calls ranged from 85.6 to
96.9% with an average of 94.2% identical genotype calls (Figure S3 C),
depending heavily on read coverage (cor = 0.9275, t
26
= 12.641, P=
1.312e-12 and cor = 0.8484, t
26
= 8.1716, P= 1.186e-08 in Aand B
samples, respectively; Figure S2 E-F). Although the correlation be-
tween the initial DNA concentration and identical genotype calls
between biological replicates was signicant (cor = 0.5990, t
26
= 3.814,
P,0.001; Figure S2 D), this seemed to mainly be due to two outlier
observations with both very low concentration and repeatability.
Identication and quantication of P. destructans
Besides our control samples, no samples showed signs of amplica-
tion of the multicopy intergenic spacer region of the rRNA gene
complex of P. destructans by 38 cycles, which is generally considered
Figure 1 Sampling locations and group-
ings for genetic sampling of Myotis
chiloensis in Chile (A). The two most
important principal components calcu-
lated from allele frequencies explain
15.8% of the total nucleotide variation
(B). Shades of red, green blue and purple
refer to different sub-regions (communa)
within the regions (Please see Table S1).
2120 | T. M. Lilley et al.
as a cut-off for the presence of the pathogen DNA in the samples
when using a qPCR-approach (Muller et al. 2013; Johnson 2014).
Therefore, we can conclude that the M. chiloensis individuals sampled
in this study did not carry P. destructans.
Population genetic analysis
Principal component analysis on 66 individuals and 5538 SNPs
without any missing values from non-duplicated samples (Figure
1 B) revealed a clear structuring of individuals according to the
sampling location indicative of strong population structure. Based on
hierarchical clustering of the two most important principal compo-
nents (Figure S4), we conrmed the four pre-determined populations
based on the natural hierarchical structuring of the data. Based on the
clustering, the sub-population assignment of one individual, sample
number 679, changed from Biobio to Maule (which was the most
common region assignment among the three nearest neighbors for
that individual). The estimation of ancestry of each sampled individual
by examining the relative contributions of ancestral populations
inhabiting regions 1-4 in the present-day revealed particularly pure
ancestral lines in the northern and southern parts of the range, with
hybridization occurring in the central part of the range (Figure 2).
Pairwise F
ST
-value estimates between the populations sampled
from the four geographical locations ranged from 0.04 (between
regions 2 and 3) to 0.113 (between the most distant regions 1 and 4,
Table 2). All estimated F
ST
values were found signicantly larger
(P,0.01) than the permuted F
ST
distributions, with the 95% condence
intervals of the null distributions between 0 and 0.0205. Both the Mantel
test approach and linear modeling between genetic distances (Euclidean
distances calculated from PC1 and PC2) and geographical distances
(latitude/longitude coordinates converted to distances in kilometres)
strongly suggested that we reject the null hypothesis of geographic
and genetic distances being unrelated. For the between-population
comparisons with F
ST
, we calculated Mantel statistic r = 0.9497
(P= 0.05) and R-squared estimate of 0.8773 (t
1,4
= 6.0630, P= 0.004)
(Figure S5). Also, for the between-individual distances, the observed
Mantel statistic r = 0.944 (P= 0.001), and R-squared estimate 0.943
(t
1,61
= 31.9940; P,0.0001) from the linear modeling, suggesting
that genetic and geographic distances are strongly positively associ-
ated (Figure 3). More detailed exploration of the between-individual
linear model revealed that the distribution of the residuals in regions
1(t
20
= -3.0679, Bonferroni P= 0.024) and 4 (t
11
¼-10.523, Bonferroni
P=,0.0001) were marginally smaller than 0, while the residuals
of individuals in regions 2 (t
15
= 3.5896; Bonferroni P= 0.0027)
and 3 (t
13
= 5.9325, Bonferroni P= 0.0002) were signicantly larger
than 0 (Figure 3).
Of the total of 47079 SNPs, 43903 were found to be in Hardy-Weinberg
equilibrium. Analyses of variance revealed statistically signicant differ-
ences between populations in observed heterozygosities [F
3,106450
=760.6,
P,2e-16], in expected heterozygosities [F
3,106450
=871.4, P,0.0001],
and in the F statistics [F
3,106450
=55.29, P,0.0001]. Both based on
condence intervals and pairwise comparisons, observed and expected
heterozygosity distributions were consistently higher (non-overlapping
95% condence intervals and adjusted P,0.05) in the southern
regions than in north, except for the two northernmost regions
(region 1 vs. region 2), where a statistically signicant difference
was not observed (Table 1, Table S2). Similarly, observed heterozy-
gosities were consistently lower than those estimated from allele
frequencies (non-overlapping 95% condence intervals and adjusted
P,0.05), which may be caused by within-region population
structure (Table 1, Table S2). Finally, pairwise comparisons of the
inbreeding coefcient distributions indicated that the excess of homo-
zygotes was greater in the north than in the south when compared to
neutral expectation based on allele frequencies. This was supported
with statistically signicant differences (non-overlapping condence
intervals and adjusted P,0.05) observed in all comparisons except in
region pairs 1-2 and 3-4 (Table 1, Table S2). This indicated that the
northern populations are inbreeding more than the southern pop-
ulations (Table 1, Table S2).
A large proportion of SNPs, 25.3%, were polymorphic in all
four regions. The fraction of SNPs unique only to one region
decreased from going north to south: while 8.6% of the SNPs were
unique to region 1, only 2.5% unique SNPs were found in region 4.
We did not nd support for secondary contact using demographic
modeling with Fastsimcoal2. Instead, the null model that included no
Figure 2 Biogeographical ancestry (admixture) analysis based on nucleotide polymorphisms. Each vertical bar represents an individual, ordered by
latitude. Red, green, blue and purple colors indicate the relative genetic contributions of ancestral populations inhabiting regions 1-4, respectively.
Volume 10 June 2020 | Population Structure in M. chiloensis |2121
migration among populations (Figure S6A) received the highest AIC
support (Table S3). Parameter priors and estimates, together with
their 95% condence intervals, from the best model are reported in
Table S4.
DISCUSSION
Our results present the rst assessment of population structure in the
widely distributed bat species, M. chiloensis, using individual-based
approach with genome-wide markers. We found that geographic
distance within the range of the species are reected in its population
structure. Although we found a clear and robust population structure
among sampling sites, population structure is correlated with geo-
graphical distance, even though populations are separated by ice
elds, mountain ranges and stretches of open water. This has
implications for the protection of populations that may be susceptible
to WNS. Our results also show that the highest genetic variability in
the species is at the southern extent of its range.
Strong population structure is rarely seen in bats, even across large
geographical scales in genera such as Myotis, with shorter dispersal
distances (Castella et al. 2000; Atterby et al. 2010; Laine et al. 2013). In
fact, geographical distance often correlates signicantly with genetic
distance in bats. This is partially due to autumn migration and
swarming behavior in Myotis species, which brings together bats
from broad geographic areas to breed and promote recombination
(Burns et al. 2014; Burns and Broders 2015). Furthermore, powered
ight allows effective dispersal, which is often male biased (Arnold
2007; Angell et al. 2013). This is reected in low xation indices in
widespread bat species, such as M. daubentonii, where individuals
separated by thousands of kilometres in Europe show low xation
indices (Laine et al. 2013). While xation indices cannot be compared
directly across species, especially when different methodological ap-
proaches are used, they do give an indication of the connectivity of
populations.
Although geographic and genetic distances were found to have
a strong positive correlation in our study, we can concur that the
southernmost population shows a higher degree of isolation com-
pared to the geographic distance to its closest comparative population
to the north. An F
ST
of 0.075 between regions 3 and 4 using whole-
genome data are already higher than F
ST
values recorded for M.
daubentonii across Europe using microsatellites (Laine et al. 2013),
and in our data, the geographic distance is only c. 1000 km. Our data,
with tens of thousands of SNPs also allowed a more precise estimate
of xation compared to a handful of microsatellites. However, due
to a limited number of individuals, it did not allow us to examine
dispersal as a function of sex, which in bats is often a male driven
function (Arnold 2007; Laine et al. 2013; Angell et al. 2013). Our
sampling may also have missed some connecting populations in
between regions 3 and 4, which couldforinstancebelocatedonthe
eastern slopes of the Andes. However, our test for relative contri-
butions of ancestral populations revealsbatsinregion4,inthe
Magallanes and Cameron (Tierra del Fuego), have no mixing of
ancestral populations with the other regions.
In the northern hemisphere, approximately 18000 years ago, at the
end of the Late Pleistocene, the ice sheets began to recede as the global
Figure 3 The correlation between geo-
graphic and genetic distances between
individuals. Geographic distances are mea-
sured in kilometres, and genetic distances
as Euclidean principal component distances
from the reference individual (sample 700).
Dashed lines represent 95% condence
interval for the linear model. The violin plot
highlights the differences between model
residuals for each study region. Residual
distributions that differ signicantly from
zero after Bonferroni correction for mul-
tiple testing are marked with
(P,0.05)
and

(P,0.0001).
2122 | T. M. Lilley et al.
climate became warmer. The biota migrated northwards following
their optimal environments (Huntley and Webb 1989). This expan-
sion of refugial populations has been associated with genetic variation
decreasing south-to-north in some species: a trend attributed to a
series of bottlenecks when the biota spread from the leading edge of
the refugial population, leading to a loss of alleles and decreasing
genetic diversity (Hewitt 1996, 1999). Contrary to what one could
expect based on these latitudinal shifts in diversity in the northern
latitude, genetic diversity in M. chiloensis appears to increase with
increasing latitude, from north to south. We presumed this counter-
intuitive pattern of heterozygosity within the species may be related to
the glaciation history of South America, where populations isolated by
glacial events could have been able to hybridize. For instance, in some
terrestrial European and Scandinavian vertebrates, the intraspecic
genealogical lineages, which formed in separate refugia, were
found to have come to secondary contact in the Fennoscandian
area (Tegelström 1987; Jaarola and Tegelström 1995; Nesbø et al.
1999; Knopp and Merilä 2009).
Myotis chiloensis is described as a vicariant species with respect
to other closely related Myotis species (M. albescens,M. nigricans,
M. levis) from South America, Ruedi et al. (2013) estimated this
isolation event at 5.5 My in late Miocene. This same time period saw
the beginning of a number of glaciations events in Patagonia with
variable intensity and duration (Rabassa et al. 2011). Glacial episodes
isolated the Patagonian forest from around middle Miocene well into
the late Quaternary, the Last Glacial Maximum (Rabassa et al. 2011,
Figure S7). During the glaciations, the forests on the Pacic coast were
most like completely suppressed, possibly with isolated small refugia.
On the Atlantic side, the forest was fragmented from 36°S southwards
(Sérsic et al. 2011). Finally, in Tierra del Fuego the forest was probably
displaced toward the current submarine shelf (Ponce et al. 2011). As
the ice retreated refugial populations may have come into secondary
contact in the southern part of current range, which could explain the
high heterozygosity as well as the small fraction of unique SNPsof
these populations. However, our coalescence analysis rejected the
secondary contact model, favoring the null model suggesting our
study populations are still largely separated after the Last Glacial
Maximum. Indeed, the F
ST
-values are high, suggesting isolation of
the populations. By contrast, our analysis for the relative contri-
butions of ancestral populations suggests mixing of populations.
One potential explanation for this apparent discrepancy is that we
derived site frequency spectra from a rather small number of individ-
uals, which may carry a signal of migration that is not strong enoughto
allow a model that includes secondary contact to be favored over a
simpler model without migration.
The spread of P. destructans via one host to another was very rapid
in North America (Blehert et al. 2009; Frick et al. 2010). This was
facilitated in part by the ecology of North American cave-hibernating
bats and the availability of suitable environment for the fungus to
propagate: limestone caves found throughout the Appalachian region
in eastern North America (Lorch et al. 2013). Furthermore, as a
consequence of down-regulation of metabolism during extended
torpor bouts, attempted immune responses fall short, and may even
contribute to mortality in hosts infected with P. destructans (Field
et al. 2015; Lilley et al. 2017, 2019). In addition to these, the massive
population declines associated with WNS in affected species (Turner
et al. 2011) were magnied by the panmictic population struc-
ture across eastern North America in the most affected species,
M. lucifugus (Miller-Butterworth et al. 2014; Vonhof et al. 2015).
Our results for M. chiloensis from austral South America suggest
the southernmost population in region 4, may be less likely to be
infected via their northerly conspecics, because of reduced contact
between the populations. Our results indicated no mixing of
ancestry in the southernmost individuals in our study, suggesting
the M. chiloensis in the Magallanes and Cameron (Tierra del Fuego)
are isolated from their mainland counterparts. To our knowledge, this
is also the only population to use extended torpor, a prerequisite for
the propagation of P. destructans and the onset of WNS (Ossa et al.
2020). Tierra del Fuego, the southern tip of Patagonia and the
continent of South America, experiences extended low winter tem-
peratures comparable to areas in North America where WNS is
manifested. Our genetic analysis for the presence of P. destructans on
the sampled bats suggests the fungal pathogen does not exist within
the distribution range of our focal species at present. Even if the
fungus were to enter the region, variability in host behavior and
environmental characteristics may be the primary factors pro-
tecting hosts from the pathology related to WNS (Zukal et al. 2014,
2016). Most strikingly large cave hibernacula with suitable, stable
environmental conditions favoring the environmental persis-
tence of the pathogen in the absence of the hosts, are very scarce
and separated by hundreds of kilometers in most of the southern
range of M. chiloensis.
The observed distribution of M. chiloensis is vast, covering a range
of forested habitats from arid Sclerophyllous to sub-Antartic (Ossa
and Rodriguez-San Pedro 2015). In this respect, taking into consid-
eration our results on clear population segregation begs to propose
thequestiononthespeciesstatusofM. chiloensis as a whole.
Indeed, M. chiloensis also appears to vary phenotypically along its
nTable 1 Individuals and samples per region
Numeric
region
Number of
individuals (A)
Number of
duplicates (B)
male /
female
Mean observed
heterozygosity (95%
condence interval)
Mean expected
heterozygosity (95%
condence interval)
Inbreeding
coefcient F
Unique
SNPs
1 20 6 16/4 0.2550 (0.2530-0.2570) 0.2793 (0.2777-0.2809) 0.0581 (0.0537-0.0624) 8.6%
2 19 6 9/10 0.2536 (0.2514-0.2557) 0.2765 (0.2749-0.2780) 0.0529 (0.0484-0.0571) 4.4%
3 14 9 7/7 0.2872 (0.2848-0.2896) 0.2993 (0.29762-0.3009) 0.0252 (0.0204-0.0301) 3.6%
4 13 9 5/8 0.3248 (0.3221-0.3275) 0.3335 (0.3316-0.3352) 0.0210 (0.0153-0.0268) 2.5%
tot. 66 30
nTable 2 Neis pairwise F
ST
and geographic distances (in italics)
between populations inhabiting the four geographic regions. The
signicance level (P<0.01) of the F
ST
statistics is denoted with

F
ST
and mean geographic distance (km)
Region 4 Region 3 Region 2 Region 1
Region 4 970.9 1550.3 2266.9
Region 3 0.075

588.7 1326.6
Region 2 0.089

0.041

757.0
Region 1 0.113

0.072

0.040

Volume 10 June 2020 | Population Structure in M. chiloensis |2123
distribution range (Mann 1978; Ossa 2016). It has been proposed that
the species was composed of three sub-species: M. atacamensis
(Larsen et al. 2012); M. ch. arescens from 29°Sto39°S; and M. ch.
chiloensis from 39°Sto53°S (Mann 1978). That classication was due
according to their coat color changes in relation to exposure to solar
radiation and ambient temperature, which is correlated to latitude
(Budyko 1969) and levels of precipitation in their habitat. They vary
from a lighter pelage to a dark brown color on a gradient from the
northern part of their range to the south (Galaz et al. 2006). This
promotes the theory, isolation by adaptation, as a driver of population
genetic structure. The genetic adaptations of an individual to their
local environment separates populations and leads to a reduced gene
ow (Orsini et al. 2013). However, the isolation by adaptation theory
negates the fact that there is a possibility of a barrier so that gene ow
is inhibited by climate or adaptations to the local environment. It is
possible to therefore state that isolation-by-dispersal limitation, and
moreover isolation-by- distance, are the more probable causes of the
observed results in the present study. Indeed, the reluctance of the
species to cross barriers, for instance the Andes, can clearly be seen by
examining population 3, where six individuals sampled from Aysén
(437, 442, 443, 167, 256, 260) are visibly isolated on the PCA plot
form individuals on the other side of the Andes, in Coyhaique, under
150 km away. This also depicts the ne scale resolution our indi-
vidual-based SNP-based approach allows. However, further studies
should focus deeper on the taxon status of different population of the
species currently recognized as M. chiloensis.
The results highlight the importance to assess the population
structure which may limit the spread of white-nose syndrome
disease. Whether P. destructans or another epizootic in the future
could spread depends largely on the population structure and con-
nectedness of hosts (Lilley et al. 2018).
ACKNOWLEDGMENTS
We thank the Rufford Foundation ((Rufford Small Grant (10502-1
and 23042-2), H2020 Marie Skłodowska-Curie Actions (706196), and
Ohio University Research Council for funding the work. We thank
Servicio Agricola y ganadero Diproren for the capture permits in
Tierra del Fuego (Res Ex: 1253/2016 and 4924/2017), Juan Carlos
Aravena from the Instituto de la Patagonia for his help, the personnel
from WCS Chile for allowing us to conduct research at Karukinka
Natural Reserve as well as for their help with eld work. We thank
Michelle Lineros and Tania Gatica from the National Health Institute
for their help to obtain the tissue samples. We thank Austin Waag for
assistance with eld work and Satu Mäkelä and Meri Lindqvist for
assistance with lab work.
LITERATURE CITED
Alexander, D. H., J. Novembre, and K. Lange, 2009 Fast model-based esti-
mation of ancestry in unrelated individuals. Genome Res. 19: 16551664.
Angell, R. L., R. K. Butlin, and J. D. Altringham, 2013 Sexual segregation
and exible mating patterns in temperate bats. PLoS One 8: e54194.
https://doi.org/10.1371/journal.pone.0054194
Arnold, B. D., 2007 Population structure and sex-biased dispersal in the forest
dwelling vespertilionid bat, Myotis septentrionalis. Am. Midl. Nat. 157: 374
384. https://doi.org/10.1674/0003-0031(2007)157[374:PSASDI]2.0.CO;2
Atterby, H., J. Aegerter, G. Smith, C. Conyers, T. Allnutt et al.,
2010 Population genetic structure of the Daubentons bat (Myotis
daubentonii) in western Europe and the associated occurrence of rabies.
Eur. J. Wildl. Res. 56: 6781. https://doi.org/10.1007/s10344-009-0292-1
Blehert, D. S., A. C. Hicks, M. Behr, C. U. Meteyer, B. M. Berlowski-Zier et al.,
2009 Bat white-nose syndrome: an emerging fungal pathogen? Science
323: 227. https://doi.org/10.1126/science.1163874
Bohonak, A. J., 1999 Dispersal, gene ow, and population structure. Q. Rev.
Biol. 74: 2145. https://doi.org/10.1086/392950
Bozinovic, F., L. Contreras, M. Rosenmann, and J. Torres-Mura,
1985 Bioenergetics of Myotis chiloensis (Quiroptera: Vespertilionidae).
Rev. Chil. Hist. Nat. 58: 3945.
Budyko, M. I., 1969 The effect of solar radiation variations on the climate of
the Earth. Tellus 21: 611619. https://doi.org/10.3402/tellusa.v21i5.10109
Burland, T. M., and J. W. Wilmer, 2001 Seeing in the dark: molecular
approaches to the study of bat populations. Biol. Rev. Camb. Philos. Soc.
76: 389409. https://doi.org/10.1017/S1464793101005747
Burns, L. E., and H. G. Broders, 2015 Who swarms with whom? Group
dynamics of Myotis bats during autumn swarming. Behav. Ecol.
26: 866876. https://doi.org/10.1093/beheco/arv017
Burns, L. E., T. R. Frasier, and H. G. Broders, 2014 Genetic connectivity
among swarming sites in the wide ranging and recently declining little
brown bat (Myotis lucifugus). Ecol. Evol. 4: 41304149. https://doi.org/
10.1002/ece3.1266
Campbell, L. J., D. P. Walsh, D. S. Blehert, and J. M. Lorch, 2020 Long-term
survival of Pseudogymnoascus destructans at elevated temperatures.
J. Wildl. Dis. 56: 278287.
Cangelosi, G., N. Freitag, and M. Riley-Buckley, 2004 From outside to inside:
environmental microorganisms as human pathogens, American Society of
Microbiology. https://www.asmscience.org/content/report/colloquia/
colloquia.14
Castella, V., M. Ruedi, L. Excofer, C. Ibanez, R. Arlettaz et al., 2000 Is the
Gibraltar Strait a barrier to gene ow for the bat Myotis myotis
(Chiroptera : Vespertilionidae)? Mol. Ecol. 9: 17611772. https://doi.org/
10.1046/j.1365-294x.2000.01069.x
Cockrum, E., 1969 Migration in the guano bat, Tadarida brasiliensis. Misc
Publ Mus Nat Hist Univ Kans. 51: 303336.
Elshire, R. J., J. C. Glaubitz, Q. Sun, J. A. Poland, K. Kawamoto et al., 2011 A
robust, simple genotyping-by-sequencing (GBS) approach for high
diversity species. PLoS ONE 6: e19379. https://doi.org/10.1371/
journal.pone.0019379
Escobar, L. E., A. Lira-Noriega, G. Medina-Vogel, and A. T. Peterson,
2014 Potential for spread of the white-nose fungus (Pseudogymnoascus
destructans) in the Americas: use of Maxent and NicheA to assure strict
model transference. Geospat. Health 9: 221229. https://doi.org/10.4081/
gh.2014.19
Excofer, L., I. Dupanloup, E. Huerta-Sánchez, V. C. Sousa, and M. Foll,
2013 Robust Demographic Inference from Genomic and SNP Data.
PLoS Genet. 9: e1003905. https://doi.org/10.1371/journal.pgen.1003905
Field, K. A., J. Johnson, T. Lilley, S. Reeder, E. Rogers et al., 2015 The white-
nose syndrome transcriptome: activation of anti-fungal host responses in
wing tissue of hibernating bats. PLoS Pathog. 11: e1005168. https://doi.org/
10.1371/journal.ppat.1005168
Frick, W. F., S. J. Puechmaille, J. R. Hoyt, B. A. Nickel, K. E. Langwig et al.,
2015 Disease alters macroecological patterns of North American bats.
Glob. Ecol. Biogeogr. 24: 741749.
Frick, W. F., J. F. Pollock, A. C. Hicks, K. E. Langwig, D. S. Reynolds et al.,
2010 An emerging disease causes regional population collapse of a
common North American bat species. Science 329: 679682. https://
doi.org/10.1126/science.1188594
Galaz, J., J. Yañez, A. Gantz, and D. Martínez, 2006 Orden Chiroptera. Pp. 6789
in Mamíferos de Chile (A. Muñoz-Pedreros and J.Yañez, eds.)., in Mamíferos
de Chile, Centro de Estudios Agrarios y Ambientales, Valdivia, Chile.
Gardner, A. (Editor), 2007 Mammals of South America, Vol. 1. University of
Chicago Press, Chicago, Illinois.
Glass, B. P., 1982 Seasonal movements of Mexican freetail bats Tadarida
brasiliensis mexicana banded in the Great Plains. Southwest. Nat. 27: 127
133. https://doi.org/10.2307/3671136
Hewitt, G. M., 1999 Post-glacial re-colonization of European biota. Biol.
J. Linn. Soc. Lond. 68: 87112. https://doi.org/10.1111/j.1095-
8312.1999.tb01160.x
Hewitt, G. M., 1996 Some genetic consequences of ice ages, and their role in
divergence and speciation. Biol. J. Linn. Soc. Lond. 58: 247276. https://
doi.org/10.1006/bijl.1996.0035
2124 | T. M. Lilley et al.
Holz, P., J. Hufschmid, W. Boardman, P. Cassey, S. Firestone et al.,
2019 Does the fungus causing white-nose syndrome pose a signicant
risk to Australian bats? Wildl. Res. 46: 657668. https://doi.org/10.1071/
WR18194
Huntley, B., and T. Webb, 1989 Migration: speciesresponse to climatic
variations caused by changes in the earths orbit. J. Biogeogr. 16: 519.
https://doi.org/10.2307/2845307
Jaarola, M., and H. Tegelström, 1995 Colonization history of north Euro-
pean eld voles (Microtus agrestis) revealed by mitochondrial DNA. Mol.
Ecol. 4: 299310. https://doi.org/10.1111/j.1365-294X.1995.tb00223.x
Johnson, J. S., D. M. Reeder, J. W. McMichael, M. B. Meierhofer, D. W. F. Stern
et al., 2014 Host, pathogen, and environmental characteristics predict
white-nose syndrome mortality in captive little brown myotis (Myotis
lucifugus). PLoS One 9: e112502.
Johnson, J. S., D. M. Reeder, T. M. Lilley, G. Á. Czirják, C. C. Voigt et al.,
2015 Antibodies to Pseudogymnoascus destructans are not sufcient for
protection against white-nose syndrome. Ecol. Evol. 5: 22032214. https://
doi.org/10.1002/ece3.1502
Knopp, T., and J. Merilä, 2009 The postglacial recolonization of Northern
Europe by Rana arvalis as revealed by microsatellite and mitochondrial
DNA analyses. Heredity 102: 174181. https://doi.org/10.1038/
hdy.2008.91
Koopman, K., 1967 The southernmost bats. J. Mammal. 48: 487488.
https://doi.org/10.2307/1377795
Laine, V. N., T. M. Lilley, K. Norrdahl, and C. R. Primmer, 2013 Population
genetics of Daubentons bat (Myotis daubentonii) in the Archipelago Sea,
SW Finland. Ann. Zool. Fenn. 50: 303315. https://doi.org/10.5735/
085.050.0505
Larsen, R. J., M. C. Knapp, H. H. Genoways, F. A. A. Khan, P. A. Larsen et al.,
2012 Genetic diversity of Neotropical Myotis (Chiroptera: Vespertilionidae)
with an emphasis on South American species. PLoS One 7: e46578.
Lemopoulos, A., S. Uusi-Heikkilä, A. Vasemägi, A. Huusko, H. Kokko et al.,
2017 Genome-wide divergence patterns support ne-scaled genetic
structuring associated with migration tendency in brown trout. Can.
J. Fish. Aquat. Sci. 75: 16801692. https://doi.org/10.1139/cjfas-2017-0014
Leopardi, S., D. Blake, and S. J. Puechmaille, 2015 White-nose syndrome
fungus introduced from Europe to North America. Curr. Biol.
25: R217R219. https://doi.org/10.1016/j.cub.2015.01.047
Lilley, T., J. Anttila, and L. Ruokolainen, 2018 Landscape structure and
ecology inuence the spread of a bat fungal disease. Funct. Ecol.
32: 24832496. https://doi.org/10.1111/1365-2435.13183
Lilley, T. M., J. M. Prokkola, A. S. Blomberg, S. Paterson, J. S. Johnson et al.,
2019 Resistance is futile: RNA-sequencing reveals differing responses to
bat fungal pathogen in Nearctic Myotis lucifugus and Palearctic Myotis
myotis. Oecologia 191: 295309. https://doi.org/10.1007/s00442-019-
04499-6
Lilley, T. M., J. M. Prokkola, J. S. Johnson, E. J. Rogers, S. Gronsky et al.,
2017 Immune responses in hibernating little brown myotis (Myotis
lucifugus) with white-nose syndrome. Proc. Biol. Sci. 284: 20162232.
https://doi.org/10.1098/rspb.2016.2232
Lorch, J. M., C. U. Meteyer, M. J. Behr, J. G. Boyles, P. M. Cryan et al.,
2011 Experimental infection of bats with Geomyces destructans causes
white-nose syndrome. Nature 480: 376378. https://doi.org/10.1038/
nature10590
Lorch, J. M., L. K. Muller, R. E. Russell, M. OConnor, D. L. Lindner et al.,
2013 Distribution and environmental persistence of the causative agent
of white-nose syndrome, Geomyces destructans, in bat hibernacula of the
Eastern United States. Appl. Environ. Microbiol. 79: 12931301. https://
doi.org/10.1128/AEM.02939-12
Mann, G., 1978 Los pequeños mamiferos de Chile. Gayana (Concepc.)
40: 1342.
Mansilla, C. A., R. D. McCulloch, and F. Morello, 2018 The vulnerability of
the Nothofagus forest-steppe ecotone to climate change: Palaeoecological
evidence from Tierra del Fuego (53°S). Palaeogeogr. Palaeoclimatol.
Palaeoecol. 508: 5970. https://doi.org/10.1016/j.palaeo.2018.07.014
McCracken, G. F., R. F. Bernard, M. Gamba-Rios, R. Wolfe, J. J. Krauel et al.,
2018 Rapid range expansion of the Brazilian free-tailed bat in the
southeastern United States, 20082016. J. Mammal. 99: 312320. https://
doi.org/10.1093/jmammal/gyx188
Meierhofer, M. B., J. S. Johnson, S. J. Leivers, B. L. Pierce, J. E. Evans et al.,
2019 habitats of bats in Texas. PLoS One 14: e0220839. https://doi.org/
10.1371/journal.pone.0220839
Miller-Butterworth, C. M., M. J. Vonhof, J. Rosenstern, G. G. Turner, and
A. L. Russell, 2014 Genetic structure of little brown bats (Myotis
lucifugus) corresponds with spread of white-nose syndrome among
hibernacula. J. Hered. 104: 354364.
Moritz, C., 1994 Dening Evolutionarily Signicant Unitsfor conservation.
Trends Ecol. Evol. 9: 373375. https://doi.org/10.1016/0169-
5347(94)90057-4
Muller, L. K., J. M. Lorch, D. L. Lindner, M. OConnor, A. Gargas et al.,
2013 Bat white-nose syndrome: a real-time TaqMan polymerase chain
reaction test targeting the intergenic spacer region of Geomyces destruc-
tans. Mycologia 105: 253259. https://doi.org/10.3852/12-242
Nesbø, C. L., T. Fossheim, L. A. Vøllestad, and K. S. Jakobsen, 1999 Genetic
divergence and phylogeographic relationships among European perch
(Perca uviatilis) populations reect glacial refugia and postglacial colo-
nization. Mol. Ecol. 8: 13871404. https://doi.org/10.1046/j.1365-
294x.1999.00699.x
Ommundsen, P., C. Lausen, and L. Matthias, 2017 First acoustic records of
the Brazilian free-tailed bat (Tadarida brasiliensis) in British Columbia.
Northwest. Nat. (Olymp. Wash.) 98: 132136. https://doi.org/10.1898/
NWN16-24.1
Orsini, L., J. Vanoverbeke, I. Swillen, J. Mergeay, and L. De Meester,
2013 Drivers of population genetic differentiation in the wild: isolation
by dispersal limitation, isolation by adaptation and isolation by coloni-
zation. Mol. Ecol. 22: 59835999. https://doi.org/10.1111/mec.12561
Ossa, G., 2016 Primer registro de la especie Myotis chiloensis, (Waterhouse,
1838) (Chiroptera, Vespertilionidae) en el Parque Nacional Alberto de
Agostini (Región de Magallanes y Antártica Chilena). An. Inst. Patagon.
44: 8588.
Ossa, G., J. S. Johnson, A. I. E. Puisto, V. Rinne, I. E. Sääksjärvi et al.,
2019 The Klingon batbugs: Morphological adaptations in the primitive
bat bugs, Bucimex chilensis and Primicimex cavernis, including updated
phylogeny of Cimicidae. Ecol. Evol. 9: 17361749. https://doi.org/10.1002/
ece3.4846
Ossa, G., T. M. Lilley, A. G. Waag, M. B. Meierhofer, and J. S. Johnson,
2020 Roosting ecology of the southernmost bats, Myotis chiloensis and
Histiotus magellanicus, in southern Tierra del Fuego, Chile. bioRxiv
2020.04.29.068130.
Ossa, G., A. Rodriguez-San Pedro, 2015 Myotis chiloensis (Chiroptera:
Vespertilionidae). Mamm. Species 47: 5156. https://doi.org/10.1093/
mspecies/sev005
Ponce, J. F., J. Rabassa, A. Coronato, and A. M. Borromei, 2011 Palaeogeographical
evolution of the Atlantic coast of Pampa and Patagonia from the last glacial
maximum to the Middle Holocene. Biol. J. Linn. Soc. Lond. 103: 363379.
https://doi.org/10.1111/j.1095-8312.2011.01653.x
Rabassa, J., A. Coronato, and O. Martínez, 2011 Late Cenozoic glaciations in
Patagonia and Tierra del Fuego: an updated review. Biol. J. Linn. Soc. Lond.
103: 316335. https://doi.org/10.1111/j.1095-8312.2011.01681.x
Raudabaugh, D. B., and A. N. Miller, 2013 Nutritional capability of and
substrate suitability for Pseudogymnoascus destructans, the causal agent of
wat white-nose syndrome. PLoS One 8: e78300. https://doi.org/10.1371/
journal.pone.0078300
Ruedi, M., B. Stadelmann, Y. Gager, E. J. P. Douzery, C. M. Francis et al.,
2013 Molecular phylogenetic reconstructions identify East Asia as the
cradle for the evolution of the cosmopolitan genus Myotis (Mammalia,
Chiroptera). Mol. Phylogenet. Evol. 69: 437449. https://doi.org/10.1016/
j.ympev.2013.08.011
Scholthof, K.-B. G., 2007 The disease triangle: pathogens, the environment
and society. Nat. Rev. Microbiol. 5: 152156. https://doi.org/10.1038/
nrmicro1596
Semenza, J. C., and B. Menne, 2009 Climate change and infectious diseases
in Europe. Lancet Infect. Dis. 9: 365375. https://doi.org/10.1016/S1473-
3099(09)70104-5
Volume 10 June 2020 | Population Structure in M. chiloensis |2125
Serre, J., 2006 Génétique des populations, Dunod, Paris.
Sérsic, A. N., A. Cosacov, A. A. Cocucci, L. A. Johnson, R. Pozner et al.,
2011 Emerging phylogeographical patterns of plants and terrestrial
vertebrates from Patagonia. Biol. J. Linn. Soc. Lond. 103: 475494. https://
doi.org/10.1111/j.1095-8312.2011.01656.x
Tegelström, H., 1987 Transfer of mitochondrial DNA from the northern
red-backed vole (Clethrionomys rutilus) to the bank vole (C. glareolus).
J. Mol. Evol. 24: 218227. https://doi.org/10.1007/BF02111235
Turbill, C., and J. A. Welbergen, 2019 Anticipating white-nose
syndrome in the Southern Hemisphere: Widespread conditions
favourable to Pseudogymnoascus destructans pose a serious risk to
Australias bat fauna. Austral Ecol. 45: 8996. https://doi.org/
10.1111/aec.12832
Turner, G. G., D. M. Reeder, and J. T. H. Coleman, 2011 A ve-year
assessment of mortality and geographic spread of white-nose syndrome in
North American bats and a look to the future. Bat Res. News 52: 1327.
Verant, M. L., J. G. Boyles, W. Waldrep, G. Wibbelt, and D. S. Blehert,
2012 Temperature-dependent growth of Geomyces destructans, the
fungus that causes bat white-nose syndrome. PLoS One 7: e46280. https://
doi.org/10.1371/journal.pone.0046280
Vonhof, M. J., A. L. Russell, and C. M. Miller-Butterworth, 2015 Range-wide
genetic analysis of little brown bat (Myotis lucifugus) populations:
estimating the risk of spread of White-nose syndrome. PLoS One 10:
e0128713. https://doi.org/10.1371/journal.pone.0128713
Warnecke, L., J. M. Turner, T. K. Bollinger, J. M. Lorch, V. Misra et al.,
2012 Inoculation of bats with European Geomyces destructans supports
the novel pathogen hypothesis for the origin of white-nose syndrome.
Proc. Natl. Acad. Sci. USA 109: 69997003. https://doi.org/10.1073/
pnas.1200374109
Wilder, A. P., T. H. Kunz, and M. D. Sorenson, 2015 Population genetic
structure of a common host predicts the spread of white-nose syndrome,
an emerging infectious disease in bats. Mol. Ecol. 24: 54955506. https://
doi.org/10.1111/mec.13396
Zukal, J., H. Bandouchova, T. Bartonicka, H. Berkova, V. Brack et al.,
2014 White-nose syndrome fungus: a generalist pathogen of
hibernating bats. PLoS One 9: e97224. https://doi.org/10.1371/
journal.pone.0097224
Zukal,J.,H.Bandouchova,J.Brichta,A.Cmokova,K.S.Jaronet al.,
2016 White-nose syndrome without borders: Pseudogymnoascus
destructans infection tolerated in Europe and Palearctic Asia but not
inNorthAmerica.Sci.Rep.6:19829.https://doi.org/10.1038/
srep19829
Communicating editor: A. Sethuraman
2126 | T. M. Lilley et al.
... Although the population structure in bats is often relatively low due to their ability to fly and cross some ecological barriers impossible for other animals (Laine et al., 2013), at large geographical scales distance can be significantly correlated with genetic distance, thus the population structure can be observed (e.g., Martins et al., 2007;Hua et al., 2013;Lilley et al., 2020). In fact, phylogeographic and population studies of bats in the Neotropical region reveal several cases of genetic structure and cryptic diversity (e.g., Martins et al., 2007;Marroig, 2016, 2017;Moras et al., 2018;Garbino et al., 2020). ...
... All extractions were performed at Instituto Aggeu Magalhães, FIOCRUZ/Pernambuco and stored at −80 • C before being sent to LUOMUS, Helsinki/Finland, for the following analyses. The amount of DNA from each sample was tested and quantified using the Thermo Scientific Nanodrop spectrophotometer, and all samples were diluted into a 12 ng/ml solution, following Lilley et al. (2020). ...
... The resulting reads were processed according to Lilley et al. (2020), and the complete scripts and pipelines are available at https://github.com/itofs/Pgymno_radseq. Demultiplexing and quality filtering were carried out using Stacks 1.48 (Catchen et al., 2013) "process_radtags." ...
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Bat caves in the Neotropical region harbor exceptional bat populations (> 100,000 individuals). These populations play a wider role in ecological interactions, are vulnerable due to their restriction to caves, and have a disproportionate conservation value. Current knowledge of bat caves in Brazil is still small. However, systematic monitoring of some bat caves in northeastern Brazil shows that they experience strong population fluctuations over short periods of time, suggesting large-scale movements between roosts and a much broader use of the landscape than previously considered. Spatio-temporal reproductive connectivity between distant populations would change our understanding of the use of roosts among bat species in Brazil, and important gaps in knowledge of long-distance bat movements in the country would be filled. Here, we used ddRADseq data to analyze the genetic structure of Pteronotus gymnonotus across nine bat caves over 700 km. Our results indicate the lack of a clear geographic structure with gene flow among all the caves analyzed, suggesting that P. gymnonotus uses a network of bat caves geographically segregated hundreds of kilometers apart. Facing strong anthropogenic impacts and an underrepresentation of caves in conservation action plans worldwide, the genetic connectivity demonstrated here confirms that bat caves are priority sites for bat and speleological conservation in Brazil and elsewhere. Moreover, our results demonstrate a warning call: the applied aspects of the environmental licensing process of the mining sector and its impact must be reviewed, not only in Brazil, but wherever this licensing process affects caves having exceptional bat populations.
... Myotis has a complex evolutionary history in the Neotropics, arising from a recent and rapid diversification caused by dispersal and isolation events (Stadelmann et al. 2007;Larsen et al. 2012b;Novaes et al. 2021a). A recent study using a double digestion RAD-seq method found a strong population structure with pronounced isolation-by-distance in M. chiloensis (sensu lato; including specimens of M. arescens), revealing high degrees of heterozygosis between southern and northern populations (Lilley et al. 2020). This study also presents a coalescent analysis indicating that M. chiloensis populations may still not have reached secondary contact after the Last Glacial Maximum (Lilley et al. 2020). ...
... A recent study using a double digestion RAD-seq method found a strong population structure with pronounced isolation-by-distance in M. chiloensis (sensu lato; including specimens of M. arescens), revealing high degrees of heterozygosis between southern and northern populations (Lilley et al. 2020). This study also presents a coalescent analysis indicating that M. chiloensis populations may still not have reached secondary contact after the Last Glacial Maximum (Lilley et al. 2020). ...
... The results of Lilley et al. (2020) corroborate our findings indicating low genetic distance between M. chiloensis and M. arescens (ca. 2%), probably caused by a recent separation. ...
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Myotis is the most diverse genus of bats in the world, with more than 30 species recognized in the Neotropics. However, many of these species represent cryptic complexes and are evidence of the existence of hidden diversity in several regions. Using an integrative approach based on molecular, morphological, and bioacoustic data, we performed a systematic review of Myotis species from Chile. Phylogenetic inference using cytochrome-b indicated the existence of three monophyletic lineages, and qualitative and quantitative morphological analyses supported these lineages as distinct and morphologically diagnosable taxa. Analysis of discriminant functions using parameters of echolocation calls also indicates the existence of three distinct bioacoustic clusters. Thus, all lines of evidence congruently indicate the existence of three distinct taxa. As a result, we recognize Myotis arescens as a valid and distinct species and define its taxonomic limits from the other species from Chile, Myotis atacamensis and Myotis chiloensis.
... The vegetative growth of P. destructans is constrained by temperature and humidity inside hibernacula [22,23] while the spread of the fungus is influenced by internal and external factors. Factors known to be associated with fungal transmission include bat species composition and abundance, population demographics [24], geography (e.g. distribution, frequency and connectivity of hibernacula) and climate [25][26][27]. ...
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Full-text available
Predicting the emergence and spread of infectious diseases is critical for the effective conservation of biodiversity. White-nose syndrome (WNS), an emerging infectious disease of bats, has resulted in high mortality in eastern North America. Because the fungal causative agent Pseudogymnoascus destructans is constrained by temperature and humidity, spread dynamics may vary by geography. Environmental conditions in the southern part of the continent are different than the northeast, where disease dynamics are typically studied, making it difficult to predict how the disease will manifest. Herein, we modelled WNS pathogen spread in Texas based on cave densities and average dispersal distances of hosts, projecting these results out to 10 years. We parameterized a predictive model of WNS epidemiology and its effects on bat populations with observed cave environmental data. Our model suggests that bat populations in northern Texas will be more affected by WNS mortality than southern Texas. As such, we recommend prioritizing the preservation of large overwintering colonies of bats in north Texas through management actions. Our model illustrates that infectious disease spread and infectious disease severity can become uncoupled over a gradient of environmental variation and highlight the importance of understanding host, pathogen and environmental conditions across a breadth of environments.
... The analysis of P. destructans isolates using microsatellites and single-nucleotide polymorphisms suggests that there are at least three distinct clades, representing geographic groupings of isolates from Far-east Asia (China), Central Asia (Mongolia) and Europe 21 (Fig. 1). 28,33,39,40,[53][54][55]68,77,[161][162][163][164][165][166] . Filled circles indicate sites where P. destructans was detected through qPCR, photographs and sampling of museum specimens and red open circles indicate sites where P. destructans was not detected (these sites in North America are not shown). ...
Article
The recent introduction of Pseudogymnoascus destructans (the fungal pathogen that causes white-nose syndrome in bats) from Eurasia to North America has resulted in the collapse of North American bat populations and restructured species communities. The long evolutionary history between P. destructans and bats in Eurasia makes understanding host life history essential to uncovering the ecology of P. destructans. In this Review, we combine information on pathogen and host biology to understand the patterns of P. destructans spread, seasonal transmission ecology, the pathogenesis of white-nose syndrome and the cross-scale impact from individual hosts to ecosystems. Collectively, this research highlights how early pathogen detection and quantification of host impacts has accelerated the understanding of this newly emerging infectious disease.
... Even if P. destructans does not manifest as WNS in warmer subtropical regions, ostensibly, if P.destructans gains a foothold in Mexican caves, it could spread bat-to-bat, cave-to-cave southward along mountainous karst regions. The virility of P. destructans in these more equatorial environments is as of yet unknown, but the recent cases of WNS indicate that cave bats in warmer temperate climates may be at higher risk than previously thought (TPWD, 2020).Further, southern regions of South America are home to cave hibernating bat species such as the Chilean Myotis (Myotis chiloensis) which may be susceptible to WNS(Lilley et al. 2020). The hardiness of this fungus, coupled with the migratory patterns of T. brasiliensis, makes it plausible that P. destructans could travel as an innocuous fungus into Mexico and southward through Central and South American karst systems, until it reaches an area with winters harsh enough for the fungus to become virulent. ...
Thesis
Full-text available
White-nose Syndrome (WNS) is caused by a fungus that has led to deaths of millions of North American bats since it was first documented in New York 2006. Since the first cases were recorded, WNS has spread rapidly across North America, and is now present in 34 US states and 7 Canadian provinces. The disease is caused by the introduced fungal pathogen Pseudogymnoascus destructans. Often, the presence of P. destructans is detected in a cave environment before signs of WNS manifest in the resident bat population-making expansion of the fungus a more reliable assessment of epidemic spread than expansion of manifested WNS. We generated a predictive model to assess the potential spread of P. destructans, the fungal causal agent of WNS, through Texas karst systems based on external features that correlate with suitable internal microclimates for fungal growth. An analysis of 43 cave microclimates across the state of Texas reveals a pattern of thermal suitability for P. destructans that correlates significantly with landscape (elevation, lithology) and external climate (mean surface temperature and precipitation). Applications of this model to external climatic variables from 2019 show seasonally varying patterns of suitability for fungal growth in select regions of Texas karst systems. Similar work conducted in Mexico surveyed 4 caves in 2 areas of varying climate and elevation. Results from these surveys show that microclimates of Mexican caves are likely able to sustain the growth of P. destructans and could act as stepping stones for the fungus, allowing it to travel southward. The resulting work will inform researchers and natural resource managers of areas of significant concern while monitoring the spread of WNS iii ACKNOWLEDGEMENTS
... Nevertheless, South American forests face extensive threats from several industries, climate change, wildlife diseases and expansion of human settlements. Therefore, an understanding of the ecologies of bat species in this region is needed to promote their conservation (Armesto et al. 1998;Bustamante & Simonetti 2005;Wilson et al. 2005;Lilley et al. 2020). ...
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Full-text available
There are few studies of day-roosting ecology of bats inhabiting the southernmost forests of South America, where cool summer temperatures and land management practices pose several challenges. The goal of the present study was to describe day-roosting habitats and patterns of thermoregulation in two bat species occurring on Tierra del Fuego, Myotis chiloensis (Chilean myotis) and Histiotus magellanicus (southern big-eared brown bat), during late austral spring. To do so, we tagged 17 bats with temperature-sensitive radio-transmitters, located 17 day-roosts, and collected 81 days of skin temperature data. We concurrently recorded ambient air temperature to determine its effect on torpor use. Both species were found roosting in large diameter (77.8 AE 6 cm), typically live, Nothofagus pumilio trees (lenga) located on the edges of forest gaps or within stands primarily composed of smaller, younger trees. Bats of both species frequently used torpor, with skin temperatures dropping below a torpor threshold on 89% of days (n = 72) and daily minimum skin temperatures averaging 16.5°C over the course of our study. Average daily air temperature was a significant predictor of torpor use, with lower skin temperatures and more time spent in torpor observed on colder days. Minimum skin temperature and time spent torpid did not vary between bat species, nor did the characteristics of day-roosts. These data show that spring ambient temperatures in Tierra del Fuego pose an energetic challenge that bats meet through frequent use of torpor and, likely, habitat selection. We recommend local conservation efforts keep these thermal challenges in mind by retaining large trees, which may provide warmer microclimates or room for social groups. Abstract in Spanish is available with online material.
Article
Full-text available
There are few studies of day-roosting ecology of bats inhabiting the southernmost forests of South America, where cool summer temperatures and land management practices pose several challenges. The goal of the present study was to describe day-roosting habitats and patterns of thermoregulation in two bat species occurring on Tierra del Fuego, Myotis chiloensis (Chilean myotis) and Histiotus magellanicus (southern big-eared brown bat), during late austral spring. To do so, we tagged 17 bats with temperature-sensitive radio-transmitters, located 17 day-roosts, and collected 81 days of skin temperature data. We concurrently recorded ambient air temperature to determine its effect on torpor use. Both species were found roosting in large diameter (77.8 AE 6 cm), typically live, Nothofagus pumilio trees (lenga) located on the edges of forest gaps or within stands primarily composed of smaller, younger trees. Bats of both species frequently used torpor, with skin temperatures dropping below a torpor threshold on 89% of days (n = 72) and daily minimum skin temperatures averaging 16.5°C over the course of our study. Average daily air temperature was a significant predictor of torpor use, with lower skin temperatures and more time spent in torpor observed on colder days. Minimum skin temperature and time spent torpid did not vary between bat species, nor did the characteristics of day-roosts. These data show that spring ambient temperatures in Tierra del Fuego pose an energetic challenge that bats meet through frequent use of torpor and, likely, habitat selection. We recommend local conservation efforts keep these thermal challenges in mind by retaining large trees, which may provide warmer microclimates or room for social groups. Abstract in Spanish is available with online material.
Preprint
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There are few studies of day-roosting ecology of bats inhabiting the southernmost forests of South America, where cool summer temperatures and land management practices pose several challenges. The goal of the present study was to describe day-roosting habitats and patterns of thermoregulation in two bat species occurring on Tierra del Fuego, Myotis chiloensis (Chilean myotis) and Histiotus magellanicus (southern big-eared brown bat), during late spring. To do so, we tagged 17 bats with temperature-sensitive radio-transmitters, located 17 day-roosts, and collected 81 days of skin temperature data. We concurrently recorded ambient air temperature to determine its effect on torpor use. Both species were found roosting in large diameter (77.8 ± 6 cm), typically live, Nothofagus pumilio trees (lenga) located on the edges of forest gaps or within stands primarily composed of smaller, younger trees. Bats of both species frequently used torpor, with skin temperatures dropping below a torpor threshold on 89% of days (n = 72) and daily minimum skin temperatures averaging 16.5 °C over the course of our study. Average daily air temperature was a significant predictor of torpor use, with lower skin temperatures and more time spent in torpor observed on colder days. Minimum skin temperature and time spent torpid did not vary between bat species, nor did the characteristics of day-roosts. These data show that spring temperatures in Tierra del Fuego pose an energetic challenge that bats meet through frequent use of torpor, and likely, habitat selection. We recommend local conservation efforts keep these thermal challenges in mind by retaining large trees, which may provide warmer microclimates or room for social groups.
Article
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White-nose syndrome is an emerging fungal disease that has devastated hibernating bat populations across eastern North America. The causal pathogen, Pseudogymnoascus destructans (PD), is a psychrophilic fungus with a known maximal growth temperature of 20 C. Although it is widely speculated that PD is primarily spread between hibernacula by the movement of bats, experimental evidence is lacking to demonstrate that PD can endure temperatures experienced by active bats for periods of time that would facilitate dispersal of viable fungus. We used an in vitro culture-based approach to study the survival of PD conidia on three artificial growth media and bat fur. The fungus was incubated at three temperatures it might realistically be exposed to on nonhibernating bats or in the environment outside of caves and mines (24 C, 30 C, and 37 C). When incubated on artificial media, we found that PD conidia were able to survive for a maximum of 150 d when exposed to temperatures of 24 C, 60 d at 30 C, and 15 d at 37 C. At all temperatures, maximal survival duration was recorded when conidia were incubated on brain-heart infusion agar with 10% volume of sheep (Ovis aries) blood. When incubated on bat fur, viable PD was recovered at 180 d, 60 d, and 5 d when exposed to temperatures of 24 C, 30 C, and 37 C, respectively. Our results suggest that viable PD conidia may be able to survive on or within the bodies of bats, which may facilitate long-distance dispersal. The long-term viability of the fungus on various fomites may differ, and therefore must be assessed for each potential substrate.
Article
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Resistance and tolerance allow organisms to cope with potentially life-threatening pathogens. Recently introduced pathogens initially induce resistance responses, but natural selection favors the development of tolerance, allowing for a commensal relationship to evolve. Mycosis by Pseudogymnoascus destructans, causing white-nose syndrome (WNS) in Nearctic hibernating bats, has resulted in population declines since 2006. The pathogen, which spread from Europe, has infected species of Palearctic Myotis for a longer period. We compared ecologically relevant responses to the fungal infection in the susceptible Nearctic M. lucifugus and less susceptible Palearctic M. myotis, to uncover factors contributing to survival differences in the two species. Samples were collected from euthermic bats during arousal from hibernation, a naturally occurring phenomenon, during which transcriptional responses are activated. We compared the whole-transcriptome responses in wild bats infected with P. destructans hibernating in their natural habitat. Our results show dramatically different local transcriptional responses to the pathogen between uninfected and infected samples from the two species. Whereas we found 1526 significantly upregulated or downregulated transcripts in infected M. lucifugus, only one transcript was downregulated in M. myotis. The upregulated response pathways in M. lucifugus include immune cell activation and migration, and inflammatory pathways, indicative of an unsuccessful attempt to resist the infection. In contrast, M. myotis appears to tolerate P. destructans infection by not activating a transcriptional response. These host-microbe interactions determine pathology, contributing to WNS susceptibility, or commensalism, promoting tolerance to fungal colonization during hibernation that favors survival. Graphic abstract Open image in new window
Article
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Few studies have described winter microclimate selection by bats in the southern United States. This is of particular importance as the cold-adapted fungus, Pseudogymnoascus destructans, which causes the fatal bat disease white-nose syndrome (WNS), continues to spread into southern United States. To better understand the suitability of winter bat habitats for the growth of P. destructans in this region, we collected roost temperature and vapor pressure deficit from 97 hibernacula in six ecoregions in Texas during winter 2016-17 and 2017-18. We also measured skin temperature of Rafinesque's big-eared bats (Corynorhinus townsendii), Townsend's big-eared bats (C. townsendii), big-brown bats (Eptesicus fuscus), southeastern myotis (Myotis austroriparius), cave myotis (M. velifer), tri-colored bats (Perimyotis subflavus), and Mexican free-tailed bats (Tadarida brasiliensis) during hibernation to study their use of torpor in these habitats. We found that temperatures within hibernacula were strongly correlated with external air temperatures and were often within the optimal range of temperatures for P. destructans growth. Hibernacula and skin temperatures differed among species, with Rafinesque's big-eared bats, southeastern myotis, and Mexican free-tailed bats occupying warmer microclimates and having higher torpid skin temperatures. For species that were broadly distributed throughout Texas, hibernacula and skin temperatures differed within species by ecoregion; Tri-colored bats and cave myotis in colder, northern regions occupied colder microclimates within hibernacula and exhibited colder skin temperatures, than individuals of the same species in warmer, southern regions. These data illustrate the variability in microclimates used as hibernacula by bats in Texas and suggest similar variation in susceptibility to WNS in the state. Thus, monitoring microclimates at winter roosts may help predict where WNS may develop, and where management efforts would be most effective.
Article
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The Cimicidae is a family of blood‐dependent ectoparasites in which dispersion capacity is greatly associated with host movements. Bats are the ancestral and most prevalent hosts for cimicids. Cimicids have a worldwide distribution matching that of their hosts, but the global classification is incomplete, especially for species outside the most common Cimicidae taxa. In this study, we place a little‐studied cimicid species, Bucimex chilensis, within a comprehensive molecular phylogeny of Cimicidae by sequencing the genomic regions of this and other closely related species. For this study, we collected B. chilensis females from Myotis chiloensis in Tierra del Fuego, 1,300 km further south than previously known southernmost distribution boundary. We also sequenced COI regions from Primicimex cavernis, a species which together with B. chilensis comprise the entire subfamily Primiciminae. Using Bayesian posterior probability and maximum‐likelihood approaches, we found that B. chilensis and P. cavernis clustered close to each other in the molecular analyses, receiving support from similar morphological features, agreeing with the morphology‐based taxonomic placement of the two species within the subfamily Primiciminae. We also describe a previously unrecognized morphological adaptation of the tarsal structure, which allows the austral bat ectoparasite, B. chilensis, to cling on to the pelage of its known host, the Chilean myotis (Myotis chiloensis). Through a morphological study and behavioral observation, we elucidate how this tarsal structure operates, and we hypothesize that by clinging in the host pelage, B. chilensis is able to disperse effectively to new areas despite low host density. This is a unique feature shared by P. cavernis, the only other species in Primiciminae.
Article
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1.White nose syndrome (WNS), affecting multiple North American bat species during the hibernation period, is a highly pathogenic disease caused by the psychrophilic fungus Pseudogymnoascus destructans (Pd). Because the fungal pathogen persists in the hibernation site environment independently of the hosts, previous theory on spatial disease dynamics cannot predict WNS epidemics. However, the ability to understand factors contributing to the spread of white‐nose syndrome (WNS) in North America is crucial to the management of infected and susceptible bat populations as well as the conservation of threatened and endangered bat species. 2.Utilizing recent theory on environmental opportunistic pathogens, we modeled the effect of 1) landscape clustering, 2) environmental conditions in hibernacula, and 3) microbial competition on the spread of WNS. We used available, already published data to construct and parameterise our model, which takes in to account the spatial distribution of hibernation sites, temperature conditions both in the outside ambient and hibernation site environment, bat population dynamics, dispersal, and infection by the pathogen, which also has its host‐independent dynamics with the environment. We also consider the effect of outside‐host competition between the pathogen and other micro‐organisms on spatial disease dynamics. 3.Our model suggests that pathogen loads accumulate in poorly connected hibernacula at short host dispersal, which can help found the epidemic. In contrast, invasion of the landscape is most successful at long host dispersal distances, with homogenous hibernation site distribution and heterogeneous between‐hibernation site temperatures. Also, increasing the mean temperature, across hibernacula increases fungal growth rate, leading to higher disease prevalence and faster invasion rate. Increasing spatial heterogeneity in hibernaculum temperatures, results in the formation of disease hot‐spots in warmer hibernacula, facilitating more effective spread of the disease in the landscape. Cold‐adapted competing microbes can prevent invasion and therefore overwintering in cold hibernacula increases probability of host survival. 4.Sites that were suboptimal for overwintering prior to WNS may have importance in preventing local extirpations. Although the model is tailored for WNS, due to pressing need for results that can assist in planning conservation measures, these novel results can be broadly applied to other environmentally transmitted diseases. This article is protected by copyright. All rights reserved.
Article
ContextPseudogymnoascus destructans is the fungus responsible for white-nose syndrome (WNS), which has killed millions of hibernating bats in North America, but also occurs in bats in Europe and China without causing large-scale population effects. This is likely to be due to differences in species susceptibility and behaviour, and environmental factors, such as temperature and humidity. Pseudogymnoascus destructans is currently believed to be absent from Australia. AimsTo ascertain the level of risk that white-nose syndrome poses for Australian bats. Methods This risk analysis examines the likelihood that P. destructans enters Australia, the likelihood of the fungus coming in contact with native bats on successful entry, and the potential consequences should this occur. Key results This risk assessment concluded that it is very likely to almost certain that P. destructans will enter Australia, and it is likely that bats will be exposed to the fungus over the next 10 years. Eight cave-dwelling bat species from southern Australia are the ones most likely to be affected. Conclusions The risk was assessed as medium for the critically endangered southern bent-winged bat (Miniopterus orianae bassanii), because any increase in mortality could affect its long-term survival. The risk to other species was deemed to range from low to very low, owing to their wider distribution, which extends beyond the P. destructans risk zone. Implications Although Australia’s milder climate may preclude the large mortality events seen in North America, the fungus could still significantly affect Australian bat populations, particularly bent-winged bats. Active surveillance is required to confirm Australia’s continuing WNS-free status, and to detect the presence of P. destructans should it enter the country. Although White-nose Syndrome Response Guidelines have been developed by Wildlife Health Australia to assist response agencies in the event of an incursion of WNS into bats in Australia, these guidelines would be strengthened by further research to characterise Australian cave temperatures and hibernating bat biology, such as length of torpor bouts and movement over winter. Risk-mitigation strategies should focus on education programs that target cavers, show-cave managers and tourists, particularly those who have visited regions where WNS is known to occur.
Article
A new Late glacial-Holocene palaeoenvironmental record from near Lago Lynch (53°54′S, 69°26′W), Tierra del Fuego is presented. The record was sampled from a mire located within the deciduous Nothofagus forest-steppe ecotone and pollen and spore analysis suggest a high degree of vulnerability of past vegetation to changes in effective moisture. AMS radiocarbon dating supplemented by the application of tephrochronology including the geochemical fingerprinting of six visible and cryptotephra layers provides robust age constraint. The Lago Lynch record commences at c.15.6 ka. The sequence of vegetation changes between c.15.6 and 14.4 ka reflect a gradual increase in temperature and humidity followed by a colder interval between c.14.4 and 13.3 ka, which is broadly coeval with the Antarctic Cold Reversal. After c.13.3 ka patches of Nothofagus forest appeared, suggesting more mesic-temperate conditions leading gradually to the establishment of an open-canopy Nothofagus forest by c.12.5 ka which marks the start of the Holocene. Moderate to strong effective moisture levels dominated during the early Holocene until c.11.0 ka, followed by a sustained period (c.11.0–6.5 ka) of drier climatic conditions, particularly two arid phases at c.10.5–10.0 ka and 8.5–6.5 ka. An eastwards expansion of the forest margin after c.6.5 ka at the site suggests a return to more humid conditions during the late Holocene. We argue that the periods of increased moisture and aridity inferred from Lago Lynch closely reflect the extent to which the southern westerly winds (SWWs) push eastwards to the drier regions of Fuego-Patagonia during the Holocene. The longitudinal variations in moisture are driven by the nature and timing of latitudinal shifts in the SWWs. More significantly the vulnerability of the forest-steppe ecotone to moisture changes amplifies the ecological impact of relatively small-scale shifts in the SWWs.