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376
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Ecology and Evolution. 2021;11:376–389.www.ecolevol.org
Received: 16 July 2020
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Revised: 11 October 2020
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Accepted: 25 O ctober 2020
DOI: 10.1002 /ece3.7055
ORIGINAL RESEARCH
DNA metabarcoding provides insights into seasonal diet
variations in Chinese mole shrew (Anourosorex squamipes) with
potential implications for evaluating crop impacts
Ke-yi Tang1 | Fei Xie1 | Hong-yi Liu2 | Ying-ting Pu1 | Dan Chen1 | Bo-xin Qin1 |
Chang-kun Fu1 | Qiong Wang1 | Shun-de Chen1 | Ke-ji Guo3
This is an op en access arti cle under the ter ms of the Creative Commons Attribution L icense, which pe rmits use, dis tribu tion and reprod uction in any med ium,
provide d the original wor k is properly cited.
© 2020 The Authors. Ecolog y and Evolution published by John Wiley & S ons Ltd.
1College of Life Sciences, Sichuan No rmal
University, Chengdu, China
2College of B iolog y and the Environment,
Nanjing Forestry Universit y, Nanjing , China
3Centra l South I nventor y and Planning
Instit ute of National Forestr y and Gr asslan d
Administration, Changsha, China
Correspondence
Shun-de Chen, College of Life Sciences,
Sichuan Normal U niversity, Chengdu
610066, China.
Email: c sd111@126. com
Ke-ji Guo, Central South Inventor y and
Plannin g Institute of National For estr y and
Grassland Administration, Changsha, Hunan
province 410014, China.
Email: guokeji@126.com
Funding information
Starting Research Fund from Sichuan
Normal U niversity, Grant/Award Num ber:
024341965; Nationa l Natural Science
Foundat ion of China, Gra nt/Award Number:
31670388, 320 01223 and 32070424;
Chengdu Municip al Science and Techno logy
Bureau p rojec t, Gra nt/Award Number:
2015-NY02–00369-NC
Abstract
Diet analysis of potential small mammals pest species is important for understand-
ing feeding ecology and evaluating their impact on crops and stored foods. Chinese
mole shrew (Anourosorex squamipes), distributed in Southwest China, has previously
been reported as a farmland pest. Effective population management of this species
requires a better understanding of its diet, which can be difficult to determine with
high taxonomic resolution using conventional microhistological methods. In this
study, we used two DNA metabarcoding assays to identify 38 animal species and
65 plant genera from shrew stomach contents, which suggest that A. squamipes is
an omnivorous generalist. Earthworms are the most prevalent (>90%) and abundant
(>80%) food items in the diverse diet of A. squamipes. Species of the Fabaceae (fre-
quency of occurrence [FO]: 88%; such as peanuts) and Poaceae (FO: 71%; such as
rice) families were the most common plant foods identified in the diet of A. squa-
mipes. Additionally, we found a seasonal decrease in the diversity and abundance
of invertebrate foods from spring and summer to winter. Chinese mole shrew has a
diverse and flexible diet throughout the year to adapt to seasonal variations in food
availability, contributing to its survival even when food resources are limited. This
study provides a higher resolution identification of the diet of A. squamipes than has
been previously described and is valuable for understanding shrew feeding ecology
as well as evaluating possible species impacts on crops.
KEYWORDS
Chinese mole shrew, ecology of pest, metabarcoding, molecular diet analysis, seasonal diet
changes
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TANG eT Al.
1 | INTRODUCTION
The Chinese mole shrew (Anourosorex squamipes Milne-
Edwards, 1872) is a small insectivore mammal (He et al., 2016;
Hoffmann, 1987; Motokawa et al., 2003), distributed in south-
western China and adjacent areas (He et al., 2016; Motokawa
et al., 2003; Motokawa & Lin, 2002; Wilson et al., 2018). Like
other house shrews (Khanam et al., 2016), A. squamipes is re-
garded as a pest in the agricultural ecosystem (Peng et al., 2018;
Zong et al., 20 17), causin g bot h direc t a nd indi re c t eff ect s (Md angi
et al., 2013). As is widely known, synanthropic species associa-
tion with human habitats widely impact agriculture and human
health through damage to crops and transmission of pathogens
(Khanam et al., 2016; Palis et al., 2007). The Chinese mole shrew
affects crops and human health in China in a multitude of ways
(Peng et al., 2018; Yang et al., 2013). For example, this shrew spe-
cies consumes and contaminates stored grains and crops (Peng
et al., 2018). In addition, it is considered to be a potential source
of various types of viruses and pathogens (Gu et al., 2016; Song
et al., 2007). A. squamipes caused severe damages to crops result-
ing from increased population sizes in Southwest China, especially
in Sichuan Basin (Yang et al., 2013; Zong et al., 2017). Moreover,
due to their peculiar food and foraging habits, existing rodent
control practices are not suitable for controlling the number of
these shrews, resulting in grain insecurity and reduced villager
livelihoods.
Diet analysis are important for understanding how animal pop-
ulations respond to resource distribution and variety as well as
how to manage them (Gordon et al., 2019). Dietary information has
been used in addition to pure feeding ecology in a variety of ap-
plied studies (Gong et al., 2017). Accurate identification of foods is a
prerequisite to fully understanding the feeding ecology of a species
and effectively controlling pest numbers (Heroldova et al., 2008).
Better understanding of the feeding habits of house shrews can
help to evaluate how growing populations of A. squamipes affect
human and agricultural systems even during resource-poor seasons
and develop more effective pests management strategies, including
targeted baits and lures (Khanam et al., 2016; Lathiya et al., 2008).
However, very few studies have described the composition and sea-
sonal variations in the Chinese mole shrew diet with higher taxo-
nomic resolution.
For natural populations, it is difficult to accurately and effi-
ciently assess wildlife diets, because of their elusive predatory
behaviors and versatile feeding habits (Gong et al., 2017; Ozaki
et al., 2018). Identifying food items with the highest taxonomic
resolution is nearly impossible with traditional microhistologi-
cal analysis of gut contents and stable isotope analysis (Jeunen
et al., 2019; Murray et al., 2016; Rytkonen et al., 2019). A major
limitation of the classical observational methods is that foods
items are often digested to a greater extent, making identifica-
tion of their remains taxonomically challenging (Berry et al., 2017;
Bessey et al., 2019). Especially in the cases of insectivorous pred-
ators, whose prey is va riable, small in size, and easily disintegrate d
in the gut, direct identification is difficult since their chyme con-
tains a mixture of degraded prey fragment s (Clare et al., 2014;
Rytkonen et al., 2019). Besides, the stable isotope approach is un-
abl e to distinguish prey at the sp ecies level (Bohmann et al., 2018).
Therefore, a broad-spectrum technique with higher taxonomic
resolution is necessar y because shrew species have highly di-
verse and flexible diets that include insects, annelids, and plants
(Churchfield et al., 2010, 2012; Haberl, 2002).
Here, DNA metabarcoding enabled us to identify food DNA (in-
cluding highly degraded DNA) in gut contents or fecal samples with
higher taxonomic resolution (Kartzinel & Pringle, 2015; Pompanon
et al., 2012). To date, among shrew species, only the diets of Asian
musk shrew (Suncus murinus) have been examined through DNA me-
tabarcoding methods (Brown et al., 2014; Khanam et al., 2016). Most
previous studies (Churchfield et al., 2010, 2012; De Pascual & De
Ascenc ao, 20 0 0; Haberl, 20 02 ; Mc Cay & Stor m, 1997) that assessed
diets in shrew species are based on microhistological identification
of insec t fragments in stomach content s or fecal pellets, resulting
in large proportions of poorly resolved plant t axa and dietary infor-
mation mainly at higher taxonomic levels. Little is known about the
invertebrate prey species and plants (especially at the species level)
consumed by Chinese mole shrew, which prevents understanding of
their feeding ecology and thus impedes effective pest control.
In this study, we attempted to characterize the Chinese mole
shrew diet across the four seasons by DNA metabarcoding of stom-
ach samples. We compared dietary richness and composition across
seasons to evaluate the impacts of this pest on crops and enhance
our understanding of dietary breadth and seasonal food preferences
in A. squamipes. Thus, this study may have implications for food niche
and management of Chinese mole shrew as well as help to develop
appropriate pest control strategies.
2 | MATERIALS AND METHODS
2.1 | Animal trapping
The animal samples of Chinese mole shrew were trapped from four
seasons (Jan, Apr, Jul and Oct) from 2018 to 2019 in Pengzhou,
Sichuan Province, southwest China. The sampling sites occupy a
range of elevations from 515 to 575 m, longitude from 103.80°E to
104.10°E , and lati tu des from 30.96°N to 31 .12° N . All col lec ted spec-
imens were identified based on external characteristics in the field
and were further confirmed according to skull morphology in the
laboratory. As soon as animal specimens were collected, the animals
were immediately stored at 0–4°C in the incubator with ice bags for
transportation. The luminal stomach contents were collected at a
super-clean bench. And stomach contents were stored at −80°C for
DNA extraction. Body mass is often used as a proxy for age in ani-
mals in previous study (Lavrinienko et al., 2018). Age identification
method for A. squamipes referred to Yang et al. (2013). We followed
the weight division criteria: Youth group (1) is less than 23.0 g, sub-
adult, group (2) was 23.1–28.0 g, adult group (3) was 28.1–38.0 g, and
378
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TAN G eT Al.
old group (4) was more than 38.0 g. A total of sevent y-two of adult
Chinese mole shrew (18 in each season) were used in this study.
2.2 | Stomach content samples and DNA extraction
The all samples of Chinese mole shrew were thawed at room tem-
perature. We obtained stomach contents (SCs) collected from 72 in-
dividuals. We try to collect the foods in the interior of SCs to avoid
the interference of host tissues or cells. The SCs samples were col-
lected according to the guidelines and approval of the Animal Ethics
Committee of Sichuan Normal University. After extracting from the
stomach, the SCs was washed with ultrapure water and wiped off
with other tissue between each extraction. Each SC was homoge-
nized and stored in 95% ethanol for DNA extraction. The SCs sam-
ples of three individuals derived from the same field or woodland
are homogeneously mixed into a mixed sample. Herein, six mixed
SCs samples are used for further sequencing in each season, and a
total of 24 mixed SCs samples are used for further molecular dietary
analysis.
We ex tracted DNA used the QIAamp Fast DNA Stool Mini Kit
(ID: 51604, QIAGEN), which is specifically developed for fecal and
gut contents samples, according to the manufacturer's instructions.
An extraction blank was included at each extraction series. The ex-
tracted DNA was further concentrated by evaporating samples in
vacuum and then was stored for metabarcoding analysis.
2.3 | Dietary DNA amplification and sequencing
PC R am p li f ic a ti on wa s car r ie d out usin g mitoc hon dri al COΙ-targeting
primer (LCO-1490/Uni-MiniBar-R), which produced a COΙ (cy-
tochrome oxidase Ι) amplicon of 177 bp (Brown et al., 2014) for
animal identification. Existing COΙ-based approaches is widely pre-
ferred to identify unknown arthropod sequences (Zeale et al., 2011).
The used primers were tested against Chinese mole shrew se-
quences to ensure no signific ant amplification of host DNA. And
the rbcL (ribulose-bisphosphate carboxylase gene) primers (h1aF
and h2aR primers) were used to identify the plant species (Pierre
et al., 2007). Sample specific barcode sequences were added to the
COΙ and rbcL primers.
PCR were performed with PCR Using Q5® High-Fidelity DNA
Polymerase (M0 491, NEB) according to the manufacturer's in-
struction. And PCR protocols were conducted primarily following
Bohmann et al. (2018). Blank extraction controls were included on
each PCR plate and for each different primer set. PCR products were
then purified using a PCR purification kit (AX YGEN). Taking the pu-
rified PCR product as the template, quantitative real-time PCR was
performed on a Microplate reader (BioTek, FLx800) using Quant-iT
PicoGreen dsDNA Assay Kit. The amplicons for each sample were
then mixed and purified according to the next high throughput se-
quencing requirements. Libraries for sequencing were constructed
using the TruSeq Nano DNA LT Library Prep Kit (Illumina, San Diego,
CA, United States) as recommended by the manufacturer. Libraries
were sequenced on an Illumina Miseq plat form (2 × 250 bp paired-
end reads) by Personalbio Bioinformatics Technology Corporation
(Shanghai, China).
2.4 | Sequence processing and data analysis
The raw reads were filtered through trimming and quality control
steps prior to taxonomic assignment according to the QIIME v.1.7.0
quality control process (Caporaso et al., 2012). Adaptor/primer re-
gions were removed, and potential chimeras were removed using
USEARCHv9.2 (Edgar, 2013). Reads were clustered at 97% into
Molecular Operational Taxonomic Unit s (MOTUs) according to the
standard setting in USEARCHv9.2 (Edgar, 2013). Rarefaction curves
were gener ated using QIIM E v.1.7.0. and reach ed st able values , indi-
cating that most of the species diversity were captured. High-quality
clean reads that passed quality filtering were queried against the full
NCBI database using BLASTn according to previous study (Berry
et al., 2017). MOTUs were resolved to species, genus, or higher,
for COΙ animals or rbcL plants primer assays based on the percent
similarity threshold: Sequences with identity ≥ 99% to a single spe-
cies were considered as a “species match,” and as a “genus match”
if sequences had ≥ 98% similarity to one or more species within
the same genus. DNA sequences in this study were deposited into
the NCBI Sequence Read Archive (SRA) under accession number:
PRJNA637184.
Alpha diversity (i.e., Chao1, Shannon and Simpson) matrices
were performed using QIIME and displayed using R v.3.3.3. soft-
ware. To evaluate the pattern of dispersion of samples within each
season, beta diversity was calculated with the euclidean distance.
Beta diversity was calculated using QIIME and visualized by two-di-
mensional principal coordinate analysis (PCoA). Diversity was com-
pared between different seasons to assess temporal differences in
diet composition. We also compared the relative abundance of food
items at various taxonomic levels and at different seasons based on
the linear discriminatory analysis (LDA) effect size (LEfSe) method
using LEfSe software.
2.5 | Statistical analyses
We used ANOVA to test for a significant difference in the dietary
composition between dif ferent seasons. We also used a nonpara-
metric statistical test (Kruskal–Wallis test) to assess the difference
in alpha diversity index between different seasons. The frequency
of occurrence (the number of pellets containing that foods divided
by the total number of pellets in the species sample, FO) and the
numbers of foods during dif ferent seasons were compared statis-
tically using Dunnett's T3 multiple comparisons test by SPSS 20.0
software. The Mann–Whitney U tes t was also adopted to assess the
difference in relative abundance of food items between different
seasons following our previous study (Tang et al., 2019). Heat maps,
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TANG eT Al.
box plots, and taxa summary bar char ts were generated using the
“ggplot2” package of R software (Wickham, 2009).
3 | RESULTS
3.1 | Overview of taxonomic assignment and
dietary diversity
In th e 24 stom ach sa mples analyze d over all se as ons, the mean num-
ber of MOTUs in animal species was 38 ± 6 for spring, 30 ± 5 for
summer, 32 ± 18 for autumn and 4 ± 1 for winter (Table 1). In plant
food items, the mean number of MOTUs was 95 ± 28 for spring,
57 ± 24 for summer, 87 ± 41 for autumn, and 120 ± 42 for win-
ter (Table 1). In total, we identified 38 potential animal food items
(spanning 26 families and 15 orders) ( Table S1) and 113 plant food
items (spanning 39 families and 23 orders) (Table S2) at species level
that are consumed by Chinese mole shrew. Seasonal dietar y changes
were detected in A. squamipes with a general shift toward low di-
etary diversity in winter. As expected, the number of animal food
items at species level decreased significantly in winter (Figure 1a).
Peak consumption of animal food items was detected in spring and
summer, which were significantly higher than those in autumn and
winter. However, we found no significant seasonal dif ferences in the
number of plant food items at genus level (Figure 1a), suggesting that
potential plant food items were constant throughout the year.
Alpha diversity indices (Chao1, Shannon, and Simpson) indicated
seasonal differences in the diversity of animal food items. There was
a significant greater Chao1 diversity index in spring and summer
compared to winter (Figure 2a; p < .01). A higher Shannon diver-
sity index was observed in autumn relative to winter (p < .05). No
significant differences were found in Simpson index of animal food
items (Figure 2a). Overall, our analysis showed a lower alpha diver-
sity of animal food items in winter. However, th e die tary alph a diver-
sity of plant food items did not differ significantly between seasons
(Figure 2b; p > .05), suggesting that the availability of plant-derived
foods were not affec ted by seasons.
The PCoA plot (Figure 3) revealed seasonal dif ferences in ani-
mal-derived diets. Animal food items in spring, summer, and autumn
weakly clustered together and were separate from diets in winter
(Figure 3a). In addition, there was dispersion within winter animal
food items, suggesting a high degree of intragroup variability. We
also obser ved a cluster of plant food items in autumn that was
separated from those in other seasons with apparent dispersion
(Figure 3b), suggesting a high degree of interindividual variability
especially during winter. It could be explained by their opportunism
and broad diet. The dominant family (Poaceae) in autumn likely con-
tributed to this separation.
3.2 | Dietary composition and seasonal variation in
animal food items
We examined seasonal variations in the diet composition of A. squa-
mipes, especially during times of resource limitation (e.g., in winter).
Based on the full year, our results showed that although some small
insect s (ants, spiders, crickets, and beetles) were consumed, the
Chinese mole shrew is primarily an earthworms-eating shrew with a
semi-fossorial foraging mode. Using order-level taxonomy only, spe-
cies of Haplotaxida, Stylommatophora, Hymenoptera, Orthoptera,
and Moniligastrida dominated the diet with species of Haplot axida
representing the highest FO (100%) and highest taxonomic rich-
ness (>74%) of consumption (Table S3). Notably, the consumption
of Haplot axida significant decreased (spring versus winter: 83% ver-
sus 45%, p = .002; summer versus winter: 81% versus 45%, p = .03;
autumn versus winter: 90% versus 45%, p = .004) during winter
(Table S3). Thus, earthworms were considered as the major food
item in the diet of A. squamipes. In addition, as the common prey of
shrews, arthropods (such as Orthoptera, Coleoptera, Dermaptera,
Diptera, and Lepidoptera) were also detected but at low frequencies
and relative abundances in A. squamipes diet (Table S3).
At the species level, the dominant (top five) animal species in
terms of both FO and relative abundance were Metaphire californica,
Amynthas morrisi, Amynthas corticis, Deroceras laeve, and Camponotus
thadeus (Figures 4a, 5 and Table 2). Among the total animal food
items, 12 dif ferent species of earthworms belonging to four families
(Megascolecidae, Enchytraeidae, Moniligastridae, and Lumbricidae)
accounted for 70%−80% of the animal-derived diet (Table 2, Figure 5
and Table S4), indicating that these soil inver tebrates are extremely
abundant and diverse in the studied region. Among them, Metaphire
californica was most frequently detected in all samples, contributing
19.8%−60% of the relative abundance of overall prey consumption
Food types
Identified
level
Spring
(Mean ± SE)
Summer
(Mean ± SE)
Autumn
(Mean ± SE)
Winter
(Mean ± SE)
Animal (COI) MOTUs 38 ± 6a30 ± 5a32 ± 18ab 4 ± 1b
Assigned to
species
12 ± 1a13 ± 3ab 9 ± 1b4 ± 1c
Plant (rbcL) MOTUs 95 ± 28 57 ± 24 87 ± 41 120 ± 42
Assigned to
species
26 ± 7 17 ± 6 30 ± 13 30 ± 9
Note: Dif ferent letters indic ate a difference between seasons (p < .05). A lack of superscript
numbers denotes no signif icant difference. SE, standard error.
TABLE 1 The number of Molecular
Operational Taxonomic Units (MOTUs)
and identified species of animal and plant
food items in the Chinese mole shrew
(Anourosorex squamipes) diet throughout
the year
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TAN G eT Al.
and peaking at 60% in autumn (Table 2 and Figure 5). The sec-
ond-richest prey (Amynthas morrisi) were eaten more frequently and
made up a larger propor tion (>34%) of the available prey in spring
and summer than in autumn and winter (<5%; Manne–Whitney U
test: p = .0 08). In addition, we found a trend in the consumption of
earthworms that shif ted from higher numbers of earthworms during
spring and summer to lower levels during autumn and the least in
winter (Figure 1b and Table S4). Thus, the relative abundances of
earthworms consumed by Chinese mole shrew during winter were
significantly decreased (p < .01; Figure 1b). Meanwhile, the relative
abundances of the all earthworm species significantly decreased
(p < .01) during winter (Figure 1b), bec ause their availability of
was reduced. Our analysis indicated the animal-derived diets of A.
squamipes contain a high prevalence and diversity of earthworms.
However, during winter, Chinese mole shrew predominantly preyed
on Camponotus thadeus and Deroceras leave with a hi gh FO (5 0%) and
in higher proportions compared to other seasons (Table 2, Figures 4a
and 5). Therefore, our study revealed that Chinese mole shrews have
FIGURE 1 Seasonal variations in the Chinese mole shrew diet. (a) Seasonal changes in animal and plant food items at dif ferent taxonomic
levels. (b) Seasonal changes in the numbers and relative abundances of earthworms at the species level. Different let ters indicate a
difference between seasons (p < .05)
0
2
4
6
8
10
12
14
16
18
0
5
10
15
20
25
30
35
Spring Summer Autumn Winter
Plant food items
Animal food items
The number of plant food items at the genus leve
l
The number of animal food items at the species level
a
a
a
a
a
a
b
c
0
1
2
3
4
5
6
7
8
0
0.4
0.8
1.2
1.6
2.0
Spring Summer Autumn Winter
a a a
b
a
ab
bc
c
Relative abundance of the earthworm specie
s
The number of the earthworm species
Relative abundance of the earthworm species
The number of the earthworm species
(a)
(b)
FIGURE 2 Box-and-whisker plot s for alpha diversity in animal (a) and plant (b) food species estimators (Chao1, Shannon, and Simpson
indices). Dif ferent letters indicate a difference bet ween seasons (p < .05)
10
20
30
40
50
Spring Summer
Chao1
1.0
1.5
2.0
2.5
3.0
Shannon
Autumn Winter Spring SummerAutumnWinter
Simpson
0.2
0.4
0.6
0.8
Spring SummerAutumnWinter
A
50
100
150
Chao1
Spring Summer Autumn Winter
2.0
2.5
3.0
3.5
Shannon
Spring SummerAutumnWinter
0.5
0.6
0.7
0.8
Simpson
Spring SummerAutumnWinter
a
a
ac
c
ab ab
ac
b
B
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381
TANG eT Al.
a broad diet comprising many different inver tebrates of various sizes
(dominantly earthworms) based on COΙ metabarcoding approaches.
3.3 | Dietary composition and seasonal variation in
plant food items
Generally, shrews are known to be small insectivorous mammals
that preferentially target invertebrate prey. Interestingly, plant food
items (especially crops) at various taxonomic levels were success-
fully detected from stomach contents of A. squamipes (Figures 6 and
7) . The spe cie s of th e Fa ba cea e fam ily (FO : 88%) we re th e mos t co m-
mon plant food items followed by Poaceae (FO: 71%) based on both
FO and relative abundance (peak value > 50 %) over the co urs e of th e
year (Table 3). The winter plant-derived diet of Chinese mole shrews
was dominated by Fabaceae species (57.2% of plants consumed),
with Arachis hypogaea (peanut) being the most frequently and abun-
dantly eaten species from this family, representing 15.2%−86.8% of
FIGURE 3 Two-dimensional principal coordinate analysis (PCoA) of MOTUs of the Chinese mole shrew diet throughout the year. (a)
represents animal food items and (b) represents plant food items. The first two principal coordinate (PC) axes are shown
−0.50
−0.25
0.00
0.25
0.50
0.75
−0.25 0.00 0.25 0.50 0.75
PC1 (50.41%)
PC2 (23.85%)
Group
Winter
Sping
Summer
Autumn
Animal food items
−0.5
0.0
0.5
−0.6 −0.30.0 0.3
PC1 (32.82%)
PC2 (15.33%)
Plant food items
Group
Winter
Sping
Summe
r
Autumn
(a) (b)
FIGURE 4 Heat map and FO of predominant animal (a) and plant (b) food items throughout the year. Each number in the heat map
indicates the relative abundance of the corresponding food. Abbreviations: Sp, spring; Su, summer; A , autumn; W, winter
0.541
0.000
0.056
0.003
0.022
0.000
0.579
0.004
0.070
0.004
0.034
0.000
0.572
0.003
0.067
0.002
0.031
0.000
0.007
0.637
0.046
0.025
0.000
0.000
0.003
0.695
0.058
0.019
0.000
0.000
0.003
0.704
0.054
0.023
0.001
0.000
0.010
0.065
0.553
0.258
0.006
0.000
0.010
0.152
0.228
0.460
0.008
0.000
0.009
0.115
0.424
0.281
0.005
0.000
0.357
0.602
0.012
0.001
0.001
0.000
0.397
0.580
0.003
0.000
0.001
0.000
0.403
0.560
0.019
0.000
0.002
0.000
0.518
0.000
0.007
0.000
0.000
0.000
0.974
0.001
0.005
0.001
0.000
0.000
0.966
0.000
0.006
0.000
0.000
0.000
0.264
0.051
0.595
0.000
0.000
0.000
0.424
0.067
0.491
0.000
0.000
0.000
0.453
0.067
0.459
0.000
0.000
0.000
0.375
0.000
0.208
0.000
0.417
0.000
0.138
0.000
0.103
0.000
0.759
0.000
0.217
0.087
0.000
0.000
0.652
0.000
0.280
0.160
0.000
0.000
0.000
0.560
0.250
0.050
0.150
0.000
0.000
0.450
0.467
0.000
0.000
0.000
0.000
0.467
Sp1306a
Sp1306b
Sp1306c
Sp1309a
Sp1309b
Sp1309c
Su1560a
Su1560b
Su1560c
Su1568a
Su1568b
Su1568c
A1003a
A1003b
A1003c
A1011a
A1011b
A1011c
W1286a
W1286b
W1286c
W1287a
W1287b
W1287c
0
0.2
0.4
0.6
0.8
Metaphire californica
Amynthas morrisi
Amynthas corticis
Deroceras laeve
Camponotus thadeus
Gryllotalpa unispina
Spring SummerAutumnWinterYearly
100% 100% 100% 100% 100%
83% 100% 67% 50% 75%
100% 100% 100% 50% 88%
100% 83% 17% 0% 50%
67% 100%0% 50% 54%
0% 0% 0% 50% 13%
Frequency of occurrence
Spring SummerAutumnWinter
0.351
0.415
0.000
0.028
0.000
0.000
0.000
0.112
0.000
0.057
0.000
0.000
0.000
0.134
0.172
0.000
0.000
0.000
0.000
0.000
0.033
0.000
0.000
0.000
0.000
0.000
0.261
0.000
0.051
0.000
0.000
0.000
0.000
0.000
0.000
0.002
0.246
0.246
0.000
0.000
0.000
0.000
0.000
0.000
0.005
0.000
0.005
0.000
0.066
0.067
0.000
0.000
0.000
0.001
0.000
0.000
0.000
0.477
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.273
0.281
0.000
0.000
0.000
0.000
0.000
0.005
0.390
0.053
0.013
0.000
0.000
0.007
0.656
0.000
0.261
0.000
0.000
0.081
0.504
0.000
0.074
0.000
0.092
0.100
0.292
0.006
0.025
0.015
0.056
0.060
0.644
0.013
0.186
0.000
0.051
0.107
0.668
0.000
0.119
0.013
0.420
0.420
0.225
0.000
0.084
0.000
0.000
0.061
0.131
0.000
0.017
0.000
0.152
0.154
0.075
0.000
0.000
0.369
0.847
0.859
0.000
0.000
0.000
0.000
0.848
0.859
0.000
0.000
0.000
0.000
0.868
0.881
0.000
0.000
0.000
0.000
Sp1306a
Sp1306b
Sp1306c
Sp1309a
Sp1309b
Sp1309c
Su1560a
Su1560b
Su1560c
Su1568a
Su1568b
Su1568c
A1003a
A1003b
A1003c
A1011a
A1011b
A1011c
W1286a
W1286b
W1286c
W1287a
W1287b
W1287c
Arachis hypogaea
Fabaceae
Poaceae
Withania frutescens
Oryza sativa
Lactuca sativa
Spring Summer Autumn Wi nter Yearly
33%83% 83%83% 71%
67%83% 100% 100% 88%
67%17% 100% 100% 71%
33%33% 50%17% 33%
33%17% 100% 100% 63%
33%33% 50%83% 50%
Spring SummerAutumnWinter
Frequency of occurrence
(a)
(b)
382
|
TAN G eT Al.
the identified plant food items (Figures 4b, 6 and Table S2). Poaceae
species were found to significant increase in relative abundance
during autumn (>53%) compared to other seasons (<8%) based on
LEfSe analysis (Table 3 and Figure 7b), suggesting that A . squamipes
feeds primarily on the seeds from Poaceae in autumn, peaking at
53% (Table 3 and Figure 4b). Oryza sativa (rice) as a commonly
eaten crop species from the family Poaceae displayed the highest
frequency (FO: 100%) and proportion (peaking at 26.1%), especially
during postharvest period (e.g., autumn and winter; Figures 4b, 6
and Table S2). In addition, the crop species Withania frutescens (bal-
sam pear) and Lactuca sativa (lettuce) were also identified during the
year but contributed a very low percent of the plant diet (Figure 4b
and Table S2). Our results confirmed that Chinese mole shrews could
cause serious damage to crops or stored grains.
Aside from crops, species from the Caryophyllaceae (31.5%) and
Lauraceae (26.7%) families were also eaten by A. squamipes in high er
proportions during spring compared to other seasons and appeared
in all of the stomach contents samples (Table 3). Chikusichloa aquatic,
which constituted the majority of Poacea e, was observe d at a signif-
icant higher frequency (FO: 100%) and proportion (36.4%) in autumn
compared to other seasons (Table S2). The Oleaceae, Asteraceae,
and Nyssaceae were frequently observed during summer, account-
ing for 19.8%, 10.6% , an d 10.4% of the identified plant diet s, resp ec-
tively ( Table 3). In total, we obser ved high diversity in Chinese mole
shrew plant-derived diet throughout the year. A wide-range foraging
mode may explain the abundant numbers of this shrew even when
food resources are limited during winter.
4 | DISCUSSION
Our study utilized high-resolution identification to explore the di-
etary compositions and seasonal diet variations of the Chinese mole
shrew present in human habitats, aiming to increase understanding
of shrew feeding ecology and evaluating their impact on the farming
system. The Chinese mole shrew tends to be an opportunistic and
generalized predator of a diverse array of invertebrates and plants,
particularly earthworms and crops. With respect to common preys
invertebrates, we confirm that Chinese mole shrew predominantly
but not exclusively feeds on earthworms with a semi-fossorial forag-
ing mode similar to other shrews in temperate habitats (Churchfield
et al., 2010, 2012; Khanam et al., 2016). Based on molecular tech-
nique, diverse plant materials at the species level were identified in
the shrew stomach contents with frequent observation of several
important crops (e.g., rice and peanut).
4.1 | Characteristics of animal-derived diet in the
Chinese mole shrew
The diets of the Chinese mole shrew in our study are similar to the
diets of other shrews (such as Sorex and Blarina) (Churchfield, 1982;
Churchfield et al., 1999, 2012; Churchfield & Rychlik, 2006;
Churchfield & Sheftel, 1994; De Pascual & De Ascencao, 2000), which
include diverse invertebrates with a preponderance of ear thworms
(Table 2, Figures 1b, 5 and Table S1). The Chinese mole shrew can
also be considered as an ear thworm-eating shrew. Using molecular
technique, we obtained a sufficiently higher taxonomic resolution of
food identification, especially earthworms (a total of 12 earthworm
species were identified), compared to previous dietary analysis of
shrews. Similar to early studies (Churchfield & Rychlik, 20 06), many
of the invertebrates eaten by A. squamipes are typical soil inhabit-
ants (e.g., Oligochaeta and Formicidae), suggesting that this species
of shrew is mainly subterranean in its foraging mode. Short-tailed
shrews are well adapted to a subterranean lifestyle and can push
through soil and leaf litter with their long proboscis and elongated
FIGURE 5 Relative abundance of the
top 10 animal food items at the species
level based on the COI metabarcoding
assay
Relative abundance (%)
020406080 100
Sp1306a
Sp1306b
Sp1306c
Sp1309a
Sp1309b
Sp1309c
Su1560a
Su1560b
Su1560c
Su1568a
Su1568b
Su1568c
A1003a
A1003b
A1003c
A1011a
A1011b
A1011c
W1286a
W1286b
W1286c
W1287a
W1287b
W1287c
Metaphire.californica
Amynthas.morrisi
Amynthas.corticis
Deroceras.laeve
Camponotus.thadeus
Gryllotalpa.unispina
Enchytraeus.japonensis
Drawida.sp..Watarase
Antrodiaetus.unicolor
Aporrectodea.aff..trapezoides.L2
Amynthas.gracilis
Drawida.koreana
Bimastos.palustris
Gryllotalpa.orientalis
Teleogryllus.emma
Harpalus.calceatus
Euborellia.femoralis
Paobius.pachypedatus
Henlea.perpusilla
Amynthas.hupeiensis
Others
Spring Summer Autumn Winter
|
383
TANG eT Al.
TABLE 2 Frequency of occurrence (FO) and relative abundance of the top 20 animal food items in the Chinese mole shrew diet
Tar get Ta xon Spring Summer Autumn Winter Yearly
Family level Species level
Relative
abundance FO (N = 6)
Relative
abundance FO (N = 6)
Relative
Abundance FO (N = 6)
Relative
Abundance FO (N = 6)
FO
(N = 24)
Megascolecidae Metaphire californica 0.284 100 % 0.19 8 100 % 0.600 10 0% 0. 288 100% 100%
Amynthas morrisi 0.340 83% 0.346 100% 0.031 67% 0.049 50% 75%
Amynthas corticis 0.059 100% 0.206 100% 0. 261 10 0% 0.077 50% 88%
Amynthas hupeiensis 0.002 50% 00% 0.001 33% 00% 21%
Amynthas gracilis 0.001 17% 0.046 67% 0.001 17% 00% 25%
Euborellia femoralis 00% 0.008 50% 00% 00% 13%
Agriolimacidae Deroceras laeve 0.015 67% 0.004 10 0% 00% 0.305 50% 54%
Formicidae Camponotus thadeus 00% 00% 00% 0.246 50% 13%
Gryllotalpida Gryllotalpa unispina 0.013 100% 0.167 83% 0.001 17% 00% 50%
Enchytraeidae Enchytraeus japonensis 0.130 100% 0.001 67% 0.001 33% 00% 50%
Harpalus calceatus 00% 00% 0.001 50% 00% 13%
Moniligastridae Drawida sp. Watarase 0.097 67% 0.001 33% 00% 00% 25%
Drawida koreana 0.022 67% 0.001 50% 0.008 17 % 00% 33%
Ocnerodrilidae sp. 3
DP-201 5
00% 0.002 50% 00% 00% 13%
Lumbricidae Bimastos palustris 0.009 100 % 0.005 50% 00% 0.007 17% 29%
Aporrectodea af f.
Trapezoides
0.002 100 % 0.012 100% 0.011 67% 0.028 33% 75%
Antrodiaetidae Antrodiaetus unicolor 00% 00% 0.058 17% 00% 4%
Anisolabididae Gryllotalpa orientalis 0.017 100% 00% 00% 00% 13%
Carabidae Harpalus calceatus 00% 00% 0.0 09 50% 00% 13%
Lithobiidae Teleogryllus emma 00% 00% 0.015 50% 00% 13%
384
|
TAN G eT Al.
claws (Churchfield & Rychlik, 20 06; Wu et al., 2011). These special
morphological adaptations help to capture earthworms and ants
depending on A. squamipes semi-fossorial foraging behavior (He
et al., 2016).
On the other hand, the preys of the Orthoptera, Formicidae,
Coleoptera, Dermaptera, Diptera, and Lepidoptera families were
occasionally observed during a particular season but only contrib-
uted a small amount of prey volume. Unlike the Chinese mole shrew,
some other shrew species have been reported to predominantly
feed on arthropods, not earthworms. For instance, Diptera (files),
Formicidae (ants) and Araneae (spiders) were the most prey species
among Southern short-tailed shrew (Blarina carolinensis; Sylvester
et al., 2012). The diet of European water shrew (Neomys fodiens
bicolor) is composed mainly of lumbricids, isopods and dipterans
(Churchfield, 2009). Isopterans (termites) and formicids were found
to be the most frequent food items in the diet of elephant shrews
(Elephantulus myurus; Churchfield, 1987). Lepidoptera larvae are
the most common prey for masked shrew (Sorex cinereus) (Bellocq
& Smith, 20 03; McC ay & Storm, 1997), followed by Coleoptera
(beetles) and Aranea (spiders). The variations in diet compositions
between different shrew species also imply that each one chooses
what types of prey to feed on, presumably in relation to their mor-
phological adaptations or according to the availability of food re-
sources (Bellocq & Smith, 20 03; De Pascual & De Ascencao, 200 0).
4.2 | Seasonal variations in animal-derived diets in
Chinese mole shrew
We also observed decreasing trends in diversity, proportions and
FO of invertebrate consumption from spring to winter (Figure 1
and Table 1). One plausible explanation is the fact that seasonal-
ity has a strong effect on the density, biomass, and reproductive
activity of the earthworm population (Kumar & Sabhlok, 2018;
Monroy et al., 20 06). For inst ance, the maximum density and mating
activity of ear thworms were achieved in spring (Biradar et al., 20 08;
Monroy et al., 2006). Furthermore, freezing weather and harsh cli-
mate conditions in winter influence the abundance and ac tivit y of
food resources that can make it challenging for organisms to obtain
sufficient amounts. For example, the activity of invertebrates is highly
temperature-dependent, and insect flight activity declines dramati-
cally as the ambient temperature drops (Churchfield et al., 2012;
Hope et al., 2014). In addition, a previous study showed that al-
though earthworms were present in the soil profile in winter, their
numbers and activity were sharply reduced (Khanam et al., 2016). In
the case of snow cover and frozen soils, earthworms become dehy-
drated and hibernate (Churchfield et al., 2012). Randolph (1973) and
Rozen (1988) also found that ear thworm biomass clearly decreases
from summer to winter. Moreover, McCay and Storm (1997) found
that earthworms and other arthropods were more abundant in irri-
gated plots during both spring and autumn, suggesting greater avail-
ability of certain foods. Thus, earthworms may not be sufficiently
abundant and available especially in winter. These findings strongly
supported our results with respect to decreases in the proportions
and numbers of earthworms consumed by A. squamipes during win-
ter (Figure 1b). With their large surface-area-to-volume ratios, short
fasting endurance, and high metabolic rates, nonhibernating shrews
need adequate food intake for maintaining endothermy and meeting
high-energy requirements at low temperatures (Brown et al., 2014;
Churchfield et al., 2010, 2012). The increased consumption of rela-
tively unpalatable and unprofitable prey, such as Deroceras laeve
and Camponotus thadeus, in winter (Table 2 and Table S1) suggests
that shrews are less preferential in winter than in summer, which
is consistent with previous findings (Churchfield et al., 2012). Thus,
the Chinese mole shrew selectively shifts its dietary preference
throughout the year to adapt to seasonal foods resource availability.
FIGURE 6 Relative abundance of the
top 10 plant food items at the species
level based on the rbcL metabarcoding
assay
Relative abundance (%)
020406080 100
Sp1306a
Sp1306b
Sp1306c
Sp1309a
Sp1309b
Sp1309c
Su1560a
Su1560b
Su1560c
Su1568a
Su1568b
Su1568c
A1003a
A1003b
A1003c
A1011a
A1011b
A1011c
W1286a
W1286b
W1286c
W1287a
W1287b
W1287c
Arachis.hypogaea
Chikusichloa.aquatica
Cinnamomum.glaucescens
Cerastium.glomeratum
Nestegis.apetala
Camptotheca.acuminata
Stellaria.media
Phytolacca.americana
Oryza.sativa
Carex.aneurocarpa
Gladiolus.x.gandavensis
Withania.frutescens
Musa.laterita
Cinnamomum.bodinieri
Mazus.reptans
Lactuca.sativa
Galinsoga.parviflora
Isopyrum.biternatum
Prunus.mongolica
Uncarina.grandidieri
Others
Spring SummerAutumn Winter
|
385
TANG eT Al.
4.3 | Crop impacts due to Chinese mole shrews
Both plant and animal foods were detected in our study, indicating
that A. squamipes may be an omnivorous generalist. No significant
differences were detected in the numbers and alpha diversity of
plant food items between the seasons (Figures 1a and 2), indicating
that the availability of plant-derived foods were balanced through-
out the year. This opportunistic forager supplemented its diet with
plant material, especially grains, in time of food shortages during
winter when invertebrate preys are scarce (Figure 1 and Table 1).
The Chinese mole shrew opts to feed on cultivated crops or stored
grains (such as peanuts and rice) more often during autumn and
winter because of the lack of more preferred prey, especially in win-
ter (Figures 4b and 6). The reason for the abundance and high FOs of
peanuts and rice in the diet may ver y well be their continued avail-
ability during autumn and winter. In southwest China, peanuts and
rice are harvested during autumn. They are the staple food grains and
stored for usage throughout the year. In addition, balsam pear and
lettuce have been detected in the diet, suggesting that the Chinese
mole shrew may cause damage to common vegetables in rural com-
munities. Plant materials were also detected in the diet of several
shrew species, such as armored shrew (Churchfield et al., 2010), and
Southern shor t-tailed shrew (Sylvester et al., 2012), and Asian musk
shrew (Brown et al., 2014). However, very few studies have repor ted
FIGURE 7 Animal (a) and plant (b) food
items at taxonomic levels significantly
differentiated between seasons as
determined by linear discriminatory
analysis (LDA) effect size (LEfSe). LDA
scores were interpreted as the degree of
difference in relative abundance
Animal food items
Plant food items
(a)
(b)
386
|
TAN G eT Al.
TABLE 3 The statistics of the top 10 plant taxa at the family level in the Chinese mole shrew diet throughout the year
Season Spring Summer Autumn Winter Yearly
Tax a
NO. of
Occur.
(N = 6)
FO
(N = 6)
Relative
abundance
NO. of
Occur.
(N = 6)
FO
(N = 6)
Relative
abundance
NO. of
Occur.
(N = 6)
FO
(N = 6)
Relative
abundance
NO. of
Occur.
(N = 6)
FO
(N = 6)
Relative
abundance
NO. of Occur.
(N = 24)
FO
(N = 24)
Fabaceae 467% 0.111 583% 0.100 610 0% 0.060 610 0% 0.572 21 88%
Poaceae 467% 0.080 117% 0.001 6100% 0.530 610 0% 0.070 17 71%
Caryophyllaceae 6100% 0.315 117 % <0.001 00% 0.000 583% 0.064 12 50%
Lauraceae 6100 % 0.267 350% <0.001 583% 0.016 6100% 0.074 20 83%
Oleaceae 233% <0.001 467% 0.198 233% <0.001 467% 0.006 12 50%
Asteraceae 350% 0.009 583% 0.10 6 467% 0.009 583% 0.053 17 71%
Nyssaceae 467% <0.001 610 0% 0.104 583% 0.069 467% 0.001 19 79%
Phytolaccaceae 00% 0.000 00% 0.000 583% 0.168 117% <0.001 625%
Iridaceae 117% <0.001 00% 0.000 00% 0.000 117% <0.001 28%
Solanaceae 233% <0.001 233% 0.076 467% <0.001 117% 0.039 938%
Cyperaceae 117% 0.016 350% 0.080 00% 0.000 117% <0.001 521%
Bold values denote relative abundance of plant taxa >0.10.
Abbreviations: FO, Frequency of occur rence; No. of Occur., Number of occurrence.
|
387
TANG eT Al.
that shrews can cause damage to and contamination in grains. In this
study, the proportional increase in crops eaten in autumn and winter
suggests that the Chinese mole shrew poses a threat to crop pro-
duction and grain stores (Figure 4b), especially in rice-based farm-
ing systems. They have a vast geographic range, occupying a wide
range of elevations from 300 to 4,000 m and latitudes from 18°N
to 35°N (He et al., 2016; Motakawa et al., 2003). The Chinese mole
shrews are abundant especially in Southwest China (He et al., 2016;
Motakawa et al., 2003; Song et al., 20 07), and the number of them
showed an increasing trend in the study area (Liao et al., 2005; Zong
et al., 2017). As a result, there may be potential negative impacts on
agricultural production and people's health due to consumption and
contamination of crops (Oyafuso, 2015). Therefore, development of
methods to control the shrew populations on farmlands is necessar y,
and dietary analysis of A. squamipes can contribute to devising suit-
able poison baits.
Over 100 plant species were identified in stomach content of
A. squamipes. Some of these may have been secondarily ingested via
consumption of many large earthworms as reported by Churchfield
et al. (2010). For A. squamipes, this dietary diversity may be a com-
pensatory strategy to meet its high-energy requirements by exploit-
ing a wider variety of plant food items. However, a previous study
also demonstrated that plant material (seeds or foliage) constitutes a
smaller proportion of the overall shrew diet (Churchfield et al., 2012)
as a result of missing data from highly digested plant foods. Thus,
further investigation of shrew diet with higher taxonomic resolution
is required to better understand the food composition of the species
and determine their actual impact.
In summary, we found that A. squamipes has a diverse diet com-
prising a range of invertebrates and plant material. The single most
important prey item, whether in terms of FOs, dietary composition
or volume contribution, was earthworms. We revealed that the diet
of this shrew contains a much higher prevalence and diversity of
earthworms than previously known. We also found that plant ma-
terials (such as rice and peanuts) were consumed more frequently
during the harvest season, implying that the Chinese mole shrew is
omnivorous and play a pest role, despite being taxonomically classi-
fied as an insectivore. Therefore, the Chinese mole shrew is capable
of shifting its dietary preferences to adapt to seasonal fluctuations
of food resources, particularly during winter when the diversity and
abundance of invertebrates are lowest. Characterizing the diet of
A. squamipes may have implications for the evaluating crop impacts
and control of this shrew species.
ACKNOWLEDGEMENTS
This research was funded by the National Natural Science Foundation
of China (31670388, 32001223, 32070424) and a Chengdu Municipal
Science and Technology Bureau project (2015-NY02–00369-NC)
as well as suppor ted by the Starting Research Fund from Sichuan
Normal Universit y (024341965).
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
AUTHOR CONTRIBUTION
Keyi Tang: Conceptualization (lead); Data curation (lead); Formal
analysis (lead); Investigation (lead); Methodology (lead); Resources
(lead); Software (lead); Writing-original draft (lead); Writing-review
& editing (equal). Fei Xie: Investigation (equal); Resources (equal).
Hongyi Liu: Data curation (supporting); Formal analysis (equal);
Software (equal); Writing-review & editing (equal). Ying-ting Pu:
Data curation (equal); Formal analysis (suppor ting); Software (equal);
Visualization (equal); Writing-original draft (equal). Dan Chen:
Investigation (equal); Methodolog y (equal); Resources (equal). Boxin
Qin: Investigation (equal); Methodolog y (equal); Resources (equal);
Validation (equal). Changkun Fu: Data curation (equal); Formal anal-
ysis (equal); Investigation (equal); Resources (equal). Qiong Wang:
Data curation (equal); Investigation (equal); Resources (equal);
Software (equal). Shunde Chen: Con ceptualization (lead); Data cura-
tion (equal); Funding acquisition (lead); Project administration (lead);
Supervision (lead); Validation (lead); Visualization (lead); Writing-
original draf t (supporting); Writing-review & editing (lead). Ke-ji Guo:
Data curation (equal); Formal analysis (suppor ting); Software (equal);
Supervision (equal); Visualization (equal); Writing-review & editing
(equal).
DATA AVAILAB ILITY STATE MEN T
DNA sequences in this study were deposited into the NCBI Sequence
Read Archive (SRA) under accession number: PRJNA637184.
(https://w ww.ncbi.nlm.nih.gov/).
ORCID
Ke-yi Tang https://orcid.org/0000-0003-4885-8260
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section.
How to cite this article: Tang K-Y, Xie F, Liu H-Y, et al. DNA
metabarcoding provides insights into seasonal diet variations
in Chinese mole shrew (Anourosorex squamipes) with potential
implications for evaluating crop impac ts. Ecol Evol.
2021;11:376–389. https://doi.org/10.1002/ece3.7055
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