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RESEARCH
Open Access
The gut of the finch: uniqueness of the gut
microbiome of the Galápagos vampire
finch
Alice J. Michel
1
, Lewis M. Ward
1
, Shana K. Goffredi
2
, Katherine S. Dawson
1,8
, Daniel T. Baldassarre
3
, Alec Brenner
1
,
Kiyoko M. Gotanda
4
, John E. McCormack
2
, Sean W. Mullin
1
, Ariel O
Neill
1
, Gabrielle S. Tender
1
, J. Albert C. Uy
5
,
Kristie Yu
1
, Victoria J. Orphan
1*
and Jaime A. Chaves
6,7*
Abstract
Background:
Darwin
s finches are a clade of 19 species of passerine birds native to the Galápagos Islands, whose
biogeography, specialized beak morphologies, and dietary choices
ranging from seeds to blood
make them a
classic example of adaptive radiation. While these iconic birds have been intensely studied, the composition of their
gut microbiome and the factors influencing it, including host species, diet, and biogeography, has not yet been
explored.
Results:
We characterized the microbial community associated with 12 species of Darwin
s finches using high-
throughput 16S rRNA sequencing of fecal s
amples from 114 individual
s across nine islands, including the unusual blood-
feeding vampire finch (
Geospiza septentrionalis
) from Darwin and Wolf Islands. The phylum-level core gut microbiome for
Darwin
s finches included the Firmicutes, Gammaproteobacteria
, and Actinobacteria, with members of the Bacteroidetes
at conspicuously low abundance. The gut microbiome wa
s surprisingly well conserved across the diversity of
finch species, with one exception
the vampire finch
which harbored bacteria that were either absent or
extremely rare in other finches, including
Fusobacterium
,
Cetobacterium
,
Ureaplasma
,
Mucispirillum
,
Campylobacter
,and
various members of the Clostridia
bacteria known from the guts of carnivorous birds and reptiles. Complementary
stable isotope analysis of feathers revealed exceptionally high
δ
15
N isotope values in the vampire finch, resembling top
marine predators. The Galápagos archipelago is also known for extreme wet and dry seasons, and we observed a
significant seasonal shift in the gut microbial community of five additional finch species sampled during both seasons.
Conclusions:
This study demonstrates the overall conservatism of the finch gut microbiome over short (< 1 Ma)
divergence timescales, except in the most extreme case o
f dietary specialization, an
d elevates the evolutionary
importance of seasonal shifts in driving not only speci
es adaptation, but also gut m
icrobiome composition.
Keywords:
Galápagos Islands, Darwin
s finches, Blood-feeding, Microbiome,
Geospiza
Background
First introduced to science by Charles Darwin in his
diaries from the voyage of the HMS Beagle [
1], Darwin
s
finches are a classic example of adaptive radiation, the
phenomenon by which species diverge from a common
ancestor as they adapt to different ecological niches. Di-
vergence of Darwin
s finches from relatives in South
America has taken place in the last ~ 1.5 My, when an-
cestral finches first colonized the islands [
2]. Since this
time, 19 formally recognized finch species have evolved
within this clade
18 in the Galápagos and 1 in the
Cocos Islands [
3, 4], with major radiations occurring in
the last 300 ka [
5, 6].
The ecological drivers of adaptation in Darwin
sfinches
have been extensively studied [
3, 7
9]. Diet has long been
recognized as a major factor in the adaptive radiation of
* Correspondence:
vorphan@gps.caltech.edu
; jachaves@usfq.edu.ec
Alice J. Michel and Lewis M. Ward contributed equally to this work.
1
Division of Geological and Planetary Sciences, California Institute of
Technology, Pasadena, CA 91125, USA
6
Colegio de Ciencias Biológicas y Ambientales, Universidad San Francisco de
Quito, Diego de Robles y Pampite, Quito, Ecuador
Full list of author information is available at the end of the article
© The Author(s). 2018
Open Access
This article is 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 you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/
) applies to the data made available in this article, unless otherwise stated.
Michel
et al. Microbiome
(2018) 6:167
https://doi.org/10.1186/s40168-018-0555-8
finches in the Galápagos, as lineages on different islands
developed beaks specialized for food sources available on
their islands, with selective pressure especially high during
the dry season when food sources are most limited [
10].
Darwin
s finches, as a group, include plant-matter con-
sumers
the seed-eating ground finches (
Geospiza fortis
,
G
.
fuliginosa
,
G
.
magnirostris
), herbivorous vegetarian
finch (
Platyspiza crassirostris
),
Opuntia
nectar- and
pollen-specializing cactus finches (
Geospiza scandens
,
G
.
conirostris
)
and insectivorous finches
the sharp-beaked
ground finches (
G
.
difficilis
,
G
.
acutirostris
), woodpecker
(
Camarhynchus pallidus
), tree finch (
C
.
parvulus
), warbler
finches (
Certhidia olivacea
,
C
.
fusca
)[3]
and the curious
blood-feeding vampire finches of Darwin and Wolf Islands
(
Geospiza septentrionalis
)[4, 11, 12]. The vampire finches
are so called because they supplement their diet with
blood harvested from Nazca and red-footed boobies (
Sula
granti
and
S
.
sula
) during the dry season when resources
are scarce on these remote islands, a unique strategy not
used by any other species [
13
15].
Growing evidence suggests that gut microbes exert a
major influence on animal nutrition, health, immunity,
and behavior [
16
18]. Reciprocally, large-scale sequen-
cing of 16S rRNA of associated gut bacteria and archaea
has revealed that gut microbial diversity can be influ-
enced by diet, host morphology, host phylogeny, or en-
vironment [
16,
19, 20].
Comparisons between gut
communities of closely related vertebrates with different
diets have yielded a range of patterns. Diet has been
shown to be a strong predictor of gut microbial compos-
ition in phyllostomid bats [
21, 22], dolphins [
23],
humans [
24], and phylogenetically diverse ant-eating
mammals [
25]. On the other hand, links between host
phylogeny and gut microbiota have been reported from
bird species [
26] and in selected mammals, such as
bears, where despite their restricted bamboo diet, pandas
maintain a microbiome similar to other bears [
27].
Other studies have identified a combination of determin-
ing factors, including phylogeny and diet (e.g., in some
birds and baleen whales [
28
30]) and biogeography and
diet (e.g., in recently radiated African cichlids and Gal-
ápagos iguanas [
31
33]). Crucially, studies have focused
on clades that diverged tens of millions of years ago,
whereas less is known about the degree of gut micro-
biome divergence in younger, relatively recent species
radiations.
Recent studies in birds have generated important data re-
garding the composition of the gut microbiome, yet there
remains an enormous gap in knowledge of most bird line-
ages [
20], particularly non-domesticated species [
34]. The
gut microbiome of the domestic chicken [
17, 35], folivorous
hoatzin [
36], various seabirds including penguin [
37]and
petrel [
38], and Passeriformes (the order that includes
Darwin
sfinches)[
39] are all dominated by members of the
Proteobacteria, Firmicutes, Ba
cteroidetes, and Actinobac-
teria, as well as other, low-abundance phyla. A variety of
factors influence the gut microbiome, but the role of diet
and environment are thought to be of paramount import-
ance in birds [
20], even when controlling for other factors,
as shown in fledglings of the brood parasite (
Molothrus
ater
), a cowbird that lays its eggs in the nests of heterospe-
cific hosts [
40].
Here, we analyzed the gut microbiota of 12 species of
Darwin
s finches, sampled across 9 of the 18 Galápagos
Islands during both the wet and dry seasons. During the
wet season, from January to early June, food is abundant,
while food becomes limiting and the birds resort to their
adaptive
diet in the dry season, from July to December
[10]. Fecal samples were collected from 114 individual
finches, including the two remote populations of vam-
pire finch (
Geospiza septentrionalis
) on Darwin and
Wolf Islands. Microbial diversity was screened using
Illumina high-throughput sequencing of the 16S rRNA
gene. A complementary analysis of stable carbon and ni-
trogen isotopes of feathers from vampire finches and a
subset of others was also performed as an independent
means
to identify dietary signatures. The diversity in diet
and lifestyle of Darwin
s finches and the extensive eco-
logical and genetic knowledge of this avian group make
them an attractive study subject to examine how host
diet, biogeography, phylogeny, and other environmental
factors affect the vertebrate gut microbial community
over relatively short evolutionary timescales.
Results and discussion
The core gut microbiome of Darwin
s finches
The 16S rRNA gene diversity of the gut microbiome of
Darwin
s finches was characterized from fecal samples
collected from 114 finches, representing 12 of 19 species
distributed across nine of the Galápagos Islands (Fig.
1;
Table
1; note that as a singleton, sequence data from the
one specimen of C. pallida was removed from subse-
quent analysis). This dataset yielded an average of
25,382 16S rRNA sequences per finch and collectively
comprised 297 unique bacterial ribotypes (OTUs; 97%
similarity level), present above a minimum threshold set
at 1% relative abundance in at least one finch in the
dataset. These OTUs primarily grouped within three
major bacterial phyla: Firmicutes, Proteobacteria, and
Actinobacteria (Fig.
2; Additional file
1: Table S1). The
number of OTUs recovered from each species was 71.6
± 9.8 (avg ± SD; Additional file
2: Table S2). Shannon di-
versity indices for the Galápagos finch microbiome data-
set ranged between 1.4 and 2.2 per finch species
(Additional file
2: Table S2), within the range of diversity
indices from other avian species (0.6
3.4) [
26].
The analysis of the core microbiome, consisting of
OTUs that were universally shared among the 113
Michel
et al. Microbiome
(2018) 6:167
Page 2 of 14
finches (after removal of the single
C
.
pallida
), revealed
a dominance of Firmicutes (50% average relative abun-
dance, by 16S rRNA recovery), Proteobacteria (40%),
and Actinobacteria (8%; Fig.
2; Additional file
3: Figure
S1). This pattern has striking resemblance to other avian
microbiomes, where surveys of diverse Neotropical birds
recovered a similar number of phylotypes (~ 201 bacter-
ial OTUs) belonging to the same three phyla (Proteobac-
teria, Firmicutes, and Actinobacteria, average relative
abundances 46%, 37%, and 1%, respectively) [
28]. Two
specific OTUs present in every finch included represen-
tatives of
Enterobacter
(17% average abundance; Gam-
maproteobacteria) and
Enterococcus
(13% average
abundance; Bacilli). A
Shigella-
affiliated OTU (8% aver-
age abundance; Gammaproteobacteria) was also detected
in all but one finch from Darwin Island.
Representatives from the Bacteroidetes and five add-
itional phyla (Chloroflexi, Cyanobacteria, Deferribacteres,
Fusobacteria, and Tenericutes) were detected, but oc-
curred in only a subset of the finches (typically at < 1%
average abundance; Fig.
2;Additionalfile
1: Table S1).
While polysaccharide-degrading Bacteroidetes represent a
significant component of the gut microbiome in many
vertebrates [
16, 38, 41, 42], they were rare, or not detected,
in Darwins
finches (< 0.5% on average in 23/113 finches,
and absent in 88 finches), with the exception of 2 finch in-
dividuals (
C
.
parvulus
, San Cristóbal, wet season and
G
.
conirostris
, Genovesa, dry season; Bacteroidetes at 12%
and 26%, respectively). Decreased Bacteroidetes presence
has been previously observed in birds; however, the previ-
ously reported relative abundance of 3
20% is comparably
higher than the vast majority of the finches analyzed in
Fig. 1
Overview of finch gut microbiome samples collected from the Galápagos Islands. Pie charts represent the number of fecal samples from
each species of finch from the different islands colored accordin
g to the legend. Cladogram of host finch phylogeny modified from
Lamichhaney et al. [
5]
Michel
et al. Microbiome
(2018) 6:167
Page 3 of 14
this study [
20, 39, 40]. Even finches specializing in a
carbohydrate-rich diet (e.g., the vegetarian and cactus
finches,
P
.
crassirostris
and
G
.
scandens
)didnotshowa
positive relationship with this bacterial phylum, with only
one
P
.
crassirostris
and one
G
.
scandens
harboring Bacter-
oidetes OTUs, < 0.02% total microbiota. It is currently un-
known which bacterial lineages may occupy a similar
niche as Bacteroidetes in the finch microbiome.
Uniqueness of the vampire finch microbiome
Perhaps the most extreme nutritional strategy among
Darwin
s finches is that of the vampire finch,
Geospiza
septentrionalis
, endemic to the remote northernmost
islands of Darwin and Wolf. During the dry season on
the Galápagos Islands, when samples for this study were
collected, vampire finches feed primarily on the blood
and eggs of boobies (
Sula
spp.; Fig.
1, inset), as well as
partially digested fish regurgitate and guano (D. Baldas-
sarre, personal observation, see Additional file
4 for in-
formation on finch feeding behavior). By contrast,
finches on other islands consume plant-based foods and
insects throughout the year [
10, 15]. Indeed, the stable
isotope values of feathers collected from vampire finches
during the dry season were notably enriched in
nitrogen-15, with
δ
15
N values between + 14.2 and +
25.1
, significantly different from the two ground finch
species,
G
.
fortis
and
G
.
fuliginosa
,
δ
15
N = + 4.9 and +
11.7
, respectively; ANOVA
p
< 0.0001; Fig.
3a.The
Table 1
Finch species sampled in this study, including information about general diet category, sampling island, and number of
specimens collected during each of the dry and wet season
Species
Common name
Diet category
Island
No. samples (season)
Dry
Wet
G
.
septentrionalis
Vampire finch
Carnivorous
2
, inc. blood
Wolf
16
0
Darwin
15
0
G
.
acutirostris
Sharp-beaked ground finch
Insectivorous
3
Genovesa
6
0
G
.
difficilis
Sharp-beaked ground finch
Insectivorous
3
Pinta
3
0
G
.
fortis
Medium ground finch
Herbivorous (seeds)
4
San Cristóbal
1
5
Santa Cruz
3
1
Santa Fé
0
1
G
.
fuliginosa
Small ground finch
Herbivorous (seeds)
4
Española
0
5
North Seymour
0
1
San Cristóbal
7
8
Santa Cruz
4
1
Santa Fé
0
1
G
.
magnirostris
Large ground finch
Herbivorous (seeds)
4
Genovesa
2
0
Pinta
3
0
G
.
conirostris
Large cactus finch
Herbivorous (
Opuntia
cactus)
Española
0
2
Genovesa
2
0
G
.
scandens
Cactus finch
Herbivorous (
Opuntia
cactus)
Santa Cruz
3
1
Santa Fé
0
2
Ce
.
olivacea
Green warbler finch
Insectivorous
Española
0
4
Pinta
3
0
C
.
parvulus
Small tree finch
Insectivorous
San Cristóbal
4
2
Santa Cruz
0
4
C
.
pallida
1
Woodpecker Finch
Insectivorous
Santa Cruz
0
1
P
.
crassirostris
Vegetarian finch
Herbivorous (leaves)
Santa Cruz
0
3
Total
12 Species
9 Islands
72
42
Genus abbreviations:
C = Camarhynchus
,
Ce = Certhidia
,
G = Geospiza
,
P = Platyspiza
1
The solitary
C
.
pallida
sample was excluded from statistical analyses
2
During the dry season,
G
.
septentrionalis
eats blood, eggs, scat of the Nazca boobies
3
Although these finch species are classically identified as seed-eaters, behavioral observations at the time of collection indicated that
G
.
difficilis
on Pinta was
foraging on 100% insects in both wet and dry seasons, and
G
.
acutirostris
on Genovesa fed 98% on insects in the wet season, and 80% on insects in the
dry season
4
While these finches predominantly eat seeds, they consume insects on occasion
Michel
et al. Microbiome
(2018) 6:167
Page 4 of 14
positively shifted
δ
15
N values of the vampire finches are
more similar to marine carnivores, including sea lions and
polar bears, than to other avian species [
43
45]. The dis-
tinct
δ
15
Nvaluesfor
G
.
septentrionalis
, consistent with a
high trophic-level marine-feeding animal, are likely the
outcome of the consumption of proteins sourced predom-
inantly from Nazca boobies, which eat fish. Alternatively,
a trend towards positive nitrogen stable isotope ratios has
also been shown to occur under starvation conditions, as-
sociated with the breakdown of muscle proteins [
46].
Given the extreme conditions during the dry season on
Darwin and Wolf Islands, this cannot be ruled out as a
contributing factor; however, the vampire finches collected
in this study had consistently high keel fat content and
weights between the wet and dry seasons (J. Chaves, per-
sonal observation), suggesting that their distinct isotopic
composition is not likely due to nutritional deprivation.
While there appears to be strong conservation in the
core gut microbiome diversity among Darwin
s finches
overall (analysis of similarities (ANOSIM)
p
> 0.05; Fig.
4;
Additional file
5: Table S3), vampire finches of Darwin
and Wolf Islands are a notable exception (Fig.
4a).
Non-metric multidimensional scaling (NMDS) ordin-
ation and ANOSIM of Bray-Curtis and UniFrac dissimi-
larities revealed that the gut microbiomes of the vampire
finches clustered separately from all other finches
(ANOSIM R 0.23
0.38,
p
= 0.002, Fig.
3b isotope
finches, 4A full dataset; Additional file
6: Figure S2A dry
season; Additional file
5: Table S3). Notably, the micro-
biome of the vampire finch,
Geospiza septentrionalis
,is
also dissimilar from the other closely related sharp-beaked
ground finches on other islands (
Geospiza difficilis sensu
lato
:
G
.
difficilis sensu stricto
on Pinta and
G
.
acutirostris
on Genovesa Island; ANOSIM R = 0.57,
p
=0.001;
Additional file
5: Table S3, Additional file
6: Figure S2B).
Carnivorous diets have been shown to be linked to gut
microbiome divergence from closely related herbivores
[16, 21, 24, 31, 47]. The divergence of the vampire finch
microbiome from other finches in our study is attributed
to the presence of several lower abundance taxa that were
absent or extremely rare in the other finches (Fig.
5).
These unique taxa included
Fusobacterium
and
Cetobac-
terium
(Fusobacteria; Fusobacteriaceae),
Ureaplasma
(Tenericutes; Mycoplasmataceae),
Mucispirillum
(Deferri-
bacteres; Deferribacteraceae),
Campylobacter
(Epsilonpro-
te
obacteria; Campylobacteraceae), and various members
of the Clostridia (Firmicutes; Clostridiaceae and Peptos-
treptococcaceae). Two Fusobacteria OTUs accounted for
~ 0.75% of the average vampire finch microbiome, but less
than 0.0004% of the relative abundance in the gut
Fig. 2
Average taxonomic composition of gut microbial communities of Darwin
s finches, from Illumina 16S rRNA gene surveys, based on OTU
clustering at 97% identity trimmed to at least 1% relative abundance in at least one finch. Data is grouped by season, island, and species. The
colored bar at the bottom, which also shows sample sizes, distinguishes finch samples from the dry (red) and wet (blue) seasons (Note that for
this analysis, Santa Cruz, and neighboring islands Santa Fé, and North Seymour are grouped together as
Santa Cruz +
). An average of 25,382
reads per finch, comprised of 297 unique OTUs (clustered at 97% similarity level), was recovered at greater than 1% relative abundance in at least
one finch across the dataset
Michel
et al. Microbiome
(2018) 6:167
Page 5 of 14
microbiomes of other finches (Mann-Whitney
U
test
p
=
0.003). Twenty-five Clostridia OTUs were collectively re-
covered from several finch species, but were significantly
more abundant in the vampire finches (1.8% vs 9.2%;
Mann-Whitney
p
= 0.000008). While the specific niche of
Fusobacteria and Clostridia in the gut community is not yet
known, the prevalence of these organisms in the guts of
vultures and alligators [
48, 49] suggests a possible relation-
ship to the unique and specific carnivore-like diet of the
vampire finch.
AB
CD
Fig. 4
Non-metric multidimensional scaling (NMDS) ordination of gut microbial communities of Darwin
s finches according to
a
species,
b
island,
c
diet, and
d
season for all finches in the sample set. Taxonomic (OTU) clustering is at 97% identity and abundance weighted by taking the
fourth-root of the OTU relative abundance in each finch. Ellipses represent 90% confidence windows following a multivariate t-distribution.
ANOSIM
p
values are shown for relationships, including and excluding the vampire finch species
G
.
septentrionalis
AB
Fig. 3
a
δ
15
N vs.
δ
13
C isotopic composition of feathers from Darwin
s finches, specifically the vampire finch (
G
.
septentrionalis
from Wolf Island;
red triangles), medium ground finch (
G
.
fortis
; San Cristóbal Island; green squares), and small ground finch (
G
.
fuliginosa
; San Cristóbal Island;
green circles), in comparison to other animal tissues (grey dots). For the finches, each symbol represents values recovered from one individual
bird. Error bars, where present, indicate the range of values obtained from two to three different feathers from the same individual.
δ
13
C for
ground finch feathers ranged from
24.1 to
18.7
vs. PDB, and from
23.8 to
16.1
vs. PDB for vampire finches.
δ
15
N ranges from + 4.9 to
+ 11.7
for ground finches and between + 14.2 and + 25.1
δ
15
N for vampire finches. The vampire finch feathers differ significantly in
δ
15
N
(ANOVA
p
< 0.0001), but not
δ
13
C (ANOVA
p
> 0.9). Isotopic values from other animals were reported in the following: [
44
46, 78
87].
b
Non-
metric multidimensional scaling (NMDS) ordination of three species of gut microbial communities of Darwin
s finches according to diet.
Taxonomic (OTU) clustering is at 97% identity and abundance weighted by taking the fourth-root of the OTU relative abundance in each finch
Michel
et al. Microbiome
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Page 6 of 14
Additional taxa that were shown to be statistically dif-
ferent in the vampire finch, relative to other finches, in-
cluded an OTU affiliated with the genus
Campylobacter
(Epsilonproteobacteria), at 0.9% average relative abun-
dance versus 0.0005% from other Darwin
s finches
(Mann-Whitney
p
= 0.0002); two
Ureaplasma
(Teneri-
cutes; Mycoplasmataceae) OTUs at 0.54% (10/31 sam-
ples) vs. 0.0001% in the other finches (3/82 samples;
Mann-Whitney
p
= 0.004); and one
Mucispirillum
(Deferribacteraceae) OTU, accounting for 0.7% of the
average vampire finch microbiome (in 17/31 specimens)
versus 0.0002% in others (2/82 samples; Mann-Whitney
p
= 0.000009). The recovered
Campylobacter
OTU was
closely related to
Campylobacter volucris
, isolated from
gulls [
50].
Campylobacter
have been reported in associ-
ation with the gut microbiome of some Passeriformes spe-
cies, as well as other birds, reptiles, and mammals [
29, 41],
and their distribution and inter-species transmission as
pathogens has been well studied [
51].
Ureaplasma
and
Mucispirillum
have also been previously described in as-
sociation with shorebirds [
52], but little is known about
their overall distribution and functional role in the host.
Given their common occurrence in the vampire finches, it
is possible that the acquisition of these lower abundance
genera may be linked to the close association of the vam-
pire finch with the co-occurring Nazca and red-footed
boobies on Darwin and Wolf Islands.
While other exclusively sanguivorous animals, such as
vampire bats and leeches, harbor
Aeromonas
as an obli-
gate gut symbiont [
53, 54], this bacterial taxon was not
recovered from the vampire finches, possibly due to the
non-obligate nature of their blood feeding. However, a
recent investigation of the microbiome of the common
vampire bat (
Desmodus rotundus
) found only very low
abundances of
Aeromonas
sp. (< 0.2%), indicating that
Aeromonas
might not be essential for obligate sanguiv-
ory, as previously presumed [
22].
Blood feeding in Darwin
s finches was highly distinct
from the other diet categories of insectivory and
plant-based diets (seeds,
Opuntia
cactus nectar, and
leaves) (ANOSIM
R
= 0.23,
p
= 0.001; Fig.
4b). The statis-
tical significance of this difference was maintained after
accounting for the larger vampire finch sample size
(ANOSIM
R
= 0.38,
p
= 0.003), sampling only in the dry
season (ANOSIM
R
= 0.34,
p
= 0.001), as well as focusing
specifically on a taxonomically narrow group of finches
Fig. 5
Heatmap showing the weighted average taxonomic composition of the gut microbial communities of Darwin
s finches grouped by
season and species. Color represents the deviation from average compositional abundance of bacterial taxa, with 0% compositional abundance
as white, average as light blue, below average as paler blue, and above average in dark blue (key at left). The bottom colored bar distinguishes
finch samples collected during the dry (red) and wet (blue) seasons, with the sample size indicated for each finch group. Dendrograms group
finches and bacterial taxa based on Euclidean distances of the compositional abundance matrix. Note the extreme deviation in the vampire
finches: the average vampire finch has 15
17 times more Deferribacteres, Fusobacteria, Tenericutes, Negativicutes, and Epsilonproteobacteria than
the average Galápagos finch overall
Michel
et al. Microbiome
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Page 7 of 14
(previously named
G
.
difficilis
), of which the vampires
belong (ANOSIM
R
= 0.57,
p
= 0.001). In the full dataset,
the distinctiveness of the vampire finch species (ANOSIM
R
=0.23,
p
= 0.001) accounts for nearly all of the variation
attributable to spec
ies differences (
R
= 0.27,
p
= 0.001;
Fig. 4a; Additional file
5: Table S3), with species differences
absent when the vampire finches are omitted (ANOSIM
R
=0,
p
= 0.5). By contrast, no discernable difference between
insectivory versus plant-based diets was detected in either
the dry season (ANOSIM
R
=0.02,
p
=0.3)orthewetsea-
son (
R
= 0.01,
p
= 0.4), or when considering the full dataset,
minus vampire finches (
R
=0,
p
= 0.9; Additional file
5:
Table S3).
Geographic isolation, host phylogeny, and unusual diet
are all possible factors influencing the composition of
the gut microbial community in the vampire finch. Darwin
and Wolf Islands are geographically isolated from the
other islands of the Galápagos (~ 300 km from Santa Cruz,
more than double the distance between all other Galápa-
gos Islands), thus limiting genetic exchange with finches
within the archipelago [
4]. Targeted comparison of the gut
microbiome of vampire finches from either Darwin or
Wolf Island, separated by a mere 40 km, found them to
differ significantly from each other (ANOSIM
R
=0.16,
p
= 0.001; Additional file
6: Figure S2C, Additional file
5:
Table S3), due to differences in the abundance of minor
taxa (Figs.
2 and 5). This suggests that geographic separ-
ation may be a factor influencing the gut microbiome.
However, preliminary analysis of a more limited sample
set across the remaining islands did not identify a trend.
For example, an analysis of
G
.
fuliginosa
and
G
.
fortis
did
not reveal a strong divergence across the islands of San
Cristóbal and Santa Cruz (~ 65 km apart; ANOSIM
R
=
0.06,
p
= 0.15). Deeper sampling of individual species
across islands will be necessary to more confidently exam-
ine the potential role of island biogeography.
Additionally, finch phylogeny appeared insignificant
for the deepest-sampled non-vampire finches (
G
.
fuligi-
nosa
and
G. fortis
,
n
=
11 each; ANOSIM
R
=0,
p
= 0.48),
suggesting that host phylogeny is not likely a major in-
fluence on the gut microbiome of Darwin
s finches. Add-
itional sampling from more distantly related and
under-sampled species is necessary to thoroughly clarify
the role of phylogeny. Nevertheless, of these potential
variables examined, a blood-feeding diet of
Geospiza sep-
tentrionalis
, independently supported by isotopic evi-
dence, appears to be the significant contributor to the
unique diversity of the gut microbiome.
Season has a significant influence on the gut microbiome
of Darwin
s finches
Investigations of Darwin
s finches provided a unique op-
portunity to compare the influence of extreme seasonal
shifts on the gut microbiome. The Galápagos Islands are
well known for strong seasonal cycles driving the avail-
ability of vegetation and other food sources utilized by
Darwin
s finches, and it is during the dry season that
natural selection for unique feeding adaptations is most
intense [
3, 55]. It is thought that food limitation during
the dry season induces specialization and speciation of
the various finch species, as species adapt to recalcitrant
resources such as
Tribulus
seeds, a process documented
in Darwin
s ground finches especially during extreme El
Niño events [
3, 10]. Plant growth and seed production
occurs during the wet season (January to June), which is
also associated with an increase in arthropod abundance
[7]. During the dry season, however, finches undergo
significant dietary stress as plant-based food sources
become limiting [
55] and it is then that speciation be-
comes observable [
3]. Season has been identified as a
significant driver of gut microbiome divergence in other
animals, including alligators, which engage in protracted
fasting during the winter [
48]. Whether the microbiome
of Darwin
s finches responds to seasonal extremes on
the Galápagos Islands is an outstanding question.
Using our dataset consisting of 72 individuals from the
dry season and 41 from the wet season, we found season
to be significantly associated with differences in micro-
bial composition. Statistical differences in the finch gut
microbiome between the wet and dry seasons occurred
across the entire dataset (ANOSIM
R
= 0.27,
p
= 0.001;
Fig. 4d), as well as when vampire finches were excluded
(ANOSIM
R
= 0.17,
p
= 0.001). Seasonal differentiation
was also detected strictly within the two finch species
sampled during both seasons (
G
.
fuliginosa
and
G
.
fortis
)
(ANOSIM
R
= 0.28,
p
= 0.001; Additional file
6: Figure
S2D, Additional file
5: Table S3). Members of the Gam-
maproteobacteria were the primary driver of the vari-
ation in finch microbiome during the dry season
(compositional abundance 48% in dry vs. 21% in wet,
Mann-Whitney
U
test
p
= 0.01), while Bacilli were dom-
inant during the wet season (35% in dry vs. 65% in wet,
Mann-Whitney
p
= 0.00008) (Fig.
2). Higher relative
abundances of Actinobacteria were also documented in
the wet season (6% vs. 12%), although this difference
was not significant (Mann-Whitney
p
= 0.5).
A significant shift in the finch gut microbial community
wi
th season may reflect temporal shifts in response to
changes in weather, food resource availability, or other fac-
tors. The seasonal variation in the gut microbiome among
all granivorous/herbivorous finches (
G
.
magnirostris
,
G
.
for-
tis
,
G
.
fuliginosa
,
G
.
scandens
,
G
.
conirostris
,
P
.
crassirostris
)
supports the ecological observations of finches opti-
mizing their feeding strategies by season (ANOSIM
R
= 0.21,
p
=0.001;
n
= 50). Omnivorous/insectivorous
finches (
G
.
difficilis
,
G
.
acutirostris
,
C
.
parvulus
,and
Ce
.
olivacea
), on the other hand, were not signifi-
cantly different by season (ANOSIM
R
= 0.08,
p
=0.2;
Michel
et al. Microbiome
(2018) 6:167
Page 8 of 14
n
= 20), consistent with lesser shifts in types of food-
stuffs between seasons. It is conceivable, however,
that either hormonal or ecological changes coincident
with season cause the microbiome to shift in com-
position, irrespective of diet. For instance, when the
finches nest during the wet season, they may come
into contact with different microorganisms, such as
those carried by the invasive nest parasite,
Philornis
downsii
[56]. Firmicutes and Proteobacteria were re-
cently reported as the dominant taxa in the micro-
biome of
P
.
downsii
on Santa Cruz Island [
57].
A preliminary assessment of diet specialization in a
limited number of these finches (broadly categorized as
granivory, omnivory, insectivory, nectarivory, and her-
bivory) revealed no obvious relationship with the gut
microbiome, despite well-documented shifts in finch
beak morphology and behavior. Other bird species have
shown similar patterns, where only the most extreme di-
ets (e.g., vultures, and other scavengers) resulted in a
marked difference in the microbiome [
20]. Most of
Darwin
s finch species have remained omnivorous, with
an emphasis on seeds; thus, the influence of diet
specialization on the gut microbiome in these species is
expected to be minor compared to other animal groups
where more extreme dietary specialization has led to shifts
in composition (e.g., phyllostomid bats [
21, 22] and cichlid
fishes [
31]). Additional sampling
particularly of the
seed-specializing ground finches,
G
.
fuliginosa
,
G
.
fortis
,
and
G
.
magnirostris
, which are distributed across multiple
islands, as well as dietary specialists such as the vegetarian
P
.
crassirostris
and insectivorous
Ce
.
olivacea
may reveal
more subtle trends driving gut microbiome composition.
Conclusions
Darwin
s finches have long been recognized as a model
system for investigating interactions between biogeog-
raphy, dietary specialization, morphology, and other as-
pects of evolutionary radiation. To this list can now be
added the interaction of these factors with the compos-
ition of the gut microbial community. The radiation of
Darwin
s finches has occurred relatively rapidly and re-
cently (~ 300 ka). During this time, it appears that the
gut microbial composition of most finch species has
remained conserved among the majority of species ex-
amined in this study. A conspicuous exception to this
conservation occurs in the case of the vampire finch,
Geospiza septentrionalis
, with a microbial community
significantly distinct from the other finches. Because of
the barrenness of Darwin and Wolf Islands, the vampire
finches undergo extreme dietary limitations during the
dry season, thus leading to blood feeding [
15]. Their
unique and carnivore-like diet likely contributes to the
distinct gut microbial composition, suggesting that cases
of extreme dietary divergence can overpower phylogenetic
inertia to drive shifts in microbiome composition, even over
short evolutionary timescales. F
uture studies in other recent
avian radiations that include dietary extremes, such as the
blood-feeding Galápag
os Hood mockingbird (
Mimus mac-
donaldi
), should help with invest
igating this hypothesis.
A robust seasonal difference was observed between
finches collected during the wet and dry seasons and
may reflect temporal shifts in response to changes in
food resource availability, weather, mating or nesting, or
other factors. Further sampling
particularly resampling
individual finches through time, across seasons
may
help to resolve these uncertainties. It will be interesting
to see whether the trends observed in this year-long
wet-to-dry cycle is reflected in longer term datasets or
with more extreme weather periods experienced, for ex-
ample, during El Niño/La Niña conditions. This goal will
be challenging given their remote nature, enhanced di-
versity, and numerous overlapping influences. Regard-
less, Darwin
s finches continue to capture our attention
and reveal new secrets after more than a century and a
half of study.
Methods
Sample collections
Individuals (
n
= 114) from 12 species of Darwin
s
finches, from nine islands over both the wet and dry sea-
sons, were captured in mist nets and their fecal material
sampled (Table
1, Fig.
1). Dry season samples were col-
lected during November
December 2015, while wet sea-
son samples were collected between March and June
2016. Fecal samples were collected by placing the bird in
a paper bag on top of a metal mesh grate above a steril-
ized weigh boat for 3-5 min [
58]. Samples were immedi-
ately transferred into ~ 2 ml of LifeGuard Preservation
Solution (MoBio Laboratories, Carlsbad, CA, USA). Fea-
ther samples from three species (
G
.
fortis
,
G
.
fuliginosa
,
and
G
.
septentrionalis
) were collected for isotopic ana-
lysis in Jan 2016 (Fig.
3a).
DNA extraction and microbial community analysis
Prior to extracting DNA, samples were centrifuged to
remove the LifeGuard preservation solution. Genomic
DNA was then recovered from the pelleted fecal mater-
ial using a PowerSoil DNA isolation kit (MoBio Labora-
tories, Carlsbad, CA, USA) following the manufacturer
s
instructions, with the exception of the addition of a
bead-beating step using a Fast Prep 120 instrument;
Thermo Electron Corporation). The V4 region of the
16S rRNA gene was PCR-amplified from each extract
using the archaeal and bacterial targeted primer set 515F
and 806R [
59], following the protocol outlined by Case
et al. [
60]. Successful PCR amplifications were pooled, in
duplicate, and barcodes were added according to the
Earth Microbiome Project protocol [
59, 61, 62]; 5
μ
lof
Michel
et al. Microbiome
(2018) 6:167
Page 9 of 14
the amplicon product from the first PCR was used as
template in a 5-cycle, 25-
μ
l reconditioning reaction with
the same EMP-recommended conditions and the full
EMP primers. Samples were mixed together in equimo-
lar amounts and purified in bulk through a Qiagen PCR
Purification kit. At all PCR steps, amplification success
and purity was checked by gel electrophoresis. Paired-
end sequences (2
x
250 basepair) were generated from
barcoded amplicon products at Laragen, Inc. on an Illu-
mina MiSeq platform. At Laragen, the raw data was
passed through a filter which demultiplexed the library
into individual samples and removed any sequences
which had > 1 basepair mismatch on the 12-basepair
barcode sequence, and assigned quality scores to each
basepair call on every sequence. At the same time,
adapter, barcode, and primer sequences were removed.
Raw reads were deposited and are available through the
Sequence Read Archive under accession number
SRP130314.
Sequence data was processed with both the DADA2
pipeline for unclustered high-
resolution exact sequence
variants (SVs) [
63] and QIIME version 1.8.0 [
64] for oper-
ational taxonomic units (OTUs) at the 97% similarity level.
In QIIME, raw sequence pairs were joined and
quality-trimmed using the default parameters. Sequences
wereclusteredintodenovoOTUsusingUCLUSTopen
reference clustering protocol [
65]. Then, the most abundant
sequence was chosen as representative for each de novo
OTU [
66]. Taxonomic identification for each representative
sequence was assigned using the Silva-119 database [
67].
For downstream analyses, the QIIME 97% OTU dataset
was trimmed to only include those OTUs representing at
least 1% of the total gut community of at least one finch
(Additional file
1: Table S1). DADA2 exact sequence vari-
ants were calculated using the published pipeline tutorial
(v1.4) in R and were analyzed the same as QIIME results,
without the 1% abundance cutoff (errors are already
accounted for in the DADA2 error model [
63]).
Sequences from the 16S rRNA region were aligned
using MAFFT [
68] and a phylogeny constructed using
FastTree [
69]. Alpha diversity was estimated using the
Shannon index, Simpson index, Fisher alpha, and Chao1
richness and by rarefaction. Diversity statistics were cal-
culated in
phyloseq
v-1.25.2 [
70] and are reported at
the 97% OTU level in Additional file
2: Table S2. Since
results across phylotype extraction methods were highly
similar, all in-text figures, values, and statistics are de-
rived from the 97% OTU dataset.
To assess the influence of various factors on the
microbiome, the 114-finch dataset was divided 19 dif-
ferent ways, each subdivision accounting for one or
more of six different ecological and biological factors.
One individual (
C
.
pallida
)wasremovedfromall
analyses because it was the sole representative of its
species, leaving 113 samples for beta diversity ana-
lysis. Each division of the dataset was balanced in
sampling within the factor in question, unless other-
wise noted. Where category sample sizes differed, the
data subdivision was randomly subset by each factor
category down to the sample size of the smallest cat-
egory, with statistics taken from the average of 10
random subsets, as described in Additional file
7:
Table S4. The influence of sampling season, island,
finch diet, finch species, and the extreme diet of
sanguivory were assessed using these subdivisions
(Additional file
7:TableS4).
Statistical analysis of the role of each factor in shap-
ing finch microbiome beta diversity was done within
each data subdivision on relative (compositional)
abundance of each OTU in each finch using non-
metric multidimensional scaling (NMDS) ordination
plots, analysis of similarities (ANOSIM) of both
Bray-Curtis and UniFrac dissimilarities, and PERMA-
NOVA (Adonis) of both Bray-Curtis and UniFrac dis-
similarities. Dissimilarity
matrices derived from both
unweighted (presence-absence) and weighted taxo-
nomic relative abundance (scaled to the fourth-root)
were calculated for data from each finch. Summary
stat
istics for all data subdivisions, dissimilarities, and
approaches listed above can be found in Add-
itional file
5: Table S3. For data subdivisions where
statistical balance was achieved via sub-setting one or
more categories within a factor, random subsets were
run 10 times and the summary statistics reported rep-
resent the average over each run.
The contribution of individual OTUs to statistically sig-
nificant grouping by factor was queried using similarity
percentage (SIMPER), and the significance of differential
abundance in individual OTUs by factor grouping were
statistically tested using two-tailed Mann-Whitney
U
tests.
In an approach similar to beta diversity tests, for data sub-
divisions where random sub-setting was necessary to
achieve equal sample sizes within each factor category,
Mann-Whitney tests were run over 100 random subsets
of the larger category size, and the average
p
value was re-
ported. Summary statistics and plots were generated in R
using the packages
phyloseq,
”“
vegan,
”“
ggplot2,
and
RColorBrewer
[70
74].
Carbon, nitrogen, and sulfur isotope analysis of feathers
The isotopic signature of an organism
including that of
δ
13
C,
δ
15
N, and
δ
34
S of proteins in animal tissues such
as hair or feathers
is primarily influenced by food
source and is frequently used to discern differences in
diet (e.g., [
75]). Feathers were collected from a subset of
finch species during the transition between dry and wet
seasons (Jan 2016). Prior to isotopic analysis, feathers
were immersed in 2:1 dichloro-methane:methanol to
Michel
et al. Microbiome
(2018) 6:167
Page 10 of 14
remove surface oils as described by Blight et al. [
76].
After air-drying, feather length and weight were re-
corded and then individual feathers were split along the
rachis and transferred to tin capsules (0.3
1.0 mg), for
parallel carbon, nitrogen, and sulfur isotope analysis.
Feather
δ
13
C and
δ
15
N and weight percent C and N
(wt.% TOC and TON) were determined via continuous
flow (He; 100 ml/min) on a Costech Instruments Elem-
ental Combustion System model 4010 by oxidation at
980 °C over chromium (III) oxide and silvered cobalt (II,
III) oxide followed by reduction over elemental copper
at 650 °C. CO
2
was subsequently passed through a water
trap and then a 5-Å molecular sieve GC column at 50 °
C to separate N
2
from CO
2
, which was diluted with he-
lium in a Conflo IV interface/open split prior to analysis.
Fast jump was calibrated and applied to measure both
CO
2
and N
2
in the same run.
δ
13
Cand
δ
15
N values were
measured on a Thermo Scientific Delta V Plus irMS.
δ
13
C and
δ
15
N values were corrected for sample size de-
pendency and then normalized to the VPDB scale with a
two-point calibration [
77]. Error was determined by ana-
lyzing sucrose (NIST 8542), acetanilide (Costech Analyt-
ical Technologies Inc.), and nitrate (IAEA-NO-3) in
combination with in-house standards (from
45.93
to
10.45
for C and
3.02
to 4.70
for N). For C
measurements, accuracy was ± 0.18
(
n
= 50) and preci-
sion was ± 0.23
(
n
= 50; 1
σ
). For N measurements, ac-
curacy was ± 0.22
(
n
= 36) and precision was ± 0.43
(
n
= 36; 1
σ
). Feather
δ
34
S and weight percent S (wt.%
TOS) was measured using continuous flow (He; 120 ml/
min) on a ThermoQuest NC2500 mass spectrometer
(ThermoQuest Italia, Milan, Italy) by oxidation at 1000 °
C over tungsten (VI) oxide and elemental copper. SO
2
was subsequently passed through a water trap and then
a 5-Å molecular sieve GC column at 85 °C. A Conflo III
interface/open split was used to introduce SO
2
to a
Thermo Scientific Delta plus XL irMS. The
δ
34
S values
were corrected for sample size dependency and the nor-
malized to the VCDT scale with a two-point calibration.
Error was determined by analyzing two silver sulfides
(IAEA-S-2 and IAEA-S-3) in combination with in-house
standards including pyrite and sulfanilamide. Accuracy
on the
δ
34
S measurement was ± 1.84
(
n
= 20) and pre-
cision was ± 0.35
(
n
= 20; 1
σ
).
Additional files
Additional file 1:
Table S1.
Taxonomic composition of Galápagos finch
gut microbial communities, from Illumina 16S rRNA gene surveys, based
on OTU clustering at 97% identity trimmed to at least 1% relative
abundance in at least one finch. (XLSX 196 kb)
Additional file 2:
Table S2.
Microbiome diversity averages for all finch
species measured in this study, along with sample sizes and diet category.
Data is based on 16S rRNA gene OTU clustering at 97% identity trimmed to
at least 1% relative abundance in at least one finch. (DOCX 71 kb)
Additional file 3:
Figure S1.
Barplot showing the compositional
(relative) abundance of microorganisms defined at the phylum to family
taxonomic scale in the fecal samples of 113 individual finches in this
study. At top, the colored bar distinguishes finch samples from the dry
(red) and wet (blue) seasons. An average of 25,382 reads per finch,
comprised of 297 unique OTUs (clustered at 97% similarity level), were
recovered at greater than 1% relative abundance in at least one finch
across the dataset. (PDF 168 kb)
Additional file 4:
Additional information regarding the observations of
finch feeding behavior, core microbiome results, and microbiome differences
between vampire finches on Darwin and Wolf islands. (DOCX 20 kb)
Additional file 5:
Table S3.
Analysis of similarities (ANOSIM) and
PERMANOVA (Adonis) significance of each grouping within each dataset
subdivision (as described in the Methods section) for weighted (to the 4
th
-
root) and unweighted (presence-absence) relative abundance. OTUs were
calculated in QIIME at 97%. Significant statistics (
p
< 0.05) are highlighted in
yellow. Adonis models used the sample size-balanced variable first when
multiple variables were tested. Bars in cel
ls indicate that variance-partitioning
was impossible, often due to the factor o
verlapping with the first factor (ex. In
12c, latitude overlaps heavily with isla
nd, with island explaining all and more
of the variation attributable to latitude). Dataset subdivisions labeled with a **
indicate small sample size; results are listed as they may show a contrasting
trend requiring further investigation. Grayed-out regions in the table are either
untestable because the dataset subdivision only has one category in that
factor (dark gray) or were not tested because they are redundant with other
data subdivisions that have better balance or sample size (light gray). Data
subdivisions are color-coded by the factor tested (green = season, blue = diet,
red = vampire, yellow = island, purple = species). (PDF 129 kb)
Additional file 6:
Figure S2.
Non-metric multidimensional scaling (NMDS)
ordination of Galápagos finch gut microbial communities according to (A)
species, during the dry season only (B) island, for the vampire finch only (C)
diet, for all medium ground finches, formerly classified as
G. difficilis
,and(D)
season, for only
G. fortis
and
G. fuliginosa
. Island was not significant, even
when grouping Santa Cruz with close neighbors to boost sampling power
(ANOSIM
p
= 0.2), thus in controlling for species and island, season is still
significant. Taxonomic (OTU) clustering is at 97% identity and abundance
weighted by taking the fourth-root of the OTU relative abundance in each
finch. Ellipses represent 90% confidence windows following a multivariate t-
distribution. (PDF 909 kb)
Additional file 7:
Table S4.
Description of dataset subdivisions for
analysis of individual factors without confounding factors, taken by
randomly sub-setting a sub-category of the dataset subdivision where
appropriate, as indicated. The influence of season was analyzed using
data subdivisions 1 and 3-8, thereby controlling for different species/island/
diet groups and the singularity of the vampire finches. Diet was assessed with
dataset subdivisions 9-12 by comparing all finch species sampled with the
exception of the vampire finches binned into diet categories of herbivorous
(including seeds, cactus, and plant matter) and omnivorous (including
insects), with control for season and island embedded in the various subsets.
Seed-eaters are not generally considered herbivores, thus these groupings
were designed broadly to encompass plant matter versus other dietary food-
stuffs. The uniqueness of sanguivory was tested in dataset subdivisions 13-15,
where season, sample size, and phylogeny (through nearest relatives) were
considered as well as a comparison with other finches whose feather
isotopes were measured. The role of finch species grouping was analyzed in
dataset subdivisions 3, 7, 12, 16, 18, and 19 to remove the confounding
influence of the extreme diet of
G. septentrionalis
and to extract the largest
groupings while avoiding potential biogeographical (island) and season bias
and to assess the ground finches alone. The influence of island was assessed
in dataset subdivisions 1-3, 6, 7, 12, and 17-19, with Santa Cruz, Santa Fé, and
North Seymour sometimes grouped together as
Santa Cruz +
since these
islands are in close geographic proximity (Fig.
1). In some cases (dataset
subdivisions 1, 2, and 12), latitude was assessed, as the northern, more isolated
islands of Pinta, Genovesa, Wolf, and Darwin versus the remaining islands of
the Galápagos. (DOCX 135 kb)
Acknowledgements
We are grateful to Sofia Carvajal, Kurt Gielow, Sarah Knutie, Andres Leon,
Simón Villamar, Angela Hansen, Sabrina McNew, Ashley Saulsberry, Carlos
Michel
et al. Microbiome
(2018) 6:167
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