DISCUSSION
Our whole-genome data reveal that invasive starling populations show
moderate genetic diversity across North America, rapid population growth
after a genetic bottleneck, negligible geographically structured genetic
differentiation, significant but quite weak isolation-by-distance, and
intriguing associations of genetic variation with environmental
parameters that might result from adaptive processes. Genetic diversity
is typically predicted to be low in invasive populations (Dlugosch &
Parker, 2008), and although RADseq tends to estimate lower genetic
diversity than the true diversity, this bias is minimal for taxa known
to be genetically depauperate (Cabe, 1998; Cariou et al., 2016).
Within invasion genetics, one area of recent focus involves the spatial
dynamics of invasion, and we can explore how genetic variation varies
across the post-invasion range of starlings in North America. Although
there is a significant (but low magnitude) signal of
isolation-by-distance, hierarchical AMOVAs find that variation within
and among individuals explains observed differentiation better than
variation among populations. There is no evidence of population
structure, and while models indicate some subtle spatial patterns of
genetic variation, these model-based inferences likely reflect sampling
artifacts (Supplementary Information). Finally, after the initial
founder effect, the effective population size has grown by a ratio of
1:100. These patterns are consistent with the expectation that extensive
gene flow—as shown by extremely low FST among
populations (Table 2)—maintains high connectivity across North
American starling populations. When interpreted within the context of a
complementary exploration of movements inferred from feather isotope
assays, this genomic survey confirms that dispersal and migration
continue to influence the genetic variation generated in just over a
century of range expansion.
Long-distance dispersal may be common in introduced starlings on
multiple continents, where rates of dispersal are determined by
demographic changes and environmental quality. In South Africa, invasive
starlings disperse when their natal environment becomes crowded or
unsuitable, as at the leading edge of their range expansion (Huiet al., 2012). In North America, the effective population size
(Ne) of the present-day invasive population has expanded
dramatically, with models indicating that current Ne is
even larger than the Ne of the founding population. This
explosive population growth from hundreds to millions of individuals may
have encouraged long-distance dispersal away from the dense populations
in eastern North America. In general, multiple lines of evidence have
shown that the dispersal rates and distances of juvenile starlings in
North America are remarkably high (Dolbeer, 1982; Cabe, 1999; Werner,
Fischer, & Hobson, 2020). Considered in concert with similar findings
in the invasive South African and Australian starling populations (Huiet al., 2012; Phair et al., 2017), it is clear that
dispersal similarly plays an ongoing and major role in the connectivity
of North American starling populations.
Although it is generally important, dispersal has not been uniform
across time or space in North American starlings. One open question in
invasion genetics is whether expanding populations maximize
spatiotemporal fitness via spatial sorting and incipient adaptation
(Williams et al. 2019). Empirical studies of some invasions suggest that
spatial sorting is common, including in common mynas (Berthouly-Salazaret al. , 2012), pumpkinseed fish (Ashenden et al., 2017),
and cane toads (Brown et al. , 2014). More empirical work is
needed to verify the conditions in which spatial sorting can lead to
lasting shifts in fitness (Lee, 2011; Phillips & Perkins, 2019;
Williams et al. , 2019). However, we do know that adaptive
dispersal strategies can facilitate range expansion in Western bluebirds
(Duckworth, 2008) and invasive beetles
(Lombaert et al., 2014; Ochocki & Miller, 2017) among other
species. For North American starlings, we can combine historical
records, isotopic and genetic evidence to make inferences about the
possibility of spatial sorting.
Feather isotope data from the same starlings sampled in this genetic
survey suggests that movements are not uniform continent-wide, and that
both juvenile dispersal and regional differences in migration could
influence genetic variation (Werner, Fischer, & Hobson, 2020). If
starling movement was the primary driver of differentiation, then we
would expect FST among populations to be directly
related to the number of birds collected and assigned to that
population. Starlings in the western U.S. appear to have differentiated
subtly from their eastern counterparts based on the higher
FST between Arizona—and to some degree, New Mexico and
Colorado—and all other sampling locations (Table 2). Birds collected
in these southwestern states are also assigned to those states by
discriminant function analysis (Werner, Fischer, & Hobson, 2020); for
example, 67% of starlings collected in New Mexico were also assigned to
that state (Table 2). We suggest that birds assigned to the same state
where they were collected may reside in that state year-round, but we
note that collecting feathers once in the lifespan of the bird does not
allow us to determine the bird’s lifelong migration and dispersal. The
highest rates of pairwise differentiation occur between states where one
of those “populations” shows a relatively high collection-state
assignment, which suggests that starling movement among sampling
locations does impact the genetic variation. This comparison is further
supported by the low but significant isolation-by-distance across the
North American range; however, environmental pressures explain genetic
variation better than a simple model that accounts for geographic
distance. In addition, assignment to a state other than the collection
state seems to have only a minor effect on FST, even
though we might expect that movement among geographically distant
sampling locations might lead to gene flow among those locations. In
Arizona—where FST is highest but still relatively
weak—Werner et al.’s model suggests that 25% of birds originated in
New Hampshire, which is approximately the percentage of birds assigned
to Arizona (28%, Table 2). Unsurprisingly, geography, environment, and
demography all appear to influence genetic variation within North
American starling, and explicit tests for selection can help to clarify
the relative weight of each factor.
This species’ general genome-wide panmixia across the continent allows
for tests of selection on loci that may be involved in local adaptation.
Because genetic diversity and differentiation are exceptionally low, it
is relatively easy to identify sites that have differentiated against
this background of low genome-wide or population-specific divergence
(Dlugosch et al., 2015). We find that almost 200 of the 15,038
RAD loci appear to be under selection using a redundancy analysis. Only
13 of these SNPs overlap with the SNPs identified by a latent-factor
mixed model, and there is no overlap between the RDA candidates and the
differentiation-based scans (BayeScEnv and Bayescan). It is unsurprising
that each test identifies different candidates for selection, because
the assumptions underlying each are very different (for more details on
our approach to selection testing, see Supplementary Information).
Rather than making inferences based on the genes identified by these
scans, we instead propose that genotype-environment associations show
that changes in precipitation and temperature can explain genetic
variation in North American starlings. However, we do suggest that rapid
evolution in genes underlying key physiological processes may have
supported the starling’s spread across North America (Supplemental
Information). Specifically, we hypothesize that aridity and cold
temperatures that are not experienced in the starling’s native range
exert enough selective pressure on North American starlings to result in
incipient local adaptation. While this finding suggests that local
adaptation may explain genetic variation within the North American
starling invasion, there are several relevant caveats to this approach.
Because there is evidence of a genetic bottleneck in the North American
starling invasion, it is possible that artifacts resulting from genetic
drift could mimic these patterns of local adaptation. We cannot rule out
allele surfing during range expansion as an explanation based on the
methods currently available for testing genotype-environment
associations in a young and expanding system like this one. However,
given how short the timescale of divergence is in North American
starlings, such drift effects are more likely if genetic variation is
(1) ancestral and (2) structured among populations. Gene flow among
starling populations is remarkably high, but we do not know if variants
putatively under selection were present in the ancestral population.
These alleles under putative selection do not approach fixation, as no
putative outlier has an allele frequency greater than 0.28. We note that
concordance among environmental and genetic distances (e.g., partial
Mantel tests) indicate that spatial autocorrelation complicates our
selection inferences. Under these conditions, any evidence for selection
is likely to be weak, and as always these selection scans can generate
false positives. However, RDA has the highest rate of true positives and
lowest of false positives, and although this method has not been tested
in such recent expansions, RDA is well-suited to systems where
FST is very low (Meirmans, 2015, Forester et al. ,
2018, Supplementary Information). Finally, we do not explicitly control
for linkage, and some allele frequency shifts could be explained by
recombination or by genetic hitchhiking.
Our study focuses on birds collected during the winter, which may limit
our inferences about population structure and selection to these
wintering populations. As discussed above, isotopic evidence suggests
that starlings in the western U.S. tend to move only regionally whereas
birds sampled in the eastern U.S. undertake longer movements (Table 2).
This in turn suggests that starlings overwintering in the western U.S.
are more likely to breed nearby, and thus the environmental conditions
may not change as dramatically among wintering and breeding ranges. In
addition, the environmental conditions that we expect to drive
selection—precipitation and temperature—vary most substantially in
the southwestern region: for example, the sampling location in Arizona
is consistently warmer (BIO1) and drier (BIO12 and BIO16) than other
locations (Figure 2, Supplementary Information). Western populations
experience these environmental conditions year-round, which could allow
selection to drive advantageous alleles toward fixation. Elsewhere in
the U.S., starlings move more freely among states: individuals within
each sampling location may come from different breeding populations, and
additional sampling could reveal stronger population structure among
true breeding populations. However, if our sampling overlooked some true
populations, we would expect some signal of population structure.
Individual-based tests of population structure—e.g., those that do not
define possible populations a priori —do not recover any signals
of population structure. This sampling strategy uses the more vagile
eastern populations as a comparison to more-resident western populations
that may also be under stronger selection. This framing may suggest that
population structure in the western U.S.—of which we find no
evidence—could explain allele frequency shifts that we infer to be
selection, but when we compare the relative importance of geographic and
environmental distances in partial Mantel tests, we find that
environmental conditions better explain genetic variation. This evidence
supports our interpretation of selection as a major driver of genetic
variation in North American starlings.
A similar project on starlings in the Australian invasion—which
colonized that continent nearly concurrently with the North American
invasion—found that geographic but not environmental distance explains
genetic patterns there (Cardilini et al., 2020). Starlings in the
Australian range show substantial population structuring and significant
patterns of isolation-by-distance. Earlier work had shown that gene flow
among Australian starling populations is low (Rollins et al.,2009), and phylogeographic patterns of mitochondrial sequence variation
confirm that starlings on the edge of the expansion front in Western
Australia have differentiated from those still living in the
introduction site (Rollins et al., 2011). In fact, starlings at
the expansion front may have rapidly adapted during the Australian
invasion (Rollins et al., 2016): the proportion of adult
starlings in Western Australia carrying a novel mitochondrial haplotype
has increased rapidly only at this range edge. A
genotyping-by-sequencing survey employing a much greater number of SNP
markers indicates three population subdivisions in Australia, where
geographic distance explains genetic differentiation in starlings better
than does environmental variation (Cardilini et al. , 2020).
Global FST across all Australian populations is an order
of magnitude higher than the equivalent FST index across
North America, despite similar areas sampled. In Australia, the strong
evidence for isolation-by-distance and founder effects complicate
attempts to disentangle selection from drift, yet despite their
differences in invasion dynamics, genotype-environment associations
reveal signatures of selection in both invasions. On both continents,
starling genetic variation can be explained by extremes in temperature
and precipitation, and preliminary results of whole-genome resequencing
of native and introduced populations confirm that variability in
temperature and precipitation may shape observed genetic variation in
starlings world-wide (Hofmeister et al., in prep ).
Our results contribute to the growing evidence of rapid adaptation in
some expanding populations, even in extremely young systems. Some
studies of rapidly expanding invasions find little evidence that
adaptation may facilitate this expansion, as in corals (Leydet et al.
2018). However, other work suggests a role for selection in supporting
rapid range expansion, such as in experimental studies of flour beetles
(Szucs et al., 2017) and empirical work in guppies
(Baltazar-Soares et al., 2019). Invasion biologists have long
highlighted propagule pressure as a driver of invasion success, but the
genetic composition may be just as important as the size of the
establishing population (Briski et al., 2018). For example,
genetic bottlenecks in monk parakeets, another avian invader now
distributed world-wide, do not seem to inhibit invasion success (Edelaaret al., 2015). Pre-adaptation in the native range or selection
during transport may facilitate the spread of invasive species, and
human commensalism may support establishment and spread, as shown in
house sparrows (Ravinet et al., 2018) and common mynas (Cohenet al., 2019), and reviewed across alien bird species (Cardador
& Blackburn, 2019). Empirical studies of invaders like the ones
described here also show how, in addition to genetic variation,
epigenetic shifts and/or plastic changes in gene expression may support
the establishment and expansion of invasive species (Marin et
al., 2019). In the well-studied house sparrow—a system quite similar
to starlings—epigenetic shifts may have supported invasions in Africa
(Liebl et al., 2013) but not necessarily in Australia (Sheldonet al., 2018). Taken together, recent work suggests that we
should consider a much wider range of demographic and ecological
processes that lead to adaptive evolution in invading populations.
Invasive populations allow us to explore the genetic consequences of
colonization and establishment in novel environments. On a background of
low genetic differentiation and diversity, we find evidence of incipient
genotype-environment associations in North American starlings. Here we
explore how genetic variation changes across the landscape, but we
cannot fully understand gene flow without studies of dispersal and
migration of the individuals that carry genes. Our results complement
other recent studies that reveal associations between climate variables
and particular loci in North American vertebrates (Schweizer et
al., 2015; Bay et al., 2018). Finally, we suggest that our study
adds to those suggesting that rapid local adaptation can evolve even in
dispersive and young populations.