Contrasting mitochondrial diversity of European starlings (Sturnus vulgaris) across three invasive continental distributions

Abstract European starlings (Sturnus vulgaris) represent one of the most widespread and problematic avian invasive species in the world. Understanding their unique population history and current population dynamics can contribute to conservation efforts and clarify evolutionary processes over short timescales. European starlings were introduced to Central Park, New York in 1890, and from a founding group of about 100 birds, they have expanded across North America with a current population of approximately 200 million. There were also multiple introductions in Australia in the mid‐19th century and at least one introduction in South Africa in the late 19th century. Independent introductions on these three continents provide a robust system to investigate invasion genetics. In this study, we compare mitochondrial diversity in European starlings from North America, Australia, and South Africa, and a portion of the native range in the United Kingdom. Of the three invasive ranges, the North American population shows the highest haplotype diversity and evidence of both sudden demographic and spatial expansion. Comparatively, the Australian population shows the lowest haplotype diversity, but also shows evidence for sudden demographic and spatial expansion. South Africa is intermediate to the other invasive populations in genetic diversity but does not show evidence of demographic expansion. In previous studies, population genetic structure was found in Australia, but not in South Africa. Here we find no evidence of population structure in North America. Although all invasive populations share haplotypes with the native range, only one haplotype is shared between invasive populations. This suggests these three invasive populations represent independent subsamples of the native range. The structure of the haplotype network implies that the native‐range sampling does not comprehensively characterize the genetic diversity there. This study represents the most geographically widespread analysis of European starling population genetics to date.


| INTRODUC TI ON
Invasive populations are useful systems to investigate responses to novel environments, providing insight into mechanisms underlying invasion success and native species' capacity to adapt to a changing world (Moran & Alexander, 2014). Despite this opportunity, these studies often examine only one introduction, reducing their power to draw robust conclusions that are broadly applicable (Packer et al., 2017). For this reason, there is a growing interest in studying invasive species that have been introduced to multiple geographically and environmentally diverse localities (Kueffer, Pyšek, & Richardson, 2013;Packer et al., 2017). In this respect, the European starling (Sturnus vulgaris) is an excellent system to investigate evolutionary responses to a wide range of introduced environments, from tropical Fiji to temperate Argentina (Pinto, 2005).
European starlings are native to the Palearctic but have been repeatedly introduced to novel environments, flourishing in their invasive ranges (Long, 1981). Starlings have now been introduced to every continent barring Antarctica (Rollins, Woolnough, & Sherwin, 2006, Figure 1). Their invasion success likely results from a suite of life-history and behavioral traits that may facilitate ecological flexibility. For example, they are often classified as diet generalists, preferring insects, but they will eat most other foods depending on availability of resources (Cabe, 1993). Another feature that likely plays a role in European starlings' ability to persist in new localities is their flexibility in patterns of seasonal migration. Although not all starling populations are migratory (e.g., in Australia and New Zealand, Higgins, Peter, & Cowling, 2006), in populations that are migratory, there is a great deal of individual variation in migratory behavior (i.e., individuals can be differentially migratory from year to year; Blem, 1981;Feare, 1984). Some research suggests that seasonal migration may be an adaptive strategy in response to seasonality; therefore, migratory flexibility in starlings may allow them to persist in seasonal environments and facilitate range expansion (Winger, Auteri, Pegan, & Weeks, 2019). This trait may also contribute to differences in population structure across introductions.
European starlings were introduced to North America in 1890 as part of an American Acclimatization Society initiative to populate Central Park with the birds from Shakespeare's plays (Cooke, 1928;Phillips, 1928). The initial introduction consisted of approximately 60 individuals released in 1890 and 40 more in 1891, leading to a total of ~100 individuals released into Central Park in New York City (Cabe, 1993). From this founding population, starlings have expanded their range across all of North America where their current population exceeds 200 million individuals, over one-third of the global population of this species (Feare, 1984). This range expansion has taken place in the last 130 years, demonstrating their ability to persist in a heterogeneous novel environment. Given the diverse environments colonized by starlings in North America, it is interesting that nuclear markers indicate that little population structure exists (allozymes, Cabe, 1998; single nucleotide polymorphisms, Hofmeister, Werner, & Lovette, 2019).
Other starling introductions from the 19th century have been previously studied, including the mid-19th century Australian introductions (Rollins et al., 2016;Rollins, Woolnough, Sinclair, Mooney, & Sherwin, 2011;Rollins, Woolnough, Wilton, Sinclair, & Sherwin, 2009) and the late 19th century South African introduction (Berthouly-Salazar et al., 2013). In Australia, up to sixteen different introduction attempts have been made with birds originating from the United Kingdom, from 1856 to 1881, with only two resulting in recorded established populations from ~165 original birds (Higgins et al., 2006;Long, 1981). Nuclear and mitochondrial markers identified concurrent population structure across the Australian range, and nuclear polymorphisms were associated with environmental variables in that population (e.g., aridity; Cardilini et al., 2020;Rollins et al., 2009Rollins et al., , 2011. In contrast to the high levels of propagule pressure in Australia, only one introduction to South Africa of ~18 birds originating from Britain in or around 1897 has been recorded (Winterbottom & Liversidge, 1954).
The South African introduction enables a powerful comparison with the North American introduction because of similarities in timing of these events (1897 and 1890, respectively). Both the Australian and South African introductions have reduced mitochondrial genetic diversity in comparison to the native source population in the UK (Berthouly-Salazar et al., 2013;Rollins et al., 2011).
Founding population sizes during introduction are often small, resulting in genetic bottlenecks and lower genetic diversity than in the native range (Baker & Stebbins, 1965;Nei, Maruyama, & Chakraborty, 1975). However, numerous insights from studies of other invasions suggest that decreased genetic diversity at introduction may not hinder these species' ability to become established in novel environments (Dlugosch, Anderson, Braasch, Cang, & Gillette, 2015;Frankham, 2005). Factors such as the number of introduction attempts, the timing of these attempts, dispersal patterns in the introduced range, and the rate of population expansion may play a larger role in shaping patterns of genetic diversity and ultimately contributing to successful colonization. A wide body of evidence suggests that adaptation in introduced ranges occurs rapidly, and this does not appear to be reliant on genetic diversity (Rollins et al., 2013).
Here, we use mitochondrial control region sequence data to examine starling population structure in North America and compare mitochondrial genetic diversity in populations from the native-range and from three established invasions: North America, Australia, and South Africa. Although the limitations of using mitochondrial DNA in population genetic analyses have been well characterized (Ballard & Whitlock, 2004;Bazin, Glémin, & Galtier, 2006), there are several benefits associated with its use. First, previous studies of starlings in Australia, South Africa, and the UK used mitochondrial control region sequence data, so the comparative strength of our study is predicated on using the same marker. Second, Australian studies that have compared population structure using mitochondrial sequence data to that of microsatellite (Rollins et al., 2011) and single nucleotide polymorphism data (Cardilini et al., 2020) found similar patterns, supporting the validity of our approach. Third, mitochondrial DNA is still one of the most reliable sources of DNA that can be extracted from historical museum specimens (Guschanski et al., 2013;Mason, Li, Helgen, & Murphy, 2011;Ramakrishnan & Hadly, 2009), and population analyses using historical specimens rely on comparable datasets from modern birds, such as this. Finally, although mitochondrial DNA cannot provide a complete evolutionary picture, it is especially useful as evidence to clarify recent changes in a population (Zink & Barrowclough, 2008). This is especially true of the noncoding control region, which has high nucleotide diversity (Saccone, Pesole, & Sbisà, 1991).
In this study, we use this unique biological system that features multiple, independent, and documented introductions to investigate how propagule pressure (e.g., the number of introductions), environmental factors, and the expansion rate in introduced ranges influence contemporary population structure and genetic diversity.
Based on previous research using nuclear markers, we predict low levels of population structure within North America. We predict that the mitochondrial diversity of the North American population will be lower than that of Australia, where multiple introductions were made (Jenkins, 1959), and these occurred prior to and had a greater number of propagules than the New York introduction (Australian introductions started in 1854; Jenkins, 1959). Further, we predict similar levels of genetic diversity in South Africa and North America, due to similarities in timing of introductions and propagule pressure.
We discuss microevolutionary changes that have occurred since the introduction of these populations across the world.  (Table 2).

| Amplification and sequencing
The primers used to amplify the mitochondrial control region in North American specimens were initially designed to analyze mitochondrial diversity of the Australian population (Rollins et al., 2011). Rollins et al. (2011 designed a series of overlapping primers to be utilized in the amplification of museum specimens or highly degraded samples (Table S1). We used these primers to sequence the control region of North American samples in four overlapping segments. Two of these primers (svCRL1 and svPheH3) amplify most of the mitochondrial control region and also were used to amplify DNA from the starling population in South Africa (Berthouly-Salazar et al., 2013).
For the PCRs, PuReTaq Ready-To-Go PCR Beads were rehydrated with 13.5 µl of molecular grade water, 5 µl of 10 µM forward and reverse primers, and 1.5 µl of DNA. The thermocycling conditions used here were identical to those described in the original paper (Rollins et al., 2011

| Population and expansion analysis
Overlapping sequences were aligned using the software Geneious 11.1.2 (Kearse et al., 2012) to generate a consensus sequence for each individual from North America. All subsequent alignments including the samples from other continents were generated on Geneious using the standard settings and the Geneious alignment algorithm (Kearse et al., 2012). Median joining haplotype networks were created using Network v10.1.0.0 (Bandelt, Forster, & Röhl, 1999) and postprocessed using the maximum parsimony calculation to remove unnecessary median vectors (Polzin & Daneshmand, 2003 FSTAT v.2.9.4 (Goudet, 2003). Mismatch analyses were also conducted in Arlequin, but with the full dataset from each invasive population (see Table S5).

| RE SULTS
The haplotype network constructed using only the North American specimens (1,181 bp sequence) included 20 haplotypes encompassing 53 polymorphic sites and did not indicate the presence of regional population structure ( Figure S1). When we included samples from all continents (928 bp sequence; Figure 2), we identified 64 haplotypes encompassing a total of 46 polymorphic sites (  Table S4).   (Table S3).
Haplotype diversity and richness were highest in the native range, followed by the North American population (Table 2)

| D ISCUSS I ON
Starlings are a highly successful invasive species occupying a wide breadth of environments across the world, resulting from introductions of varying age and intensity. This system enables a unique opportunity to study molecular evolution and adaptation.
Here we use mitochondrial sequence data to compare the population genetic structure and diversity of the three best-studied starling invasions: North America, Australia, and South Africa. Overall, our findings and those from data of other studies included here suggest that low genetic diversity is not an obstacle for this species' rapid  (Dlugosch & Parker, 2008;Rollins et al., 2013).
As expected, the invasive populations had lower genetic diversity than the population in the native range, likely caused by genetic bottlenecks at introduction. The highest haplotype richness (which accounts for differences in sample size) was found in the UK (R = 30.0); although only 45 individuals were sampled, we identified 30 haplotypes in this population. Surprisingly, despite higher propagule pressure in Australia as compared to that of North America or South Africa, Australia harbored the lowest haplotype richness (R = 7.7). The North American population, which was intermediate in terms of propagule pressure, has retained the most genetic diversity (R = 14.7). Given the timescales involved, this is unlikely to be caused by novel mutations arising in North America (but see Rollins et al., 2016). However, it could be caused by differences in genetic diversity of founders or by higher levels of differential survival between haplotypes in Australian or South African starlings as compared to those from North America. It may be that some haplotypes have been lost in the native range since founders were collected.
Differences in population expansion rates in novel environments also could be responsible for the differences in genetic diversity we found, with faster expansion resulting in higher haplotype diversity and lower nucleotide diversity (Halliburton & Halliburton, 2004).
The haplotype network including all populations (Figure 2) revealed some interesting relationships among haplotypes. South African starlings are genetically distinct from those of North America and Australia, suggesting that the founders for this population may have been sourced from a different region of the UK. North American and Australian starlings are genetically similar (intermixed in the network), but only shared a single haplotype (H_25), suggesting that the founders for these populations may have been sourced from the same region of the UK, but were likely to have been genetically distinct. As expected, UK samples were well distributed across the network, but many of the invasive haplotypes were not found in UK samples, highlighting the paucity of information that exists about starlings in their native range and making it difficult to further interpret sources of founding populations. For this reason, and because European starling populations are in decline in their native range (Heldbjerg et al., 2019), it may be important to further characterize this population.
Previous studies have investigated population structure within introduced populations of starlings. Within Australia, genetically distinct groups of starlings have been characterized using nuclear and mitochondrial markers (Rollins et al., 2009(Rollins et al., , 2011 and evidence of local adaptation to the Australian environment has been described (Cardilini, Buchanan, Sherman, Cassey, & Symonds, 2016;Cardilini et al., 2020). However, in South Africa, no evidence of population structure was found (Berthouly-Salazar et al., 2013).
The regional analysis conducted within North America in the present study also found little evidence of population structure in this invasive population. We did see a slight (F ST = 0.04) albeit statistically significant difference between Central and Western samples but this may be due to the low sample size from the Central United States (N = 20). Overall, our findings are consistent with an earlier investigation of this population, which utilized allozyme data (Cabe, 1998), and a recent study using genome-wide SNPs (Hofmeister et al., 2019). However, the latter indicated that there are genotypes associated with specific environmental features such as precipitation and/or temperature. This may imply that over time, population structure could develop in this invasive population, despite apparent high levels of dispersal. Interestingly, migration rates between Central and Western sites differ (Hofmeister et al., 2019) and banding data in North America have shown that the starlings are found to migrate in unpredictable ways, not always in the North and South direction, but also in the East and West directions (Brewer, 2000). Therefore, the genetic pattern we found may be due to the high dispersal rates and these unpredictable and latitudinal migration patterns.
When we investigated genetic differentiation across continents, we found that invasive populations were genetically divergent (F ST ranged from 0.17 to 0.26, all statistically significant) and all significantly different from populations in the native range (F ST ranged from 0.06 to 0.17). North America was most similar to the UK and Australia was least similar. These differences are likely caused by a combination of discrete introduction sources and founder effects.
However, this could also be due to differences in timing of introductions; the Australian introduction occurred earlier than the others (mid-19th century) so it is possible that these differences reflect shifts that occurred in the native range in the latter half of the 19th century.
Not surprisingly, we found genetic evidence of spatial expansion in all three invasive populations. While there was genetic support for demographic expansion in both North America and Australia, the mismatch analysis of South African data did not support the sudden (demographic) expansion model (Figure 4). This may mean that the South African starling population may still be in the "lag phase", which typically occurs following introduction (Sakai et al., 2001).
Neither Tajima's D nor Fu's F s values supported the presence of population expansion in any of the invasive populations. However, Fu's F s was significantly negative in the native range, which suggests that this population may either be undergoing expansion or that it has an excess of recent mutations (Fu, 1997). Given observations of population decline in the native range (described above), this might be a signal of directional selection, which could be a response to novel environmental stressors resulting from land use changes in the UK (Heldbjerg et al., 2019).
It is also interesting to consider that differences in the environments of each of the three invasive ranges studies here may have influenced population expansion rates. The United Kingdom and surrounding parts of Europe (native range) are largely classified as temperate with a hot or warm summer (Beck et al., 2018).
Temperate areas similar to the native range are the regions where most starling invasive range expansion has occurred. The starling population in North America is about the same latitude as that of the native range between 40°-55°N, whereas the invasive populations in Australia and South Africa occur at about 30°-35°S (Sullivan et al., 2009). In Australia and South Africa, starlings have not expanded to cover the same area that they have in a comparable amount of time in North America. In North America, starlings spread from New York to Alaska from 1890 to 1970, which represents 80 years and a rate of 90 km/year (Bitton & Graham, 2014).
In Australia, starlings rapidly expanded their range into south-eastern Australia and were in Western Australia by the 1970s. However, starlings have not colonized the arid center (Higgins et al., 2006) of the continent, where the highest temperatures and lowest rainfall occur (Jones, Wang, & Fawcett, 2009)