This study applied empirical evidence to assess how an urban landscape has impacted the connectivity and genetic structure of T. recurvata. In accordance with the hypothesis of this study, the urban T. recurvata populations exhibited high global SGS within Alfenas city. However, this was not the result of isolation by distance (IBD). All populations showed genetic differentiation, as well as relatively high levels of inbreeding and moderate genetic diversity. A significant departure from the Hardy-Weinberg Equilibrium (HWE) was observed across all populations. According to the HWE, several elements may have interacted to determine this outcome, including: small population size, genetic drift, gene flow, breeding system, natural selection and mutation (Lachance 2016).
Population size can function as a baseline for the aforementioned conditions required for the maintenance of HWE (Lanchance 2016). In this study, T. recurvata was found in small populations, isolated to multiple clusters of low tree density throughout Alfenas city. This species has also been known to inhabit power cables and non-organic substrates, although these populations are found in much smaller quantities (Wester and Zotz 2010). The low tree density in Alfenas city was driven by land-use change and deforestation for urban expansion, which likely led to the fragmentation of a previously continuous landscape (Defries et al. 2010), and also by the low number of planted trees in its streets and squares (Monalisa-Francisco and Ramos 2019). This trend has been mirrored across many urban areas (Arshad et al. 2020). Evidence suggests that this fragmentation may have reduced the size of a previously large and interconnected population of T. recurvata (Toledo-Aceves et al. 2014), primarily due to dispersal limitations (Cascante-Marín et al. 2009). A genetic bottleneck most likely occurred within these condensed T. recurvata populations, which translated into reduced genetic diversity as a result of small gene pool size (Ackerman and Zimmerman 1994). Genetic drift then possibly ensued from this bottleneck and altered the frequency of alleles overtime within the finite T. recurvata populations (Chaves et al. 2021). This resulted in the genetic differentiation and high SGS that were observed in this study (Nussbaum 2016) and have indicated that genetic drift has dictated the connectivity between urban T. recurvata populations. Furthermore, the short life span of T. recurvata (approx. six years; Chaves et al. 2021) and the extended period of urban expansion in Alfenas (founded in 1805), both suggest that multiple generations of this epiphyte have experienced genetic drift overtime and amplified the differentiation between populations (Nussbaum 2016).
The role of low tree density and small population size on genetic differentiation, as well as genetic drift, were reinforced through a recent simulation of T. recurvata spreading dynamics in an orchard landscape (Chaves et al. 2021). Chaves et al. (2021) generated a TREC model that used an individual-based approach, with the simulated spreading dynamics of T. recurvata adjusted relative to empirical genetic data. The findings of Chaves et al. (2021) revealed genetic differentiation and high SGS between T. recurvata populations, which the TREC model attributed to low tree density. A greater differentiation was also observed at the stage of early colonisation (Chaves et al. 2021). Specifically, they found that each tree formed individual habitat units, which encouraged distinct groups of multi-locus genotypes (MLGs) through genetic drift, resulting from founder events on each host tree (Chaves et al. 2021). The random combination of alleles that formed these MLGs were sufficiently diverse to maintain moderate genetic diversity and mitigate strong evidence of IBD (Chaves et al. 2021). The aforementioned findings of Chaves et al. (2021) were supported by the observations of this study and have reinforced the influence of genetic drift, after the founder events on each studied population, on the connectivity of urban T. recurvata populations.
Genetic drift has also been recognised as a key factor in the differentiation of T. recurvata in the wider literature (Soltis et al. 1987), as well as in another species of Tillandsia (González-Astorga et al. 2004). Similarly, several other species of plants show evidence of differentiation as a result of genetic drift in isolated populations, many of which share comparable characteristics to those of T. recurvata (selfing, rapid and clonal growth, epiphytic life cycle, high abundance) (Trapnell et al. 2004; Vekemans et al. 2004; Barluenga et al. 2011; Pettengill et al. 2016; Atwater et al. 2018; Torres et al. 2018; Mota et al. 2020; Chaves et al. 2021). The evidence presents, therefore, a strong case that genetic drift has driven the random differentiation and high SGS of T. recurvata populations in this study.
Genetic drift, high SGS and population differentiation all indicate low gene flow between the T. recurvata populations in this study (Ramos et al. 2016; Chaves et al. 2021). T. recurvata maintains gene flow exclusively through wind dispersed seeds (Chilpa-Galván et al. 2018), since it has spontaneous self-pollination on its cleistogamous flowers (Bianchi and Vesprini 2013). The exact range of T. recurvata seed dispersal remains unquantified, although the small, light seeds of this species and low seed terminal velocity are both suggestive of a high dispersal potential (Chilpa-Galván et al. 2018). Nonetheless, the limitations of wind dispersal, specifically restricted movement within dense and enclosed landscapes, have frequently been documented over long distances (Barrett et al. 1991; Nathan et al. 2002; Vergara-Torres et al. 2010; Belinchón et al. 2017). As such, the confined and complex landscape of urban architecture within Alfenas may have limited T. recurvata seed dispersal in this study (Ramos et al. 2016); amplified by the range between the urban tree patches (330m-5km) and the low density of planted trees in the streets and squares. For instance, the air currents surrounding moving traffic and buildings may have dictated the flow of wind dispersed seeds (Lippe et al. 2013). Equally, human-mediated mechanical removal of this opportunistic epiphyte may have influenced population size and subsequent gene flow (Chaparro et al. 2011). Under the pressure of sustained dispersal limitations, evidence has dictated that isolated species become more likely to experience inbreeding depression and IBD, as well as a subsequent reduction in genetic diversity and population fitness (Wright et al. 2013). In contrast, the moderate genetic diversity observed in this study has contradicted this predicted outcome, and suggests that an alternative factor has influenced the gene flow and connectivity of this urban landscape.
Further contemplation of the spreading dynamics of T. recurvata may offer an insight into this controlling factor, which has determined the connectivity of these urban populations. Specifically, the adaptability and dominance of T. recurvata within fragmented landscapes (Orozco-Ibarrola et al. 2014), and its ability to grow in isolated trees (Chaves et al. 2021) or in “harsh” habitats, such as pastures (Elias et al. 2021), enables the accumulation of dense populations. These areas of high density then act as sources for the spread of T. recurvata propagules to the surrounding landscape, facilitating the transmission of several multi-locus lineages (MLLs) (Chaves et al. 2021) and gene flow to more isolated populations. In view of these spreading dynamics, the pasture trees and forest fragments that surround Alfenas city must be incorporated within our current perspective of this urban landscape. Trees in pasture fragments are common in the surrounding land around Alfenas, in particular, and have been shown to host a high abundance of T. recurvata (Carvalho et al. 2015; Monalisa-Francisco 2015; Elias et al. 2021). This would create a metapopulation dynamic, with external pasture trees facilitating gene flow to isolated populations across the city and preventing IBD. Under a metapopulation structure, and in combination with the previously described influence of small population size and genetic drift, the results of this study conform to the expected findings of high SGS and population differentiation (Chaves et al. 2021). We were prevented from collecting T. recurvata samples from pasture fragments outside of Alfenas city by coronavirus restrictions and were; therefore, unable to test this hypothesis within this study.
Although, in contrast to the findings of this study, low genetic diversity was also anticipated within populations, as a result of genetic drift from consecutive founder events on each isolated urban host tree (Ward 2006). The size of population required to mitigate this genetic drift has not been quantified in the Tillandsia genus, although work on other species of similar selfing plants have proposed a local effective population size of 150–200 individuals (Goldringer et al. 2001; Siol et al. 2007). The local urban T. recurvata populations in this study were substantially smaller than this predicted value, which has reinforced the expected outcome of low genetic diversity within these populations.
In order to maintain genetic diversity within a fragmented landscape or urban ecosystem, T. recurvata must be adaptable to the consequences of small population size and genetic drift- namely inbreeding as a result of low gene flow (Agrawal and Whitlock 2012). Inbreeding depression has the capacity to reduce population fitness through the accumulation of harmful homozygous recessive mutations, amassed across multiple generations of inbred progeny (Agrawal and Whitlock 2012). However, T. recurvata and other selfing plants are able to lower the proportion of accumulated lethal mutations, through the purging of recessive deleterious alleles in their homozygous state- as a result of natural selection (Byers and Waller 1999; Bosse et al. 2019). As such, the self-fertilisation strategy of T. recurvata gives reproductive assurance (Ingvarsson 2002), despite the constraints of potentially consecutive founder events (Eckert et al. 2006). A number of papers have reinforced this concept across several species of selfing plants (Lloyd 1992; Johnston and Schoen 1996; Aguilar et al. 2008; Honnay and Jacquemyn 2007; Vandepitte et al. 2010; Cutter 2019; Lander et al. 2019). Thus, the ability of selfing plants to moderate the potentially lethal consequences of isolation, offers a partial justification for the moderate diversity that was maintained within the urban T. recurvata populations of this study.
The reproductive assurance of self-fertilisation has made this strategy a common and valuable characteristic amongst angiosperm epiphytes (Fenster and Marten-Rodriguez 2007; Rios and Cascante-Marin 2016). Thus, the response of several selfing species to urban ecosystems, especially those that share a similar life history to T. recurvata, can be inferred from the data of this study. For example, the maintenance of genetic diversity within isolated landscapes through reproductive strategy, as was previously discussed, suggests that selfing species are more likely to dominate epiphyte communities in urban areas- allowing greater reproductive success in fragmented landscapes (Herlihy and Eckert 2002; Goodwillie et al. 2005; Bhatt et al. 2015; Furtado and Neto 2015; Prather et al. 2018). The wider literature also suggests that outcrossing epiphytes, in contrast, will be unable to maintain sufficient genetic diversity or persist in isolated urban landscapes, especially in the absence of an efficient pollination strategy (Ksiazek-Mikenas et al. 2019).
Although self-fertilisation offers reproductive assurance in fragmented landscapes, this study has highlighted, although not tested, the potential importance of external pasture trees for sustained connectivity between isolated urban epiphyte populations. The removal of these pasture trees, through land-use change or urban expansion, may eradicate any connectivity between populations (Ingvarsson 2002). Evidence has shown that this can result in population decline of selfing epiphytes or more substantial evolutionary consequences (Wright et al. 2013). Moreover, pasture trees contain a greater diversity of epiphytes than urban landscapes (Poltz and Zotz 2011), largely due to limited opportunity for effective colonisation in the fragmented habitat of urban ecosystems (Bhatt et al. 2015). The loss of pasture trees through urbanisation; therefore, also has direct implications for broader epiphyte diversity, as opportunistic epiphytes that are better adapted to capitalise on limited establishment opportunities within urban landscapes (such as T. recurvata) outcompete species that do not possess these same advantages (Poltz and Zotz 2011).