Evolution of phenology
Our results clearly demonstrate a strong latitudinal cline for the number of days required to reach anthesis and the amount of biomass produced by M. vimineum populations collected from throughout its invasive range. Plants from higher latitudes flowered earlier and produced less biomass than plants from more southern populations. Growing plants in a common environment in growth chambers allowed us to demonstrate that these traits are most likely under genetic control, while replication of the experiment under two distinct light regimes confirmed that these trends are generalized findings, independent of specific local light regimes. Possible alternative explanations for the phenotypic patterns we recorded here include maternal or epigenetic effects. However, we believe these kinds of effects to be unlikely in this case. As Montague et al. (2007) noted, maternal effects are unlikely for the types of traits measured in this experiment. Maternal effects are only expected when seed dispersal distances are less than pollen movement (Galloway, 2005), a prerequisite clearly not met by the highly selfing M. vimineum. In addition, maternal effects are most likely expressed as early life-history traits such as propagule quality (Rossiter, 1996) as opposed to the late life-history traits studied here (days to anthesis and biomass production). However, to demonstrate that maternal effects are unlikely for the materials used in this study, we did examine early life-history traits in seven of the seed lots for which we had ample seed material. For these seven populations, there were no significant differences in germination or seed weights based on seed origin (P > 0.05, see also Flory et al., 2011). To date, the heritable epigenetic effects known to exist in plants are primarily triggered by various forms of biotic and abiotic stresses and generally lead to changes in genomic stability, such as increased transpon activity (Hauser et al., 2011). Although heritable epigenetic effects could have adaptive significance in some cases, it is difficult to imagine a scenario in which they could be responsible for the phenotypic cline observed here. In any case, even an epigenetically mediated effect that becomes a stably heritable element still represents a form of adaptive evolution. As such, our observed population differences indicate divergent phenological and biomass allocation characters under genetic control. Moreover, we conclude that clinal variation in the traits we measured is most likely due to adaptive evolution, as a result of differing selective pressure to complete flowering and seed maturity before the end of the growing season (i.e. cold temperatures arresting seed maturity) at differing latitudes. We suggest that such evolution of phenological traits has permitted the rapid expansion of M. vimineum invasions into more northern habitats and such evolutionary patterns may be a common trait of invasive plant species.
Although we could not measure fitness consequences of flowering time and biomass variation directly in this study, adaptive evolution of phenological timing is the most likely explanation for what we observed here. The only other possible explanations to explain such clinal patterns would be a nonadaptive process such as isolation-by-distance (IBD) or that native M. vimineum propagules were transported, via multiple introductions, from latitudes in Asia to environmentally equivalent latitudes in North America (preadaptation). IBD could result from the northern phenotype (i.e. shorter time to flowering at lower biomass) being introduced in the northern United States and the southern phenotype (i.e. longer time to flowering at higher biomass) being introduced in the southern United States. If these two phenotypes dispersed towards each other, eventually overlapped and began crossing (and these traits were neutral), we could expect to observe such a cline as a result of IBD. However, it is unlikely that flowering timing and biomass determination are neutral traits, though direct measurement of fitness or reciprocal transplant experiments would be required to confirm nonneutrality. The introduction and dispersal patterns that would be necessary in an IBD or preadaption scenario are also unlikely given the available herbarium records in North America. The plant was first noticed in the southeastern United States by the 1910s, and then radiated northward and westward (Fairbrothers & Gray, 1972). Though we cannot preclude the possibility of multiple introductions, even such introductions would almost certainly have been discrete events, located at major shipping locations, of plants from a limited latitudinal distribution in Asia, as the plant has been reported to be introduced as packing material for ceramics imported to North America from central China (Dorman, 2008). Furthermore, steady range expansion of M. vimineum across North America, particularly northward, has been noted in recent years (Mehrhoff, 2000).
Most interestingly, the evolutionary patterns we observed here must have arisen over a 100 year period or less. We are aware of three other genera of invasive plants for which similar flowering phenology clines have developed after invasion: Lythrum salicaria in North America (Montague et al., 2007), two Solidago species in Europe (Weber & Schmid, 1998) and Impatiens glandulifera in Europe (Kollmann & Bañuelos, 2004). Interestingly, both the invasive Solidago species and L. salicaria are self-incompatible, whereas I. glandulifera is self-compatible but protandrous (i.e. male flowers maturing before female flowers, often to promote outcrossing). Therefore, M. vimineum is the first invasive plant species identified that has evolved clinal flowering time phenology in its invasive range but does not possess biology favoring, or requiring, outcrossing. In fact, M. vimineum's biology promotes selfing due to cleistogamy; although, we should note that Maron et al. (2004) observed clines in biomass and fecundity for the invasive apomict Hypericum perforatum. Microstegium vimineum may also have evolved clinal phenology in a shorter period of time than Solidago spp., L. salicaria and I. glandulifera, which were all introduced in their invasive ranges by the early 1800s (Weber & Schmid, 1998; Blossey et al., 2001; Kollmann & Bañuelos, 2004). Furthermore, unlike these other species, M. vimineum is a wind-pollinated, monocot. With the addition of M. vimineum to the examples of invasive plants that have evolved flowering phenology in their invasive ranges, we now have evidence of such evolution in a wide variety of plant families and orders (Lythraceae, Myrtales; Balsaminaceae, Ericales; Asteraceae, Asterales; Poaceae, Poales), various reproductive biologies (highly selfing to obligate-outcrossing, and both wind and insect pollination), and several different habitat types (field, wetland and forest understory; e.g. Weber & Schmid, 1998; Kollmann & Bañuelos, 2004; Montague et al., 2007). This may indicate the presence of a general trend of phenological evolution during processes of plant invasion.
Microstegium vimineum seems to have undergone a lag phase from the time it was introduced in the early 1900s until it was recognized as an invasive species in the late 1980s (Barden, 1987). As a fecund annual with a relatively short-lived seed bank, M. vimineum possesses the potential for rapid adaptation, given adequate genetic diversity. At the minimum, it has cycled through approximately 100 generations in the invasive range, though evolution is likely to have taken place over a much shorter period of time in areas where the plant has only existed for a few decades (e.g. New England). Apparently, a tendency to inbreed has not impaired this species' ability to evolve clinal phenology, as it has done so even more rapidly than the outbreeding species that have evolved similar patterns. Interestingly, in our results there was a general trend towards smaller variance in days to anthesis for populations from the extremes of the invasive range, compared with the centre of the range (Table 1 and Fig. 1a,b). This could be a result of limited genetic diversity at the edges of the range, possibly due to decreased gene flow or stronger selection under the more extreme climate regimes expected at the northernmost range extents.
We have also conducted microsatellite (Simple Sequence Repeat) marker analysis on over 30 populations of M. vimineum from its native and invasive ranges (preliminary data published in Novy et al., 2012). We found that genetic diversity, measured both by heterozygosity and effective number of alleles, was lower in the invasive range, a clear indication of at least some degree of founder effect. Therefore, despite an initial bottleneck and high levels of inbreeding, M. vimineum has been able to evolve clinal variation in phenology over approximately 100 generations.
Both above- and below-ground M. vimineum biomass decreased with increasing latitudinal origin of populations. Because M. vimineum biomass is strongly correlated with seed production (total chasmogamous and cleistogamous seeds, r2 = 0.90, n = 24, S. L. Flory, unpublished data), reduced biomass from more northern populations probably reflects decreased seed production relative to more southern populations, which was also found for invasive populations of L. salicaria (Colautti et al., 2010). It has long been appreciated that for short-day flowering plants, local survival of a plant species depends on the production of viable seed before frost, or other inhospitable climate conditions, arrests metabolism (e.g. Allard, 1932). As optimal flowering time, where reproductive output is maximized before seasonal climatic conditions become unfavourable, will vary with photoperiod latitudinally, short-day flowering plants must evolve appropriate critical photoperiods for each local habitat to maximize reproductive success. For M. vimineum, this suggests evolution of a life-history trade-off between flowering time and size at reproduction.