The Environment and Life Cycle Changes

Pauses: Dormancy. In the life cycles of most organisms there are one or more reversible stages characterized by reduced metabolic activity and related changes. These periods of delay in development are variously termed dormancy (plants), ‘shut-down’ (microbes), diapause (insects), hibernation (certain mammals) or estivation (certain birds, mammals, and lungfishes). While obviously each is unique in detail, all share the attribute of quiescence and for simplicity will be construed here broadly as dormancy.

Entry into such a phase is typically associated with a morphological transition such as the dropping of leaves by deciduous plants, or the formation of resistant propagules such as spores, seeds, or cysts by numerous taxa. In a life cycle context, dormancy frequently coincides with the dispersal stage and is often closely associated with genetic recombination. This is unlikely due to happenstance, as discussed later. A major consequence is that one or more of the emerging products of meiosis and genetic fusion (new genets) are suited to the new environment. Dormancy may also appear as an interruption of the growth phase, as in the annual cycle of vegetative expansion, growth cessation, and bud set in trees.

Dormancy is usually though not always (see e.g., Simons 2014) interpreted as a bet-hedging strategy to adverse conditions (Childs et al. 2010; Scott and Otto 2014; Rajon et al. 2014). Levins (1969) points out that it may arise instead as an adaptation simply to extreme environmental uncertainty. A developmental delay is in effect a ‘cost-averaging’ mechanism that spreads reproductive output over time and across environments. Adversities may occur regularly or irregularly and can involve abiotic factors such as drought and temperature extremes, or biotic factors such as extreme predation (for an unusual example with bdelloid rotifers, see Wilson and Sherman 2013) and scarcity of resources. Microbial life in oligotrophic environments such as the open seas, deep sub-seafloor, and soil is largely one of varying degrees of starvation, dormancy, and physiological quiescence where metabolic rates and turnover times are several orders of magnitude lower than in nutrient-rich environments (Morita 1997; Hoehler and Jorgensen 2013).

Relevant timescales there are centuries to millennia. Attempts to interpret the evolution of dormancy should recognize that while in some cases it may have arisen in response to a particular adversity or uncertainty, dormancy may now be maintained by selection for other reasons. For example, the internal physiological environment during insect diapause is complex and, where diapause is obligate, the phenomenon may have become a developmental necessity and operate under the control of factors different from those having driven its inception (Levins 1969).

Thus, if environments were always benign and predictable, would quiescence as a trait be maintained? This is difficult if not impossible to test because other than within the artificial confines of a laboratory setting (e.g., unrestricted bacterial growth in a chemostat) these situations are rare, though occasionally we see such glimmers (Wolda 1987, 1989). Harper (1977, p. 74) points out that seed development and germination are continuous in some tropical maritime mangroves. The prominence of physical factors as potential phenological cues is attenuated in lowland tropical rain forests, where leaf production and leaf senescence of most tree species may “appear to follow a free-running periodicity (i.e., no annual biological clock) [sic] based on internal regulation” (Reich 1995, p. 168).

However, even the moist tropics have some modest seasonality, for example with respect to irradiance and the amount of precipitation. Moreover, changes in a physical cue tend to be transmitted and frequently amplified among community members because of species interactions in food and energy webs (Chap. 3 and Wolda 1987; van Schaik et al. 1993). For example, phenological changes in the plant community imposed by the abiotic environment are often reflected in their consumer populations, which switch their diet, change breeding pattern, or their range (van Schaik et al. 1993).

In most if not all organisms, including even at least some prokaryotes, numerous biological physiological processes such as patterns of gene expression oscillate with a standardized periodicity of activity. (Note that these cyclic changes are in contrast to plasticity resulting from noise and stochastic switching—as exemplified by bacterial persisters, see later discussions and Ackermann 2015.) When the cycles occur in about 24-h increments they comprise what is known as circadian rhythm.

The circadian clock may be set by an environmental cue, such as the well-known light:dark cycle, or it may run irrespective of an environmental signal (free-running rhythm) (Bell-Pedersen et al. 2005). Though varying extensively in complexity, all circadian systems are based on an internal oscillator system of negative and positive components that compose an autonomous, internal regulatory feedback loop used to generate the periodicity (for mechanism and examples, see Bell-Pedersen et al. 2005).

Among the processes regulated by a circadian clock, depending on the organism, are photosynthesis (even in cyanobacteria; Cohen and Golden 2015), solar-tracking (heliotropism) in sunflowers (Atamian et al. 2016), cell division, uptake and other metabolic activities, locomotion, and melatonin levels. The unicellular cyanobacterium Prochlorococcus (discussed in Chap. 3) lives throughout the euphotic zone in tropical and subtropical oceans (Zinser et al. 2009; Biller et al. 2015). It is an oxygenic phototroph and so, not surprisingly, its daily activities are closely coordinated with the light:dark cycle. In a detailed study, Zinser et al. (2009) report on patterns of cell physiology (e.g., cell cycle; C-fixation; nutrient acquisition and assimilation, etc.) and gene expression patterns.

They examined the entire transcriptome and found that 80% of the annotated genes exhibited cyclic expression over the diel cycle in tightly scripted fashion, with expression activity for most peaking at sunrise or sunset. New cells were born usually late in the day or at night; when sunlight appeared at dawn they were fully metaboli- cally primed for maximum photosynthesis and biomass increase. This tiny unicellular creature illustrates beautifully how the ability of organisms to coordinate biological processes with daily environmental cycles has adaptive value.

Where there are seasonally recurring hazards, dormancy tends to be initiated synchronously with and typically somewhat in advance of the changes based on an external clock such as photoperiod. In the case of seeds, germination is more reliably triggered by photoperiod than by temperature, which, because of its vagaries, can be an unreliable predictor (e.g., hazards of a late frost). There is considerable theoretical work on optimal clock timing, presumably set by natural selection (Chap. 3 in Harper 1977; Scott and Otto 2014). A photoperiod-based clock may not allow the organism to take advantage of an extraordinarily early season but would prevent a catastrophic loss associated with being unprepared for an exceptionally late lethal event. Using photoperiod rather than temperature as the cue means that efficient, advance preparation is possible rather than having to adjust to events themselves and usually being too late.

Thus, the physical form of the signal and the response may bear no relationship (Levins 1968; Harper 1977) and the less seasonal is the environmental change, the less useful is an external clock (e.g., unpredictable disasters such as hurricanes; see comments on adaptive phenotypic plasticity in Simons [2014] and imposed stochastic switching related to bacterial bet-hedging by Beaumont et al. [2009]). While the cyclical synchrony between environmental adversity and organism preparations may give the appearance of prediction, it is important to remember that the pattern is ingrained because of the actions of natural selection acting over countless past generations on the individual's ancestors (recall discussion on adaptation in Chap. 1).

Some form of developmental delay is phylogenetically widespread and continues to be maintained by natural selection in organisms that physiologically could have shorter generation times than they in fact have (Levins 1969). This suggests that dormancy is beneficial especially as it is not a cost-free transition. Organisms usually have to make investments in switching to a resistant form; in the maintenance of the dormant state where, for example, environmental signals must be transduced; and in the process of resuscitation (Dworkin and Shah 2010; Lennon and Jones 2011).

These costs vary substantially with the degree of shut-down and other internal and external factors such as temperature (Price and Sowers 2004), but are rarely if ever nonexistent. They have been examined in numerous papers by Maughan and colleagues (e.g., 2009) for the spore-forming microbe Bacillus subtilis. In principle, costs are theoretically lower for adaptation based on a purely stochastic switching mechanism (no sensing; involves population heterogeneity) than a responsive switching mechanism (involves direct response to an external cue) (Kussell and Leibler 2005). This leads to the conclusion that the cost of a responsive mechanism is justified where environmental uncertainty is higher, whereas the stochastic switching is favored where change is relatively infrequent.

Dormancy also comes with the ecological cost that by becoming quiescent the organism is opting out of active competition for resources, territory, and contributing its genes to the gene pool. As such, Harper (1977, p. 62) views dormancy, at least in plants, as a “weak solution to the problem of adaptation to a changing environment” (for one example of such costs, see Janzen and Wilson 1974). An alternative in a cyclically unfavorable environment is potentially to migrate. To a degree, organisms adapted for dispersal combine both options because dispersed propagules typically are dormant. At the extremes, Harper (1977, p. 82) views two strategies for dealing with a deteriorating local habitat: “escape to somewhere else” (i.e., migration), or “wait until the right habitat reappears” (dormancy).

An intriguing idea is that mobility and the more versatile physiological adaptation of many animals to changing environments have largely replaced the need for dormancy (Bonner 1958). An intermediate option apparently is to transition to a different but metabolically still relatively active phenotype. A case in point is the alternation between large- and small-leaved forms in certain plants in seasonal wet and dry seasons; this is one example of seasonal dimorphism (Chap. 3 in Harper 1977; Palacio et al. 2006).

Bacterial 'persisters'. In meeting potential environmental adversity, clonal organisms, by virtue of their growth form (Chap. 5), can do things that unitary organisms cannot: They put a fraction of their ramets on a standby condition, while others continue active growth. This would be effectively a form of bet-hedging and there is some evidence from clonal plants for this strategy (de Kroon et al. 2005; Magyar et al. 2007). This option does not involve sensing but rather simply population heterogeneity and has been discussed by Kussell and Leibler (2005) under the semantics of stochastic switching.

There is even better evidence for such a strategy in the microbial world, particularly for bacteria. Certain bacteria exhibit standard forms of dormancy closely analogous to seeds based on production of a resistant, frequently long-lived propagule (e.g., endospore; McKenney et al. 2013). Of more interest, however, is that all or virtually all bacteria produce at very low frequency a subpopulation of slow- or nongrowing cells called 'persisters'. Other species segregate a population into ‘sporulators’ and ‘diauxic growers’ (Veening et al. 2008a). Not only is the persister phenomenon ecologically significant from the standpoint of survival, it has important medical implications because traditionally defined persisters are metabolically more-or-less inactive and as such are usually tolerant to antibiotics that kill replicating cells.

Persistence has been recognized almost from the inception of the antibiotic era (early 1940s) and a classic example is the protracted regimen of multiple antibiotics necessary to control diseases such as tuberculosis caused by Mycobacterium tuberculosis (Hu and Coates 2003; Zhang 2014; see further comments on mycobacteria below). Persisters are a specific example of the ability of bacterial populations to exhibit ‘bistability’, which refers to the bifurcation of a system into two dynamically stable states, in this case phenotypically distinct subpopulations (Dubnau and Losick 2006; Veening et al. 2008b). The cells in the original metapopulation are clonal, effectively genetically identical, and grown under effectively homogeneous, ‘identical’ environments. Bistability can arise from stochastic, epigenetic alteration in gene expression (generally referred to as ‘noise, noted at the outset and with further discussion later).

The persister phenomenon was investigated elegantly by Balaban et al. (2004) who directly monitored the time course of population dynamics in wild-type (wt) and per- sister-enhanced mutants of E. coli cells. Growth was followed microscopically in an ingenious microfluidic device where narrow grooves provided a system whereby the descendants of each bacterial cell formed a separate, linear microcolony. The authors found that the wt population consisted of three components: (i) persisters that were continuously generated by spontaneous, stochastic phenotype-switching; (ii) persisters that were triggered to form by responsive switching during stationary phase (for bacterial growth phases, see Finkel 2006); and (iii) typical, rapidly growing cells. This implies that the original clonal bacterial population became at some level heterogeneous and cells could switch reversibly between persister and normal growth. (The broader issue of individual cell phenotypic heterogeneity is discussed by Avery 2006 and Hashimoto et al. 2016.) Persisters also can be induced by various triggers. In numerous recent reports in this rapidly emerging field, several endogenous and exogenous ‘stress’ cues have been identified, among them starvation, DNA damage, and antibiotic treatment (Cohen et al. 2013; Amato et al. 2014; Zhang 2014).

It now appears that some forms of persistence may not be attributable to simply nondividing persisters, but rather to balanced division and death in this subpopulation; or to quiescence in most cellular activities but selective activity in others such as antibiotic efflux systems (Pu et al. 2016; however, this seems to confuse conventional resistance mechanisms with the persister phenomenon. See Chap. 2 Sidebar and Nikaido 2009). With respect to balanced division and death, using similar microfluidic devices and time-lapse microscopy, in this case of Mycobacterium smegmatis, Wakamoto et al. (2013) describe stochastic pulses of the enzyme catalase-peroxidase (KatG) that activates a drug (isoniazid), which kills the bacterium. Hence, such bursts are negatively correlated with cell survival. Pulsing and death are not a function of single-cell growth rates but apparently are determined by molecular noise (see following section).

Persistent subpopulations (termed cell lineages) of M. smegmatis may survive because of infrequent pulses of the enzyme, which in turn fail to activate isoniazid to lethal levels. Notable also is that mycobacteria are distinctive in several attributes: their unique cell-wall characteristics; their ability to extensively restructure the cell envelope in deterministic fashion based on environmental triggers; and their enlargement and division processes that result in asymmetric daughter cells (Aldridge et al. 2012; Kieser and Rubin 2014). The populations resulting from asymmetry have different growth rates and antibiotic sensitivity (Richardson et al. 2016). In other words, in mycobacteria these basic phenomena contributing to substantial phenotypic plasticity are supplemented by stochastic changes in gene expression.

To summarize, the persister phenomenon, whether arising from a truly quiescent or a dynamically maintained cell birth:death state, has important survival and evolutionary implications. It may actually be an ‘evolutionary reservoir’ for emergence of resistant cells (Cohen et al. 2013). It appears to be one of several examples of a bet-hedging strategy by bacteria (e.g., Veening et al. 2008b). While in origin, persistence, like mutation, may be inevitable and nothing more than the result of various errors (Johnson and Levin 2013), it is intuitive that the consequences have survival value. A clone possessing such a property would have an advantage over one that did not or that produced persisters less frequently. This remains to be seen; presently, some work supports such an interpretation (Kussell et al. 2005) and some authors argue otherwise (Johnson and Levin 2013).