The spatially patchy nature of Trichodesmium blooms in the lagoon (Fig. 2a–c), and the rapid temporal modifications in water-column abundance of filaments and colonies probably induced by physical drivers (turbulence and wind-stress), complicate in situ sampling when targeting changes in specific biomass. To overcome this, we collected Trichodesmium populations from the surface midday bloom and examined the physiological, biochemical, and gene expression changes occurring with time until the biomass crashed after ∼ 24 h (see methods Sect. 2.2) (Figs. 2 and 3). In these enclosed microcosms, Trichodesmium 16S copies comprised > 90 % of total copies (Fig. 3), enabling the use of Chl a to follow changes in its biomass (Fig. 2f). Maximal Chl a concentrations in the incubations (> 150 ± 80 µg L-1; n=6) were measured at the start of the incubation soon after the biomass collection and resuspension in FSW. These Trichodesmium populations collapsed swiftly over the next day, with Chl a concentrations declining to 24 and 11 µg L-1 Chl a after 10 and 22 h, respectively (Fig. 2f).

In experiment 1 we characterized the microbial community associated with the Trichodesmium biomass within the microcosms by 16S rRNA gene sequencing from two replicate bottles (experiment 1). At T0 94 and 93 % of the obtained 16S tags in both replicates (Fig. 3) were of the Oscillatoriales order (phylum Cyanobacteria), with 99.9 % of these sequences classified as Trichodesmium spp. (Fig. 3). In both replicates, the temporal decline of Trichodesmium biomass coincided with an increase in Alteromonas 16S tags, but this development temporally lagged in replicate 1 compared to replicate 2 (Fig. 3). Six hours (T6) after the surface bloom was originally sampled (T0), over 80 % of the 16S tags from replicate 1 were characterized as Trichodesmium. Fourteen hours after T0, Alteromonadales and Vibrionales replaced Trichodesmium, thereafter constituting only 9 % of 16S tags (Fig. 3). In replicate 2, Trichodesmium declined by 80 % 6 h after T0, with Alteromonadales and Flavobacteriales comprising the bulk of the biomass 18 h after the start of incubations (Fig. 3).

The rate of decline in Trichodesmium biomass within the 4.6 L microcosms paralleled that of Trichodesmium collected from the surface accumulations at 17:00 and incubated in 20 L carboys under ambient conditions for > 72 h (defined hereafter as experiment 2: Fig. 4). Here, Trichodesmium biomass decreased by > 80 % within 24 h of incubations with trichome abundance declining from ∼ 2500 trichomes L-1 at bloom collection to ∼ 495 trichomes L-1 (Fig. 4a). No direct correlation was observed between the decline of Trichodesmium and viral populations. VLP abundance at the time of the surface bloom sampling was at a maximum of 8.2 × 106 mL-1 (Fig. 4a), decreasing to 5.7 × 106 mL-1 in the next 4 h and then remaining stable throughout the crash period (within the next 42 h), averaging ∼ 5 × 106 ± 0.7 mL-1 (Fig. 4a).

As Trichodesmium crashed in the experimental incubations, high values of NH4+ were measured (Fig. 4b). In experiment 2, NH4+ increased exponentially from 73 ± 0.4 nmol NH4+ L-1 when the surface bloom was collected and placed in the carboys (17:00) to 1490 ± 686 after 24 h and values > 5000 nmol L-1 42 h after the incubation start (Fig. 4b). The high ammonia declined somewhat by the end of the experiment (after 72 h), yet was still high at 3494 ± 834 nmol L-1. Concurrently with the high NH4+ concentrations, and despite the dying Trichodesmium, we measured an increase N2-fixation rates. N2 fixation rose from 1.5 at T0 to 3.5 ± 2.8 nmol N L-1 h-1 8 h after incubations began and 11.7 ± 3.4 nmol N L-1 h-1 24 h later (Fig. 4b). These high values represent other diazotrophs including UCYN types and diatom–diazotroph associations that flourished after the Trichodesmium biomass had declined in the carboys (Bonnet et al., 2016b; K. Turk-Kubo, personal communication, 2016). POC and PON, representing the fraction of C and N incorporated into biomass, ranged between 5.2 and 11.2 µmol C L-1 and 0.6 and 1.1 µmol N L-1 during pre-bloom periods (Fig. 1c–d) and 12.6 ± 4.6 µmol C L-1 and 1.3 ± 0.5 µmol N L-1 when the surface bloom was sampled (Fig. 4b–c). Twenty-four hours after collection of bloom biomass, POC increased ∼ 6-fold to 63.2 ± 15 µmol C L-1 and PON increased 10-fold to 10 ± 3.3 µmol N L-1 (Fig. 4b–c). After 72 h, total POC was 62 ± 4 µmol C L-1 (Fig. 4c) and PON increased to 14.1 ± 6 µmol N L-1 (Fig. 4b).

Organic carbon in the form of TEP is secreted when Trichodesmium is stressed and undergoing PCD (Bar-Zeev et al., 2013; Berman-Frank et al., 2004). TEP concentrations in the lagoon waters during the pre-bloom period (first 20 days) fluctuated around ∼ 350 µg GX L-1 (Fig. 1d) that increased to ∼ 500 µg GX L-1 on day 22 (Fig. 1d). During the time of biomass collection from the surface bloom TEP concentration exceeded 700 µg GX L- 1 (Fig. 4c). After biomass enclosure (experiment 2) TEP concentrations declined to 420 ± 35 µg GX L-1 and subsequently to 180 ± 25 µg GX L-1 42 h and 72 h after T0 (Fig. 4c).

Metatranscriptomic analyses of the Trichodesmium biomass were conducted in samples from experiment 1, at T0, T8, and T22 (Fig. S1). We examined differential expression during this period by investigating a manually curated gene suite including specific pathways involved in P and Fe uptake and assimilation, PCD, or gas vesicle synthesis. Genes involved in the acquisition and transport of inorganic and organic P sources were upregulated, concomitant with biomass demise; significantly higher expression levels were evident at T8 and T22 compared to T0 (Table S1). Abundance of alkaline phosphatase transcripts, encoded by the phoA gene (Orchard et al., 2003), increased significantly (∼ 5-fold) from T0 to T22 (Fig. 5a). The transcript abundance of phosphonate transporters and C-P lyase genes (phnC, phnE, phnH, phnI, phnL, and phnM) increased significantly (5–12-fold) between T0 and both T8 and T22 (Fig. 5a, Table S1). Of the phosphite uptake genes, only ptxA involved in the phosphite (reduced inorganic phosphorus compound) uptake system, and recently found to operate in Trichodesmium (Martínez et al., 2012; Polyviou et al., 2015), was significantly upregulated at both T8 and T22 compared to T0 (4.5- and 7-fold change, respectively). The two additional genes involved in phosphite uptake, ptxB and ptxC, did not change significantly, as Trichodesmium biomass crashed (Fig. 5a).

As Fe limitation induces PCD in Trichodesmium (Berman-Frank et al., 2004, 2007), we examined genetic markers of Fe stress. At the time of surface bloom sampling (experiment 1, T0), Fe stress was indicated by higher differential expression of several genes. The isiB gene encodes flavodoxin and serves as a common diagnostic indicator of Fe stress in Trichodesmium, since it may substitute for Fe–S-containing ferredoxin (Bar-Zeev et al., 2013; Chappell and Webb, 2010). Transcripts of isiB were significantly higher at T0 (3-fold) than at T8 and T22 (Fig. 5b, Table S1). The chlorophyll-binding protein IsiA is induced in cyanobacterial species under Fe or oxidative stress to prevent oxidative damage (Laudenbach and Straus, 1988). Here isiA transcripts increased 2- and 3-fold from T0 to T8 and T22, respectively (Fig. 5b, Table S1). The Fe transporter gene idiA showed a transient higher transcript accumulation only at T8. As the health of Trichodesmium declined, transcripts of the Fe-storage protein ferritin (Dps) decreased by > 70 % at T22 (Fig. 5b, Table S1)

Our earlier work demonstrating PCD in Trichodesmium allowed us to utilize two independent biomarkers to investigate PCD induction during Trichodesmium demise, namely changes in catalytic rates of caspase-specific activity (Berman-Frank et al., 2004, 2007) and levels of metacaspase transcript expression (Bar-Zeev et al., 2013). When the surface bloom was sampled (experiment 1, T0), protein normalized caspase-specific activity was very low at 0.23 ± 0.2 pmol L-1 mg protein-1 min-1 (Fig. 6a). After a slight decline in the first 2 h, caspase activity increased throughout the experiment with 10-fold higher values (2.9 ± 1.5 pmol L-1 mg protein-1 min-1) obtained over the next 22 h as the bloom crashed (Fig. 6a).

We followed transcript abundance over the demise period for the 12 identified metacaspase genes in Trichodesmium (Asplund-Samuelsson et al., 2012; Asplund-Samuelsson, 2015; Berman-Frank et al., 2004): TeMC1 (Tery_2077), TeMC2 (Tery_2689), TeMC3 (Tery_3869), TeMC4 (Tery_2471), TeMC5 (Tery_2760), TeMC6 (Tery_2058), TeMC7 (Tery_1841), TeMC8 (Tery_0382), TeMC9 (Tery_4625), TeMC10 (Tery_2624), TeMC11 (Tery_2158), and TeMC12 (Tery_2963) (Fig. 6b, Table S1). A subset of these genes was previously shown to be involved in PCD of Trichodesmium cultures in response to Fe and light stress (Bar-Zeev et al., 2013, 2004; Bidle, 2015). Here, we interrogated the entire suite of metacaspases in natural Trichodesmium populations. As the biomass crashed from T0 to T22, 7 out of 12 metacaspases (TeMC1, TeMC3, TeMC4, TeMC7, TeMC8, TeMC9, and TeMC11) were significantly upregulated 8 and 22 h after T0 (Fig. 6b). For these genes, transcript abundance increased 2.3- to 5.3-fold 8 h after T0 and 3.5–6.2-fold 22 h after T0 (Fig. 6b, Table S1) TeMC5 and TeMC10 transcripts increased significantly after 22 h by 2.9- and 3.2-fold, respectively. TeMC6 was upregulated 2.9-fold after 8 h. TeMC2 transcripts did not significantly change over time. We did not detect any expression of TeMC12 throughout the experiment.

Export flux can be enhanced by PCD-induced sinking (Bar-Zeev et al., 2013) as PCD in Trichodesmium results in degradation of internal components, especially gas vesicles that are required for buoyancy (Berman-Frank et al., 2004). Although we did not measure changes in buoyancy itself, we observed rapid sinking of the Trichodesmium biomass in the bottles and carboys. The metatranscriptomic analyses demonstrated that, excluding one copy of gvpL/gvpF, gas vesicle protein (gvp) genes involved in gas vesicle formation (gvpA, gvpN, gvpK, gvpG, and gvpL/gcpF) were all significantly downregulated relative to T0 (Fig. 7, Table S1).

Our microcosm incubations allowed us to specifically focus on the loss factors and show the involvement of biotic and abiotic stressors in inducing PCD and mechanistically impacting the demise and fate of a natural Trichodesmium bloom. We recognize that the enclosure and incubation of collected biomass in bottles and carboys may accelerate cellular processes compared to the natural lagoon setting. However, the published rates of Trichodesmium mortality from field studies (Rodier and Le Borgne, 2010) indicate that these can parallel our loss rates with natural bloom demise occurring 24–48 h after peak of biomass.

We focused initially on biotic factors that could impact the incubated Trichodesmium biomass. The low number of harpacticoid zooplankton specific to Trichodesmium (O'Neil and Roman, 1994; O'Neil, 1998) in the lagoon (Hunt et al., 2016) and especially those in the bottles (personal observation) refutes the possibility that grazing caused the massive mortality of Trichodesmium biomass in our experimental incubations.

Viruses have been increasingly invoked as key agents terminating phytoplankton blooms (Brussaard et al., 2005; Jacquet et al., 2002; Lehahn et al., 2014; Tarutani et al., 2000; Vardi et al., 2012). Infection by phages has been invoked as the mechanism of Trichodesmium bloom crashes, but it has yet to be unequivocally demonstrated (Hewson et al., 2004; Ohki, 1999); indeed, no specific Trichodesmium phage has been isolated or characterized to date (Brown et al., 2013). Here, total VLP abundance was highest at the time of sampling from the surface Trichodesmium bloom and at the start of the incubation (∼ 8 × 106 VLP mL-1). It actually declined 2-fold in the first 8 h of incubation before increasing over the next 32 h (Fig. 4a). While our method of analysis cannot distinguish between phages infecting Trichodesmium from those infecting other marine bacteria, it argues against a massive, phage-induced lytic event of Trichodesmium. Such an event would have yielded a notable burst of VLP upon bloom crash, especially considering the high Trichodesmium biomass observed. The coincidence between the maximal abundance of VLP and highest Trichodesmium biomass is counter to viruses serving as the mechanism of mortality in our incubation experiments. Nonetheless, virus infection itself may be a stimulant for community N2 fixation perhaps by releasing key nutrients (i.e., P or Fe) upon lysis of surrounding microbes (Weitz and Wilhelm, 2012). Although we did not characterize them here, it is indeed possible that Trichodesmium-specific phages were present in our incubation experiments and they may have exerted additional physiological stress on resident populations, facilitating PCD induction. Virus infection has been shown to increase the cellular production of reactive oxygen species (Evans et al., 2006; Vardi et al., 2012), which in turn can stimulate PCD in algal cells (Berman-Frank et al., 2004; Bidle, 2015; Thamatrakoln et al., 2012). Viral attack can also directly trigger PCD as part of an antiviral defense system activated to limit virus production and prevent massive viral infection (Bidle and Falkowski, 2004; Bidle, 2015; Georgiou et al., 1998).

Nutrient stress can be acute or chronic to which organisms may acclimate on different timescales. Thus, for example, the consistently low DIP concentrations measured in the lagoon during the 22 days preceding the Trichodesmium surface bloom probably enabled acclimation responses such as induction of APA and other P acquisition systems. Trichodesmium has the ability to obtain P via inorganic and organic sources, including methylphosphonate, ethylphosphonate, and 2-aminoethylphosphonate (Beversdorf et al., 2010; Dyhrman et al., 2006), and via a phosphite uptake system (PtxABC) that accesses P via the reduced inorganic compound phosphite (Martínez et al., 2012; Polyviou et al., 2015). Our metatranscriptomic data demonstrated upregulated expression of genes related to all three of these uptake systems (DIP, phosphonates, phosphites) 8 and 22 h after incubation began, accompanying biomass demise (Fig. 5a). This included one gene for phosphite uptake (ptxA) and several genes from the phosphonate uptake operon (phnDCEEGHIJKLM) (Hove-Jensen et al., 2014). Upregulated expression of phnD, phnC, phnE, phnH, phnI, phnJ, phnK, phnL, and phnM occurred as the Trichodesmium biomass crashed (Fig. 5a, Table S1), consistent with previous results demonstrating that phnD and phnJ expression levels increased during DIP depletion (Hove-Jensen et al., 2014). It is likely that, during bloom demise, the C-P lyase pathway of remaining living cells was induced when DIP sources were extremely low, while POP and DOP increased along with the decaying organic matter. The ability to use phosphonates or phosphites as a P source can provide a competitive advantage for phytoplankton and bacteria in P-depleted waters (Coleman and Chisholm, 2010; Martinez et al., 2010). Thus, it is puzzling why dying cells would upregulate phn genes or phoA transcripts after 22 h incubation (Fig. 5a). A more detailed temporal resolution of the metatranscriptomic analyses may elucidate the expression dynamics of these genes and their regulating factors. Alternatively, in PCD-induced populations, a small percentage of cells remain viable and resistant as either cysts (Vardi et al., 1999) or hormogonia (Berman-Frank et al., 2004) that can serve as the inoculum for future blooms. It is plausible that the observed upregulation signal was attributable to these subpopulations.

The concentrations of dissolved and bioavailable Fe were not measured in the lagoon water during the experimental period as Fe is typically replete in the lagoon (Jacquet et al., 2006). However, even in Fe-replete environments such as the New Caledonian lagoon, dense patches of cyanobacterial or algal biomass can deplete available resources and cause limited microenvironments (Shaked, 2002). We obtained evidence for Fe stress using several proxy genes demonstrating that enhanced cellular Fe demand occurred during the bloom crash (Table S1). Trichodesmium's strategies of obtaining and maintaining sufficient Fe involves genes such as isiB. isiB was highly expressed when biomass accumulated on the surface waters, indicative for higher Fe demand at this biomass load (Bar-Zeev et al., 2013; Chappell and Webb, 2010), yet expression declined significantly with the dying biomass. Transcripts for chlorophyll-binding, Fe-stress-induced protein A (IsiA) increased (albeit not significantly) 3-fold over 22 h of bloom demise (Fig. 5b, Table S1). In many cyanobacteria, isiA expression is stimulated under Fe stress (Laudenbach and Straus, 1988) and oxidative stress (Jeanjean et al., 2003) and functions to prevent high light-induced oxidative damage by increasing cyclic electron flow around the photosynthetic reaction center photosystem I (Havaux et al., 2005; Latifi et al., 2005; Michel and Pistorius, 2004). Dense surface blooms of Trichodesmium are exposed to high irradiance (on day 23 average photosynthetically active radiation was 3000 µmol photons m-2 s-1). It is possible that high Fe demand combined with the oxidative stress of the high irradiance induced the higher expression of isiA (Fig. 5b). As cell density and associated self-shading of Trichodesmium filaments decreased during bloom crash, light-induced oxidative stress is likely the principal driver for elevated isiA expression.

The gene idiA is another environmental Fe stress biomarker that allows acquisition and transfer of Fe through the periplasm into the cytoplasm (Chappell and Webb, 2010). In our incubation, upregulated expression of idiA (an ABC Fe+3 transporter) was evident after 8 h. This is consistent with increasing Fe limitation, as Trichodesmium abundance (measured via 16S rRNA gene sequencing) was still high at T6 (after 6 h of incubation) (replicate 1). These findings are consistent with proteomics analyses from depleted iron (0 µM Fe) Trichodesmium cultures which revealed an increase in IdiA protein expression (Snow et al., 2015). Lastly, our metatranscriptomic data highlighted a reduction in Fe storage and utilization, as the expression of Fe-rich ferritin-like DPS proteins (Castruita et al., 2006), encoded by dpsA, decreased ∼ 5-fold by the time that most of the biomass had crashed (T22) (Fig. 5b, Table S1). dpsA was also downregulated under Fe-replete conditions in Synechococcus (Mackey et al., 2015), but the downregulation observed here is more likely related to Trichodesmium cells dying and downregulating Fe-demanding processes such as photosynthesis and N2 fixation.

The physiological and morphological evidence of PCD in Trichodesmium has been previously documented in both laboratory (Bar-Zeev et al., 2013; Berman-Frank et al., 2004) and environmental cultures collected from surface waters around New Caledonia (Berman-Frank et al., 2004). Here, we confirmed characteristic features of autocatalytic PCD in Trichodesmium such as increased caspase-specific activity (Fig. 6a), globally enhanced metacaspase expression (Fig. 6b), and decreased expression of gas vesicle maintenance (Fig. 7). Metatranscriptomic snapshots interrogating expression changes in all of the annotated Trichodesmium metacaspases (Fig. 6b) generally portrayed upregulated expression concomitant with biomass decline. Our results are consistent with previous observations that Fe-depleted PCD-induced laboratory cultures of Trichodesmium IMS101 had higher expression levels of TeMC1 and TeMC9 compared to healthy Fe-replete cultures (Bar-Zeev et al., 2013; Berman-Frank et al., 2004). To our knowledge, this is the first study examining expression levels of metacaspases in environmental Trichodesmium samples during a natural bloom. Eleven of the 12 metacaspases in Trichodesmium were expressed in all three metatranscriptomes from the surface bloom. To date, no specific function has been determined for these metacaspases in Trichodesmium other than their association with cellular stress and death. Efforts are underway to elucidate the specific cellular functions, regulation, and protein interactions of these Trichodesmium metacaspases (Bar-Zeev et al., 2013; Pfreundt et al., 2014; D. Spungin, personal communication, 2016).

In cultures and isolated natural populations of Trichodesmium, high caspase-like specific activity is correlated with the initial induction stages of PCD with activity declining as the biomass crashes (Bar-Zeev et al., 2013; Berman-Frank et al., 2004, 2007). Here, caspase-like activity increased with the crashing populations of Trichodesmium (Fig. 6a). Notably, maximal caspase activities were recorded at T22, after which most Trichodesmium biomass had collapsed. The high protein-normalized caspase-specific activity may be a result of a very stressed and dying subpopulation of Trichodesmium that had not yet succumbed to PCD (Berman-Frank et al., 2004). Alternatively, the high caspase-like activity may be attributed to the large population of Alteromonas bacteria that were associated with the remaining detrital Trichodesmium biomass. However, currently, we are unaware of any publications demonstrating high cellular caspase-specific activity in clades of γ-Proteobacteria.

Gas vesicles are internal structures essential for maintaining buoyancy of Trichodesmium populations in the upper surface waters enabling them to vertically migrate and respond to light and nutrient requirements (Capone et al., 1997; Walsby, 1978). Mortality via PCD causes a decline in the number and size of cellular gas vesicles in Trichodesmium (Berman-Frank et al., 2004) and results in an enhanced vertical flux of trichomes and colonies to depth (Bar-Zeev et al., 2013). Our metatranscriptomic data supported the subcellular divestment from gas vesicle production during bloom decline, as the expression of vesicle-related genes was downregulated (Fig. 7). In parallel, TEP production and concentration increased to > 800 µg GX L-1, a 2-fold increase from pre-bloom periods (Figs. 1d and 4c). When nutrient uptake is limited, but CO2 and light are sufficient, uncoupling occurs between photosynthesis and growth (Berman-Frank and Dubinsky, 1999), leading to increased production of excess polysaccharides (such as TEP) and corresponding with high TEP found in bloom decline phases rather than during the increase in population density (Engel, 2000; Smetacek, 1985). In earlier studies we demonstrated that PCD-induced demise in Trichodesmium is characterized by an increase in excreted TEP (Berman-Frank et al., 2007) and enhanced sinking of particulate organic matter (Bar-Zeev et al., 2013). TEP may be positively buoyant (Azetsu-Scott and Passow, 2004), yet their stickiness causes aggregation and clumping of cells and detritus, ultimately enhancing sinking rates of large aggregates, including dying Trichodesmium (Bar-Zeev et al., 2013).

In the incubations, other diazotrophic populations succeeded the declining Trichodesmium biomass as indicated by increasing N2-fixation rates, POC, and PON (Fig. 4b). In experiment 2, based on qPCR of targeted diazotrophic phylotypes, the diazotroph community composition shifted from being dominated by Trichodesmium spp. and unicellular groups UCYN-A1, UCYN-A2, and UCYN-B (T0) to one dominated by diatom–diazotroph associations Het-1 and Het-2 (T72) (Bonnet et al., 2016b; K. Turk-Kubo, personal communication, 2016). In experiment 1 heterotrophic bacteria thrived and increased in abundance as the Trichodesmium biomass crashed (Fig. 3).

Trichodesmium colonies host a wide diversity of microorganisms, including specific epibionts, viruses, bacteria, eukaryotic microorganisms, and metazoans (Hewson et al., 2009; Hmelo et al., 2012; Ohki, 1999; Paerl et al., 1989; Sheridan et al., 2002; Siddiqui et al., 1992; Zehr, 1995). Associated epibiont bacterial abundance in dilute and exponentially growing laboratory cultures of Trichodesmium is relatively limited (Spungin et al., 2014) compared to bloom conditions (Hewson et al., 2009; Hmelo et al., 2012). Proliferation of Alteromonas and other γ-Proteobacteria during biomass collapse (Fig. 3) confirms their reputation as opportunistic microorganisms (Allers et al., 2008; Hewson et al., 2009; Frydenborg et al., 2014; Pichon et al., 2013). Such organisms can thrive on the influx of organic nutrient sources from the decaying Trichodesmium as we observed (Fig. 3). Furthermore, the increase in organic matter including TEP produced by the stressed Trichodesmium (Figs. 1d and 4c) probably stimulated growth of these copiotrophs. Moreover, as the Trichodesmium biomass declined in the carboys, the high concentrations of NH4+ (> 5000 nmol L-1) (Fig. 4b) sustained both autotrophic and heterotrophic organisms (Berthelot et al., 2015; Bonnet et al., 2016b, c). Thus, the increase in volumetric N2 fixation and PON that was measured in the incubation bottles right after the Trichodesmium crash in experiment 2 (Fig. 4b) probably reflects both the enhanced activity of other diazotrophs (see above and Bonnet et al., 2016b) and the resistant residual Trichodesmium trichomes (Berman-Frank et al., 2004) with increased cell-specific N2 fixation. This scenario is consistent with the hypothesis that PCD induction and death of a fraction of the population confers favorable conditions for survival and growth of individual cells (Bidle and Falkowski, 2004; Bidle, 2015).