Philippa Kaur

For this study we use the University of Victoria Earth System Climate Model (UVic ESCM) version 2.940,41,42. The UVic ESCM is an intermediate-complexity earth system model with a resolution of 1.8(^circ ) latitude by 3.6(^circ ) longitude and 19 ocean depth levels. The surface ocean level is 50 m deep. The model contains a two dimensional energy moisture-balance model of the atmosphere, as well as representations of sea ice, ocean circulation and sediments, and terrestrial carbon. The particular biogeochemical version used here includes three phytoplankton functional types, namely diazotrophs (DZ), mixed phytoplankton (PH), and small phytoplankton and calcifiers (CO)43. The model pre-industrial climate has been previously described43, as has its response to business-as-usual atmospheric (hbox{CO}_2) forcing44. The following sections describe the MP model. A model schematic is presented in Fig. 6.
Figure 6

Microplastic model schematic. Marine snow is produced as a fixed fraction of the free detritus (DET) pool. MP aggregates with this marine snow, entering the (hbox{MP}_A) (marine snow entrained MP) pool. (hbox{MP}_A) held in aggregates sinks at the aggregate rate, with a fraction reaching the seafloor considered to be lost from the ocean. Detrital remineralisation releases the (hbox{MP}_A) from marine snow aggregates at the rate of detrital remineralisation. MP is also grazed by zooplankton and excreted into a pellet-bound (hbox{MP}_Z) pool. Pellet-bound (hbox{MP}_Z) sinks and is released back to the free MP pool at the rate of detrital remineralisation, but some is also lost at the seafloor. Details on the biogeochemical aspects of the model are previously described43.

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Model description
The base model43 was modified in order to quantify the roles of two of the three theorised biological export pathways on MP (aggregation in marine snow and zooplankton ingestion; for now, we neglect an explicit representation of biofouling29). We distinguish between detritus that becomes faecal pellets, and the physical aggregation of marine snow, by introducing a new faecal pellet tracer to divert 50% of zooplankton particulate losses into a separate detrital pool27. For simplicity, this new pellet detrital pool has the same sinking parameterisation as the original detritus. Using the same sinking rates for both detrital classes produces ocean biogeochemistry that is identical to the previously published versions of the model. In the model, plastic particles only interact passively with marine snow (they do not, for example, modify aggregate sinking rates), but they interact actively with zooplankton grazing (described below). Plastic particles have been observed to both increase and decrease the sinking rates of marine snow21,22 and decrease the sinking rates of faecal pellets20,38, but for simplicity and as a first approximation we neglect these effects in our model.
Three MP compartments are introduced; “free” (unattached) microplastic (MP), microplastic aggregated in marine snow ((hbox{MP}_A)), and microplastic in zooplankton faecal pellets ((hbox{MP}_Z)). All MP are considered to represent particles within a biologically active size range, but this size (and the particles’ composition) is never made explicit. These assumptions could bias modelled MP towards polymer types favoured by generic zooplankton34 and particle sizes in the lower end33,34 of the defined range of microplastic size. However, our parameter sensitivity testing in the Supplemental Information tests various fractional uptake rates that implicitly consider size and particle composition in how biologically “active” the MP pool is. As with all model ocean tracers, microplastic concentrations (MP) vary according to:

$$begin{aligned} frac{dmathrm {MP}}{dt} = T + S(mathrm {MP}) end{aligned}$$
(1)

With T including all transport terms and S representing all source minus sink terms. The source and sink terms for free microplastic are:

$$begin{aligned} S(mathrm {MP}) = Emis – S(mathrm {MP}_A) – S(mathrm {MP}_Z) + w_p frac{delta mathrm {MP} times F_R}{delta z} end{aligned}$$
(2)

Microplastic is emitted to the ocean (Emis) along coastlines and major shipping routes using a scaling against regional (hbox{CO}_2) emissions (a dataset provided with the standard UVic ESCM version 2.9 package download), in order to approximate degree of industrialisation and population density in this first version of this model. The rate of emission is a proportion of the total annual plastic waste generation ((F_T))45. For now, abiotic degradation of macroplastics as a source of microplastics to the ocean is neglected to keep the model simple and focus on biological transport. The MP then exchanges with the marine snow ((hbox{MP}_A)) and zooplankton faecal pellet ((hbox{MP}_Z)) pools. A fast particle rising rate ((w_p)) of 1.9 cm per second46 is prescribed to a fraction ((F_R)) of the free MP in each grid cell below the surface level as an approximation of positive buoyancy. An alternative approach would be to assign a uniform rise rate to all MP particles, and to subject the value of the rise rate to sensitivity testing. However, a weakness of this alternative approach is that the many types of plastic in the ocean have different characteristic buoyancies, which could produce unique particle pathways18. In this alternative approach it would be more appropriate to explicitly simulate multiple MP types in the model (which we sought to avoid in this first modelling effort for the sake of simplicity). Nevertheless, we conducted a sensitivity test using several different rise rates, and the effect of reducing the mean rise rate was similar to reducing the fraction assigned a rise rate.
In the current model version there are no abiotic breakdown rates (i.e., photo-degradation39) or respiration losses47 removing MP from circulation.
MP is modelled to aggregate in marine snow as:

$$begin{aligned} S(mathrm {MP}_A) = A_{upt} – A_{rel} – w_Dfrac{delta mathrm {MP}_A}{delta z} end{aligned}$$
(3)

MP particles are taken up ((A_{upt})) via a Monod function applied to the rate of marine snow formation (sources of detritus; (D_A) in nitrogen units, multiplied by an aggregation fraction, (F_A)) in order to approximate an increased likelihood of MP/marine snow encounter with increasing MP concentrations that approaches a level of saturation at high MP concentrations:

$$begin{aligned} A_{upt} = frac{mathrm {MP}}{k_P + mathrm {MP}} times source(D_A) times F_A end{aligned}$$
(4)

The uptake constant ((k_P)) is subjected to sensitivity testing, as is the fraction of marine snow aggregation ((F_A)). In this parameterisation, the aggregation of MP in marine snow represents the net uptake of MP into aggregates by both aggregation and biofouling processes. Biofouling occurs mostly in the upper 50 m35, which is the entire surface layer grid cell in our model. The entrainment-release cycle of biofouling is implicit in our parameterisation via the microbial loop, which is temperature-dependent. Sensitivity testing of the (k_P) and (F_A) parameters therefore represent testing of the net aggregation due to non-zooplankton biological aggregation effects. MP is released ((A_{rel})) from marine snow at the rate of detrital remineralisation ((mu _D)). This rate is temperature-dependent and results in higher rates of release in the low latitudes.

$$begin{aligned} A_{rel} = mu _D mathrm {MP}_A end{aligned}$$
(5)

A particle sinking term ((w_D)) applies to marine snow-associated MP, and has the same value as sinking detritus. The base unit of all MP tracers is number of plastic particles. As a first approximation we assume that all marine snow aggregates forming from free detritus have the characteristic of diatom aggregates (8.8 (upmu )g C per aggregate48). Model detritus in mmol N is converted to mmol C using Redfield stoichiometry, which is then converted to (upmu )g C to calculate the maximum number of aggregates. The maximum number of aggregates is then multiplied by the aggregation fraction (F_A), to calculate (hbox{MP}_A) source and sink rates. MP is conserved for all MP tracers when surface flux balances sedimentary loss rate. What fraction of MP particles reaching the seafloor via aggregate and faecal pellet ballasting are returned to the water column ((F_B)) is tested. For simplicity and as a first approximation, detritus ballasted by calcite, and calcite43, are assumed to not aggregate with microplastic.
Similarly, for MP associated with zooplankton, sources and sinks are:

$$begin{aligned} S(mathrm {MP}_Z) = P_{upt} – P_{rel} – w_Dfrac{delta mathrm {MP}_Z}{delta z} end{aligned}$$
(6)

The calculation of MP particle ingestion rate ((P_{upt})) is the same as for other food sources37. A grazing preference ((psi _{MP})) for MP is subjected to sensitivity testing. This sensitivity testing implicitly examines effects such as biofouling altering the grazing preference of zooplankton for MP. It is assumed that 100% of ingested MP will be egested as faecal pellets and released ((P_{rel})) to the “free” MP pool at the rate of faecal pellet remineralisation, with no plastic remaining in the gut and no plastic being metabolised by the zooplankton. Ingesting MP also results in a reduced zooplankton carbon uptake rate19, with implications for primary and export production (although, Redfield ratios are conserved). Pellet-bound (hbox{MP}_Z) is considered to sink at the rate of faecal pellets ((w_D)).
Plastic is eaten by zooplankton in this model. The Holling II grazing formulation37 is extended to include MP. Grazing of MP ((G_{MP})) is calculated as:

$$begin{aligned} begin{aligned} G_{MP}&= mu _Z^{max} times Z times mathrm {MP}times R_{M:P}times R_{F:MP}times R_{N:F}times psi _{MP}\&quad times ,(psi _{CO}CO+psi _{PH}PH+psi _{DZ}DZ+psi _{Detr_{tot}}Detr_{tot}\&quad +,psi_{Z}Z+psi _{MP}mathrm {MP}times R_{M:P}times R_{F:MP}times R_{N:F} + k_Z)^{-1} end{aligned} end{aligned}$$
(7)

The maximum potential grazing rate ((mu _Z^{max})) is scaled by zooplankton population (Z) and MP availability (MP), and weighted by a food preference ((psi _{MP})), total prey (CO, PH, DZ, (hbox{Detr}_{{tot}})), and Z representing the food sources described in44 and a half saturation constant for zooplankton ingestion ((k_z)). Grazing preferences must always sum to 1 in the model, so sensitivity testing of (psi _{MP}) requires that all grazing preferences must also be adjusted. This is done by varying (psi _{MP}) but requiring (psi _{DZ}) always be set to 0.1 (on the basis that diazotrophs are a poor food source, and to minimize disruption to the nitrogen cycle). The remaining allowance is equally split by the other (psi ) terms. The calculation occurs in N units, so MP is first converted to grams of MP using the MP particle-to-mass conversion of 236E3 tonnes MP = 51.2E12 particles MP ((R_{M:P}))4. It is assumed that 1 g MP will roughly replace 1 g of food (at Redfield ratios; (R_{N:F}) is the conversion from mol Food to mol N) in the zooplankton’s diet, and MP is thus converted to mmol N for the grazing calculation. However, we subject this ratio ((R_{F:MP})) to sensitivity testing. Zooplankton uptake of plastic is therefore:

$$begin{aligned} P_{upt} = frac{G_{MP}}{R_{M:P}times R_{F:MP}times R_{N:F}} end{aligned}$$
(8)

MP particles are released from faecal pellets via remineralisation, which occurs at the same rate as the remineralisation of aggregates:

$$begin{aligned} P_{rel} = mu _D mathrm {MP}_Z end{aligned}$$
(9)

Model forcing
The model was integrated at year 1765 boundary conditions (including agricultural greenhouse forcing and land ice) for more than 10,000 years until equilibration was achieved. From year 1765 to 1950, historical (hbox{CO}_2) concentration forcing, and geostrophically adjusted wind anomalies are applied. From 1950 to 2100 the model is forced with a combination of historical (hbox{CO}_2) concentration forcing (to 2000) and a business-as-usual high atmospheric (hbox{CO}_2) concentration projection RCP8.549,50. MP emissions start from 2 million metric tonnes in year 1950 (a total plastic waste generation estimate45), increasing at a rate of 8.4% per year. (hbox{CO}_2) and MP forcing is summarized in Fig. 7. It has been estimated that about 4% of total plastic waste generated enters the ocean30, but that the microplastic mass found at the sea surface represents only about 1% of the annual plastic input to the ocean4. We test a range of input fractions (see Table 2), after applying a mass conversion from tonnes to number of MP particles4. Using a considerable over-estimation of MP pollution rate also implicitly accounts for abiotic degradation of larger plastics.
Figure 7

Model forcing from years 1950–2100. Atmospheric (hbox{CO}_2) follows RCP8.5 (panel a). Plastic flux into the ocean is assumed to be some fraction of the total historical and projected plastic waste generation estimate (panel b), with a continuing rate of increase of 8.4% per year45, converted to MP particles using a mass conversion4. Previous estimates of actual total plastic mass flux into the ocean is only about 4% of the total plastic waste generation30, with the MP fraction being a small proportion of that.

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Table 2 Microplastic model parameters and range tested.
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Experimental setup
A 700-member Latin Hypercube51 was used to test the microplastic parameter space of the model using the forcing described in the previous section. While biological model parameters might also influence microplastic uptake and transport, we limited our initial tests to the new parameters introduced above. A range of values was prescribed to the parameters listed in Table 2, in which the parameter space was randomly sampled with a normal distribution. The objective was to see what can be learned about plastic accumulation in the ocean, when very little is known about plastic/particle interactions and basic processes are still poorly understood. An analysis of the full Latin Hypercube parameter search is provided as Supplemental Information.
We adopted an incremental approach to increasing model complexity. We started with a control Hypercube where biology was not allowed to take up plastic, in order to first test the physical parameters ((F_T) and (F_R), the fraction of total annual plastic produced entering the ocean as MP, and the fraction assigned a rise rate, respectively). One hundred simulations were performed in this configuration, with the results analysed in the Supplemental Information. We next included passive plastic aggregation in marine snow (MP plus the (hbox{MP}_A) tracer) in a 300 simulation Hypercube, spread across the (k_P) (marine snow uptake coefficient) parameter space (0–1, 1–100, 100–1000 particles (hbox{m}^{-3}), each with 100 Hypercube simulations) in a normal distribution. These 300 simulations explored the 5 relevant MP model parameters: (F_T), (F_R), (F_A) (marine snow aggregation fraction), (k_P), and (F_B) (fractional return to ocean at the seafloor). These results are also provided in the Supplemental Information. Finally, we added active zooplankton-associated plastic (MP, plus (hbox{MP}_A) and (hbox{MP}_Z) tracers) as a third 300-individual Hypercube set. This third Hypercube is similarly split across the (k_P) parameter space in a normal distribution, but with the addition of grazing parameters (psi _{MP}) (MP grazing preference) and (R_{F:MP}) (the food to MP substitution ratio; 7 parameters in total). More

In this study, we generated a new time-series dataset to investigate the response of dark-synchronized MT+ cultures to SIP− filtrate (6 time points, 0–9 h, Fig. 2a) and to compare expression patterns with two existing datasets on the response of MT− to SIP+ [16, 18] (4 time points, 0–10 h, Fig. 2b). Here, we first report the results of a conventional DE analysis for each dataset separately. Next, we use these results to discover genes or biological processes related to sexual reproduction, either based on their predicted functional annotation or the current literature. Finally, we integrate the results of both the novel and publicly available datasets in an integrative analysis that aims at identifying key genes involved in either a single or both mating types.
Fig. 2: Transcriptional responses induced by SIP treatment of Seminavis robusta.

a Multidimensional scaling (MDS) plot for MT+ expression data (0 h–9 h), and (b) MDS plots of two MT− expression datasets (0 h–3 h and 10 h, respectively). Distances between samples in the MDS plot approximate the log2 fold changes of the top 500 genes. c Number of significant DE genes between control and SIP treated cultures for each time point in both mating types. Each dataset was analyzed on a 5% overall FDR (OFDR) level, i.e., the fraction of false positive genes over all rejected genes. The color represents mating type (MT+: red, MT−: blue) and the shade denotes direction of change (dark: downregulated after SIP treatment, light: upregulated after SIP treatment). d Number of significantly enriched GO terms on a 5% FDR level for each time point. The color represents mating type (MT+: red, MT−: blue). A high number of GO terms are discovered in the early time points for MT+, in comparison to the number of DE genes.

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General transcriptional response and identification of key SIP responsive genes
Multidimensional scaling plots of the RNA-seq data (Fig. 2a, b) showed that in both mating types the dark-to-light transition and time since illumination were the major drivers of gene expression change throughout the experiments. However, as time progresses, the effect of SIP becomes more pronounced. This is supported by DE analysis showing that the number of significant genes increased markedly at later time points (Fig. 2c). Overall, most genes are significantly DE in only a single time point on a 5% overall FDR (OFDR) level (Supplementary Fig. 1B). Combined, on a 5% OFDR level, 4037 genes are DE in response to SIP treatment for MT+, while 5486 genes are found to be DE in MT− in the first 3 h and 6079 genes after 10 h. The stronger response during the first 3 time points in MT− versus MT+ may be the result of the use of a chromatographic fraction of SIP+ for MT− while MT+ cultures were treated with a SIP− containing filtrate. Gene sets for many biological processes were significantly enriched in the conventional lists of DE genes: a total of 1081 and 740 enriched biological process terms were discovered for the response to sex pheromones in MT+ and MT−, respectively (Fig. 2d, Supplemenatry Fig. 2).
We discovered key genes by developing a statistical integrative analysis workflow that is capable of testing for equivalent, i.e., non-DE, expression between conditions. Coupling equivalence testing in one mating type with DE calls in the other allowed for the discovery of key genes exhibiting mating type specific responses to SIP, while DE calls in both datasets found key genes responsive in both mating types. This workflow revealed 52 key genes responding to SIP in both mating types (SRBs), 12 genes uniquely responding in MT+ (SRPs) and 70 genes uniquely responding in MT− (SRMs) (Fig. 3a, Supplementary Figs. 3, 4, 5). Similar to the conventional DE analysis, the response of MT− was more pronounced compared to MT+, likely due to technical differences such as different protocols for pheromone administration. Remarkably, while in MT− we discovered a comparable number of down- and upregulated SRMs, we only found upregulated SRPs and SRBs, possibly indicating that sexual processes induced by SIPs are mainly driven by the induction of key genes rather than the downregulation of inhibitory genes (Fig. 3a).
Fig. 3: Visualization and main results of the integrative workflow.

a Schematic representation of the integrative workflow indicating how SIP responsive genes with a shared response (SRBs) or mating type specific response (SRPs, SRMs) were discovered. Non-responsive genes consist of genes that are equivalently expressed after SIP treatment versus control, or that are very lowly/not expressed (filtered). A log fold change (LFC) cutoff of ±log(3) was used to define responsive (differentially expressed) genes and equivalent genes. At the right side of the panel, log2 fold changes of SRMs, SRPs and SRBs in both mating types are plotted. Each gene is plotted for the time point at which they are differentially expressed. Genes which were not expressed (“filtered”) in the non-responsive mating type are plotted as diamonds. The red horizontal lines represent the fold change cutoff used to determine equivalence and differential expression. The number of discovered genes is indicated in the top left corner of each plot. b Expression of a selection of SIP responsive genes (SRMs, SRPs, SRBs). For each gene, counts per million (CPM) are plotted as a function of time for both mating types. The data points correspond to gene expression of the replicates in each time point and the solid line represents the mean. Data points and lines are colored according to condition, i.e., black for control condition and orange for SIP treatment. c Expression of the five genes belonging to the gene family of SRP12 (Sro2882_g339270). Data are presented in the plots in the same manner as in (b).

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In what follows we will discuss in more detail the genes and pathways that are responding to SIP in both mating types or uniquely in one mating type and link these changes to physiological events in the mating process. In each section, we first discuss key genes highlighted by the integrative analysis (Fig. 3), after which we discuss results from the conventional DE analysis, focussing on selected biological processes (Fig. 4).
Fig. 4: Gene expression of Seminavis robusta genes involved in mating-related processes.

a Heatmap of genes related to mitotic and meiotic cell cycle progression, which are differentially expressed (DE) in both mating types in the conventional DE analysis. Each gene is plotted for control and SIP treated conditions in both S. robusta mating types. Genes are specified as row names and are scaled relative to the mean expression, amounting to counts per million (CPM) standardized to zero mean and unit variance for each gene in each mating type separately. Blue indicates low expression, while red indicates high expression. b Expression of genes related to diproline synthesis and reactive oxygen species (ROS) production, which are significantly DE in only one mating type in the conventional DE analysis. CPM are plotted as a function of time for both mating types. The data points correspond to gene expression of the replicates in each time point while the solid line represents the mean. Data points and lines are colored according to condition, i.e., black for control condition and orange for SIP treatment. P5CS = Δ1-pyrroline-5-carboxylate synthetase; P5CR = Δ1-pyrroline-5-carboxylate reductase.

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Responses to SIP conserved in both mating types
Integrative analysis reveals key genes responsive in both mating types
A large fraction (22/52) of SRBs, i.e., key genes with a strong response to SIP in both mating types, lack any functional annotation and homology to sequenced genomes of other diatoms (Supplementary Table 2), suggesting that the molecular mechanisms underlying early mating are highly species-specific. The remaining 30 SRBs can be linked to energy metabolism, ROS signaling and meiosis, amongst others. Pyruvate kinase (Sro373_g129070) and isocitrate dehydrogenase (Sro492_g153950), respectively involved in glycolysis and the citric acid cycle, are strongly upregulated in both mating types (Fig. 3b), suggesting an increased energy demand. Interestingly, pyruvate kinase is also upregulated during gametogenesis in the brown alga Saccharina latissima [26] and the parasite Plasmodium berghei [27]. In addition, two enzymes from the pentose phosphate pathway (PPP) are among the SRBs: transketolase (Sro524_g159900) and transaldolase (Sro196_g083630) (Supplementary Fig. 3). The PPP generates NADPH, a reductive compound needed in various metabolic reactions and involved in detoxification of ROS by regenerating glutathione [28, 29]. Furthermore, one SRB encoding a heme peroxidase (Sro1252_g256250) exhibited strong upregulation upon SIP treatment (Fig. 3b). Upregulation of heme peroxidases was also reported during sexual reproduction in other eukaryotes, e.g., mosquitoes (Anopheles gambiae) [30] and fungi [31, 32]. Heme peroxidases promote substrate oxidation in various metabolic pathways and are essential for the detoxification of ROS [33], suggesting that ROS signaling plays a role in the response to SIP, as seen in the green algae Volvox carteri, where high ROS levels trigger sex [34]. Finally, a highly expressed SRB encodes a transmembrane protein containing an Epidermal Growth Factor-like (EGF-like) domain (Sro65_g036830, Fig. 3b), with potential orthologs encoded in pennate and centric diatoms including P. tricornutum and T. pseudonana (BLASTp, E  More

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Our work was conducted under a permit from the University of North Florida Institutional Animal Care and Use Committee (permit #18-005).
Field methods
We performed nighttime spotlight surveys with an outboard motorboat throughout 2019 to determine relative alligator abundance, distribution, and habitat selection. This technique is an established method for estimating relative population sizes in crocodilians across heterogeneous habitat32. However, a limitation of spotlight surveys is the variation in detection probability caused by different environmental conditions or observers29. To control for these effects, we implemented a standardized survey protocol33,34. All surveys covered the first 8 km of nine tributaries within the lower St. Johns River system, starting at the point where each tributary meets the main channel of the river (Fig. 1). We limited our surveys to the first 8 km because some tributaries contained low bridges that blocked boat access after this point. We chose tributaries that were surrounded by different amounts of urban land cover such that our surveys spanned an urbanization gradient from approximately 5 to 80% urban land cover within 1 km of the river’s edge (Fig. 2). GIS analyses also revealed that land use patterns around the St. Johns River are dynamic, with different urban land cover proportions at 0.1, 1, 3, and 5 km from the water’s edge for each tributary (Fig. 2). To reduce temporal bias, we conducted surveys over the span of 1 year and segregated sampling periods into four distinct seasons (winter [Dec–Feb], spring [Mar–May], summer [Jun–Aug], and fall [Sep–Nov]. We surveyed each tributary one time during the middle month of each season, resulting in a total of four surveys per tributary. We surveyed the tributaries in a quasi-random fashion because the tributaries closest to the mouth of the St. Johns River are under significant tidal influence, so we timed surveys of those tributaries during periods of high tide in order to access the full survey area. We only performed surveys when rainfall was absent and wind speeds were below 16 km/h since these factors have been shown to affect alligator detection probability24. Quasi-random sampling over the span of a year was best suited to randomize environmental conditions that affect nighttime spotlight survey counts, such as water level, temperature, moon phase, and moon illumination24,31,35,36.
Figure 1

Map of the tributaries surrounding the lower St. Johns River that were surveyed as part of our study (white areas). From northeast to south: Clapboard Creek, Dunn Creek, Broward River, Trout River, Arlington River, Ortega River, Doctors Lake, Julington Creek, and Black Creek. The map was created with ArcGIS Pro 2.6 (https://arcgis.pro/).

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Figure 2

Levels of urban development (FLUCCS code 1000) surrounding the tributaries of the St. Johns River that were surveyed in this study. Land use was quantified using 0.1, 1, 3, and 5 km buffers around each tributary transect.

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We began all surveys no earlier than 30 min after sunset and we maintained a constant boat speed of 10–12 km/h. At the start and end of each survey we recorded moon phase, weather conditions, visibility, ambient light, air temperature, water temperature, and salinity. We detected alligator eyeshine primarily using two 1200 lm handheld spotlights, but we also used additional handheld lights (6000 lumens) often throughout the surveys. As soon as we detected eyeshine we approached the alligator at reduced speed. We placed each individual into a size class (30–90 cm [juvenile], 90–180 cm [sub-adult], 180–270 cm [adult], 270–360 cm [large adult], + 360 cm [largest adult]) by estimating the distance between the eyes and the tip of the snout37,38. If an alligator submerged before size estimation could take place, we recorded its length as unknown or simply larger or smaller than 180 cm. At each sighting we recorded global positioning system location using the on-deck boat navigation unit. We measured environmental characteristics at each sighting using a YSI meter (Pro2030; YSI; Yellow Springs, Ohio, USA), a thermometer, and a sky quality meter (SQM; Unihedron; Grimsby, Ontario, Canada).
We recorded information about habitat characteristics for each sighting following previous studies13,26. We first visually characterized habitat in a 10 m radius circle centered on the alligator sighting location (“used habitat”). We recorded the proportion of open water, emergent vegetation, floating vegetation, anthropogenic structure, and dry ground within the circle, as well as the alligator’s distance from shore, vegetation, and anthropogenic structure. We then visually classified the same habitat characteristics in a 20 × 100 m plot centered on the alligator sighting location and stretching along the shoreline (“available habitat”). If an alligator sighting occurred entirely in open water, then we shifted the available habitat plot to the closest shoreline. For each used habitat circle and available habitat plot, we classified the respective shorelines as natural, hardened, or mixed, depending on if the shore was totally vegetated, subject to anthropogenic armoring, or a mixture of the two types, respectively. We also estimated the proportion of shoreline found within these areas that were covered in naturally growing vegetation rather than anthropogenically altered lawns.
Land use classification
We used ArcGIS Pro (ESRI; Redlands, CA, USA) for all spatial data manipulation and visualization. We acquired land use and cover data from the St. Johns River Water Management District (SJRWMD) via the Florida Geographic Data Library. For all analyses we used data from the most recent SJRWMD dataset, which was from 2014.
We split a 100 k definition polygon of the St. Johns River to create smaller units representing each tributary transect. The resulting features consisted of the main portion of each tributary surveyed where lower order streams that were not surveyed were deleted. Because the extent to which alligators respond to land use changes was not known a priori, we buffered the transect polygon feature for each tributary to 0.1, 1, 3, and 5 km to further clip the SJRWMD land cover and use data layer. By creating four buffers for each of the nine tributaries, we generated a total of 36 land cover and use layers.
We classified land use types through the Florida Land Use and Cover Classification System (FLUCCS), as cited in SJRWMD metadata documentation. This hierarchical coding scheme contains four levels, of which we used the highest level (level 1) designation. This particular level classifies land use into nine distinct categories. These categories included urban and built-up; agriculture; upland nonforested; upland forests; water; wetlands; barren land; transportation, communication, and utilities; and special classification. For the purposes of this study, we only included defined terrestrial land use types in statistical analyses. These land use types were urban and built-up (e.g., residential, industrial, and recreational areas), agriculture (e.g., cropland, pastures, aquaculture), upland nonforested (e.g., shrub and brushland), upland forests (e.g., coniferous forests, hardwood forests, tree plantations), wetlands (e.g., freshwater/saltwater marshes, mangrove swamps, wet prairies), barren land (e.g., beaches other than swimming beaches, borrow areas, spoil areas), and transportation, communication and utilities (e.g., highways, electrical power facilities, wastewater treatment facilities). We calculated the proportions of each land use type using each respective land use shape area divided by total shape area.
Statistical analyses
To determine if environmental conditions and/or land use characteristics affect broad scale alligator distribution, we performed multiple analyses using SPSS (IBM; Armonk, NY, USA). We included all alligator sightings in our analyses, but we did not apply population estimate correction equations to the alligator counts because they tend to underestimate population numbers in crocodilians39. Sighting data used in statistical analyses therefore represent relative alligator abundance, not estimates of true alligator population size. We first checked normality for each variable using Kolmogorov–Smirnov and Shapiro–Wilk tests to determine if parametric or nonparametric tests were appropriate. Normality varied greatly across the suite of variables; therefore, Spearman’s rho and Pearson’s correlation coefficient were used when appropriate. We then performed simple linear regression to determine if there were any direct relationships between relative alligator abundance and individual variables. We performed these tests for alligator counts in each tributary by season and for the average number of sightings per tributary across seasons. We also averaged environmental variables for each tributary by season and for the average value per tributary across seasons. We tested for the effect of land use at all four buffer sizes for each tributary, including all relevant terrestrial land use types.
We then performed multiple linear regression analyses in a stepwise manner. This modeling system excluded variables found to be highly correlated with other variables (multicollinear) and retained variables that significantly contributed to the model (P ≤ 0.05). We then performed these tests on modified datasets that did not contain the two most saline tributaries to further validate preliminary findings. When more than one significant model was produced for a given data set, we calculated AICc values to rank models while penalizing model complexity and accounting for our small sample sizes.
To evaluate habitat selection, we compared percent shoreline vegetation and the proportions of habitat characteristics found in the 10 m radius circle to those found in the remaining areas of each respective 20 × 100 m plot using the Wilcoxon signed rank test. When comparisons could be made between two normally distributed groups of data, we used a paired sample t test instead. While comparing used to available habitat data was the basis of the tests, the amount of data per analysis differed between analysis groups. The first group was composed of all habitat selection data across time and space. This “global” dataset was the most robust in terms of sample size but may have been biased by double counting individuals across time. The second group was divided by season, so analyses were performed on all data collected within a season across space. This group removed the bias of double counting individuals but may be affected by variation in the number of sightings per season and tributary. More

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