Experimental Design and Methodologies
This document outlines the comprehensive experimental setup, culture conditions, imaging techniques, quantitative assays, fieldwork, genomic analyses, and behavioral studies conducted. General laboratory methods involved using dilution percentages as volume/volume (v/v), with sample sizes not predetermined by statistical methods, and without the utilization of blinding or randomization.
C. flexa clonal monoxenic cultures (strain ChoPs7, referred to as strain 1) were cultivated in either 1% seawater complete (SWC) medium or 5% cereal grass medium 3 (CGM3), both diluted in artificial seawater (ASW).
Cellular Imaging and Microscopy Techniques
Single-Cell Tracking and Time-Lapse Imaging
Single-cell division of C. flexa was meticulously tracked by diluting cultures to a density of 1 cell/µl. Imaging was performed using a Zeiss Axio Observer Z.1 inverted microscope equipped with a ×20 objective and tile scan mode. Time-lapse imaging captured the dynamics of mixed clonal-aggregative multicellularity, with colonies imaged every 5 minutes using differential interference contrast (DIC) microscopy and a ×63 objective. Further observations of aggregation dynamics were conducted through low-magnification (×5) and high-magnification (×63) time-lapse videos of dissociated C. flexa cultures over an overnight period, with all experiments performed in four independent biological replicates.
Staining and Airyscan Imaging
For detailed cellular visualization, cells were incubated for various durations, subsequently fixed with paraformaldehyde (PFA), permeabilized using Triton X-100, and then stained with FM 4-64X and Alexa Fluor 488 Phalloidin. Samples were then imaged using a Zeiss LSM900 Airyscan 2, enabling high-resolution visualization.
Chimeric Aggregates and Cyst Formation
Dual-labelled chimeric aggregates were created by staining C. flexa cells with CellTrace CFSE (green) and CellTrace Far Red (magenta). These stained populations were mixed, incubated overnight, fixed, and then stained with Alexa Fluor Plus 405 Phalloidin before Airyscan imaging. Cyst formation was monitored daily for four days using DIC microscopy, induced by gradual evaporation and controlled temperature changes and lid adjustments, increasing salinity from 60 ppt to 110 ppt. The resulting cysts were fixed, permeabilized, and triple-stained with FM 4-64X, Alexa Fluor 488 Phalloidin, and Hoechst for Airyscan imaging.
Prey Capture Efficiency
Prey capture efficiency was quantified by staining Halopseudomonas oceani food pellets with BactoView-Live Green. These stained pellets were then added to C. flexa cultures, fixed, and imaged using both DIC and green fluorescence microscopy.
Quantitative Assays and Behavioral Studies
Aggregation and Growth Rate Analysis
Aggregation in aphidicolin-treated cells was quantified by exposing C. flexa cultures to either aphidicolin or a DMSO control, followed by imaging every 30 minutes for a total of two hours. The aggregation behavior of fixed versus live cells was compared by either fixing cells immediately or leaving them live, then subjecting them to an orbital shaker or static conditions for 24 hours before imaging. Normalized growth rates and aggregation efficiency were calculated under two distinct salinity conditions: 1× and 2×. The efficiency of clonality and aggregation was also rigorously assessed across a range of cell densities (10^2 to 10^5 cells/ml) after incubation periods of 24, 48, and 72 hours.
Sheet Characterization and Inversion
Aggregative and control sheets were characterized through detailed staining and Airyscan imaging. Control sheets were specifically grown at low density to favor clonal formation, while aggregative sheets were induced at high density in 2× ASW. Both types were fixed, stained, and imaged, allowing for the quantification of cell counts, collar–collar angles, and circularity. Light-regulated inversion in aggregative and control sheets was investigated using confocal microscopy with light-to-dark stimulation, and changes in colony area during inversion were precisely measured.
Fieldwork and Environmental Sampling
Curaçao Expeditions
Fieldwork was conducted in Shete Boka National Park, Curaçao, spanning three expeditions during July–August 2023 and July–August 2024.
- Exped-A: Involved collecting seawater from 79 splash pools, with 15 of these pools monitored daily for evaporation and refilling cycles. In-situ parameters such as salinity, temperature, and depth were meticulously measured, and GPS coordinates along with photographs were recorded for each site.
- Exped-B: Focused on random sampling within a 10m x 4m area upstream of Boka Wandomi, where 71 splash pools were collected and analyzed. This expedition successfully led to the isolation of new C. flexa strains (referred to as strains 2 and 3).
- Exped-C: Sampled 12 splash pools located near Boka Wandomi and Boka Pistol. Salinity was measured prior to collection to maximize the likelihood of finding C. flexa sheets.
Soil Sample Analysis and Artificial Evaporation
Soil samples from 32 splash pools across Exped-A and Exped-C were rehydrated in sterile-filtered seawater in the laboratory and subsequently monitored for the presence of C. flexa sheets. Artificial evaporation experiments were performed in the lab by exposing C. flexa cultures in six-well plates to gradual evaporation over nine days at 30 °C, with salinity measurements and cell counting performed at each timepoint.
Genomic Sequencing and Analysis
Reference Genome Assembly and Annotation
The reference genome for C. flexa (strain ChoPs8) was sequenced using Omni-C+PacBio technology. The genome assembly spanned 56.4 Mbp across 528 scaffolds, boasting an N50 of 1.3 Mbp and a GC content of 50.83%. Gene annotation utilized an integrated pipeline combining predictions from Augustus, SNAP, and GlimmerHMM, C. flexa transcriptomic data, and other choanoflagellate proteomes. This process resulted in the annotation of 14,084 genes with an impressive 82.8% BUSCO completeness score. Bacterial contaminant sequences, specifically from H. oceani, were meticulously identified and removed.
Whole-Genome Sequencing and Variant Calling
Whole-genome short-read sequencing (Illumina) was performed for C. flexa strains 1, 2, and 3. Reads underwent thorough quality assessment, followed by alignment to the reference genome using BWA-MEM. Variants were subsequently called using GATK HaplotypeCaller.
Phylogenomics and Diversifying Selection
A phylogenomic tree encompassing all samples was constructed using IQ-TREE based on homozygous single-nucleotide polymorphisms (SNPs). Polymorphic sites under putative diversifying selection were identified by calculating K_a/K_s ratios (dN/dS) between strains, with functional enrichment analysis of InterPro signatures conducted in regions exhibiting high K_a/K_s ratios.
Kin Recognition Experiments
Kin recognition experiments involved differential staining of strains 1, 2, and 3 with CellTrace CFSE (green) and CellTrace Far Red (magenta). Green- and magenta-labelled populations from each strain were mixed in 1:1 ratios, incubated overnight, and subsequently imaged. Green and magenta cells were manually counted, and a segregation index was calculated to quantitatively assess kin recognition.
Statistical Methods
Statistical analyses were rigorously conducted in R (v.4.4.1) utilizing the base stats package. The non-parametric Mann–Whitney U-test was employed for pairwise comparisons, while the Shapiro–Wilk normality test and F-test were used to evaluate data normality and differences in variance, respectively.