Interspecific variation in the use of chemical defenses in Prionostemma Opiliones
Data files
Jun 15, 2026 version files 45.69 KB
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README.md
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secretion_data_raw.csv
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Abstract
We quantified chemical secretion behavior in 11 sympatric species of Prionostemma within the understudied arachnid order Opiliones. We surveyed species in two distinct tropical forests in Costa Rica and tested their secretion propensity when stimulated, and whether such differences correspond to morphology, leg loss, or social behavior. We found that species varied in their tendency to release chemical secretions, forming a continuous gradient from taxa that rarely secreted to those that secreted in nearly all encounters. However, this variation was not explained by body size or leg loss. Species with similar morphology or comparable leg loss displayed contrasting patterns of secretion. Among the seven sympatric species with available social data, aggregation patterns showed no linear association with secretion propensity: species spanned the full range of secretion behavior regardless of whether they frequently formed roosting aggregations with conspecifics or heterospecifics, or roosted alone. Instead, each species combined secretion propensity, leg loss, body size, and aggregation tendencies in distinct ways, indicating a mosaic of defensive strategies rather than a single axis of trait covariation.
Dataset DOI: 10.5061/dryad.wdbrv1644
Dataset and Code for:
Chemical secretion use varies among species but lacks a straightforward relationship with other defenses in Neotropical Opiliones
Authors: Freire, Marilia; Prachand, Sejal V; Skelly, Noah; and Escalante, Ignacio
Corresponding author: I. Escalante; iem@uic.edu
This repository contains the data and R code required to reproduce all quantitative analyses, statistical models, tables, and data-derived figures presented in the manuscript.
Study Overview
This repository includes the raw data used to investigate interspecific variation in defensive secretion behavior, autotomy, body size (leg length). To reproduce aggregation-related analyses, users must download the aggregation dataset from the cited Dryad repository (Escalante et. al 2022) and place it in the same directory as the analysis script.
Illustrative graphical elements were added in Adobe Illustrator after exporting the quantitative figures from R. These aesthetic additions do not alter any statistical results or underlying data.
Description of the data and file structure
File Inventory
secretion_data_raw.csv
Analysis Script Freire_2026.R
Variable Definitions
Site
Field site where specimen was collected.
Type: categorical.
Collected_from
Microhabitat from which specimen was collected.
Type: text.
Collection_date
Date of specimen collection.
Type: date.
Trial_date
Date of behavioral trial.
Type: date.
Trials_time
Time of day of trial.
Type: time.
species
Species identifier.
Type: categorical.
Description: We studied 11 currently undescribed species, all in the genus *Prionostemma*. Hence, this variable refers to those. For instance, "sp.10" refers to an individual of* Prionostemma* sp.10, and so on.
Number_legs
Number of legs observed at trial.
Type: integer.
Missing_legs
Number of missing legs.
Type: integer.
Released_secretion_when_squeezed_3_s
Binary response to 3-second squeeze stimulus (“yes”/“no”).
Type: binary.
Leg_IV_length_mm
Length of leg IV.
Units: millimeters.
Note:
NAs represent "not available values", of either missing legs we did not record, or leg length values we did not measure.
Aggregation Dataset Provenance
The aggregation dataset used in this study was originally published in:
Escalante I, Domínguez M, Gómez-Ruiz DA, Machado G (2022)
Benefits and Costs of Mixed-Species Aggregations in Harvestmen (Arachnida: Opiliones).
Frontiers in Ecology and Evolution Volume 9-2021: https://doi.org/10.3389/fevo.2021.766323
Dryad DOI: https://doi.org/10.5061/dryad.zs7h44j99
The original dataset is not redeposited here to avoid duplication. Users should obtain the raw aggregation data directly from the original repository.
Statistical Analyses
Secretion Differences Among Species
- Species by secretion contingency table
- Pearson chi-square test
- Monte Carlo simulated chi-square test (10,000 replicates)
- Firth logistic regression using logistf
- Reference species: sp.4
- Odds ratios with 95% confidence intervals
- Model-predicted probabilities per species
Clustering Analyses
Species were clustered based on predicted secretion probabilities using:
- Distance metric: Euclidean
- Linkage method: Ward.D2
- Silhouette analysis for k = 2–4
Autotomy and Secretion Relationship
Species-level Pearson and Spearman correlations were calculated between:
- Autotomy percentage
- Model-predicted secretion probability
Leg Length Analyses
- Species-level summary statistics
- ANOVA for interspecific differences
- Kruskal–Wallis test (non-parametric alternative)
- Correlation between mean leg length and predicted secretion probability
Species-Level Trait Heatmap
A comparative heatmap was generated using the following traits:
- Secretion probability
- Autotomy percentage
- Mean leg length
- MSA percentage
- SSA percentage
- Solitary percentage
Z-scores were calculated independently for each trait column as: Z = (x − mean_trait) / sd_trait
Aggregation data were available only for species sp.5–sp.11. Missing aggregation values for other species are represented as NA and appear as grey cells in the heatmap.
Software and Package Versions
Analyses were conducted in R version 4.4.3
Required packages:
- readxl
- dplyr
- tidyr
- ggplot2
- logistf
- emmeans
- cluster
- factoextra
- car
- ggrepel
We conducted fieldwork in May and June 2025 at two Neotropical rainforest sites in Costa Rica: La Selva Research Station (10.4315° N, 84.0031° W; 50 m elevation), a lowland rainforest, and Las Cruces Research Station (8.7844° N, 82.9594° W; 1200 m elevation), a premontane forest. Our study focused on 11 Prionostemma species (Opiliones: Eupnoi: Sclerosomatidae), here referred to as sp.1-sp.11. Species sp.1-sp.4 occur at La Selva and species sp.5-sp.11 at Las Cruces.
Collected individuals were transported for less than 1 hour to a laboratory at the field station and housed for less than 24 hours in temporary mesocosms containing leaf litter and small branches. Containers were kept humid, and animals were provided ad libitum food (cucumber and carrot). All procedures followed established protocols and guidelines for the care and welfare of invertebrates in research (Escalante & O’Brien 2024, ASAB 2025) and were conducted in accordance with Costa Rican governmental regulations for animal research (permit number: R-032-2025-OT-CONAGEBIO).
Chemical secretion assayChemical secretion was quantified using a standardized assay intended to mimic an acute predatory attack (Raspotnig et al. 2015; 2022). Each individual was restrained by gently securing as many legs as possible with both gloved hands while maintaining the dorsal surface upward. The dorsal body was then subjected to gentle pressure with the experimenter’s thumb for approximately three seconds, or until secretion occurred. Secretion release was recorded as a binary response (yes/no). A trial was scored as “yes” when a visible droplet of fluid emerged from one or both ozopores, typically accompanied by a detectable odor, and as “no” when no visible droplet emerged, and no odor was detected. Each individual was tested once, marked on the dorsal surface with a non-toxic Posca marker to prevent resampling, and released at the site of capture. All assays were conducted within 24 hours of collection.
Morphological measurements and autotomy scoringEach individual was inspected for autotomy immediately after the secretion assay. Autotomy was scored as a binary variable (yes/no), with “yes” recorded when one or more legs were missing and “no” when all legs were intact.
Body size was quantified by measuring the length of the fourth leg (Leg IV) on one side of the body using digital calipers (0.01 mm precision), a standard body size proxy in Prionostemma. Measurements were taken while gently restraining the animal to ensure the leg was fully extended without applying force.