DOI: 10.5061/dryad.2bvq83c0b
title: Data from: Effects of manakin gut passage on germination of a neotropical melastome shrub (Melastomataceae)

description: Gut passage is an important but understudied component of animal-mediated
 seed dispersal that can impact germination and survival. Melastomataceae
 plants are abundant and ecologically important throughout the tropics, but
 studies have obtained contrasting results regarding effects of avian gut
 passage on melastome germination. We conducted a paired germination
 experiment to test how gut passage by four species of manakins—key avian
 dispersers of melastomes—affects germination of the pioneer melastome
 shrub Miconia rubescens. Manakin gut passage accelerated M. rubescens
 germination, with gut-passed seeds germinating an average of 5.1 days
 earlier than controls, and percent germination of gut-passed seeds was
 significantly higher at 20 and 40 days post-planting. Interestingly,
 manakin species varied in their gut passage effects, with L. velutina
 having stronger overall germination effects than M. manacus. Within
 species, greater body mass was correlated with higher percent germination
 and shorter times to first germination. Within M. manacus, seeds passed by
 females had significantly shorter times to first germination than seeds
 passed by males. Because control seeds were manually depulped in the
 experiment, the observed gut passage effects are likely attributable to
 minor scarification of the seed coat during gut transit. Our results
 suggest manakin gut passage can have biologically meaningful impacts on
 melastome germination, and the magnitude of these effects can vary based
 on manakin species, mass, and sex. This study refines our understanding of
 the ‘quality’ component of seed disperser effectiveness in an iconic
 dispersal mutualism, with implications for plant community composition and
 recovery of deforested neotropical ecosystems.
description: <strong>Study Area</strong> This
 study took place in the Chocó biogeographic zone of northwestern Ecuador,
 a global hotspot for biodiversity experiencing extreme habitat loss and
 declining species abundance due to deforestation and other anthropogenic
 activity (Myers et al., 2000). Field work was conducted at Reserva
 FCAT<em> </em>(Fundación para la Conservación de los Andes
 Tropicales; 00◦ 23′ 28′′ N, 79◦ 41′ 05′′ W), a 700-ha private reserve
 within the Mache-Chindul Ecological Reserve in Esmeraldas Province. The
 average temperature in the area ranges between 23-25ºC and annual
 precipitation is approximately 2,500-3,000 mm, with the wet-season
 occurring from late December to June (Clark et al., 2006). At our site,
 agricultural crops (e.g., cacao, plantain) and pasturelands are prevalent
 and increasing, although intact and successional forests also remain
 relatively common (Van Der Hoek, 2017). However, heavy deforestation and
 fragmentation has occurred in the Mache Chindul Reserve, disrupting
 connectivity and species interactions (Van Der Hoek, 2017).
 <strong>Study System</strong> Our
 study species, <em>Miconia rubescens </em>(Gamba and Almaeda,
 2018; formerly <em>M. neomicrantha</em> (Judd & Skean,
 1991) and <em>Octopleura rubescens </em>(Triana, 1872)), is a
 woody shrub producing fruits that are small (mean height x width: 6.04 x
 2.99 mm; mean weight = 0.11 g), round, watery, and white when ripe (mean
 Brix = 4.88%). This species is the most geographically widespread species
 in the Octopleura clade, occurring in 13 countries over Central and South
 America (Gamba & Almeda, 2014, 2018). Fruit produced by
 <em>M. rubescens </em>are consumed by numerous neotropical
 frugivorous birds, particularly manakins, tanagers, thrushes, and
 brush-finches (Kessler-Rios & Kattan, 2012; Loiselle &
 Blake, 1999; Stiles & Rosselli, 1993; Wheelwright et al.,
 1984). Manakins are small passerines that constitute
 some of the most abundant frugivores in South and Central American
 rainforests, making them prominent agents of endozoochorous seed dispersal
 in these habitats (Cestari & Pizo 2012, 2013). In this study, the
 following manakin species were used in gut passage trials:
 <em>Manacus manacus</em>, <em>Lepidothrix
 velutina</em>, <em>Ceratopipra mentalis, </em>and
 <em>Cryptopipo litae. </em> Manakins at our field site are
 regularly observed eating whole fruits of <em>M.
 rubescens</em>. This is especially true of <em>M.
 manacus</em>, which tend to situate their lek sites in disturbed
 secondary forest and along forest edges where <em>M.
 rubescens</em> is relatively abundant.
 <strong>Gut passage trials</strong>
 Gut passage trials were performed on wild-caught birds over the
 course of eight weeks, from early June to late July 2022. Passive mist
 netting took place in regenerating forest surrounding known lek sites of
 <em>M. manacus </em>using 2–7 nets, each 12m in length, that
 remained open in the morning hours until manakins were captured; we paused
 netting for subsequent stages of the trials. Birds were fasted for 45
 minutes after capture to ensure that the majority of previously consumed
 seeds had left the bird’s digestive tract (Levey, 1986; Worthington,
 1989). During the fasting period, manakins were extracted from nets,
 weighed, processed, and banded for identification. Once processing was
 complete, they were placed back into a fabric mist netting bag in the
 shade until the fasting period was complete.
 Ripe<em> </em>fruits (determined by color and
 softness) were collected from the field &lt; 24 hr prior to use in gut
 passage trials<em> </em>and stored in dry and dark conditions.
 Each paired trial in the experiment corresponded to fruits collected from
 a different <em>M. rubescens</em> individual (n = 41), which
 were marked to avoid resampling. Following the fasting period, birds were
 hand fed 1–4 whole <em>M. rubescens</em> fruits that had been
 previously collected from a single plant. The number of fruits fed to a
 given bird varied depending on the individual’s cooperation and condition.
 Immediately after feeding, birds were placed into a small pet carrier with
 a perch, covered for 45–60 minutes, then released. Fecal samples
 containing gut-passed seeds were collected from a piece of white printer
 paper at the bottom of the carrier, placed in sterilized marked
 containers, and processed for the experimental germination procedure later
 the same day (below). <strong>Germination
 experiment</strong> <em> </em>We
 compared percent germination of gut-passed and non-digested (control)
 seeds using a paired experimental design. For a given experimental trial,
 gut-passed and control fruits were sourced from the same plant to account
 for potential individual variation in germination rate or success among
 plants. To minimize handling effects during extraction from feces
 (gut-passed treatment) or fruits (non-digested treatment), seeds were
 suspended in a small amount of sterile water and gently removed with a
 sterile metal spatula. Extracted seeds were then placed on sterile
 germination paper inside plastic petri dishes. For each paired trial, the
 same number of seeds were placed in control and gut-passed petri dishes.
 However, due to variation in the number of seeds that were recovered
 following gut passage, the number of seeds obtained varied among trials
 (mean: 31.83 ± 1.72 SEM, range = 6–42). Petri dish pairs were placed
 immediately adjacent to one another indoors where temperatures ranged from
 13–22 ºC  and situated near a window to allow exposure to indirect natural
 sunlight on a natural 12 hr light/12 hr dark cycle. Germination paper was
 kept damp as needed using a spray bottle filled with sterile water.
 Control seeds received the same amount of water as paired gut-passed seeds
 and were watered at the same times. All petri dishes were monitored
 together at least 1–2 times per week to record the number of germinated
 seeds. <strong>Statistical
 Analysis</strong> To compare the effect of gut
 passage on germination times across all seeds, we fit a linear
 mixed-effects model using the <em>nlme</em> package in R
 (Pinheiro et al., 2017). The model included time elapsed until germination
 as a response variable (square root transformed to improve residual
 normality), treatment (i.e., gut-passed vs. non-digested) as a predictor
 variable, and dish within trial as a nested random effect. This nested
 random effect structure accounted for the fact that seeds belonging to the
 same trial (i.e., paired gut-passed and non-digested seeds) were derived
 from the same <em>M. rubescens </em>plant, and seeds in the
 same dish (in the case of the gut-passed treatment) passed through the
 same manakin. To test whether gut passage influenced variation in seed
 germination time, we calculated the within-dish variance
 (σ<sup>2</sup>) in time to germination for all gut-passed and
 non-digested seeds in each trial separately. We then conducted paired
 t-tests to compare the variances in germination times between treatments
 for all manakins, for <em>L. velutina </em>only, and for
 <em>M. manacus </em>only; variable transformations were
 applied as necessary to meet normality assumptions. To test whether gut
 passage influenced percent germination, we fit a model in
 <em>lme4</em> (Bates et al., 2015) with final percent
 germination at the end of data collection as a response variable,
 treatment as a fixed effect, and bird ID and species as random
 intercepts. To examine germination success at various
 timepoints throughout the experiment, we conducted Wilcoxon signed-rank
 tests comparing the percent of gut-passed (GP) versus non-digested (ND)
 seeds that germinated at 20, 40, 60, and 80 days after planting. Because
 seeds were not checked for germination on a daily basis, we used the
 percent germination recorded closest to the desired 20-day interval for
 analyses (20-day timepoint mean ± SD: 20.17 ± 1.14 days; 40-day timepoint:
 40.32 ± 1.60 days; 60-day timepoint: 60.80 ± 2.01 days; 80-day timepoint:
 80.20 ± 2.69 days). All seeds were checked for germination on the same
 days, so the distance from a given 20-day interval did not differ within
 an experimental-control pair. We compared percent germination of
 gut-passed seeds relative to paired controls for each of the following
 groups: all manakin species together, <em>L. velutina
 </em>only, and <em>M. manacus</em>
 only<em>.</em> Gut passage by <em>C. mentalis
 </em>and <em>C. litae</em> was not analyzed separately
 due to small sample sizes<em>. </em> We fit
 additional linear mixed-effects models in <em>nlme</em> to
 investigate how bird mass and sex influenced germination parameters. Due
 to the small sample sizes of <em>C. mentalis</em> and
 <em>C. litae</em>, only <em>L. velutina </em>and
 <em>M. manacus </em>were included in these analyses, and only
 trials that reached 80 days post-planting (n = 30) were considered. To
 investigate the effects of mass, sex, and species on germination success,
 we fit a model that included the difference in percent germination between
 gut-passed and control seeds (D percent germination) as a response
 variable; mass, sex, and species as fixed effects; and individual bird ID
 as a random intercept. To investigate the effects of bird mass, sex, and
 species on germination rate, we fit a second model with the same fixed and
 random effects as the first, but a response variable of D days until first
 germination (i.e., the difference between gut-passed and control
 treatments in days elapsed prior to the first occurrence of germination).
 This model also included a fixed variance structure to account for
 heteroskedasticity. Finally, within <em>M. manacus</em> (the
 species for which we had the greatest number of known-sex individuals), we
 used a Wilcoxon test to compare D days until first germination between
 known males and females. Statistical tests and figures were conducted and
 generated in RStudio Version 4.4.1 (RStudio Team, 2024) and JMP Version
 17.00 (JMP, 2021). All descriptive statistics are presented as means ± 1
 SEM.
description: # Effects of manakin gut passage on germination of a neotropical melastome
 shrub (Melastomataceae) *Biotropica* resubmission of Rompf et al. 2024
 [https://doi.org/10.5061/dryad.2bvq83c0b](https://doi.org/10.5061/dryad.2bvq83c0b) ## Description of the data and file structure **Study synopsis:** The goal of this study was to investigate how gut passage by manakins, *Pipridae*, influence the germination of a prominent, likely-pioneer, shrub species, *Miconia rubescens* (Melastomataceae). We conducted a comparative germination experiment in northwestern Ecuador, where we compared the germination between non-digested seeds (extracted from *M. rubescens *fruits), and seeds digested by manakins (collected from feces during field-based gut passage trials). We tested two hypotheses for how gut passage by four manakin species would influence seed germination compared to control (non-digested) seeds. 1) manakin gut passage will increase percent germination (i.e., proportion of seeds germinated) and accelerate germination times of *M. rubescens *seeds. 2) there will be no differences in gut-passage effects according to manakin species, mass, or sex, due to the relatively similar body sizes and digestive morphologies across manakins (Worthington, 1982). We found that manakin gut passage accelerated germination, with gut-passed seeds germinating an average of 5.1 days earlier than controls, and percent germination of gut-passed seeds was significantly higher at 20 and 40 days post-planting. We suspect that the mechanism by which manakin gut passage accelerates *M. rubescens* germination is micro-scarification of the seed coat that occurs during digestive processing, increasing the seeds’ porosity to nutrients, water, and sunlight. Contrary to our second hypothesis, manakin species varied in their gut passage effects, with *L. velutina *having stronger overall germination effects than M. manacus*. Within species, body mass was positively correlated with higher percent germination and earlier occurrences of first germination. *Additionally, within *M. manacus*, time to first germination was shorter in seeds passed by females compared to males. This study refines our understanding of the ‘quality’ component of seed disperser effectiveness in an iconic dispersal mutualism, with implications for plant community composition and recovery of deforested neotropical ecosystems. ### Files and variables **Description of the files and data structure:** The files herein contain raw data from our germination experiment, where we observed the proportion of seeds germinated over time between paired trials of gut-passed treatment seeds (digested by manakin species: *Manacus manacus*, *Lepidothrix velutina*, *Ceratopipra mentalis*, and *Cryptopipo litae*) and non-digested treatment seeds, of *M. rubescens.* The first file (“Rompf et al.2024 Supporting Data”) contains two sheets: “Germination over Time-original data”, and “Complete 20-day Intervals”.  The first sheet “Germ over time-original data”, from the file “Rompf et al.2024 Supporting Data”, contains germination data, in which the proportion of seeds germinated in each petri dish was recorded every few days. This sheet also contains information associated with each petri dish and seed-counting day. The complete columns listed in “Germ over time-original data” are defined as follows: *  Bird Species: The manakin species associated with the gut-passed treatment (indicates that species digested those particular seeds) within a paired trial. - Bird ID: The individual identification of each manakin used in gut passage trials, as defined by the colored band codes (Black = B, Blue = U, G = Green, O = Orange, Red = R, Yellow = Y) or numbered, metal bird ID bands. - Trial #: Denotes which paired trial, numbered from 1 – 43 (not all trials were used in final analysis due to removal of replicates with the same manakin individual), of gut-passed and non-digested treatment seeds, each column of information is associated with. - Solo or mixed- Denotes whether a gut-passage trial was completed using solely *Miconia rubescens *fruits (“SOLO”) to feed birds, or a combination of *M. rubescens and* Solanum sp. fruits (“MIXED”). Analyses revealed no significant differences in germination outcomes between the two, therefore all data associated with gut-passage trials involving *M. rubescens, *were treated identically in our reported analyses (for comparing germination outcomes between the “gut-passed” treatment and “non-digested” treatment). * Treatment: Denotes whether seeds in each trial were associated with the ND (non-digested) treatment, which were extracted from fruits, or the GP (gut-passed) treatment, in which seeds were extracted from manakin feces during field gut passage trials. - Plant- Denotes which species of fruits were fed to manakins for each gut-passage trial. * Date: Which calendar day (month, day, year: 00/00/00) each paired trial was performed (a gut-passage trial, in which seeds were extracted from feces, and seeds extracted from the same *Miconia* plant, for the non-digested treatment). - Total # Seeds Germinated: The number of seeds that had germinated in a petri dish on a given seed-counting day. * Percent Germination: The proportion of seeds that had germinated on each petri dish corresponding to a paired trial. The second sheet “Complete 20-Day Intervals” within the file “Rompf et al.2024 Supporting Data”, connects percent germination from sheet 1 (“Germ over time-original data”), to the days elapsed since seeds were planted in each petri dish. The data was then narrowed down so that the number of days elapsed since planting (with that counting day’s associated percent germination at that time) could be classified into the closest temporal categories of either: 20 days post-planting, 40 days post-planting, 60 days post-planting, or 80 days post-planting (where “post-planting” means any time following when seeds from each treatment were put into their designated petri dish for a given paired trial. Since seeds were not counted on a daily basis, this allowed for the percent germination to be analyzed between treatments over time in consistent intervals of time across trials. The columns in “Complete 20-Day Intervals” are defined as follows: * Date: defined identically to previous sheet (above) - Trial Code: a code for each row with a combination of the following information in the format “Trial number - Treatment (either GP for gut-passed or ND for non-digested – Plant (Miconia)”. For example, “1-GP-Miconia”. * Treatment: defined identically to previous sheet (above) - Sex/Plumage: Listed as either the known sex of the manakin used in a given gut passage trial (always “Adult Male”, based on characteristic mature male plumage) or “Green”, as either immature manakins or mature females have solid green plumage. - Bird Species: defined identically to previous sheet (above) - Percent Germination: defined identically to previous sheet (above) - Time Elapsed: The number of days that had passed since seeds from a paired trial (a gut passage trial and seed extraction from fruit) occurred and were planted in petri dishes. - 20-Day Interval: Because seeds were not checked for germination on a daily basis, for analysis, we used percent germination recorded closest to the desired 20-day interval for analyses (20-day time point mean ± SD: 20.17 ± 1.14 days; 40-day time point: 40.32 ± 1.60 days; 60-day time point: 60.80 ± 2.01 days; 80-day time point: 80.20 ± 2.69 days). All seeds were checked for germination on the same days, so the distance from a given 20-day interval did not differ within an experimental-control pair.           Missing data: NA ## Code/software To see the R code used to generate results from the above data files, please see the included file titled: GP_Code (Rompf et a;. 2024).Rmd.