Author names and contact details:

Vanya G. Rohwer 1 [vgr7@cornell.edu],  Jennifer L. Houtz 2,4 [jhoutz@allegheny.edu], Maren N. Vitousek 2 [mnv6@cornell.edu], Robyn L. Bailey 3 [rb644@cornell.edu], Eliot T. Miller 3 [etm45@cornell.edu]

1) Cornell University Museum of Vertebrates, Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY, 14850

2) Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY, 14850

3) Cornell Lab of Ornithology, Ithaca, NY, 14850

4) Department of Biology, Allegheny College, Meadville, PA, 16335

Brief summary of study:

This study examines a unique breeding behavior in birds: the incorporation of shed snake skin into nests. We probe this behavior using both comparative and experimental approaches. Briefly, this behavior is largely restricted to passerines and species that nest in cavities are reported to use this material most often. Experimental approaches revealed that shed snake reduced nest predation in cavity, but not open cup nests. This suggests that the fitness benefits of using shed snake skin may be unequal across nest morphologies, helping to explain why this behavior is most commonly observed in cavity nesting species.

Personnel responsible for analyses and data collection:

Vanya G. Rohwer: performed experiments, gathered data and conducted analyses for predation analyses, nest ectoparasite analyses, nest record card analyses, and social signaling analyses.

Jennifer L. Houtz: performed experiments, gathered data and conducted analyses for nest microbiome analyses.

Eliot T. Miller: gathered data and conducted analyses for comparative analyses.

Summary of files:

Below, we describe analyses, data files and their columns, and scripts used in analyses. ANALYSES are indicated by capitalized bold font, data files and scripts for each analysis are indicated by lower case bold font, and column names for data files are indicated by italic font. Scripts have in-line annotations.

PREDATION ANALYSES

This analysis experimentally tests if the effect of shed snake skin in reducing nest predation differs between cavity and open cup nests. Data for this experiment come from artificial nests.

R script: predation.R

This is the R script used for nest predation analyses.

Data file: predation.csv

nest_id: unique identifier for each nest in this experiment.

type: describes nest type used in this experiment as either cavity or cup.

treatment: EXP nests received 20-30 cm of shed snake skin; CON nests received no shed snake skin.

treatmentczeo: binary coding for EXP and CON nests where EXP=1 and CON=0.

fate_1dep_0suc: binary coding for nest survival where 1=nests that were depredated and 0=nests that were not depredated and assigned as successful.

days_till_dep: the number of days between the start of the experiment and when a nest was depredated; 14 days represents successful nests.

height: a continuous variable for cup nests only; height is measured in inches above the ground; all nest boxes were placed 4-5 meters above the ground.

NEST ECTOPARASITE ANALYSES

This analysis experimentally tests for differences in the number of ectoparasites in cavity nests with or without shed snake skin. The species used in this analyses, Tree Swallow and Eastern Bluebird, do not normally use shed snake skin in nest construction.

R script: ectoparasites.R

This is the R script used for nest ectoparasite analyses.

Data file: ectoparasites.csv

nest_id: unique identifier for each nest in this experiment.

clutch_size: number of eggs in nest.

ss: the treatment column. Did the nest receive 20-30 cm of shed snake skin: Yes or No; in other words yes=EXP (nest received snake skin), no=CON (nest did not receive snake skin).

species: EABL=Eastern Bluebird, TRES=Tree Swallow.

first_egg_date: date the first egg was laid.

day_of_year: date the first egg was laid converted to day of year calendar (where January 1st = day 1).

nest_survive_support_parasites: sorting column; if a nest survived past nestling day 4 it is coded as “yes” and included in our analysis. Nests that did not survive beyond nestling day 4 were coded as “no” and not included in our analysis.

numb_fleas: this column summarizes the total number of fleas found in nest dissections.

numb_ticks: this column summarizes the total number of ticks found in nest dissections.

numb_big_blowfly: this column summarizes blowfly larvae in their 3rd and older larval instars and pupated larvae.

total_blowflies: this column summarizes numbers of all blowfly larvae found within a nest.

NEST RECORD CARD ANALYSES (proportion of snake skin use in nests among cavity- vs cup-nesting species)

This analysis examines for differences in the proportion of (unmanipulated) nests containing shed snake skin between open cup and cavity nesting birds, all of which have been reported to use shed snake skin in nest construction. All data for this analysis come from nest record cards at the Western Foundation of Vertebrate Zoology.

R script: nest_record_cards.R

This is the R script used to analyze nest record cards.

Data file: nest_record_cards.csv

species: Focal species of bird.

total_cards: The total number of scanned nest record cards (1 card per nest) we examined and includes nests with and without descriptions of nesting materials.

usable_cards: The number of cards that included a description of nesting materials. This column represents the total number of cards we used to calculate the frequency of snake skin use for a given species.

cards_with_SS: The number of cards that included shed snake or reptile skin in the description of nesting materials.

frequency: This column gives the frequency that a species was reported to use shed snake skin in nest construction and was calculated as: (cards.with.SS/usable.cards).

nest_type: This column summarizes if a species nests in cavities or constructs a cup nest.

SOCIAL SIGNALING ANALYSES

This analysis compares clutch sizes between (unmanipulated) nests with and without shed snake skin, among species all of which have been reported to use shed snake skin in nest construction. All data for this analysis come from nest record cards at the Western Foundation of Vertebrate Zoology.

R script: social_signaling.R

This is the R script used for social signaling analyses.

Data file: social_signaling.csv

This spreadsheet contains three columns per species: “catalog_species code”, “snake_skin_species code”, and “clutch_size_species code”. The species codes are as follows: SLSP = Slaty Spinetail; BEWR = Bewick’s Wren; BLGR = Blue Grosbeak; GCFL=Great Crested Flycatcher; HOWR=House Wren; TUTI=Tufted Titmouse.

As an example for one species, SLSP (Slaty Spinetail):

catalog_SLSP:  This column provides the unique ID# from the Western Foundation of Vertebrate Zoology for each nest.

snake_skin_SLSP:  This column summarizes if snake skin was reported in the nest (yes or no).

clutch_size_SLSP:  This column provides clutch size data from nest record cards for each nest.

 

COMPARATIVE ANALYSES

These analyses explore intraspecific variation in the proportion of nests that contain snake skin, using a subset of North American species, all of which have been reported to use snake skin in nest construction.

R script: script_corHMM_plusExtras.R

This is the R script that all comparative analyses are conducted with the following data files.

Data file: Stage2_Hackett_MCC_no_neg.tre

This is the tree file used in our comparative analyses; the tree comes from BirdTree.org, Jetz et al. 2012.

Data file: cavities.csv

This file is a csv of the "cavities" worksheet from van der Hoek 2017’s database of cavity nesting birds. Columns are as follows:

Order: Order to which individual bird species belongs.

Family: Family to which individual bird species belongs.

Name: Common English name of species.

Scientific Name: Scientific name of species.

Size (cm):  Size range of cavities used for breeding.

Cavity nester type: categorical variable describing if a species excavates their nesting cavity, uses existing cavities, etc.

Reference: Sources used by van der Hoek 2017.

Obligate or Facultative: categorical variable describing if a species always nest in cavities (i.e., obligate).

Occasional: Sorting column indicating if a species nest “occasionally” in cavities.

Excavated: Sorting column indicating species that excavate their cavities.

Decay-formed: Sorting column indicating species that nest in natural, decay-formed cavities (i.e., cavity formed naturally by a rotten tree limb).

Excavator species: Indicates potential species/groups of birds that initially excavated cavities that non-excavating species use.

Status: Categorical variable assessing population health (i.e., vulnerable, threatened, etc.).

Nearctic: Geographic area a species is found.

Palearctic: Geographic area a species is found.

Neotropical: Geographic area a species is found.

Afrotropical: Geographic area a species is found.

Oriental: Geographic area a species is found.

Australasia: Geographic area a species is found.

Data file: fixed.csv

This file summarizes 79 mismatches in taxonomy between the cavities data set (described above) and eBird taxonomy. Columns are as follows:

db.name: Scientific name reported for a species in van der Hoek (2017)’s cavities datafile.

change.to: Scientific name reported for the same species but using the eBird taxonomy.

direct.to.ebird: Mismatches in species status between taxonomies.

Data file: fixedCavities.csv

This file largely mirrors the "cavities" database from van der Hoek 2017 (above) except mismatches in species names between taxonomies are now fixed. Column names follow those described above for datafile “cavities.csv” with the following additions:

ebird.name: Scientific name recognized by eBird.

jetz.name: Scientific name recognized by Jetz et al. 2012.

 
Data file: currentJetzEbird3Feb2019.csv

This file helps reconcile conflicting taxonomies between: BirdTree.org (Jetz et al. 2012), the International Ornithological Congress’s world bird list (IOC), and eBird.

jetz.etm.assigned: Avian taxonomy from Jetz et al. 2012.

IOC_7.3: Avian taxonomy from IOC.

ebird_v2017_18Aug2017: Avian taxonomy from eBird.

marshall.notes: taxonomy notes column from master taxonomist, Marshall Ilif.

Data file: ebird_taxonomy_v2022.csv

This file is eBird’s avian taxonomy data file.

TAXON_ORDER: A numerical value assigned to each species; used for sorting.

CATEGORY: Categorical variable describing species status (i.e., “species” = species, “issf” = subspecies, “spuh” = species group only (i.e., ducks), etc.).

SPECIES_CODE: 6-letter code for each species within eBird.

PRIMARY_COM_NAME: Common name assigned to species.

SCI_NAME: Scientific name assigned to species.

ORDER1: Order to which species belong.

FAMILY: Family to which species belong.

SPECIES_GROUP: Grouping variable by species category (i.e., all Rhea species are assigned “Rheas”, all Duck species are assigned “Ducks”, etc.).

REPORT_AS: Species code for subspecies that lack official species status.

Data file: HBW3.0-ebirdv2018match4Feb2019.csv

This data file helps reconcile differences in taxonomy between Handbook of Birds of the World (HBW)/eBird (Clements) and Avibase.

orig_sort: Sorting column based on numerical value.

AvibaseID: Unique identifier for each bird species within the Avibase taxonomy.

Avibase_latin_name: Scientific name for bird species as assigned in Avibase.

cat_hbw: Categorical variable describing species status between Avibase and Clements taxonomy (i.e., grp = group (as in “Ducks”), sp = species (as in “Northern Pintail”), etc.).

TAXON_nid: Unique numerical identifier for each bird species.

hbw_latin: Scientific name for species assigned in Handbook of Birds of the World (HBW).

hbw_english_for_eBird: Common name for species assigned in HBW.

HBW-Clem: taxonomic congruency between HBW and Clements taxonomy.

match: sorting column indicating matches in taxonomic congruency between HBW and Clements taxonomy.

clements_latin: Scientific name for species assigned by Clements.

clements_english: English names for species assigned by Clements.

eBird code: unique, species identifiers in eBird taxonomy.

cat_Clements: species status within Clements taxonomy (i.e., grp = group, sp = species, hybr = hybrid, etc.).

cl_sort: unique identifier for sorting Clements taxonomy.

species (HBW or Clements): sorting column to identify species recognized by HBW or Clements (1 = recognized species).

Data file: fromVanya23Mar2023.csv

This file summarizes data for all the species reported to use shed snake skin in bird nests.

Species: Common English names of birds.

Scientific names: Scientific names of birds.

Family: Family to which birds belongs.

Suboscines/Oscines: categorical variable; both clades belong to the Passeriformes order and are distinguished by how they learn/inherit their songs.

nest type: Categorical variable describing the type of nest constructed by each species.

placement of skin: Categorical variable describing where shed snake skin is placed within a nest.

type of skin: Categorical variable describing source of skin (snake or lizard).

sex building nest: If known, which member of the pair builds the nest (male, female, both).

estimate of frequency of skin use: If provided in the literature, the frequency of nests within a species reported to use snake skin.

source: Literature documenting use of snake skin for each species.

Data file: snakeSppToFix.csv

This file summarizes 20 species reported to use shed snake skin (from above data file “fromVanya23Mar2023.csv”) where taxonomies between HBW and Jetz et al. 2012 are inconsistent. This data file has only a single column to ID the species that need updates to their names.

x: A column summarizing species with mismatches in taxonomy.

Data file: snakeSppFixed.csv

This file provides the updated taxonomies for the 20 species reported in “snakeSppToFix.csv”.

orig: Initial taxonomy that was inconsistent with Jetz et al. 2012.

new: Corrected taxonomy for these 20 species.

MICROBIOME ANALYSES

This analysis explores how the nest microbiome differs across a) unmanipulated nests of multiple bird species that vary in their nest type, b) different nest types in a single species (American Robin), and c) nests of Eastern Bluebirds and Tree Swallows that had snake skins experimentally placed in the nest.

 
QIIME2 script: microbiome_qiime2_code.txt

This file contains the code that is used in terminal/command line in QIIME2 (v 2021.11) for the first half of microbiome analyses.

R script: microbiome.R

This is the R script that is used after the QIIME2 steps for the remainder of the nest microbiome analyses.

 
Data files for microbiome analyses in QIIME2:

 
FASTQ microbiome sequences

These are the raw demultiplexed forward and reverse sequences for each microbiome sample are available as fastq.gz files. These are uploaded to QIIME2 using the microbiome_sequence_manifest.csv file.

 
Data file: microbiome_sequence_manifest.csv

This is the manifest file that matches the file names of the raw FASTQ sequences to the sample names for importing them into QIIME2. Columns are as follows:

sample-id: sample name that corresponds to samples names in the microbiome metadata file "microbiome.csv" that is used in QIIME2 and R analyses.

absolute-filepath: where the user will input the absolute filepath name for each sequence.

direction: direction of the read (forward or reverse).

Data files for microbiome analyses in R:

Data file: unrarefied_ASV_table.csv

This is the amplicon sequence variant (ASV) table that is outputted from QIIME2 and uploaded to R using the phyloseq package. Each column represents a sample name.

ASVID: unique code assigned to each ASV.

Data file: taxonomy_microbiome.csv

This is the taxonomy assigned to each ASV that is outputted from QIIME2 and uploaded to R using the phyloseq package.

ASVID: unique code assigned to each ASV.

Taxon: microbial taxonomic assignments (phylum to genus) for each ASV

Data file: tree_microbiome.nwk

This is the microbial rooted tree that is outputted from QIIME2 and uploaded to R using the phyloseq package.

Data file: microbiome.csv

This is the metadata for each microbiome sample (rows are sample names that correspond to names of sequences found in the "microbiome_sequence_manifest.csv" file). Columns are as follows:

sample_name: sample name that corresponds to raw sequences within the "microbiome_manifest.csv" file.

nest: unique identifier for each nest in this experiment.

incubation_time_point: early (EI) or late (LI) incubation stage at the time sample was collected.

treatment_incubation_timepoint: combination of treatment of snake skin (EXP) or control (CON) for nests in the snake skin placement experiment and early (EI) or late (LI) incubation stage at the time the sample was collected.

date_extracted: date that DNA was extracted from the swab.

sample_or_control: indicates if the sample is a real sample or a negative control (used for decontamination step in R).

quant_reading: DNA quantification (ng/ul) (used for decontamination step in R).

site: field site where the nest was located.

species: bird species of the nest where the swab was taken.

experiment: "species_swab" refers to nests that were only swabbed once without any snake skin manipulation; "snake_skin_experiment" refers to Eastern Bluebird and Tree Swallow nests that were part of the snake skin manipulation experiment.

treatment: treatment of snake skin (EXP) or control (CON) for nests in snake skin placement experiment

nest_type: summarizes if a species nests in cavities or constructs a cup nest.

placement: where the nest was physically placed by the birds.

egg_number: number of eggs within the nest where the swab was taken.

swab_date: date that the sample was collected.

date_found: date that the nest was found.